Generation and characterization of antibodies for proteomics ...275849/...KARIN LARSSON iii Karin Larsson (2009): Generation and characterization of antibodies for proteomics research

Generation and characterization of antibodies for proteomics research

Karin Larsson

Royal Institute of Technology School of Biotechnology

Stockholm 2009

ii GENERATION AND CHARACTERIZATION OF ANTIBODIES FOR PROTEOMICS RESEARCH

© Karin Larsson Stockholm 2009 Royal Institute of Technology School of Biotechnology Division of Proteomics AlbaNova University Center SE-106 91 Stockholm Sweden Printed by Universitetsservice US-AB Drottning Kristinas väg 53B SE-100 44 Stockholm Sweden ISBN 978-91-7415-439-9

TRITA-BIO Report 2009:21 ISSN 1654-2312

Cover illustration: Confocal image of immunofluorescently stained A-431 cells, Western blot image and immunohistochemical staining of colon.

KARIN LARSSON iii

Karin Larsson (2009): Generation and characterization of antibodies for proteomics

research. School of Biotechnology, Royal Institute of Technology (KTH), Sweden.

Abstract Specific antibodies are invaluable tools for proteomics research. The availability of thoroughly validated antibodies will help to improve our understanding of protein expression, localization and function; fundamental processes and features of all living organisms. The objectives of the studies in this thesis were to develop high-throughput methods to facilitate the generation and purification of monospecific antibodies, and to address problems associated with antigen selection for difficult target proteins and subsequent validation issues.

In the first of the studies, it was demonstrated that antibodies specific to human proteins could be generated in a high-throughput manner using protein epitope signature tags (PrESTs) as both antigens and affinity ligands. A previously developed purification process was adapted to a high-throughput format and this, in combination with the development of a protein microarray assay, resulted in monospecific antibodies that were used for profiling protein expression in 48 human tissues. Data obtained in these analyses suggest that a complete Human Protein Atlas should be attainable within the next ten years. In order to reduce the number of animals needed for such a massive project, and improve the cost-efficiency of antibody generation, a multiplex immunization strategy was developed in a further study. Antisera from rabbits immunized with mixtures of two, three, five and up to ten different PrESTs were successfully purified and analyzed for specificity using protein arrays. Almost 80% of the animals immunized with up to three PrESTs yielded antibodies towards all the PrESTs administered, and they yielded comparable immunohistochemical staining patterns (of consecutive human tissue sections) to those of antibodies obtained from traditional single PrEST immunizations.

Proteins with highly similar sequences to other proteins present a major challenge for the proteome-wide generation of antibodies. In another study, Cytokeratin-17 which displays high sequence similarity to closely related members of the intermediate filament family, was used as a model and the specificity and cross-reactivity of antibodies generated against this target were investigated using epitope mapping in combination with comparative IHC analyses. Antibodies identified by epitope mapping as binding to the most unique parts of the Cytokeratin-17 PrESTs also showed the most Cytokeratin-17-like staining pattern, thus further supporting the strategy of using sequence identity scores as the main criteria for PrEST design.

An alternative antigen design strategy was investigated for use in raising antibodies towards G-protein-coupled receptors (GPCRs). The extracellular loops and N-terminus of each of three selected GPCRs were assembled to form single antigens and the resulting antibodies were analyzed by flow cytometric and confocal microscopic analyses of cell lines over-expressing the respective receptors. The results from both flow cytometric and immunofluorescence analyses showed that the antibodies were able to bind to their targets. In addition, the antibodies were used successfully for the in situ analysis of human brain and pancreatic islet cells.

Keywords: antibody, antibody validation, immunohistochemistry, immunofluorescence, tissue

microarray, Western blot, epitope mapping, antigen design

© Karin Larsson

iv GENERATION AND CHARACTERIZATION OF ANTIBODIES FOR PROTEOMICS RESEARCH

KARIN LARSSON v

List of publications This thesis is based upon the following four papers, which are referred to in the text by the corresponding roman numerals (I-IV). The papers are included in the Appendix.

I Nilsson P., Paavilainen L.*, Larsson K.*, Ödling J.*, Sundberg M.

Andersson A., Kampf C., Persson A., Al-Khalili Szigyarto C., Ottosson J., Björling E., Hober S., Wernérus H., Wester K., Pontén F. and Uhlén M. (2005) Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling. Proteomics. 5, p. 4327-4337

II Larsson K., Wester K., Nilsson P., Uhlén M., Hober S., Wernérus H.

(2006). Multiplexed PrEST-immunizations for high-throughput affinity proteomics. J. Immunol. Methods. 315, p. 110-120

III Larsson K., Eriksson C., Schwenk J.M., Berglund L., Wester K., Uhlén

M., Hober S., Wernérus H. (2009). Characterization of PrEST-based antibodies towards human Cytokeratin-17. J. Immunol. Methods. 342 p. 20-32

IV Larsson K., Hofström C., Lindskog C., Hansson M., Angelidou P.,

Hökfelt T., Uhlén M., Wernérus H., Gräslund T., Hober S. (2009) Novel antigen design for the generation of G-protein-coupled receptor antibodies. Manuscript

*These authors contributed equally to this work. All papers are reproduced with the kind permission of the respective copyright holders.

vi GENERATION AND CHARACTERIZATION OF ANTIBODIES FOR PROTEOMICS RESEARCH

Related publication

Uhlén M., Björling E., Agaton C., Szigyarto C. A., Amini B., Andersen E., Andersson A. C., Angelidou P., Asplund A., Asplund C., Berglund L., Bergström K., Brumer H., Cerjan D., Ekström M., Elobeid A., Eriksson C., Fagerberg L., Falk R., Fall J., Forsberg M., Björklund M. G., Gumbel K., Halimi A., Hallin I., Hamsten C., Hansson M., Hedhammar M., Hercules G., Kampf C., Larsson K., Lindskog M., Lodewyckx W., Lund J., Lundeberg J., Magnusson K., Malm E., Nilsson P., Ödling J., Oksvold P., Olsson I., Öster E., Ottosson J., Paavilainen L., Persson A., Rimini R., Rockberg J., Runeson M., Sivertsson A., Sköllermo A., Steen J., Stenvall M., Sterky F., Strömberg S., Sundberg M., Tegel H., Tourle S., Wahlund E., Waldén A., Wan J., Wernérus H., Westberg J., Wester K., Wrethagen U., Xu L. L., Hober S., Pontén F. (2005) A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol Cell Proteomics. 4(12) p.1920-1932

KARIN LARSSON vii

Contents

Introduction .............................................................................................................................................. 1

1. Proteomics........................................................................................................................................ 2

1.1 Mass spectrometry-based proteomics ................................................................................... 3

1.2 Affinity-based proteomics ....................................................................................................... 4

1.2.1 Human Proteome Resource............................................................................... 5

1.2.2 Other affinity-based proteomics projects ........................................................ 7

2. Affinity reagents............................................................................................................................. 10

2.1 Antibodies ................................................................................................................................ 11

2.1.1 Polyclonal antibodies ........................................................................................ 12

2.1.2 Monoclonal antibodies ..................................................................................... 14

2.1.3 Recombinant antibodies ................................................................................... 14

2.2 Alternative protein scaffolds ................................................................................................. 16

Experimental techniques....................................................................................................................... 19

3. Methodology .................................................................................................................................. 20

3.1 Antibody purification ............................................................................................................. 20

3.1.1 Fc-binding proteins ........................................................................................... 20

3.1.2 Antigen-based affinity purification ................................................................. 21

3.2 Methods for antibody characterization and their use in proteomics research .............. 23

3.2.1 ELISA.................................................................................................................. 23

3.2.2 Western blotting ................................................................................................ 25

3.2.3 Immunohistochemistry and Tissue Microarrays .......................................... 25

3.2.4 Immunofluorescence microscopy................................................................... 27

3.2.5 Flow cytometry .................................................................................................. 28

3.2.6 Epitope mapping ............................................................................................... 28

Present Investigation ............................................................................................................................. 31

4. Objective......................................................................................................................................... 32

5. High-throughput generation of monospecific antibodies (Paper I) ...................................... 34

6. An alternative immunization strategy using multiplexed PrEST-antigens (Paper II)......... 38

7. Generation and validation strategies for antibodies raised towards targets with highly

similar sequences (Paper III) ....................................................................................................... 43

8. An alternative strategy for designing antigens for membrane-bound receptors

(Paper IV) ....................................................................................................................................... 48

viii GENERATION AND CHARACTERIZATION OF ANTIBODIES FOR PROTEOMICS RESEARCH

9. Concluding remarks ...................................................................................................................... 53

References ............................................................................................................................................... 57

Acknowledgements................................................................................................................................ 64

KARIN LARSSON ix

Abbreviations 2-DE Two-dimensional gel electrophoresis ABP Albumin binding protein AP Alkaline phosphatase CDR Complementarity-determining region ELISA Enzyme-linked immunosorbent assay Fab Fragment, antigen binding Fc Fragment, crystallizable FACS Fluorescence-activated cell sorting HAMA Human anti-mouse antibody HIAR Heat-induced antigen retrieval HPR Human Proteome Resource HRP Horseradish peroxidase HUPO Human Proteome Organisation IEF Isoelectric focusing IF Immunofluorescence IHC Immunohistochemistry IMAC Immobilized metal ion affinity chromatography IPG Immobilized pH gradient mAb Monoclonal antibody MALDI Matrix-assisted laser desorption/ionization MS Mass spectrometry msAb Monospecific antibody pAb Polyclonal antibody pI Isoelectric point PrEST Protein Epitope Signature Tag PTM Post-translational modification RT-PCR Reverse transcriptase polymerase chain reaction ScFv Single-chain variable fragment SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SpA Staphylococcal Protein A SpG Streptococcal Protein G TOF Time-of-flight TMA Tissue Microarray

x GENERATION AND CHARACTERIZATION OF ANTIBODIES FOR PROTEOMICS RESEARCH

KARIN LARSSON 1

Introduction

2 GENERATION AND CHARACTERIZATION OF ANTIBODIES FOR PROTEOMICS RESEARCH

1. Proteomics

The completion of the human genome sequence [4, 5] has allowed genome-wide studies,

which have uncovered major features of our genetic code (e.g. less than 2% is protein-

encoding and different structural variations such as single-nucleotide polymorphism)

furthering our understanding of biology. The study of gene expression, transcriptomics, has

added further to this knowledge through discoveries such as non-coding RNA and small

RNAs with regulatory properties [6]. Even though this has generated important information

about the causes of and/or susceptibility of individuals to many diseases [7, 8], further studies

of protein function are required to fully understand cell organization and function. This is

because the products of gene expression, the proteins, are the effectors of most tasks in the

cell, in addition to constituting about 80% of all drug targets [9].

The term proteome was first used by Mark Wilkins, in 1996 [10], for the entire set of proteins

expressed by a cell, tissue or organism at a given time point. Proteomics is the study of the

proteome, and involves the large-scale study of proteins, particularly their structures, biological

functions and interactions with other proteins. Currently, there are two distinct branches of

proteomics; mass spectrometry-based proteomics and affinity-based proteomics.

Huge financial and technical resources have been expended in sequencing the human genome,

leading to a number of breakthroughs (e.g. new sequencing technologies), but deciphering the

function of all proteins is even more challenging. Many factors influence the proteome that

cannot be identified or characterized by whole genome or transcriptome analysis, including

mRNA degradation and translational efficiency, alternative splicing, post-translational

modifications (PTMs) and protein degradation rates [11]. The number of protein-coding genes

is estimated to be around 20500 [12, 13]. If alternative splicing and PTMs are included there

are estimated to be over 100 000 proteins variants [14], greatly increasing the complexity.

Other problems facing proteomics researchers trying to analyze the whole proteome are posed

by the huge range of abundances of proteins, spanning several orders (7-8) of magnitude in

tissues and even more (>10) in plasma samples [11, 15]. Abundant proteins, such as albumin in

plasma, tend to mask those that are less abundant in analyses by many techniques. Therefore,

there are substantial challenges when using currently available proteome profiling and

detection techniques. Indeed, prefractionation and removal of highly abundant proteins is a

requirement when applying existing methods. In addition, unlike nucleic acids, proteins have

KARIN LARSSON 3

high biochemical heterogeneity, making analysis of all proteins by any single method

unfeasible.

1.1 Mass spectrometry-based proteomics

Two-dimensional gel electrophoresis (2-DE), in combination with mass spectrometry, is highly

associated with proteomics research. It was in 1975 that separation of proteins in complex

mixtures was made possible with the introduction of high-resolution two-dimensional gel

electrophoresis (2-DE) [16-18]. Subsequent technical improvements, like the introduction of

immobilized pH gradients (IPGs) [19, 20], have allowed the resolution of over 5000 proteins

simultaneously with current technologies [21]. 2-DE provides scientists with knowledge of

protein expression levels, post-translational modifications and protein isoforms. In the first

dimension proteins are separated using isoelectric focusing (IEF) based on differences in their

isoelectric points (pI) and then separated in the second dimension, according to their masses

using SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis). Following

visualization of protein spots with suitable staining methods (e.g. Coomassie blue or silver

staining), spots are excised from the gel, enzymatically digested and extracted into a liquid

phase. The protein digests obtained are then analyzed by mass spectrometry. A major

advantage with 2-DE is its ability to simultaneously analyze the expression of proteins in

complex protein mixtures, such as whole cell lysates and tissue lysates. Additionally there is the

possibility of detecting changes in protein expression levels, protein isoforms and

posttranslational modifications, such as phosphorylations and glycosylations [21]. Limitations

with this technology include difficulties in separating very hydrophobic proteins like cell

surface receptors, automation problems and the need for time-consuming optimization,

depending on the type of proteins to be studied [22]. Moreover, it is necessary to reduce the

complexity of samples using prefractionation methods and to deplete them of highly abundant

proteins in order to detect the fractions containing less abundant proteins.

Mass spectrometry (MS) provides highly sensitive and accurate mass determinations of

biomolecules. It is a versatile tool in proteomics research with a wide range of applications,

including protein identification, quality control of recombinant proteins and studies of post-

translational modifications [23]. The most prominent use of MS is in combination with 2-DE,

in which masses of enzymatic digests of proteins are determined and peptide mass

“fingerprints” are obtained [24-26] for comparison with entries in available protein databases.


Various combinations of ionization techniques, mass analyzers and detectors may be used. The

ionization sources used for protein research are electrospray [27] and matrix-assisted laser

desorption/ionization (MALDI) systems [28], since their relatively gentle modes of action

permit analysis of large polypeptides. These ionization systems are combined with different

mass analyzers in order to separate the ions formed, depending on the application. This mass

separation can be accomplished with time-of-flight (TOF), quadrupole, or ion trap mass

spectrometers, as well as combinations of these instruments.

Mass spectrometry is also used without 2-DE in proteomics research sometimes, e.g. in

shotgun proteomics [29], a method devoid of time-consuming gel handling, in which whole

protein samples are enzymatically digested in solution with no prior separation on gels.

Following digestion, the peptides are separated by liquid chromatography and then detected

using tandem mass spectrometry. Other applications of MS include detection of protein-

protein interactions, where tagged proteins are precipitated and their binding partners

identified according to accurate mass determinations, taking advantage of affinity interactions

but not to the target protein [23, 30].

1.2 Affinity-based proteomics

Although two-dimensional gel electrophoresis combined with mass spectrometry has been

historically synonymous with proteomics research, other approaches, such as affinity-based

proteomics are becoming increasingly important. In affinity-based proteomics the systematic

exploration of the proteome is undertaken by high-throughput generation of various types of

affinity reagents. These binders are valuable tools in a wide range of analyses and applications,

such as: the detection of target proteins in various assays (e.g. Western blots, ELISA, protein

array analyses and immunohistochemical assays) and the purification or pull-down of proteins

or protein complexes from complex mixtures (e.g. serum and tissue extracts) for use in, for

example, protein structure determinations [1].

Creating affinity-binders to the human proteome is considered an overwhelming task. Several

hundred thousand binders would be needed in order to achieve complete coverage of the

proteome, especially if protein splice variants and post-translational modifications and the

possible need for several binders to each target (in order to perform ‘sandwich’ assays and for

validation purposes) is taken into account. Therefore, ProteomeBinders, a European

KARIN LARSSON 5

consortium was formed (amongst others) with the aim of coordinating and standardizing

independent efforts to produce well-validated affinity binders for proteomics research [31].

A key issue in affinity proteomics is to ensure the quality of the binders generated. This can be

addressed in various ways. Bioinformatics can be used to minimize possible cross-reactivity by

selecting antigens that are not similar to other human proteins, by performing Blast searches (if

working with peptides or parts or proteins as antigens) [32, 33] or by selecting antibodies via

epitope mapping that bind to dissimilar regions (Paper III). Analytical techniques like Western

blotting are also useful for determining whether developed affinity-reagents bind specifically to

their full-length target proteins. In situ hybridization permits comparison of levels of RNA and

protein expression. Although large data sets have already been published, RNA and protein

levels do not necessary correlate [11, 34, 35]. Comparative protein expression analysis using

tissue microarrays and several binders to the same target is perhaps the most favorable strategy

available to validate novel affinity reagents, especially when combined with literature searches.

In particular, when working with binders of proteins of unknown function and cellular

distribution, a careful approach to ensure quality is to produce several binders to non-

overlapping epitopes and compare results obtained from different binding assays [36, 37].

Access for the scientific community to well-characterized affinity binders to all human proteins

would greatly improve and facilitate research on protein function and biomarker discovery.

1.2.1 Human Proteome Resource

The overall objective of the Human Proteome Resource (HPR), an antibody-based proteomics

program, is to generate affinity-binders (monospecific antibodies) to all non-redundant

proteins in the human proteome, i.e. one representative protein per gene locus, and to compile

a publicly available database with data showing the expression and localization of these

proteins in a wide range of human tissues, cell lines and cancer types [38]. Generation of

monospecific antibodies (msAbs) is carried out using Protein Epitope Signature Tags (PrESTs)

as antigens [39]. The PrESTs are about 100-150 amino acid long polypeptide sequences

derived from the human genome sequence retrieved from the Ensembl database [13]. A

suitable region of the open reading frame is selected, using a bioinformatics tool [32], based on

a set of criteria such as: low identity to other human proteins and absence of regions of low

complexity, transmembrane regions, signal peptides and restriction sites. Once a PrEST has

been selected, primers are designed and the gene fragments are amplified using the reverse

transcriptase polymerase chain reaction (RT-PCR) utilizing different mRNA pools. Following


Figure 1. An overview of the production flow in the Human Proteome Resource (HPR) project. Protein

Epitope Signature Tags (PrESTs) are designed using a bioinformatics tool, cloned into an expression

vector, produced in E. coli and used to immunize New Zealand White rabbits. Monospecific antibodies

are then generated in a two-step purification method followed by thorough validation. The quality-

assured antibodies are subsequently used for expression and localization studies by tissue microarray

analyses and immunofluorescence microscopy.

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cloning of the amplicon into a destination vector and sequence verification, the PrESTs are

expressed in Escherichia coli as fusions with a hexa-histidine (His6) tag, for purification of the

antigen, and an albumin binding protein (ABP) for enhanced solubility [40]. Purification of the

His6-tagged proteins is performed with a semi-automated approach [41] using Immobilized

Metal ion Affinity Chromatography (IMAC) [42]. In addition, the fusion partner, His6-ABP, is

expressed and purified for later use in purification of the antibody. Following antigen

preparation, the His6ABP-PrEST fusions are used to immunize rabbits and the resultant

antisera are affinity-purified using a two-step protocol (see Figure 1). First, tag-specific

antibodies are depleted from the antisera using His6ABP-coupled affinity columns and the

flow-through is subsequently purified on a second affinity column coupled with the respective

His6ABP-PrEST fusion protein previously used for immunization [43]. The monospecific

antibodies are initially analyzed on protein arrays consisting of 384 purified PrEST fusions in

order to ensure that no detectable/low cross-reactivity towards other proteins or the fusion tag

remains. Antibodies passing the PrEST array screening are further validated by Western blots

utilizing two cell lines; RT-4 and U-251MG and two human tissues; tonsil and liver, as well as

human plasma. Furthermore, the antibodies are used for protein expression profiling on tissue

microarrays (TMAs), first on a smaller test array and then on arrays consisting of 48 normal

tissues, 20 different cancer types and 47 cell lines. The tissue and cancer moieties of the arrays

are manually annotated by pathologists [44] and the cell arrays are annotated automatically by

TMAx software [45]. Finally, immunofluorescence (IF) analysis using confocal microscopy is

performed utilizing the monospecific antibodies to localize expression of particular proteins to

specific cell compartments. Three different cell lines are used: A-431, U-2OS and U-251MG

[46]. Based on the results obtained from Western blot, IHC and IF analyses, a validation score

is appointed to the antibody and published together with the rest of the results on the Protein

Atlas web site (www.proteinatlas.org).

In addition to creating a map of the human proteome, the enormous volume of data and

images on the Protein Atlas has great potential utility in biomarker research aimed at finding

cell- and tissue-specific protein expression. This could be used for the diagnosis, prognosis and

monitoring of diseases.

1.2.2 Other affinity-based proteomics projects

In addition to the Human Proteome Resource program, other initiatives have been started

with the common goal of producing validated affinity reagents to be made available to the


proteomics community. In this section, I will give a brief summary of current affinity-based

proteomics projects.

The Sanger “Atlas of protein expression” initiative published an article in 2007 in which they

described the selection and validation of a vast number of recombinant antibodies using phage

display and IHC [47]. A phage-antibody library was constructed containing over 1010 clones.

Proteins used as targets for subsequent selection were produced both in bacterial and

mammalian expression systems [48, 49]. A total number of 38000 antibody clones, recognising

290 target proteins, were selected and screened. Following DNA sequencing to reduce

redundancy, the number was reduced to 7200 unique antibody clones and 4400 were selected

for further evaluation. A selection success rate of over 70% enabled generation of 25

antibodies for each target protein. When an antibody had been successfully selected and found

to meet various selection criteria it could be transferred into different expression systems,

allowing a renewable source of antibodies. The recombinant antibodies produced were finally

used to perform immunohistochemical staining of tissue microarrays. Even though time-

consuming and costly screening of the numerous clones was required, preparation of

numerous affinity-binders to the same target protein represents a great advantage in the

validation of these molecules, since protein expression patterns in IHC analyses can be

compared and evaluated as discussed above (section 1.2).

A completely different strategy was employed in a high-throughput antibody generation

project in China [50]. The general idea was to immunize mice against fractionated plasma or

tissue samples in order to produce monoclonal antibodies to proteins of high- and medium-

abundance, thus avoiding the need to produce recombinant proteins. Initially, plasma was

separated by liquid chromatography into three fractions, which were then injected into mice.

Spleen cells from the immunized animals were homogenized and fused to myeloma cells.

Hybridoma cells were selected and screened, and then in subsequent rounds of immunization

the antibodies generated in the first or previous immunizations were utilized in order to

deplete the highly abundant proteins in the plasma fractions. Since the monoclonal antibodies

were produced using unidentified protein mixtures the target proteins then needed to be

identified. This was accomplished with the use of ELISA and Western blot analyses as well as

immunoprecipitation combined with MS. In this project, four immunizations generated 200

strains of monoclonal antibodies and over 30% of these antibodies recognized antigens of

different sizes.

Among other initiatives is the Antibody Factory, a German national collaborative project

aimed at developing in vitro methods for the generation of recombinant antibody fragments

KARIN LARSSON 9

(scFv), and the American Clinical Proteomic Technologies for Cancer Reagents and

Resources, funded by the US National Cancer Institute (NCI) aimed at the high-throughput

generation of monoclonal antibodies. Other new projects aim to coordinate and promote the

generation of well-characterized antibodies, including the HUPO Antibody Initiative and

ProteomeBinders (as mentioned earlier). Also, the National Institutes of Health recently

commenced the Protein Capture Tools program, which strives for high-throughput generation

of monoclonal antibodies [31, 51-53].


2. Affinity reagents

In order to study the functions of proteins and their cellular organization in a systematic

manner, efficient tools are needed for their high-throughput capture, detection and analysis.

Invaluable tools in proteomics research are affinity reagents (i.e. molecules that bind

specifically to intended target proteins) of various types, such as antibodies, antibody

fragments and other non-antibody binders (summarized in Table 1). Access to affinity-binders

is important for many methods for protein analysis such as immunoprecipitation of proteins

and protein complexes, protein localization studies, development of new diagnostic assays and

as potential drugs (e.g. for the inhibition/activation of receptors and delivery of toxins).

A good affinity reagent should be specific for its target, and should not bind to other proteins.

They should also have suitable (usually high) affinity for the target protein and be functional in

different technical platforms. In addition, it is highly desirable for them to be sufficiently stable

to couple to effector molecules such as fluorophores and compatible with available secondary

reagents.

Antibodies with natural variability, stability and high affinity, have historically been the most

widely used affinity reagents. Technical advances in combinatorial display technologies [54]

and the compilation of large antibody libraries have made it possible to select recombinant

Affinity reagent Origin Numbers of epitopes Size

(kDa)

Polyclonal antibody Animal serum Several 150

Mono-specific antibody Affinity-purified animal

serum Several 150

Monoclonal antibody Hybridoma cells Single 150

Recombinant full-length

antibody Combinatorial libraries Single 150

Recombinant antibody

fragments Combinatorial libraries Single 15-55

Alternative scaffolds Combinatorial libraries Single 5-20

Table 1. Summary of affinity reagents discussed. Modified from [1].

KARIN LARSSON 11

antibodies to most proteins and these, therefore, raise the possibility of an alternative source of

high-affinity binders. An advantage of these molecules over polyclonal and monoclonal

antibodies is that no animals are needed for their production, since this has been a limiting

factor when generating binders at the whole proteome scale. Moreover, engineered antibody

fragments could be promising alternatives to entire antibodies, since they still have the same

binding properties, but are much easier to produce in high yield and at low cost in, for

example, bacterial hosts [2]. In addition, protein engineering has provided tools to develop

binders that completely lack the globulin domain, here denoted alternative scaffolds, enabling

innovative design of the binders depending on intended uses and targets.

2.1 Antibodies

Animals are exposed to diverse threats, ranging from invading pathogenic microorganisms to

cancer (i.e. altered self-cells.). As a means of defense the immune system has evolved a

remarkable set of mechanisms. The innate branch of the immune system is the first line of

defense, comprising many different components, such as phagocytic cells, the skin and

antimicrobial substances. It recognizes classes of molecules present on common pathogens

opposed to the more specific, adaptive part of the immune system, which is activated upon

such antigenic challenge and can be divided into cell-mediated and humoral immunity.

Figure 2. Schematic representation of an antibody (IgG). Two heavy chains and two light chains are

connected by disulfide bridges forming a bivalent molecule. The constant fragment (Fc) is responsible

for effector functions while the antigen-binding fragment (Fab), primarily the variable fragment (Fv), is

responsible for antigen binding.

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Effector T cells execute cell-mediated responses by activating phagocytic cells, as well as killing

altered self-cells, such as tumor cells and virally infected cells. Humoral responses rely on B-

cell-antigen interactions leading to proliferation of the B-cells and their differentiation into

antibody-secreting plasma cells, upon activation by T-helper cells. Antibodies or

immunoglobulins exist both as membrane-bound molecules conferring specificity to B-cells,

and as secreted molecules circulating in the blood stream that detect, neutralize or mark

foreign antigens for elimination from the body [55].

An antibody consists of two identical light chains and two identical heavy chains forming a

characteristic Y-shaped molecule held together by disulfide bonds (Figure 2). The ‘arms’ of the

Y-shaped molecule are responsible for antigen binding whereas the ‘foot’ of the molecule

functions as an effector of various significant aspects of the immune response, such as

complement activation, and as a receptor for phagocytic cells. The N-terminal domains of the

heavy and light chains form the most variable part of the antibody and, therefore, denote the

variable (V) region. Most of the amino acid variability is concentrated in three loops of each

chain called complementarity-determining regions (CDRs). The remaining domains of the

heavy and light chains, forming the constant (C) region, show less sequence variability.

Immunoglobulins are divided into different subclasses depending on their heavy chain

sequence. In humans, five different types of heavy chains are present (designated α, δ, ε, γ and

µ), giving rise to five different subclasses: IgA, IgD, IgE, IgG and IgM, respectively. IgG is the

most abundant type of immunoglobulin and hereafter when antibody is mentioned it will refer

to IgG. In addition adaptive immunity has the remarkable feature of immunological ‘memory’,

leading to a fast onset of the defense mechanism after a second exposure to a pathogen. Also,

incredible variability results in the ability to create binders to most immunogenic substances.

This is due to various events which lead to diversification, such as the rearrangement of

somatic gene segments, junctional flexibility and somatic hypermutation [56, 57].

2.1.1 Polyclonal antibodies

A mixture of antibodies with varying affinities and specificities for the immunized antigen is

present in polyclonal antisera. These are derived from non-identical B-cells. Before antibodies

had been discovered the ability of polyclonal antisera to transmit passive immunity from an

immunized to a non-immunized individual was demonstrated by Behring and Kitasato in 1890.

KARIN LARSSON 13

This major breakthrough led to Behring being awarded the first Nobel Prize for research in

immunology in 1901.

Polyclonal antibodies (pAbs) are produced by injecting an antigen into an animal such as a

mouse, rabbit, goat or hen. The choice of animal depends on how much of the antibody is

needed, as well as the phylogenetic relationship between the immunized animal and the

original source of antigen. The bigger an animal the bigger its blood volume and thus the

greater volume of antiserum will be obtained. In addition, the more distant the relationship

between host and antigen, the more immunogenic the antigen should be. The antigen is usually

administered together with an adjuvant such as Freund’s adjuvant, in order to enhance the

immune response and the adjuvant-antigen mixture can be injected through various routes, for

example subcutaneously or intramuscularly. After primary injection and possibly further

booster injections, the blood is collected and centrifuged to remove blood cells and clotting

factors. For egg yolk, several fractionation processes are available such as filtration,

centrifugation and precipitation in order to remove lipids and proteins. In polyclonal antisera,

antibodies specific to the antigen used for immunization comprise approximately 1% of the

total antibody population [14]. To purify these antibodies, the 99% of antibodies that are non-

specific and unwanted can be removed using one or more of the purification strategies

discussed in section 3.1. If the antigen used for immunization is used as an affinity ligand in

purification processes, a mixture of antibodies with varying specificities will still be present in

the purified antiserum, but they will only recognize the target protein and be monospecific, in

terms of their binding to the antigen.

Polyclonal antibodies work well in many technical applications [14]. Since they are able to

recognize several different epitopes on the same target protein, because they contain a mixture

of antibodies, problems with masked or denatured epitopes can be avoided. This kind of

problem occurs during immunohistochemical staining of tissues where cross-linking of

proteins often leads to antibody binding sites being inaccessible [58, 59]. Also, in SDS-PAGE

dependent applications, such as Western blotting, most proteins are denatured, thus destroying

many epitopes.

However, the fact that pAbs are mixtures of antibodies is a major drawback and possibly

hindrance for potential therapeutic applications. The heterogeneity of any polyclonal antiserum

means it is difficult to reproduce it with exactly the same composition. In therapeutics,

therefore, polyclonal antisera are mainly used in replacement therapies where patients are

suffering from immunodeficiency diseases, or for the neutralization of toxins and viruses [60,

61].


2.1.2 Monoclonal antibodies

A major milestone in immunological research was the development of monoclonal antibodies

by Köhler and Milstein in 1975; work for which they were later awarded the Nobel Prize in

Physiology or Medicine [62]. They fused a mouse myeloma cell line with mouse spleen cells

from an immunized donor. The resultant hybridoma cell line possessed the immortal growth

properties of the cancerous plasma cells and the antibody secreting properties of the normal B-

cells, thus creating an indefinitely growing cell line that produced large quantities of antibodies

with the same specificity, i.e. recognizing a single epitope.

Monoclonal antibodies (mAbs) are produced by initially immunizing mice with the target

protein. The subsequent fusion of spleen B-cells and myeloma cells is conducted in the

presence of polyethylene glycol then the resultant cells are grown in a selective medium in

which only hybridoma cells can proliferate [63]. Clones are subsequently screened for

specificity using appropriate analytical methods (e.g. ELISA) and good antibody-producers are

selected. For large-scale production of mAbs the hybridomas can be grown as cell cultures or

injected into mice, where they produce tumors secreting antibody-rich fluid (ascites fluid). The

benefits of monoclonal antibodies include the fact that they can be infinitely available and their

single-epitope binding characteristics make them appropriate for therapeutic uses. Indeed, they

are currently the most commonly used immunoreagents for diagnostic applications [64].

Limitations with mAb use include the time-consuming screening of the hybrid cells during

their production and later problems with immune reactions when they are used in humans

because of their murine origin. [63]. In addition, their single epitope recognition site means

that they may have limited use in some applications in which antibody binding-sites may be

disrupted or hidden, such as Western blotting.

2.1.3 Recombinant antibodies

As discussed above, the monospecific binding properties of monoclonal antibodies make them

good reagents for therapeutic use. However, the murine origin of the antibodies has been

shown to generate problems associated with poor effector function and immunogenicity, i.e.

upon repeated administration human anti-monoclonal antibodies (HAMA) frequently develop

[63, 65, 66]. Many attempts have been made to produce human hybridoma cells, but technical

difficulties and problems with their stability and the efficiency of subsequent antibody

production have hampered their development [67]. However, the introduction of recombinant

KARIN LARSSON 15

full-length antibodies (e.g. chimeric antibodies, humanized antibodies and fully human

antibodies) has reduced these problems [2]. Initially, efforts were made to exchange the

constant part of the mouse mAb with constant regions of human origin and thereby create a

chimeric mouse-human mAb [68, 69]. Antibodies with the constant part originating from

humans would lead to improved effector functions as well as a decrease in immunogenicity

compared to the murine equivalents. However, problems with anti-idiotypic reactions were

reported for some chimeric monoclonal antibodies [70], leading to the development of

humanized antibodies, in which as well as the constant region parts of the variable region are

replaced by their human counterparts [71]. Humanization of the variable regions has been

conducted by CDR grafting [72, 73]. In CDR grafting, the hypervariable loops of the mouse

antibody are implanted onto the human framework and constant regions, thus reducing the

murine content of the antibody considerably, leading to less potential immunogenicity than

with the chimeric variants.

Intact antibodies have both advantages and disadvantages. They are bivalent binders,

increasing their apparent affinity or avidity due to longer retention times on cell-surface

receptors and on antigens with repeated motifs. They also maintain effector functions, as

discussed above. These features are not always required for diagnostic and therapeutic

purposes and they may lead to, for example, inappropriate complement activation [2]. The

engineering of antibody fragments is an emerging field, offering potentially interesting

alternatives to full-length antibodies. They may be more easily produced and fused successfully

to different compounds (e.g. toxins and enzymes). A vast range of antibody formats is

available, as summarized in Figure 3.

Antibody Fab and single-chain Fv fragments consist of both VH and VL domains, joined by a

flexible peptide linker in scFvs. These monovalent binders maintain the antigen-binding

affinity of the parent IgG, but show improvements in, for example, tissue penetration. Single

variable domains, especially from camels (VhH) and sharks (V-NAR), possess long surface

loops which are able to penetrate cavities on antigens (e.g. viruses and enzyme active sites) that

have been difficult to raise antibodies against by other means [74]. Unlike those produced by

humans and mice, these single variable domains are soluble and stable, but their potential

immunogenicity must be considered if used in vivo. Multivalent binders can be formed from

Fab and scFv fragments, such as Fab2 [75] and diabodies [76], triabodies [77] and tetrabodies,

in order to obtain desired avidities [78]. Combining scFvs or Fabs with different specificities

allows the formation of bispecific and even trispecific binders [79, 80].


2.2 Alternative protein scaffolds

Recent advances in combinatorial engineering and library display technologies have allowed

novel classes of binding molecules to be developed based on alternative scaffolds [81]. These

binding molecules, typically selected through phage [82, 83], bacterial [84, 85] or ribosomal [86]

display technologies, often show both high specificity and affinity for their targets. Antibody

molecules have complex structures with long half-lives in serum, and they are also difficult and

expensive to produce. Alternative scaffolds, on the other hand, are usually small, single-chain

proteins lacking disulfide bonds or free cysteines, enabling their production in environments

such as the bacterial cytoplasm [87]. The absence of free cysteines also permits the

introduction of cysteines at desired positions allowing, for example, site-specific incorporation

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Figure 3. Schematic representation of intact IgG monovalent binders (Fab, Fv and ScFv), multivalent

binders (diabody, triabody and tetrabody), bispecific molecules (Fab2 and diabody) and single domain

binders (VhH and V-NAR). Colored and uncolored ovals symbolize variable domains and constant

domains, respectively, while circles represent antigen-binding sites. Black lines in single chain fragments

and Fab2 fragments correspond to linkers. Modified from [2] and [3].

KARIN LARSSON 17

of fluorophores [88]. The small size of many alternative scaffolds also enhances tissue

penetration and body clearance, which are advantageous features in bioimaging, and reduce

their half-life in serum [87, 89]. However, the non-human origin of most alternative scaffolds

means that if the binders are to be used in therapeutic applications, immunogenicity issues

must be considered.

The affibody molecule is a three-helix bundle derived from an engineered domain of

Staphylococcus aureus Protein A [90]. The binding surface of this 58-amino acid peptide consists

of 13 amino acids distributed on two helices forming a relatively flat binding surface suitable

for binding to proteins. Through randomization of these 13 amino acids a combinatorial

library of about 109 binders was created, permitting (for example) the selection of binders of

HER2 [91], and the amyloid beta peptide [92]. This cysteine-free scaffold can be selected by

phage or bacterial display, it can also be produced by solid-phase synthesis [93]. It has been

demonstrated to be a stable scaffold, well suited for the purification, detection and targeting of

proteins [94, 95].

Designed ankyrin-repeat proteins (DARPins) have been developed from naturally occurring

mediators of protein-protein interactions [96]. These proteins are composed of 33 amino acid

repeats, a β-turn followed by two antiparallel α-helices and a loop linking to the next repeat.

The diversity of the DARPins comes both from the randomization of the interaction surface,

and from variation in the number of repeats. Hence, this modularity allows the scaffold to be

adapted to different binding surfaces. High-affinity molecules that recognize proteins such as

the maltose binding protein have been documented [97].

The fibronectin type III domain has an antibody-like structure and is also a natural protein-

protein interaction mediator, occurring in about 2% of all proteins. The 94 amino acid β-

sandwich is similar to the VH domain of antibodies, but is not dependent on disulfide bridges

to fold. Its variability lies in the randomization of the CDR-like loops [98]. A high-affinity

binder to TNF-α has been selected using mRNA display [99].

Anticalins [100, 101] are derived from lipocalins, a family of proteins involved in the storage

and transport of biological compounds. Although diverse in nature, lipocalins have a

consensus structure; a barrel of eight antiparallel β-strands connected by four loops that form

a cavity responsible for ligand binding. In anticalins 16 residues in the binding pocket are

randomized in order to bind a diverse set of small molecules. Examples of such binders,

selected with phage display, are the hapten fluorescein [100] and the toxin digoxigenin [102].


KARIN LARSSON 19

Experimental techniques


3. Methodology

The purpose of this section is to summarize the techniques generally used, in particular those

of central importance for the projects in this thesis, for the purification, validation and

subsequent use of antibodies in affinity-based proteomics research.

3.1 Antibody purification

Regardless of the choice of affinity-binding molecule, purification increases the success rate of

subsequent analyses. The removal of contaminating serum proteins and nonspecific host

antibodies from, for example, polyclonal antisera decreases cross-reactivity and nonspecific

binding, thus enhancing the quality of data obtained.

Affinity chromatography offers an excellent technique for antibody purification. The principles

of affinity chromatography depend on the reversible interaction between a binding protein

(e.g. antibody) and a ligand (e.g. antigen or Protein A). One of the interacting molecules is

coupled to a solid support. The binding between antibodies and respective antigens involves a

number of non-covalent interactions, including van der Waals forces, ionic bonds, hydrogen

bonds and/or hydrophobic interactions. This enables elution of antibodies in a concentrated

form by changing the conditions in the buffer (e.g. pH, ionic strength or polarity) used or

introduction of a molecule that competes with the ligand for specific binding sites [103].

Various affinity purification methods are available, of which Fc binding protein purification

and antigen-specific purification are the most frequently used, and will therefore be discussed

in more detail below.

3.1.1 Fc-binding proteins

Staphylococcal Protein A (SpA), a protein found in the cell wall of Staphylococcus aureus, has

been used for many years to purify antibodies, due to its natural affinity for the Fc part of

different subclasses of immunoglobulin G derived from a variety of species. Coupling of SpA

to a solid support enables efficient purification of IgG from crude protein samples, such as

serum, ascites fluid or cell culture supernatants using affinity chromatography with the elution

of bound IgG at low pH [104]. The low affinity of SpA for human IgG3 and immunoglobulins

KARIN LARSSON 21

of some other species (e.g. rat) has also led to the use of other Fc-binding proteins. Among

these is the streptococcal Protein G (SpG) [105]. Compared to Protein A, Protein G shows a

wider ability to bind to IgGs from different species and the subclasses of human IgG [106].

The complementary binding of these two proteins inspired the development of a chimeric

antibody-binding protein containing the IgG-binding domains from both Protein A and

Protein G. This fusion protein possesses the additive properties of both parental proteins, thus

making it an excellent choice for antibody purification [107]. Since Fc-binding proteins are

unable to separate antibodies with different specificities, their use in affinity-purification is

principally for the purification of monoclonal antibodies with single clonality, as opposed to

polyclonal antiserum consisting of a pool of antibodies with varying epitope recognition

properties.

3.1.2 Antigen-based affinity purification

Even though Fc-binding proteins like SpA are versatile purification reagents that are widely

used in research facilities, both in industry and academia, other purification strategies are often

preferred. As previously mentioned, Fc-binding proteins like SpA or SpG do not discriminate

between antibodies with different epitope specificities, so they are used to separate the

complete IgG pool from other host proteins. For the purification of polyclonal antisera

containing more than 99% host-derived antibodies that are not specific for the antigen used

for immunization, this is often not satisfactory. In addition, if the target protein or peptide is

coupled to a fusion tag, for purification or immunogenicity purposes, there may be antibodies

directed towards the fusion tag, resulting in unexpected cross-reactivity in subsequent assays

[43]. Therefore, antigen-specific purification, using the target protein as an affinity ligand, is

often a preferred choice when working with pAbs.

Many different purification methods are available, most of which involve the target protein

being covalently coupled to a solid support. The most commonly used matrix for affinity

media preparation is beaded agarose [108]. Coupling of the ligand is enabled by derivatization

of the hydroxyl group on the agarose sugar residue to appropriate activated groups. These

activated groups are then covalently attached to different groups on the ligand molecule (e.g.

primary amines, thiol, carboxyl or hydroxyl groups).

If the antigen used for immunization is a fusion protein containing, for example, a

hexahistidine tag (His6) used for antigen purification, antibodies will be produced that are


directed towards the tag and this must be taken into consideration when planning a

purification procedure. Tag-specific antibodies can be removed from the serum prior to the

purification of target-specific antibodies simply by coupling the tag protein to an initial column

and purifying the serum in two steps: the first aimed at removing antibodies with unwanted

specificity and the second designed to elute desired target specific antibodies, i.e. monospecific

antibodies (see Figure 4) [43].

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Figure 4. Three antibody purification strategies. (A) In strategy 1, the column is coupled to Protein A

(SpA) and all antibodies regardless of specificity are separated from other serum proteins. (B) In strategy

2, the protein used for immunization (in this case a fusion protein) is immobilized on the column and

antibodies specific for the antigen (both to target and tag) will be separated from host antibodies and

serum proteins. (C) In strategy 3, the antiserum is purified in two subsequent steps, the first using a

column coupled with solely the fusion tag, and the second using a column coupled to the target protein.

The tag-specific antibodies are eluted from the first column and the eluant is then purified on the second

column, enabling elution of only the target-specific, i.e. monospecific, antibodies.

KARIN LARSSON 23

3.2 Methods for antibody characterization and their use in proteomics research

Having access to carefully validated antibodies is of great importance when carrying out

proteomics research. Otherwise, interpreting data obtained from immunohistochemical

analyses and immunofluorescence microscopy is difficult and false conclusions may be drawn

concerning protein expression and localization. In particular, when characterizing unknown

proteins, annotating tissue microarrays becomes a complex task if antibodies used for analyses

are nonspecific. Ideally, two or more antibodies to each target protein should be used, to help

decide which results are most reliable. Methods used in applications of antibodies are

described below, and their uses are summarized in Table 2.

3.2.1 ELISA

Enzyme-linked immunosorbent assay, or ELISA, is an immunoassay for the detection and

quantification of antigens or antibodies in samples, which has numerous analytical and clinical

applications. The technique was first developed by a group at Stockholm University in 1971

[109] and has since been widely applied for many clinical purposes, including the development

Method Epitope stage Aim

Protein assays (ELISA,

protein arrays, etc.) Native (assay dependent) Relative amounts

Western blot Partially denatured

(detergent)

Size determination

Identification

Immunohistochemistry Partially denatured/native

(formaldehyde/frozen)

Expression

Localization

Tissue profiling

Immunofluorescence

(confocal microscopy)

Partially denatured

(formaldehyde, methanol) Subcellular localization

Flow cytometry Native

Cell surface expression

analysis

Cell identification

Table 2. Summary of applications of antibodies. Modified from [1].


of a tremendous number of diagnostic assays, such as HIV and allergy tests, in which

antibodies in serum are detected. In ELISA antibodies are covalently linked to an enzyme to

detect the analyte. After addition of a substrate the amount of analyte can be visualized by a

color change or fluorescence. Several different assay designs can be used, including indirect

and sandwich ELISA. In indirect ELISA (Figure 5A), wells are coated with antigen and the

antibody to be analyzed is added. After subsequent washing steps and addition of a secondary

enzyme-labeled antibody, the substrate to be assayed is introduced. Formerly, chromophores

were used to induce a measurable color change, but now fluorophores are more commonly

used. Many methods and techniques employ the principles of indirect ELISA, including

Western blotting, IHC, Gyrolab and Luminex technology. Sandwich ELISAs [110] have a high

degree of specificity, since two antibodies to the same antigen are used (Figure 5B). The wells

are coated with one antibody and the second added after the analyte, hence an antigen-

antibody sandwich is formed. However, a limitation when using sandwich ELISAs in

proteome-wide analyses is that it requires two antibodies to each target, while producing one

suitable antibody to each target is in itself difficult to achieve.

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Figure 5. (A) In indirect ELISA, a primary antigen-specific antibody is added to antigen-coated wells. An

enzyme-conjugated secondary antibody specific for the primary antibody is then added, followed by a

substrate producing a color change. (B) In sandwich ELISA, the wells are coated with one antibody and

a second enzyme labeled antibody is added subsequent to antigen addition.

KARIN LARSSON 25

3.2.2 Western blotting

Western blotting can be used for the identification of proteins in complex mixtures, such as

tissue samples and serum [111, 112]. It is also an established method for validating that

antibodies bind to a target protein of the expected size, as determined from amino acid

sequences. It can also be used to detect specific antibodies in serum which is useful when, for

example, confirming HIV test results. In addition, posttranslational modifications like

phosphorylations can be studied. In SDS-PAGE, which is the most common form of gel

electrophoresis, samples are boiled in buffer containing sodium dodecyl sulfate (SDS) and β-

mercaptoethanol in order to break any secondary or tertiary structures and reduce disulfide

bonds respectively (although separation under native conditions can also be performed). The

reduced samples (e.g. tissue homogenates or plasma) are then separated on a polyacrylamide

gel according to the lengths of their polypeptide chains, i.e. molecular weights. The separated

proteins are then typically transferred to a nitrocellulose or polyvinylidene fluoride (PVDF)

membrane, either by capillary blotting or by applying an electric field across a gel/membrane

sandwich. Nonspecific binding to the membrane is blocked, using a suitable blocking buffer,

and the target protein is detected in a similar manner as in indirect ELISA. The primary

antibody, either to be validated or used to detect a protein, is applied to the membrane and

after sufficient washing a secondary antibody (conjugated to a chromophore or

chemiluminiscent enzyme) is incubated with the membrane for detection purposes. Although

it is a very versatile method for both antibody validation and protein research, there are

limitations with this method. Since SDS-PAGE is usually run under denaturing conditions, the

epitopes of the proteins are linear and this can be a problem if monoclonal antibodies are used

for analysis. Moreover, estimations of band size are not clear-cut and shifts in apparent

molecular weight are not unusual. In addition, analyses of membrane-bound proteins, such as

cell-surface receptors, are difficult and often fail, due to poor solubility, (especially of integral

membrane proteins) and low abundance [113].

3.2.3 Immunohistochemistry and Tissue Microarrays

Information on protein expression in tissues, at both cellular and subcellular levels, can be

provided by immunohistochemical (IHC) analysis. Due to technical developments in tissue

microarray preparation [44, 114], automated staining procedures and image acquisition, IHC is

nowadays a high-throughput technique that is widely used in tissue protein expression

profiling and proteomics research [36].


Before any immunohistochemical staining can be performed, the tissue sample must be frozen

in liquid nitrogen or fixed in order to stop protein degradation and to preserve tissue

morphology. Fixation can be achieved by immersion of the tissue in a cross-linking fixative,

such as formaldehyde. Following fixation the tissues are dehydrated using ethanol or acetone

in increasing concentrations. Finally, the tissues are washed in xylene in order to remove the

alcohol and embedded in paraffin wax to create a tissue block. Thin sections of the formalin-

fixed, paraffin-embedded tissue sample block may then be prepared using a microtome and

IHC analysis can be applied to detect proteins that have been expressed in the sample. By

combining tissue cores from different paraffin blocks a tissue microarray (TMA) can be

formed, enabling high-throughput histological analysis of many samples (e.g. tissues, tumors or

cells) simultaneously. Before TMAs can be used for staining with antibodies they must be

conditioned. Initially the paraffin blocks must be sectioned to an appropriate size, then

deparaffinized. After this, tissue sections are rehydrated using decreasing concentrations of

ethanol. Endogenous peroxidase is then blocked in order to avoid interference with the

enzymes used for protein detection. The proteins in the tissue sections are cross-linked by

fixation, which might hide the epitopes, therefore a procedure called antigen retrieval is usually

performed. This can be executed using enzymes, but is much more commonly carried out

using heat (heat induced antigen retrieval; HIAR) typically in a microwave oven or pressure

cooker. This procedure breaks the cross-linking, but also denatures the proteins. The sections

are now ready for staining with antibodies in an indirect ELISA-like procedure using a specific

primary antibody and a secondary enzyme-labeled antibody (e.g. enzyme-linked dextran

polymers) to produce a color change in positively stained tissues. Commonly used enzymes

include horseradish peroxidase (HRP) and alkaline phosphatase (AP). The numerous and often

harsh steps involved in immunohistochemical staining procedures of tissue sections, affect

proteins and their epitopes and this must be taken into consideration when choosing the

antibody to use.

The TMA technique is dependent on specific, well-validated antibodies. When working with

unknown proteins, this is of great importance. Unspecific antibodies can lead to positive

staining even when there is no target protein present or give misleading subcellular

localizations of proteins. False negative results are often caused by epitopes becoming

inaccessible due to cross-linking or denaturation. This is more often a problem when using

monoclonal antibodies rather than polyclonal antibodies, due to their intrinsic single epitope

recognition site [36]. Benefits with TMA compared to traditional IHC analysis of tissue

sections are firstly, that less material is needed, which is especially important when using rare

tissue specimens, secondly, that the technique is less time-consuming and thirdly, that a semi

quantitative analysis can be accomplished due to identical/similar treatment of all samples on

KARIN LARSSON 27

the same slide. The major drawback is that since smaller amounts of samples are used a less

representative picture of the tissue is obtained, so important information may be lost in the

process.

3.2.4 Immunofluorescence microscopy

Even though immunohistochemical analysis reveals some information about subcellular

localization of proteins, the output is limited. Often, discrimination between nuclear or

cytoplasmic staining is achievable and in some cases membranous staining can be recognized.

By using confocal microscopy, on the other hand, high-resolution images can be obtained and

proteins can be localized to a wide range of sub-cellular compartments (e.g. the nucleolus,

Golgi or mitochondrion). The reason why the subcellular localization of proteins is of interest

is to improve understanding of the functions of individual proteins within the cell. Confocal

microscopy uses either live cells [115] or fixed cells [46] for the immunofluorescent detection

of protein expression and localization. Live cells have the advantage that momentary protein

expression patterns can be observed, whereas in fixed material proteins can only be studied at

particular end-points [46]. When working with large molecules like antibodies, cells must be

permeabilized in order for the molecules to penetrate the cell and find their targets, this

requires cell fixation.

The most commonly used fixative is paraformaldehyde and cells are regularly permeabilized

using a detergent such as Triton-X 100. Cell samples can be mounted on glass slides or

prepared in glass-bottomed micro-titer plates. Following fixation and permeabilization, cells

are stained with a target-specific primary antibody and fluorophore-labeled secondary

antibody. Cells are usually counterstained with a nuclear probe (e.g. DAPI) and several other

cellular probes (e.g. phalloidin for actin filament staining and MitoTracker for staining of

mitochondria) can be used in order to help in the annotation process.

Since strong fixative agents are also used in immunofluorescent analysis, problems with

denatured epitopes and poor antibody binding may arise, as previously discussed for IHC

analysis. However, cross-linking of proteins occurs to a lesser extent since much shorter

incubation times are required in fixatives. In addition, epitopes on membrane-bound proteins

may be disturbed during permeabilization of the plasma membrane, posing a potential

problem in antibody-based immunofluorescence microscopy, since this could lead to reduced


antibody recognition and false negative results. In such cases live cells might be a better

alternative.

3.2.5 Flow cytometry

Flow cytometry is a valuable technique that is used to obtain information about the physical

characteristics of cells such as their size, shape and, more importantly (in molecular biology),

proteins expressed on their surface. The first fluorescence-based flow cytometer was

constructed by Wolfgang Göhde in 1968, and since then the technique has been extensively

used for a wide range of diagnostic and clinical applications. An example of the routine use of

flow cytometry is in the immunophenotyping of leukemia. The subtyping, i.e. analysis of

surface marker expression of lymphocytes, helps to determine the prognosis of the disease and

the appropriate course of treatment [116]. Another application is the monitoring of CD4+

lymphocyte levels in HIV patients, used as an indicator of disease progression, responses to

therapy and susceptibility to infections [117].

The flow cytometer analyzes cells in suspension. The cells are streamed through the flow

cytometer one by one, passing through a laser light beam focused on the stream. Detectors

measure scattered light and emitted light, giving information about the size and inner

complexity of the cells, and cell-surface antigens, respectively. An additional feature of some

flow cytometers is their ability to sort cells based on their fluorescence and light scattering

properties. This is called fluorescence activated cell sorting (FACS) and it is a versatile method

for the enrichment of subpopulations in complex samples that exhibit certain characteristics.

By fluorescently labeling antibodies, the cell surface expression of proteins can be monitored.

By using a cell line with known surface expression, flow cytometry can also be used to evaluate

the ability of antibodies to bind to native proteins.

3.2.6 Epitope mapping

An epitope, also called an antigenic determinant, is the part or parts of an antigen recognized

by the immune system. The corresponding part of the antibody responsible for binding to the

antigen is termed a paratope. There are two types of epitopes, termed linear or structural

epitopes [118]. Linear or sequential epitopes correspond to the amino acid sequence of the

protein (primary protein structure) while structural or conformational epitopes are

KARIN LARSSON 29

discontinuous in terms of the amino acid sequence, but are brought together in space by the

natural folding of the protein (tertiary protein structure). There is, however, no clear-cut

distinction between these two types of antigenic determinants. Conformational epitopes may

have sequential elements in them and linear stretches of amino acid sequences may both have

structural features (e.g. loops in proteins) and be discontinuous in nature (e.g. not all amino

acids in the stretch may be involved in the antigen-antibody interaction) [119].

Epitope mapping is a method used to determine the minimum part of an antigen needed for

antibody binding. Characterization of the antibody-binding site is desirable for research and a

necessity in applications such as vaccine development. Several methods are available for

mapping structural epitopes, including X-ray crystallography [120], mutagenesis of the antigen

[121] and site-directed masking of epitopes [122]. Although these methods have all been

shown to work well, high-throughput determination of structural epitopes is not feasible. Both

mutagenesis and site-directed masking of antigens depend on structural data for making

appropriate decisions about which amino acids to modify. Due to these issues, epitope-

mapping efforts have mainly focused on linear epitopes, [123] even though most epitopes are

thought to be structural [124]. A wide range of methods is available for mapping sequential

epitopes. The most common approaches involve measuring binding of the antibody to peptide

fragments. These can be generated either from the protein’s primary sequence using an overlap

covering the whole sequence at single amino acid resolution [125, 126], or using peptide

libraries (random or antigen-derived) expressed by phage or bacterial display, among other

methods [127-130]. Although mapping of linear epitopes can be considered a high-throughput

method, peptide synthesis remains expensive and efforts have been made to predict linear

epitopes in silico since the beginning of the 1980s [131]. Amino acid sequences of peptides has

been used to create propensity profiles, based on various characteristics such as

hydrophobicity, but a recent study found that the correlation between known linear epitopes

and propensity profile peaks was only slightly better than random [123].


KARIN LARSSON 31

Present Investigation


4. Objective

The main objective of the studies this thesis is based upon was to develop methods to facilitate

the high-throughput generation and subsequent purification of monospecific antibodies

(Papers I and II). In addition, some of the challenges associated with generating antibodies

directed towards targets with highly similar sequences and their validation were investigated

(Paper III), and finally an alternative antigen design strategy was investigated for generating

antibodies towards G-protein-coupled receptors (Paper IV). All of these studies were

performed as part of the Human Protein Atlas program.

Before the Human Protein Atlas program was officially started, while its details were still being

refined, monospecific antibodies were purified by incubating antisera with Western blot

membranes containing the target protein and cutting out the areas where antibodies apparently

binding to target proteins were detected, these antibodies were then extracted from the

membrane by eluting them at low pH [132]. Following subsequent developments, a system

with peristaltic pumps and PrESTs immobilized on affinity columns was used, enabling two

antibodies to be purified per pump in a single day [43]. Soon after the project was launched,

the peristaltic pumps were upgraded to ÄKTA explorer systems, resulting in a doubling in the

output of purified antibodies. Even though less hands-on time was then needed per antibody,

the purification rate was still not sufficient for the requirements of the HPR project. However,

the introduction of the ÄKTAxpress system greatly boosted the throughput (as described in

Paper I), leading to the current output of about 600 purified antibodies per month. Methods

for analyzing the specificity of these antibodies have been similarly enhanced, by using protein

arrays with 384 spotted PrESTs rather than low-throughput manually manufactured dot blots

with four PrESTs.

An alternative immunization strategy was also developed (Paper II), in which multiplexed

PrEST antigens (consisting of two or more PrESTs) were used, providing both economic and

ethical benefits. The use of PrESTs as both antigens and affinity-purification ligands allows

both high-throughput antigen production [41] and antibody purification, in a format that can

be readily adapted to previously described methods (Paper I).

The validation of antibodies is important but not always straightforward, especially for

antibodies raised against difficult targets, such as proteins with highly similar sequences to

other proteins. Paper III, describing the generation of ten antibodies to such a target,

Cytokeratin-17, illustrates these issues. The resulting antibodies were characterized using a

KARIN LARSSON 33

combination of epitope mapping and comparative IHC analysis, in addition to the analyses

generally used in HPR procedures (e.g. protein array and Western blot analyses).

In Paper IV, a novel antigen design strategy for the generation of GPCR-specific antibodies is

described. The structure of GPCRs (which have an extracellular N-terminal domain, seven

transmembrane helices connected by short loops and an intracellular C-terminal tail) makes

them difficult to produce in the expression systems normally used for antigen production.

Here, an alternative approach was investigated, involving fusion of the extracellular loops and

N-terminal to create an antigen that could be used for immunization and antibody purification.


5. High-throughput generation of monospecific antibodies (Paper I)

As discussed above, high-throughput generation of affinity reagents is of great importance for

proteomics research, but achieving it presents major challenges, hence specific antibodies are

only currently available for a subset of the human proteome. Prior to the studies presented in

this thesis, a strategy had been developed for the generation of monospecific antibodies based

on conventional immunization of rabbits with recombinantly produced protein fragments,

denoted Protein Epitope Signature Tags (PrESTs) followed by stringent affinity purification of

the resulting polyclonal antisera [43]. In the study presented in Paper I this procedure was

adapted for high-throughput applications, by applying a modular semi-automated

chromatography system for the following operations: (i) depletion of unwanted tag-specific

antibodies; (ii) capture of antibodies with the desired specificity and (iii) buffer exchange using

gel filtration. Each module allows the automated, serial purification of up to four samples and

Abs

orba

nce [m

AU]

Retention [ml]

(b) (c)

(a)

A.

216 116 87

48

32 26

18

B. 1. 2. 3.

Figure 6. Chromatogram and Western blot illustrating the outcome of the unit operations applied in the

purification of an antibody specific for the estrogen receptor (ER-α) and subsequent characterization of

isolated fractions using an MCF-7 protein lysate. (A) Chromatogram showing flow-through (a), the

elution of anti-tag antibodies (b) and anti-ER-α antibodies (c). (B) Western blot membrane lanes

showing detected proteins in MCF-7 cell extract: in a sample following primary incubation with

unpurified antisera (panel 1), the eluate from the His6-ABP column (panel 2, corresponding to elution

peak (b)) and the eluate from the PrEST-coupled column (panel 3, corresponding to elution peak (c)).

KARIN LARSSON 35

the modularity of the system allows up to 12 modules to be controlled by a single computer,

thus enabling true high-throughput purification of polyclonal antisera. Monospecific antibodies

(msAb) were validated with a newly developed PrEST array format as well as by Western

blotting, and were then used to localize proteins in normal and diseased tissue.

In order to generate monospecific antibodies, a stringent affinity purification procedure was

applied [43]. Initially, the antiserum was depleted of fusion tag-specific (anti-His6-ABP)

antibodies by using an affinity column with immobilized tag protein. Eluted antibodies were

discarded and the flow-through containing PrEST-specific antibodies was collected for use in

subsequent purification steps. This depletion step also effectively captured most of the rabbit

serum albumin [43]. In order to capture the target-specific antibodies in the depleted flow-

through, affinity chromatography was applied using a matrix coupled with the same PrEST-tag

fusion protein that had been used for immunization. For the second unit operation a

chromatographic system, with the capacity to purify four samples per module in series was

used (which also provided the possibility of increasing the number of modules operated in

parallel), followed by buffer exchange. Since all antibodies towards the tag moiety of the

immobilized fusion protein were depleted in the first step, only PrEST-specific antibodies were

eluted with a low pH buffer and, therefore, captured by this procedure (Figure 6). Finally, the

buffer was changed to a suitable storage buffer using a desalting column. In this study four

modules of the chromatographic system were used, allowing 16 different antisera to be

purified in less than 6 h. Antibodies in different fractions were analyzed by Western blotting

against whole cell protein lysate (Figure 6). The stringency of the purification method and the

specificity of antibodies obtained was assessed (for quality assurance) using a newly developed

protein array method in which the specificity of the binding of acquired antibodies to the

cognate PrEST was tested by examining its binding to a large set of unrelated PrESTs. The

results indicated whether the PrEST-design had been valid and, most importantly, whether tag-

specific antibodies had been removed (Figure 7). A dual-color dye labeling system was used for

detecting PrEST-antibody interactions (and any possible cross-reactivity) and to ensure that all

PrESTs were present on the array. This was accomplished by mixing tag-specific antibodies

generated in hens together with the rabbit-derived PrEST-specific antibodies during the

primary incubation step. A secondary incubation step then followed with anti-hen and anti-

rabbit antibodies, which had been labeled with two different fluorophores. Using this

procedure, 12 msAbs could be analyzed for their specificity and cross-reactivity towards 72

different PrESTs spotted on a single glass slide. This was accomplished by using an adhesive

silicone mask with 12 wells, each containing 72 different PrESTs spotted in duplicate,

including the PrEST corresponding to the antibody being analyzed. Ninety-six percent (446) of


the 464 affinity-purified antibodies included in this study showed satisfactory specificity in this

protein array analysis.

A.

B.

C.

Figure 7. Protein array analysis of an antibody directed to the leukocyte common antigen (LCA). Red

bars indicate binding to the target antigen (spotted in duplicate) and grey bars signify binding to

nonrelated antigens. The green line shows the presence of spotted proteins on the glass slide. (A)

Analysis of unpurified polyclonal serum demonstrates binding of tag-specific antibodies to the His6ABP

protein fused to all PrESTs. (B) Protein array analysis of depleted serum, illustrating the importance of

the first purification step. (C) Analysis of PrEST-specific antibodies eluted from the last unit.

KARIN LARSSON 37

One hundred and ninety of the 446 antibodies that passed this specificity test were further

assessed in terms of their ability to localize proteins in immunohistochemically stained tissue

microarrays (TMAs). In total, 48 normal tissue samples were analyzed, in triplicate, with each

antibody, and some of the antibodies were also tested using TMAs containing a set of samples

representing some of the most common cancers. Pathologists manually annotated the TMAs,

and 81% of the antibodies produced staining that appeared to be specific, as judged from

staining intensity and distribution. In addition, further specificity checks were carried out for

25 of the antibodies using an adsorption assay in which they were pre-incubated with their

respective PrEST-antigens, prior to immunostaining (Figure 8). Immunostaining with all the

tested antibodies disappeared after this procedure, further supporting the conclusion that the

antibodies were specific for their respective targets. The tissue distribution patterns obtained,

using a subset of the antibodies, were also compared to results obtained with clinically

approved monoclonal antibodies, and there was a high degree of concordance between results.

In conclusion, the stringent, high-throughput purification scheme, in combination with an

efficient protein array-based validation method described, enabled the systematic generation of

antibodies with a high rate of success and these monospecific antibodies were shown to be

highly specific in several immunoassays.

B A

Figure 8. Results of adsorption test demonstrating that specific patterns of immunoreactivity were

quenched after pre-incubation of the antibodies with the corresponding PrEST-antigen. Staining of

glandular mucosa with a specific antibody to zinc finger protein 183 shows strong nuclear staining

(brown) (A), which completely disappears after adsorption with PrEST (B).


6. An alternative immunization strategy using multiplexed PrEST-antigens (Paper II)

Using traditional immunization methods the generation of both monoclonal and polyclonal

antibodies on a whole proteome scale would require massive immunization programs.

Therefore, from both ethical and economic perspectives, alternative immunization strategies

are desirable, in order to reduce the number of animals needed for such large-scale production

of antibodies, but to date there have been few reported attempts to develop suitable strategies,

and most of those that have been published involve monoclonal antibody production [50,

133]. In addition to these ethical considerations, the overall potential to automate methods, as

well as their complexity and cost-efficiency, are important considerations for all high-

throughput projects.

Figure 9. Schematic diagram of the multiplexed immunization strategy for the generation of

monospecific antibodies; from antigen preparation to affinity purification of individual antibody pools.

KARIN LARSSON 39

In this study, a multiplexed PrEST-based immunization strategy for generating polyclonal

antibodies was investigated. Antigens composed of two, three, five or up to ten PrESTs in

different combinations were used to immunize a total of 28 different rabbits and the resulting

polyclonal antisera were purified using a modified two-step affinity purification method and

the ÄKTA explorer system (Figure 9). After depleting tag-specific antibodies, PrEST-specific

purification was performed by connecting affinity columns in series for loading and washing

samples, then eluting individual columns at low pH, one-by-one, to obtain pure, monospecific

antibodies. This method could easily be adapted for purification using the ÄKTAxpress

chromatography system, by collecting the flow-through from one column and loading it onto

the following columns, thus in effect individual PrEST-specific antibodies from a multiplexed

immunization could be purified one at a time in an iterative procedure with increased

throughput.

The concept of using multiplexed antigen was initially evaluated using a combination of two

different PrESTs for immunization. The total amount of administered antigen remained

constant, irrespective of the degree of multiplexing. Hence, in a double immunization the

amount of each participating PrEST administered was half the amount administered in

traditional, single PrEST immunizations. This strategy was necessary to meet national

guidelines regarding the use of animals for scientific purposes, preventing the administration of

such high doses for immunization that harmful reactions may result. In total, 13 rabbits were

immunized with two different PrESTs in various combinations and in 10 of these (77%) the

resultant antisera contained antibodies towards both PrESTs, while in two cases (15%) only

one of the PrESTs gave rise to a detectable, specific immune response. Following one of the

immunizations no specific immune response could be detected towards either of the

participating PrESTs (Table 3). All of the affinity-purified antibodies were thoroughly tested

using both protein arrays and Western blots against a larger set of both positive and negative

control PrESTs. Any potential cross-reactivity resulting from the multiplexing was shown to

have been effectively removed, resulting in pure monospecific antibodies that were

comparable to corresponding antibodies obtained from traditional, single PrEST-

immunizations. This was further corroborated by immunohistochemical staining of

consecutive human tissue sections, which resulted in very similar immunoreactivity patterns

for antibodies produced by single and multiplex immunizations.

Encouraged by these findings, the feasibility of increasing the degree of multiplexing was

investigated in trials in which 28 rabbits, in total, were immunized with two, three, five or up

to ten different PrESTs in various combinations. In order to accelerate the analysis of the

resulting immune responses, PrEST array analysis was also used for pre-screening antisera that


had been depleted of tag-specific antibodies prior to affinity purification. The screening of

depleted sera showed that 80% of the animals immunized with antigens composed of two or

three PrESTs generated antibodies towards all of the administered PrESTs. Of the animals

immunized with five PrESTs, 25% produced antisera recognizing all targets and 50%

produced antibodies towards four out of the five PrESTs. None of the immunizations with

ten PrESTs resulted in antibodies towards all the targets (Table 3).

Illustrative results from the analysis and purification of an antiserum resulting from one of the

immunizations with five different PrESTs are shown in Figure 10. Initial screening of the

immune response was performed by PrEST-array analysis of serum that had been depleted of

tag-specific antibodies, which showed that antibodies towards all five PrESTs could be

detected in the antisera (Figure 10A). Chromatograms obtained from samples following the

subsequent purification showed that the total amounts of antibodies harvested corresponded

well to intensities in PrEST-array analysis, suggesting that this method could be used as a

screening tool to investigate the immune response and possibly to facilitate subsequent

purification of individual antibodies (Figure 10B). Following purification, all antibodies were

analyzed by Western blotting, which demonstrated that all detectable cross-reactivity resulting

Table 3. Immune responses determined by PrEST-array analysis of depleted antisera in rabbits

immunized against multiplexed PrEST antigens.

Degree of Multiplexing Recognized

PrESTs 2 PrESTs 3 PrESTs 5 PrESTs 10 PrESTs

0 1 - - -

1 2 - - -

2 10 2 - -

3 7 1 -

4 2 -

5 1 -

6 1

7 1

8 -

9 -

10 -

No. immunized rabbits 13 9 4 2

KARIN LARSSON 41

α-HT432

α-HT2323

α-HT3

α-HT2389

α-HT1524

M 1 2 3 4 5 6

210 120 84 60 39 28 18

α-HT432

α-HT2323

α-HT3

α-HT2389

α-HT1524

PrEST

Inte

nsity

α-HT432

α-HT2323

α-HT3

α-HT2389

α-HT1524

Retention [ml]

Inte

nsity

[mA

U]

A.

B.

C.

Figure 10. Purification and analysis of an antiserum derived from a multiplex immunization with five

unique PrESTs. The serum was pre-screened using a PrEST-array, followed by affinity purification and

Western blot analysis. (A) Intensity plot from PrEST-array analysis of depleted sera. Bars illustrate

interactions of antibodies with PrESTs, solid lines show the amounts of immobilized proteins. (B)

Elution profile from an ÄKTA explorer system showing antibody purification. (C) Western blots of

PrESTs in the multiplexed antigen, using the respective purified antibodies.


from the multiplexing had been effectively removed (Figure 10C). The amounts of antibodies

obtained, from each PrEST, following these multiplexed immunizations were generally lower

than amounts obtained from traditional single immunizations, and the antibody titers were

PrEST (probably due to intermolecular antigen competition) as well as animal-dependent

factors. The lower antibody yields from multiplexed immunizations were probably due to the

lower amounts of each PrEST present in the multiplexed antigens, compared to

immunizations with single PrEST antigens. As mentioned earlier, the total amount of protein

was kept constant regardless of the number of PrESTs, hence the higher the degree of

multiplexing, the lower the amount of each administered PrEST. This is probably the main

reason why the success rate decreased with increases in the degree of multiplexing.

To summarize, a PrEST-based multiplex immunization strategy was developed in this study

for the high-throughput generation of monospecific antibodies, providing both ethical and

economic benefits due to a reduction in the numbers of animals needed. The method provides

an efficient means of producing antibodies against up to three antigens simultaneously, with

the possibility of further increasing multiplexing, although this would be at the expense of

antibody yields. It would also result in a more complex purification process, lowering the

throughput, although this could be partially overcome by recent technical improvements, e.g.

use of the ÄKTAxpress purification system. Importantly, the antibodies obtained from these

immunizations against multiplexed proteins showed excellent specificity in a number of assays

(protein array analyses and Western blots), they also compared well with the corresponding

antibodies obtained from traditional immunization procedures in immunohistochemical

analyses of human tissue sections.

KARIN LARSSON 43

7. Generation and validation strategies for antibodies raised towards targets with highly similar sequences (Paper III)

In affinity-based proteomics the aim should be to generate at least one specific binder for each

non-redundant protein of the human proteome, defined here as a protein representing each

gene locus. Families of proteins exhibiting high degrees of similarity in their amino acid

sequences can complicate this endeavor. Such similarities make it difficult to find unique

regions that are appropriate for use as antigens and result in strict requirements for the

validation of the generated antibodies. Here, Cytokeratin-17, a member of the intermediate

filament family of proteins, which show a high degree of sequence similarity and play

important roles in cell morphology, was chosen as a model for two main reasons. Firstly, to

illustrate the challenges faced in raising antibodies to such proteins, and secondly to study

batch-to-batch variations in antibodies produced from repeated immunizations to the same

target. Two non-overlapping PrESTs were designed, expressed, and each used to immunize

five rabbits. The resulting antibodies were purified and analyzed using protein arrays, Western

blotting, immunofluorescence-based confocal microscopy and immunohistochemical staining

of consecutive tissue sections. In addition, to characterize the antibodies obtained further,

epitope mapping was performed using both the Gyrolab platform, which incorporates

microfluidic structures in a CD-format for analyzing proteins at the nanoliter level, and a bead-

based Luminex suspension array, which allows true multiplexing of the assays.

There are about 130 genes containing InterPro keratin domains (defining the intermediate

filament family and the cytokeratins) in the Ensembl database [134]. Hence, the standard

PrEST-design criteria; a threshold value of 60% identical amino acids over a 50 amino-acid

window and no local sequence with eight out of ten amino acids identical to each other, were

difficult to fulfill. This is illustrated by a bioinformatics analysis, which found >90% identity in

domain and epitope-sized sequences between Cytokeratin-17 and other members of the family

across its whole primary sequence (Figure 11A) [32]. However, if two genes predicted as

paralogues to Cytokeratin-17 (ENSG00000128422 and ENSG00000131885) were excluded

from the analysis the antigen design was considerably eased (Figure 11B). When all genes

containing an InterPro keratin domain were excluded (Figure 11C) the sequence identity plot

showed an identity well below 60% over the whole protein, for the 50-aa window.


The two Cytokeratin-17 PrESTs, denoted here as PrEST-A and PrEST-B, were recombinantly

produced in Escherichia coli as His6ABP-fusions, then each of them was used to immunize five

New Zealand white rabbits. Following bleeding and serum collection, monospecific antibodies

were obtained using the standard two-step immunoaffinity-based protocol described in Paper

I. Initial specificity analysis was performed using planar protein arrays, which showed specific

binding to the respective antigens, with no detectable background binding or other unwanted

Figure 11. PrEST location and sequence identity plots including domain and epitope sized (50 and 10

amino acids respectively) sequence similarity comparisons of Cytokeratin-17 to all human genes (A), to

all genes except two genes predicted as paralogs to Cytokeratin-17 (B) and to all non Inter Pro keratin

domain containing proteins (C).

A.

B.

C.

KARIN LARSSON 45

cross-reactivity. Further analysis by Western blotting showed that all of the antibodies were

able to recognize a protein of the size corresponding to Cytokeratin-17 (48kDa), with little

variation within or between the respective PrEST-groups.

A thorough analysis of linear epitopes was performed for all antibodies using synthetic

overlapping peptides (15 amino acids long with a 10 amino acid overlap) with two different

systems, the Gyrolab platform and the Luminex. Mapping was carried out to assess differences

between immune responses when different animals were immunized with the same PrEST,

and to account for observed discrepancies in results of Western and immunohistochemical

analyses. Using both systems, mapping showed that antibodies bound principally to the C-

terminal peptides of PrEST-A and PrEST-B, possibly due to their flexibility and exposure to

the immune system (in contrast to their N-termini, which are fused to the rather large His6-

ABP fusion tag). This was especially evident for the five antibodies generated against PrEST-B,

for which almost all the detectable binding was to the last six C-terminal peptides, with the

PrEST-A AbA1

AbA2

AbA3

AbA4

AbA5

AbB1

AbB2

AbB3

AbB4

AbB5

PrEST-B

Figure 12. Epitope mapping results from affinity-purified antibodies generated towards PrEST-A and

PrEST-B of Cytokeratin-17. The binding intensity was measured as total integrated volume (TIV)

towards each synthetic peptide. (A) Results obtained with antibodies AbA1-AbA5 generated using

PrEST-A as antigen. (B) Results obtained using PrEST-B specific antibodies (AbB1-AbB5).


exception of a few outliers. A subset of the PrEST-A antibodies exhibited additional binding

to the N-terminus (Figure 12).

To investigate how observed differences in epitope recognition between antibodies might

influence the results when used to stain samples of human tissue, they were applied in

immunohistochemical analyses of consecutive tissue sections cut from tissue microarray blocks

of 48 tissue samples with normal morphology. The results, summarized in Figure 13, showed

that all PrEST-A antibodies apart from one (AbA2) yielded similar staining patterns, with

strong staining of a number of epithelial tissues, some of which was expected and some

unexpected according to the literature [135, 136]. The PrEST-A antibody, AbA2, yielded a

Cytokeratin-17-like staining pattern and four of the PrEST-B antibodies (AbB1-AbB4)

exhibited this pattern of staining even more strongly. AbB5 stained most epithelial tissues

intensely (Figure 13). When the PrEST sequences and their similarity to other cytokeratins

were analyzed in more detail, it was found that epitopes recognized by AbA1 and AbA3-AbA5

were situated in a region of PrEST-A that shared 24 adjacent amino acids in common with

Cytokeratin-19, for example. This is a cytokeratin that is widely distributed in simple

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Figure 13. Tissue or cell type distribution of immunohistochemical staining obtained with ten antibodies

generated towards two Cytokeratin-17 PrESTs. Red spots represent strong staining, orange spots weak

to moderate staining and white spots no detectable staining.

KARIN LARSSON 47

epithelium [135]. Antibody AbA2, which only bound to the last peptide (A23), only shared

three out of six amino acids with Cytokeratin-19, hence generating a more Cytokeratin-17-like

staining pattern in the TMA. Interestingly, the C-terminal part of PrEST-B, where most of the

PrEST-B antibody binding was observed, does not share any identical sequence with other

cytokeratins, thus explaining the more Cytokeratin-17-like staining pattern obtained for these

antibodies. All antibodies were further analyzed using immunofluorescent confocal

microscopy with a Cytokeratin-17 positive cell line, A-431. All PrEST-B antibodies yielded the

expected sub-cellular localization pattern of immunoreactivity with distinctive intermediate

filament staining, thus further validating the Western blot and IHC findings. For PrEST-A

antibodies results were less supportive since more diffuse cytoskeletal staining was observed,

and in one case clear nuclear staining was evident.

In conclusion, the possibility of generating antibodies towards targets which have highly

similar sequences to other proteins was investigated, and a validation strategy was

demonstrated, based on Western blotting, IF and comparative IHC of several binders to the

same target combined with epitope mapping. The results showed that antibodies (including

most of the PrEST-B antibodies) binding to the most sequence-unique part of Cytokeratin-17,

the C-terminal region, also exhibited the most Cytokeratin-17-like staining pattern in IHC

analyses. However, PrEST-A antibodies displayed additional binding, possibly to other

cytokeratins in the analyzed tissues, in accordance with the higher degree of sequence similarity

between PrEST-A and other members of the cytokeratin family observed in sequence analyses.

Furthermore, the binding patterns (with relatively few distinct epitopes) of antibodies

generated by the immunization of different animals with the same antigen were similar,

although not identical. These data support the strategy of producing antigens with little

sequence similarity to other proteins as an important criterion. They also suggest that for

affinity-based proteomics projects use of at least two, and preferably more, binders raised to

non-overlapping regions of the same target protein is desirable.


8. An alternative strategy for designing antigens for membrane-bound receptors (Paper IV)

G-protein-coupled receptors (GPCRs) constitute a large family of important cell-surface

receptors. These are proteins with seven transmembrane domains that play key roles in

regulating cellular functions via interactions with a wide range of ligands (e.g.

neurotransmitters and hormones). These regulators of cell functions include a great number of

important drug-binding sites, accounting for 60-70% of current pharmaceutical research and

development targets. Well-validated antibodies towards GPCRs are potentially important tools

which can be used for the discovery of novel drugs and for research aimed at increasing

understanding of these endogenous receptors [137]. In addition to the seven transmembrane

Figure 14. Schematic representation of a G-protein-coupled receptor and the antigen used for antibody

generation. (A) GPCRs consist of a seven transmembrane domain core connected by loops, an

extracellular N-terminus and an intracellular C-terminus. (B) The extracellular loops and N-terminus are

assembled into a single antigen separated by a polar linker, and fused to His6-ABP.

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KARIN LARSSON 49

domains, all GPCRs contain three extracellular loops and an N-terminal tail, which are

responsible for binding their respective ligands, as well as three intracellular loops and a C-

terminus responsible for intracellular signaling (Figure 14A). In this study the possibility of

producing GPCR-specific antibodies by combining the N-terminus and extracellular loops to

form a single antigen was investigated.

To test this approach, three GPCRs were selected: Neuropeptide Y type 5 receptor (NPY5R),

beta 2 adrenergic receptor (B2AR) and the glucagon-like peptide 1 receptor (GLP1R). In each

case, using solid-phase gene assembly [138, 139] the extracellular loops and the N-terminus

were cloned to form a single antigen separated by a polar linker (GSSSG), which was then

fused to a hexahistidine and an albumin binding protein (His6-ABP) tag, and expressed in E.

coli (Figure 14B). The resulting antigens were each used to immunize two rabbits, and stringent

affinity-purification of the resulting antibodies was performed using previously described

methods (Paper I). Since GPCRs are generally expressed at very low levels [140], cell lines that

over-expressed the receptors were generated with a lentiviral gene delivery system. Flow

cytometry and immunofluorescence-based confocal microscopy were then applied to the over-

expressing cell lines to characterize the binding of the antibodies obtained to intact receptors.

Epitope mapping using synthetic peptides corresponding to the extracellular loops and the N-

terminal sequence of the three GPCRs was used to characterize antibody-antigen interactions.

Once stable expression in the A-431 cells had been established, all the antibodies generated, as

well as a number of commercial antibodies, were analyzed using a flow cytometer. All the

antibodies towards NPY5R (Figure 15A) and GLP1R (Figure 15C) produced an increase in

fluorescence, indicative of binding to their cognate full-length receptor, but only one of the

B2AR-specific antibodies (B2AR:1) showed fluorescence suggestive of specific binding (Figure

15B). Only one of the antibodies (the commercial antibody, MAB28141) raised against

GLP1R, exhibited nonspecific binding to control A-431 cells. Immunofluorescence analysis

was carried out for further validation of binding to full-length proteins and to assure

specificity; staining to cell compartments other than the membrane or membrane-associated

compartments would suggest that antibodies are not sufficiently specific. In general, the

analyzed antibodies produced the expected pattern of plasma membrane staining, which was

more pronounced at the cell junctions. Additional staining in the Golgi could be observed with

all the NPY5R antibodies, and the only B2AR antibody (B2AR:1) that generated any staining

also stained this compartment. Since the Golgi apparatus modifies proteins before they are

transported to the plasma membrane this was to be expected. The GLP1R antibodies also

weakly stained the nuclear membrane. NPY5R:1 and the commercial anti-GLP1R antibody,

GA1940, stained the nuclei of normal A-431 cells nonspecifically, but not the nuclei of cell


over-expressing the respective receptors. Epitope mapping did not reveal any general pattern

of immunoreactivity. The results showed that antibody NPY5R:2 bound strongly to the N-

terminal region of NPY5R, while antibody NPY5R:1 bound to most parts of the antigen with

lower signal intensities. Both B2AR antibodies showed binding with high signal intensities:

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Figure 15. Flow cytometric analysis of antibodies specific for NPY5R (A), B2AR (B) and GLP1R (C).

X-axes indicate cell counts and y-axes fluorescence intensity. Each histogram shows data obtained from

three samples: wild A-431 cells incubated solely with secondary antibody (thin lines), A-431 cells

incubated with both primary and secondary antibodies (dashed lines) and over-expressing cell lines

immunostained with the respective primary antibody and a secondary antibody.

KARIN LARSSON 51

B2AR:1 to the N-terminus and second extracellular loop, and B2AR:2 to the N-terminus as

well as to both the second and third extracellular loops. Interestingly, B2AR:2 bound with very

strong signal intensity in the Luminex assay used for epitope mapping, but failed to generate

any detectable staining in immunofluorescence assays or flow cytometry analyses. Due to the

long (120 amino acids) N-terminal tail of GLP1R no peptides were synthesized to represent

this part of the antigen, so no possible binding to this receptor region could be determined in

these experiments. GLP1R:1 and GLP1R:2 showed binding to the first and third extracellular

loops, respectively, but with weak intensity of detected signals in the former case.

In addition, some of the antibodies were used for in situ staining of human tissue. The antibody

NPY5R:2 were used for immunofluorescence staining of unfixed cryostat sections of human

brain tissue. A large number of fluorescent processes were seen in the dorsal part of the

periaqueductal gray in the human mesencephalon, staining that was lost after pre-incubation

with the antigen used for immunization, thus indicating specific staining of the antibody to the

target receptor. No cell bodies were visible (Figure 16). Moreover, the antibodies generated

towards GLP1R were used for IHC staining of formalin-fixed paraffin embedded normal

human pancreas. The GLP1R:1 antibody exhibited positive staining of the plasma membrane

Figure 16. Immunofluorescence micrograph taken from the dorsal part of the periaqueductal gray in the

human mesencephalon. Unfixed cryostat sections incubated with the antibody NPY5N2 and stained

with green fluorescein isothiocyanate (FITC). Bar indicates µm.


in islets and no staining in exocrine tissue (Figure 17). GLP1R:2 also stained the plasma

membrane of islet cells but with additional weak cytoplasmic staining in exocrine cells. Neither

of the antibodies GA1940, AB9433 or MAB28141 gave rise to staining in the plasma

membrane, but rather weak to strong cytoplasmic staining both in islet and exocrine cells.

AB9433 and MAB28141 also exhibited nuclear staining in islet cells as well as exocrine cells.

In summary, antibodies towards G-protein-coupled receptors are invaluable tools for studies

of their sub-cellular localization, posttranslational modifications, interaction partners and

molecular identity [141]. The paper describes a novel antigen design strategy for the

straightforward generation of GPCR-specific antibodies. Antigen production is considerably

eased when the N-terminus and extracellular loops are combined to form single antigens rather

than using whole receptor molecules. In addition, use of an antigen with several receptor

domains (rather than a single synthetic peptide) increases the likelihood of inducing an

immune response. The results showed that the antibodies generated in this manner work well

across several technical platforms, and that they recognize both denatured and native targets in

over expressing cell lines and in human tissue samples.

Figure 17. Immunohistochemical staining of normal human pancreas using an antibody generated

towards GPL1R (GLP1R:1). A moderate staining of the plasma membrane of islet cells was shown

(depicted in brown), but no staining in exocrine tissue.

KARIN LARSSON 53

9. Concluding remarks

Antibodies are invaluable tools for use in proteomics research, since they exhibit both

remarkable variability and specificity for their target proteins. The systematic generation of

specific, well-validated antibodies to gain complete coverage of the proteome and hence

facilitate increased understanding of protein expression, localization and function, is an

important challenge for scientists. The main objectives of the studies presented in this thesis

were to develop methods facilitating the high-throughput generation and subsequent

purification of monospecific antibodies, and to address problems associated with validating

antibodies.

Paper I describes a high-throughput approach for proteome-wide generation of monospecific

antibodies (to non-redundant proteins) and their validation with a newly developed protein

array assay. The antibodies generated were used for parallel profiling of protein expression and

sub-cellular localization. The data obtained suggest that monospecific antibodies could be used

to generate a comprehensive atlas of the human proteome. Today, around 600 antibodies are

purified each month within the Protein Atlas program and a first draft of the human proteome

is expected in 2014. More than 90% of human genes are currently in the HPR pipeline. The

biggest current challenges are to generate antigens to gene products for which previous

attempts have failed somewhere in the process, and to design antigens to targets that are highly

similar to other proteins, and to targets with short sequence stretches that are suitable for

PrEST generation.

An assessment of an alternative immunization strategy using multiplexed PrESTs antigens (for

ethical and economic reasons) is described in paper II. It was shown that it is feasible to

generate antibodies towards two to three different protein targets in a single animal and,

furthermore, that these antibodies could be separated in subsequent purification steps, without

compromising quality or throughput. It would be interesting to investigate the PrESTs that

failed to evoke an immune response further by using them in different combinations for

immunizations, to see if this resulted in different patterns of immunoreactivity. Antibody titers

could also be investigated and the possibility that there are immunodominant PrESTs (and, if

so, their amino acid sequences). In addition, immunizations using the same amount of each

PrEST as used in single immunizations would be of interest, to assess whether the low

antibody titers observed in this study were, in fact, a consequence of lower antigen

concentrations.


Proteins with highly similar sequences to other proteins, related or unrelated, pose problems,

both for the high-throughput generation of antibodies and their subsequent validation.

Cytokeratin-17 belongs to the intermediate filament family, a group with closely related

members, and was therefore chosen as a target in the studies described in Paper III. In these

studies ten antibodies towards two non-overlapping PrESTs were produced and characterized.

Epitope mapping revealed an immune preference for the C-terminal and the more flexible part

of both PrESTs, especially for the PrEST designed from the C-terminus of Cytokeratin-17.

These findings explain observed differences in the results of comparative

immunohistochemical analyses with these antibodies. The C-terminus, corresponding to one

of the PrESTs, is the most unique part of Cytokeratin-17 and the antibodies that bound

principally to this region showed the most Cytokeratin-17-like staining patterns in IHC.

Robust antigen design is, therefore, of utmost importance and the strategy of using regions

with the least sequence similarity to other proteins as the main criterion is justified. For

Cytokeratin-17, immunization with shorter peptides designed from the C-terminus would

probably improve results. Alternatively, subjecting antibodies to a second round of purification

with the C-terminal peptide as an affinity ligand could be attempted, to see if the results

obtained in subsequent analyses change.

G-protein-coupled receptors are important targets for the development of new drugs since

they regulate numerous cellular functions. In Paper IV, a novel antigen design strategy for

generating specific antibodies to three different GPCRs is described and evaluated. The

extracellular loops and the N-terminus were combined into a single antigen and the resulting

antibodies showed specificity for their respective GPCR targets in cell lines over-expressing

them. This was assessed by several techniques, including flow cytometric analysis and

immunofluorescent staining. Two of the antibodies also produced positive staining of tissues

expected to express the respective targets: human brain tissue (NPY5R) and pancreatic islets

(GLP1R). This approach to the generation of antibodies towards GPCRs should also be

applicable to other receptors (both nuclear and plasma membrane-associated) for which

antigen design is difficult since they have short stretches of exposed amino acid residues. The

protocol could also be adapted to purification processes that are already available by using

fusion tag proteins other than His6-ABP.

Large numbers of antibodies and other binders are being generated in various proteomics

initiatives, and validation of these affinity reagents is, as stated earlier, an important but

difficult task. Widely used techniques, such as immunohistochemistry, have drawbacks in

terms of standardization and reproducibility [142]. In order to ascertain that the results

obtained in IHC are accurate, the use of several binders to non-overlapping regions of the

KARIN LARSSON 55

protein under investigation is helpful. However, there are many proteins for which not even

one specific binder is available, so obtaining two or more will consequently be difficult.

Therefore, the use of other, complementary methods would be beneficial to the validation

process. One alternative approach could be to use knockout mice, if they are available, and if

the binder investigated is functional in mice. Other options, applicable to a more vide range of

targets, include RNA interference (RNAi) of expression, following which a reduction of signal

in immunofluorescence or Western blot analyses would provide strong evidence that the

applied antibodies are specific for their respective targets. Using an arsenal of well-

characterized antibodies, proteomics research will ultimately be able to increase our

understanding of protein function and biological processes substantially.


KARIN LARSSON 57

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Acknowledgements

Nu har jag faktiskt kommit till det allra sista! När jag började doktorera trodde jag nog aldrig

att jag skulle komma hit, eller det kändes i alla fall oändligt långt borta. En massa år senare så

sitter jag här och undrar hur det gick till. Utan all hjälp och allt stöd skulle jag inte ha kommit

speciellt långt det är jag ganska säker på. Därför vill jag passa på att tacka alla (hoppas verkligen

inte att jag glömmer någon) som har gjort sitt till för att den här avhandlingen skulle bli av.

Jag vill börja med att tacka mina handledare:

Henrik, du har varit både en jättebra chef när jag jobbade i Immunotech gruppen och en

superbra handledare sedan jag började doktorera. Jag skriver lite för kort, du lägger till så

brukar jag ta bort lite och sen blir det ofta rätt bra på slutet. Att du dessutom är AIKare är bara

ett plus i kanten. Särskilt tack för att du så imponerande snabbt kastade dig in i

avhandlingsarbetet när du kom tillbaka från pappaledigheten.

Sophia, efter varje möte vi haft känner jag mig alltid supertaggad och full med energi. Du är

alltid så positiv och har en förmåga att sprida din entusiasm när jag har motivationssvackor.

Sedan kommer du ofta med väldigt bra råd, både jobbmässiga och annat som rör livet i stort.

Ditt inskolningstips räddade förra hösten!

Sedan vill jag såklart tacka övriga Professorer och PIs för att ni gör den här avdelningen till en

rolig, minnesvär och intressant plats att jobba på. Jag misstänker att det är ganska ovanligt med

en så stor avdelning (numera tre avdelningar) där alla på något sätt samarbetar, känner och har

så roligt tillsammans.

Tack alla som jag har samarbetat med under den här tiden. Extra tack till Peter Nilsson och

din grupp för hjälp med protein array spottning, Tobbe och Camilla för GPCR samarbetet

och för att ni lärde mig allt om cell odling och FACSande, Cilla, Jochen och Lisa för hjälp,

data och sunpunkter på Cytokeratinpeket och Pia för att du bla hjälpte till med kloning och

odling när jag var mammaleding. Ett speciellt tack till Kennet Wester för trevliga avbrott när

jag kommit till Uppsala och för alltid lika fina IHC bilder och roliga mikroskoptittningar.

Alla i HPR projektet! Proteinfabriken och Molbio för hjälp, buffertdealar och svar på en

massa frågor. Konfokal gruppen: Emma för svar på alla mina frågor om immunofluorescence,

KARIN LARSSON 65

speciellt under avhandlingsskrivandet, Marie för att du kan alltid tar dig tid att svara på allt

från om jag borde splitta mina celler till hur man blandar PFA och för nya A-431 celler när jag

råkat blasticidina mina, Charlotte för att du hjälpte mig framkalla finfina konfokalbilder. Alla i

Uppsala för roliga julfester mm. Nya och gamla immunotechare för att jag alltid får rena på

xpresserna och för att jag fått sticka emellan och framkalla mina blottar: Pia förutom all hjälp

för att vi är original immunotechare! Erik för att du höjde stämningen och villigt svetsade i vår

xbox, Cajsa för alla snygga blotbilder mm du skickat under avhandlingsskrivandet och för en

hel del skvallrande och snack på ditt nya kontor;-)

Gamla och nya rumskompisar för alla roliga pratstunder och behövliga pauser: Tove, Emma,

Cecilia L, Esther, Pawel (för att du är ett bajenfan och för att det alltid är lika roligt att höra

om alla konspirationer som finns mot hammarby), Julia, Erik (har varit lite ont om

djurgårdare att tracka sen du slutade), Johan L, Markus, bonus-Danne, Joel, Ulrika,

Sebastian, Johanna (för hjälp med stavning när inte ens Word förstått vilket ord jag vill skriva

och för lite sista-minuten-nödlösningar) och Sverker (för hjälp med diverse Illustrator

problem).

Jag vill även tacka Ronny och Stisse för excellent exjobbshandledning (känns som att det var

ganska länge sen nu…)

Johanna för att det är mycket roligare när du är här och lite tomt när du är borta. Det har

blivit många roliga luncher på Phiphi, boxnings/avreageringspass på KTH-hallen (även om det

är väldigt länge sen nu), i alla fall en konferens (värsta flygresan) och en mammaledighet ihop.

Cilla och Hanna bla för roligt sällskap på diverse konferenser och för att ni fick mig att förstå

hur roligt det är att sticka och Anna för lite skitsnack i korridorerna och för att du (väldigt

målande) förklarat ungefär hundra ggr hur man kalibrerar Luminexen, Marica och Anna för

att ni är så trevliga och också varit här nästan lika länge som jag;-)

Fia och Johan för alltid lika roliga fikapauser, luncher mm. Det har känts bra att ha er nära, vi

har ju faktiskt som Johan påpekade känt varandra mer än en tredjedel av våra liv…


Alla andra på plan 3 för att det alltid är roligt när jag kommer till jobbet även när labbande och

skrivande har känts mindre upplyftande.

Och såklart mina vänner utanför jobbet som förgyller min fritid (även om det inte funnits så

mycket av den varan på sista tiden). Det är alltid lika roligt att träffas!

Och sist men absolut inte minst familjen: Mamma och Pappa för massor av stöd och

uppmuntrande under den här tiden, speciellt under hösten, och tack för alla dagishämtningar

och inhopp när jag har behövt jobba. Alla mina syskon: Git, Tobbe, Jenny och Pelle för att

ni har varit sådana superbra föregångare. Min egen lilla familj: Oggi för att du alltid lugnar mig

när paniken kryper närmare och för att du kommer med konstruktiva idéer och lösningar på

mina ofta påhittade och ibland verkliga problem och William för att du kom och lärde mig

vad som är viktigt i livet!

Documents

Generation and characterization of antibodies for proteomics ...275849/...KARIN LARSSON iii Karin Larsson (2009): Generation and characterization of antibodies for proteomics research