Dr Frank Hills

Outline Applications

Qualitative: immunodiffusion, IHC, Western blotting

Quantitative: immunoassay nephelometry, turbimetry, FACS, cell sorting

Pitfalls

Specificity/cross-reactivity

Speed of analysis

Refinements

Monoclonal

Chimeric - FAb fragments

Non equilibrium assay

Recent developments

Assay chip

Sample prep for mass spectrometry

Antibody structure

Antibodies

Aka immunogloboluns (IgG, IgM etc.)

Antibody specificity

Cortisol

MW = 362

Stress hormone

Used to monitor adrenal function

Diagnosis of adrenal function

e.g. Cushing’s, Addisons

CH3

CH3

17β-Estradiol

MW = 272

Female sex hormone

Used to monitor ovarian function

Diagnosis of impaired ovarian function

e.g. amenorrhoea, menopause,

IVF

Testosterone

MW = 288

Male sex hormone

Used to monitor testicular function

Diagnosis of impaired testicular function

e.g. Klinefelters syndrome, anorchia, IVF,

hirsutism, polycystic ovary syndrome

CH3

CH3

CH3

Cortisol

MW = 362

Stress hormone

Used to monitor adrenal function

Diagnosis of adrenal function

e.g. Cushing’s, Addisons

CH3

CH3

17β-Estradiol

MW = 272

Female sex hormone

Used to monitor ovarian function

Diagnosis of impaired ovarian function

e.g. amenorrhoea, menopause,

IVF

Testosterone

MW = 288

Male sex hormone

Used to monitor testicular function

Diagnosis of impaired testicular function

e.g. Klinefelters syndrome, anorchia, IVF,

hirsutism, polycystic ovary syndrome

CH3

CH3

CH3

3 Double bonds 1 double bond

Antibody specificity

Antibody generation

Animal injected with

molecule to be

measured

Animal immune system (B

lymphocytes) generates

specific antibodies

Collect blood

Extract

antibody to

molecule

Immunisation

Collection

Immune response improved by:

Attaching larger, foreign target

Injecting adjuvant

Affinity chromatography

Serum added to affinity

column containing molecule to

be detected attached to solid

beads

Some proteins do not

bind to molecule on

beads and pass through

column

Some bind loosely and

others bind tightly

Specific antibodies are

washed off using low pH

buffer

Early applications

Antibodies first used to treat disease in the late 19th

century (diptheria)

Immunodiffusion (1960s)

Ouchterlony

Radial

Rocket

Immunohistochemistry

Western blotting

Gel Diffusion Antibody or antigen added

to center well.

Known sample added to

outer wells.

Wait for bands to form.

QUALITATIVE only

Antibody mixed with agar

Sample added to wells.

Antigen will diffuse out and

form precipitin ring.

Diameter proportional to

concentration.

Immunohistochemistry

Add substrate

•Colour reaction only where the antibody is present

Enz

** *

Enz Enz

Brown area:

Coloured substrate

present

enzyme present

2nd antibody present

1st antibody present

Molecule present

Blue colour - counterstain

Syndecan-1 Isotype control

Term placenta

Hills et al (2014)

Western blotting

Reveals only the protein of interest

SDS-PAGE separates proteins by size

Proteins blotted on membrane

Antibodies bind to protein of interest

Application

Heparin blocks apoptosis

•Effects of glycosaminoglycans on villous trophoblast function

Heparin stimulates EGF receptor activation

Hills et al (2006)

Quantitative techniques

Immunoassay

Competitive (RIA)

Direct (ELISA)

Flow cytometry (FACS)

Cell sorting (MACS)

Capture antibody adsorbed onto plate

Serum sample added. Antigen binds to antibody

Enzyme-coupled detector antibody added.

Enzyme-linked immunosorbant assay (ELISA)

Substrate added

Colour reaction

Ab

so

rba

nc

e

Antigen

Applications of immunoassay

Anim-Nyame et al 2000

Hills et al 2014

Flow cytometry

Cell sorting (MACS)Mixture of different cell types

Antibody attached to magnetic bead

Antibody binds to protein only expressed

by one cell type

Cells passed through magnet

Cells which bind antibody are

retained by magnet

Magnet removed

Cells which bind antibody are

removed

Hills et al 2012

MACS to isolate villous trophoblasts from placenta

HLA class I,II,III -ve immunoselection

Confirmed by immunocytochemistry

Pitfalls Specificity

Quality of antibody

Amount of antibody

Amount of antibody

Antibody

bound

Specific binding

Non-specific

binding

Refinements• Specificity (monoclonal and polyclonal antibodies)

• Fusing antibody-producing cells with cancer cells

• Hybridomas produce multiple copies of identical antibodies

• Rapid immunoassays

Polyclonal vs. monoclonalPolyclonal

• Cheap to produce

• Mixed population of antibodies

• May bind to different areas of

target molecule

• Tolerant of small changes in

protein structure (denaturation,

dimerisation, phosphorylation)

Monoclonal

• Expensive to produce

• Single antibody species

• Will only bind single specific

site

• May only recognise a particular

protein form (phosphorylation,

dimersied)

• Infinitely renewable

Polyclonal antibodiesMonoclonal antibodies

Rapid immunoassays

Ab + Ag AbAg

0

10

20

30

40

50

60

0 20 40 60 80 100 120

Ab

Ag

co

mp

lex

Time (minutes)

non-

equilibrium

equilibrium

• Automated accurate timing allows

rapid non-equilibrium reactions

•Nephelometry and turbimetry

(colour reaction not required)

Detector

*

*

New techniques

Assay chips

IPP for Proteomic/mass spectrometry analysis

Nanodot array luminometric immunoassay (NALIA)

• Antigens or antibodies adsorbed onto underside of membrane on 96 well plate

• 5 x 5 array allows measurement of 10 analytes in duplicate

• In this case,

• autoantibodies analysed

• serum added, drains through membrane

• Autoandibodies bind array

•hrp labelled anti human IgG added

•Chemiluminescent substrate Patient sample

Autoantibodies

Antigen array

Nanodot array luminometric immunoassay (NALIA)

Controls in central

region - + - + -

Array (La, Ro etc

elsewhere)

Economical platform

for a wide range of

multiple analyte

applications

Immunoprecipitation for proteomics

Ab mixed with protein A

coated bead

Bound Ab added to

serum/cell lysate etc

Target protein immobilised

on bead and applied to

SDS-PAGE

Protein of

interest

Application – glycan analysis

MALDI glycan sequencing

-ve mode

Zip tips

HILIC (hydrophilic peptides)

C18 (hydrophobic)

• Improving the predictive value of biomarkers

• Isoforms e.g. glycan analysis

• Combines Ab technology with mass spectrometry

Protein of

interest

Trypsin

wash

elute

Cut out bands In-gel digest Further clean-up

MALDI Ionisation Technique

Sample mixed with matrix

Laser vaporises and ionises

sample

Ions migrate down a tube

Speed is inversely

proportional to their m/z

ratioMALDI Glycan

sequencing (-ve mode)

Summary

• Antibody technology has been in use for many years

• New technologies are continually developing

• Continue to incorporate antibodies specificity and ease of use

•In our labs we are using antibodies in a variety of ways to:• Identify novel biomarkers for disease

•Understand mechanisms of actions of specific molecules in order to

• identify novel treatments

Thank you

Assay chips

Figure 3: The biosensor developed. (a) With the protein G layer. From bottom to top: The golden substrate,

SAM layer, protein G layer and antibodies with attached biomarker, (b) the sensor coated with the antibody Fab

fragment which couples to the electrode through its C-terminal SH group.

• Antibody immobilised via linker to gold or graphene surface

of biosensor

• Antigen binding causes changes in electrical impedance

• Can be multiplexed

• PoC device