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Section Section T – Techniques in CelT – Techniques in Cell and Molecular Biologyl and Molecular Biology
ContentsContents
T1 Optical microscopyT1 Optical microscopyT2 Phase-contrast microscopyT2 Phase-contrast microscopyT3 Fluorescence microscopyT3 Fluorescence microscopyT4 Transmission electron microscopyT4 Transmission electron microscopy T5 Confocal microscopy T5 Confocal microscopy T6 The Use of radioisotopesT6 The Use of radioisotopesT7 Cell cultureT7 Cell cultureT8 The Fractionation of a cell’s contents T8 The Fractionation of a cell’s contents
by centrifugationby centrifugation
ContentsContents
T9 X-ray diffraction analysisT9 X-ray diffraction analysisT10 Measurements of proteins and of nucT10 Measurements of proteins and of nuc
leic acid concentration by spectrophotleic acid concentration by spectrophotometry ometry
T11 Ultracentrifugation T11 Ultracentrifugation T12 Nucleic acid hybridization T12 Nucleic acid hybridization T13 Recombinant DNA and T13 Recombinant DNA and
technologytechnologyT14 Gene transfer in plants and animalsT14 Gene transfer in plants and animalsT15 The use of antibodiesT15 The use of antibodies
For all scientific researches, theory needs and impels development of techniques, application of techniques to researches will push theory forward.
Questions:Who can give a few techniques applied in researches on molecular biology ?
Requirement for studying this ChapterUnderstand common techniques used in Molecular Biology.
T1 Optical microscopy
Microscopes allow Microscopes allow us to see living us to see living organisms which are organisms which are too small to be seen too small to be seen by the naked eyeby the naked eye
T2 Phase-contrast microscopyDesigned on basis for
differences of different parts of an object in their effect on light.
Who knows the advantages of use this technique?
Suitable for observation of Suitable for observation of small,unstained specimens such as a small,unstained specimens such as a living cell. living cell.
Most useful for examining intracellular Most useful for examining intracellular components of living cells.components of living cells.
Only suitable for observing single cells Only suitable for observing single cells or thin cell layer.or thin cell layer.
It has optical handicaps that result in It has optical handicaps that result in loss of resolution,and the image loss of resolution,and the image suffers from interfering halos and suffers from interfering halos and shading .shading .
Ctenoid Fish Scale
Positive Phase Contrast
Negative Phase Contrast
T3 Fluorescence microscopy
Certain compounds(called fluorochromes 荧光素 or fluorophores 荧光团 ) absorb invisible,ultraviolet radiation and release a portion of the energy in the longer,visible wavelengths, this phenomenon is called fluorescence.
Most common application －－－ immunofluorescence （免疫荧光） :
A fluorochrome 荧光染料 (such as rodamine 罗丹明 or flurescein 荧光素 ) is covalently linked to an antibody to produce a flurescent antibody that can be used to determine the location of a specific protein within the cells. This technique is called immunofluorescence.
嵌合体
波形纤维蛋白
Fluorescence in situ Hybridization
(FISH)
Fluorescencestereomicrosco
py
T4 Transmission electron
microscopy
The electron microscope uses The electron microscope uses beams of beams of electronselectrons rather than rather than lightlight to illuminate the specimen to illuminate the specimen
A beam of electrons has an A beam of electrons has an effective wavelength of less effective wavelength of less than than 1 nm1 nm so it can be used to so it can be used to resolve small sub-cellular ultra-resolve small sub-cellular ultra-structurestructure
Allowed biologists to view the Allowed biologists to view the organelles within a cell for the organelles within a cell for the first timefirst time
Transmission microscopeTransmission microscope (SEM)(SEM)
Scanning electron microscopeScanning electron microscope(( TEM)TEM)
TEMTEM It has been exploited most widely in the It has been exploited most widely in the
examination of the internal structure of cexamination of the internal structure of cells. ells.
Works like a light microscope, it Works like a light microscope, it transmits a beam of electrons transmits a beam of electrons through a thin specimenthrough a thin specimen
Then focussing the electrons to Then focussing the electrons to form an image on a screenform an image on a screen
This is the most common form of This is the most common form of electron microscope and gives electron microscope and gives good resolution.good resolution.
SEM SEM It is utilized primarily to examine thIt is utilized primarily to examine th
e surfaces of objects.e surfaces of objects. This scans a fine beam of This scans a fine beam of
electron onto specimen and electron onto specimen and collects electrons scattered by collects electrons scattered by surfacesurface
This has poor resolution but This has poor resolution but gives good 3-D imagesgives good 3-D images
Sizes of cells and their components drawn on a logarithmic scale, indicating the range of readily resolvable objects in the light and electron microscope.
Light microscope
Electron microscope
Organically Deposited Iron Oxide
The deer tick( 壁虱 ), responsible for transmitting Lyme Disease
Disadvantages of the Disadvantages of the electron microscopeelectron microscope
The specimens must be fixed The specimens must be fixed in plastic and viewed in a in plastic and viewed in a vacuum and so they must be vacuum and so they must be deaddead
Sometimes specimens can be Sometimes specimens can be damaged by the electron beam damaged by the electron beam and must be stained with an and must be stained with an electron-dense chemicalelectron-dense chemical
T5 T5 Confocal microscopy Confocal microscopy
laser scanning confocal microscope (LSCM) offers several advantages over conventional optical microscopy, including
1 ） shallow depth of field, 2 ） elimination of out-of-focus glare（眩目光，或瞪眼现象） , 3 ） the ability to collect serial optical sections from thick specimens.
Being common in the biomedical sciences. For imaging either fixed or living cells and tissues that have usually been labeled with one or more fluorescent probes.
Application
LSCM is somewhat better than in the conventional widefield optical microscope, but still considerably less than that of the transmission electron microscope, it has in some ways bridged the gap between the two more commonly used techniques.
The information flow in a modern laser scanning confocal microscope is diagramed in the Figure.
T6 The use of radioisotopes
Tracers are often used to localize and monitor molecules during the course of an experiment.
Molecular tracingMolecular tracing
Fluorescently labledFluorescently labled Spin labeled (Spin labeled ( 自旋标记自旋标记 )) Density labeled(Density labeled( 密度标记密度标记 )) Radioactively labeledRadioactively labeled
T7 Cell culture
Growing cells outside the organism.
1) Animal cell culture, such as
human embryos .
2) Plant cell culture.
Points for attentionPoints for attentionDesign and equipment Design and equipment
for the cell culture for the cell culture laboratorylaboratory
Safety aspects of cell Safety aspects of cell cultureculture
Sourcing of cell linesSourcing of cell lines Main types of cell cultureMain types of cell culture
The cell environmentThe cell environment Cryopreservation(Cryopreservation( 深低温保藏深低温保藏 ) and ) and storage of cell linesstorage of cell linesGood cell banking practicesGood cell banking practicesQuality control considerationsQuality control considerations Authentication(Authentication( 真实性真实性 ) of cell line) of cell linessAlternative cell culture systemsAlternative cell culture systems
Key factors of cell/tissue culture
Media
Sources of cell / tissue
Attached Cell LinesName Species and tissue of origin Morphology
MRC-5 (Prod. No. 84101801) Human lung FibroblastHELA (Prod. No. 93021013) Human cervix EpithelialVERO (Prod. No. 84113001) African Green Monkey Kidney Epithelial
NIH 3T3 (Prod. No. 93061524) Mouse embryo Fibroblast
L929 (Prod. No. 85011425) Mouse connective tissue FibroblastCHO (Prod. No. 85050302) Chinese Hamster Ovary FibroblastBHK-21 (Prod. No. 85011433) Syrian Hamster Kidney Fibroblast
HEK 293 (Prod. No. 85120602) Human Kidney Epithelial
HEPG2 (Prod. No. 85011430) Human Liver Epithelial
BAE-1 (Prod. No. 88031149) Bovine aorta EndothelialSuspension Cell LinesName Species and tissue of origin Morphology
NSO (Prod. No. 85110503) Mouse myeloma Lymphoblastoid-like
U937 (Prod. No. 85011440) Human Hystiocytic Lymphoma Lymphoblastoid
Namalwa (Prod. No. 87060801) Human Lymphoma Lymphoblastoid
HL60 (Prod. No. 98070106) Human Leukaemia Lymphoblastoid-like
WEHI 231 (Prod. No. 85022107)
Mouse B-cell Lymphoma Lymphoblastoid
YAC 1 (Prod. No. 86022801) Mouse Lymphoma LymphoblastoidU 266B1 (Prod. No. 85051003) Human Myeloma Lymphoblastoid
SH-SY5Y (Prod. No. 94030304) Human neuroblastoma Neuroblast
Plant cell culture
Preparation of protoplasts
Growing the protoplasts
Obtaining an undifferentiated clump of cells,called callus
Inducing to develop shoots
Using some tissues
Plant cell suspension culture
Primary culture
Scale-up of cultures
T8 The fractionation of
a cell’s contents by
centrifugationCentrifugation is a process used to separate or concentrate materials suspended in a liquid medium.
We often meet centrifugation to study a particular function of different organelles ,such as mitochondria, or particular components within cells, such as enzymes within the Golgi complex.
Two methods of the cell fractionation:
1) Differential centrifugation.
2) Buoyant density centrifugation.
Based on differences of of organelles or molecules in gravity caused by differences in their sizes, shapes and densities
Principle of differential centrifugation
A test tube containing homogenization before differential centrifugation.
After the centrifugation
This diagram shows the break down of the cell parts relating to the speed during differential centrifuge.
The fractionation of rat liver is an example of how this process works.
Isolate mitochondria from cauliflower. This will be done by two centrifugations, resulting in two pellets and two supernatant fractions:
The sucrose gradient and the buoyant density centrifugation.
The buoyant density centrifugation involves viruses with densities of 1.1-1.2 g/cm and a sucrose gradient. The cell suspension is added to the top of the sucrose gradient. In this centrifugation the densest components move fastest down the tube and stops at the sucrose density equal to its own. The sucrose gradient bands at the bottom contain cell components with high buoyant densities and the components at the top have low buoyant densities.
T7 Isolation ,purification ,and fractionation of proteins
We must obtain a relatively pure proteins to investigate their functions.
Key problems for doing this:
Effectively remove the contaminants from the target protein.
Challenge missions:
1) A cell contain thousands of different proteins.
2) Some among them have similar properties.
3) Others are present in the cell at low concentrations.
Strategies and methodsStrategies and methods Selective precipitationSelective precipitation Liquid column chromatograpghy(Liquid column chromatograpghy( 液相柱液相柱层析层析 ))
Chromatography is a term of techniques in whicChromatography is a term of techniques in which a mixture of dissolved components is fractionh a mixture of dissolved components is fractionatedated （（分级分离分级分离） ） as it move through some of pas it move through some of porous matrix.orous matrix.
1)1) Gel filtration chromatography Gel filtration chromatography 2)2) Affinity chromatography Affinity chromatography
Figure 1. Loading affinity column.
Figure 2. Proteins sieve through matrix
of affinity beads.
Figure 3. Proteins interact with affinity ligand with some binding loosely and others t
ightly.
Figure 4. Wash off proteins that do
not bind.
Figure 5. Wash off proteins that bind
loosely.
Figure 6. Elute proteins that bind tightly to ligand and collect
purified protein of interest.
Polyacrylamide gel electrophoresis(PAGE)Polyacrylamide gel electrophoresis(PAGE) 1) Isoelectric focusing1) Isoelectric focusing 2) Sodium dodecyl sulfate (SDS)-PAGE2) Sodium dodecyl sulfate (SDS)-PAGE 3) Two-dimensional gel electrophoresis3) Two-dimensional gel electrophoresis
PAGE systemPAGE system
PAGE systemPAGE system
T8 Determination of T8 Determination of protein structure by X-protein structure by X-ray diffraction analysisray diffraction analysis
If you want to investigate a If you want to investigate a protein structure , the X-ray protein structure , the X-ray diffraction technique is the diffraction technique is the best choice.best choice.
X-ray diffraction is ideally X-ray diffraction is ideally suited for determining the suited for determining the structure of soluble proteins.structure of soluble proteins.
Principle
The sample, as a single The sample, as a single crystal or ground to powder, crystal or ground to powder, is exposed to X-rays at is exposed to X-rays at various angles; the diffraction various angles; the diffraction patterns produced are then patterns produced are then compared with reference compared with reference standards for identification.standards for identification.
Note:
X-ray diffraction is less appropriate for study of complex molecular structures,such as ribosomes or proteasomes ( 蛋白酶体 ),or for membrane proteins ， where it is difficult to obtain the three-dimensioned crystals required for analysis. Structural analysis of these types of specimens is often conducted using alternate technique known as electron cryomicroscopy( 冷冻电镜技术 ).
T9 Purification and T9 Purification and fractionation of nucleic fractionation of nucleic
acid acid
You must obtain purified DNA You must obtain purified DNA or RNA molecules with or RNA molecules with different sizes in order to different sizes in order to conduct the downstream conduct the downstream researches ,such as analysis of researches ,such as analysis of gene expression, and gene expression, and identification of gene functions.identification of gene functions.
Usually, we encounter separation of DNA or RNA from a mixture of DNA ,RNAs ,and proteins.
Separation of DNA restriction fragments by gel electrophoresis
Here is an example how to separate DNA by gel electrophoresis in the lab. Gel electrophoresis is also widely used to separate nucleic acids.
Unlike proteins, all nucleic acid molecules ,regardless of their length, have a similar charge density,and therefore, all have an equivalent potential for migration in an electric field.
So, fractionation of nucleic fractionation of nucleic acids is easier and simpler than acids is easier and simpler than that of proteins. that of proteins.
T10 Measurements of proteinT10 Measurements of proteins and of nucleic acid concents and of nucleic acid concentration by spectrophotometry ration by spectrophotometry
Based on maximal light Based on maximal light absorbance:absorbance:
For proteins, at 280nm.For proteins, at 280nm.
For nucleic acids,at 260nm.For nucleic acids,at 260nm.
T11 Ultracentrifugation T11 Ultracentrifugation
The technique for the The technique for the sedimentation of particles sedimentation of particles subjected to extremely rapid subjected to extremely rapid rotation to overcome the effects rotation to overcome the effects of diffusion and cause of diffusion and cause macromolecules to sediment macromolecules to sediment toward the bottom of a toward the bottom of a centrifuge.centrifuge.
During ultracentrifugation, During ultracentrifugation, centrifugal forces as high centrifugal forces as high as 500,000 times the as 500,000 times the force of gravity can be force of gravity can be generated.generated.
Differential centrifugation
Rate zonal centrifugation
( 差速区带离心 )
Isopycnic centrifugation
( 等密度离心 )
T12 Nucleic acid T12 Nucleic acid hybridization hybridization
Principle Principle
A technique used to A technique used to identify and locate identify and locate DNA sequences DNA sequences which are which are complementary to complementary to another piece of another piece of DNA called a probe.DNA called a probe.
Southern blot
Northern blotNorthern blot
A technique used A technique used to identify and to identify and locate mRNA locate mRNA sequences that sequences that are are complementary to complementary to a piece of DNA a piece of DNA called a probe. called a probe.
FISH - (Fluoresence in situ hybridiFISH - (Fluoresence in situ hybridization)zation)
A process which viviA process which vividly(dly( 生动地生动地 ) paints c) paints chromosomes or porthromosomes or portions of chromosomeions of chromosomes with fluorescent ms with fluorescent molecules. This techniolecules. This technique is useful for idenque is useful for identifying chromosomal tifying chromosomal abnormalities and gabnormalities and gene mapping. ene mapping.
Recombinant DNA are molecules Recombinant DNA are molecules containing DNA sequences derivecontaining DNA sequences derived from more than one source.d from more than one source.
Recombinant DNAs can be formeRecombinant DNAs can be formed in a variety of ways or techniqud in a variety of ways or techniques.es.
T13 Recombinant DNA T13 Recombinant DNA and and
technologytechnology
TechnologyTechnology DNA cloningDNA cloning Construction of a DNA libraryConstruction of a DNA library Coning larger DNA fragments in Coning larger DNA fragments in
specialized cloning vectorsspecialized cloning vectors Chemical synthesis and site-Chemical synthesis and site-
directed mutagenesisdirected mutagenesis Cloning of genes by PCRCloning of genes by PCR … …
Construction of a genomic Construction of a genomic librarylibrary
Genomic library Genomic library comprises a set of comprises a set of bacteria, each carrying a bacteria, each carrying a different small fragment different small fragment of human DNA. For of human DNA. For simplicity, cloning of just simplicity, cloning of just a few representative a few representative fragments (colored) is fragments (colored) is shown. In reality, all the shown. In reality, all the gray DNA fragments will gray DNA fragments will also be cloned. also be cloned.
Construction of a Construction of a YAC libraryYAC library
YAC :Yeast artificial YAC :Yeast artificial
chromosome .chromosome .
A YAC can be considered as a functional artificial A YAC can be considered as a functional artificial chromosome (self replicating element), since it chromosome (self replicating element), since it includes three specific DNA sequences that enaincludes three specific DNA sequences that enable it to propagate from one cell to its offspring:ble it to propagate from one cell to its offspring:
TELTEL: The telomere which is located at each chro: The telomere which is located at each chromosome end, protects the linear DNA from degrmosome end, protects the linear DNA from degradation by nucleases. adation by nucleases.
CENCEN: The centromere which is the attachment s: The centromere which is the attachment site for mitotic spindle fibers, "pulls" one copy oite for mitotic spindle fibers, "pulls" one copy of each duplicated chromosome into each new df each duplicated chromosome into each new daughter cell. aughter cell.
ORIORI: Replication origin sequences which are sp: Replication origin sequences which are specific DNA sequences that allow the DNA replicaecific DNA sequences that allow the DNA replication machinery to assemble on the DNA and motion machinery to assemble on the DNA and move at the ve at the replication forksreplication forks. .
ExampleExample
Cloning into a plasmidCloning into a plasmid
Process by Process by which a plasmid which a plasmid is used to import is used to import recombinant recombinant DNA into a host DNA into a host cell for cloning.cell for cloning.
Whole genome Whole genome sequencing sequencing
Hierarchical(Hierarchical( 梯级梯级 ) Shotgun ) Shotgun SequencingSequencing
Shotgun sequencingShotgun sequencing
Hierarchical(Hierarchical( 梯级梯级 ) sh) shotgun sequencingotgun sequencing
11 ）） The DNA was cut intThe DNA was cut int
o 150 Mb fragments and o 150 Mb fragments and arranged into overlappiarranged into overlapping fragments. ng fragments.
22 ）） These contigsThese contigs （（重叠重叠群群） ） were cut into smallwere cut into smaller pieces and sequenceer pieces and sequenced completely.d completely. Schematic diagram of Schematic diagram of
sequencing strategy used by sequencing strategy used by the publicly funded Human the publicly funded Human Genome Project.Genome Project.
Shotgun sequencingShotgun sequencing The method developed and preferred by The method developed and preferred by
Celera is simply called shotgun sequencing. Celera is simply called shotgun sequencing. This approach was developed and perfected on This approach was developed and perfected on
prokaryotic genomes which are smaller in size prokaryotic genomes which are smaller in size and contain less repetitive DNA.and contain less repetitive DNA.
Shotgun sequencing randomly shears Shotgun sequencing randomly shears
genomic DNA into small pieces which are genomic DNA into small pieces which are cloned into plasmids and sequenced on both cloned into plasmids and sequenced on both strands, thus eliminating the BAC step from the strands, thus eliminating the BAC step from the HGP's approach. Once the sequences are HGP's approach. Once the sequences are obtained, they are aligned and assembled into obtained, they are aligned and assembled into finished sequence. finished sequence.
Schematic diagram of shSchematic diagram of shorugun sequencing .orugun sequencing .
The DNA was cut into smaThe DNA was cut into sma
ll pieces and sequenced cll pieces and sequenced completely. ompletely.
These fragments were orgThese fragments were organized into contigs based anized into contigs based on overlapping sequenceon overlapping sequences.s.
T14 Gene transfer in T14 Gene transfer in plants and animalsplants and animals
Introduction of DNA into Introduction of DNA into animal cellsanimal cells
Example 1Example 1 :A eukaryotic gene of i :A eukaryotic gene of interest is cloned in a plasmid conterest is cloned in a plasmid containing a drug resistance markentaining a drug resistance marker that can be selected for in cultur that can be selected for in cultured animal cells. The plasmid DNred animal cells. The plasmid DNA is introduced into cultured cellA is introduced into cultured cells as a calcium phosphate coprecis as a calcium phosphate coprecipitatepitate （（磷酸钙沉淀物磷酸钙沉淀物）） , which is , which is taken up and expressed by a fractaken up and expressed by a fraction of the cells for a few days (trtion of the cells for a few days (transient expression). Stably transansient expression). Stably transformed cells, in which the plasmiformed cells, in which the plasmid DNA becomes integrated into cd DNA becomes integrated into chromosomal DNA, can be selectehromosomal DNA, can be selected for by their ability to grow in dd for by their ability to grow in drugrug －－ containing medium. containing medium.
Example 2 :Example 2 : Cloned genes can also be introduced int Cloned genes can also be introduced into the germ line of multicellular organisms, allowing o the germ line of multicellular organisms, allowing them to be studied in the context of the intact animthem to be studied in the context of the intact animal rather than in cultured cells.al rather than in cultured cells.
Production of transgenic miceProduction of transgenic mice DNA is microinjected into a pronucleus DNA is microinjected into a pronucleus of a fertilized mouse egg (fertilized eggs contain two pronuclei, one of a fertilized mouse egg (fertilized eggs contain two pronuclei, one from the egg and one from the sperm). The microinjected eggs are tfrom the egg and one from the sperm). The microinjected eggs are then transferred to foster mothershen transferred to foster mothers （（代孕母亲代孕母亲） ） and allowed to devand allowed to develop. Some of the offspring (transgenic) have incorporated the injecelop. Some of the offspring (transgenic) have incorporated the injected DNA into their genome. ted DNA into their genome.
Introduction of genes Introduction of genes into plant cells via the into plant cells via the
Ti plasmidTi plasmid The Ti plasmid contains the T region, wThe Ti plasmid contains the T region, w
hich is transferred to infected plant cellhich is transferred to infected plant cells, and virulence (s, and virulence (virvir) genes, which func) genes, which function in T DNA transfer. In Ti plasmid vection in T DNA transfer. In Ti plasmid vectors, foreign DNA is inserted into the T rtors, foreign DNA is inserted into the T region. The recombinant plasmid is intregion. The recombinant plasmid is introduced into oduced into Agrobacterium tumefacienAgrobacterium tumefacienss, which is then used to infect cultured , which is then used to infect cultured cells. The T region of the plasmid (carrycells. The T region of the plasmid (carrying the inserted DNA) is transferred to ting the inserted DNA) is transferred to the plant cells and integrates into chrohe plant cells and integrates into chromosomal DNA. A transgenic plant can tmosomal DNA. A transgenic plant can then be generated from the transformehen be generated from the transformed cells. d cells.
T15 The use of antibodiesT15 The use of antibodies
Antibodies function as markers, binding to the antigen so that the antigen molecules can be recognized and destroyed by phagocytes.
A summary of the process of monoclonal antibody production. (Source: Biotech, 1989)
杂交瘤细胞
Uses of monoclonal Uses of monoclonal antibodiesantibodies
Monoclonal bodies have a variety Monoclonal bodies have a variety of academic, medical and of academic, medical and commercial uses. It would be commercial uses. It would be impossible to list all of these here. impossible to list all of these here. But the following list should But the following list should indicate how ubiquitous indicate how ubiquitous monoclonal antibody technology monoclonal antibody technology has become in biotechnology.has become in biotechnology.
Diagnostic tests to detect small amouDiagnostic tests to detect small amounts of drugs, toxins or hormones.nts of drugs, toxins or hormones.
e.g. monoclonal antibodies to human e.g. monoclonal antibodies to human chorionic gonadotropin (HCG;chorionic gonadotropin (HCG; 绒毛促性绒毛促性腺激素腺激素 ) are used in pregnancy test kits ) are used in pregnancy test kits (Biotech, 1989). Another diagnostic us(Biotech, 1989). Another diagnostic uses of antibodies is the diagnosis of AIDes of antibodies is the diagnosis of AIDS by the ELISA test. S by the ELISA test.
Classify strains of a single pathoClassify strains of a single pathogengen
e.g. e.g. Neisseria gonorrhoeaeNeisseria gonorrhoeae can can be typed using monoclonal antibe typed using monoclonal antibodies (Wang et al, 1977). bodies (Wang et al, 1977).
RadioimmunodetectionRadioimmunodetection and and radioimradioimmunotherapymunotherapy of cancer, and some ne of cancer, and some new methods can even target only the cw methods can even target only the cell membranes of cancerous cells ell membranes of cancerous cells (Ch(Chaudhari et al, 1994)audhari et al, 1994). .
A new cancer drug based on monoclonal A new cancer drug based on monoclonal antibody technology is Ritoxin, approved antibody technology is Ritoxin, approved by the FDA in November 1997 (Orrs, 1997). by the FDA in November 1997 (Orrs, 1997).
Treat viral diseases, Treat viral diseases, traditionally considered traditionally considered "untreatable". "untreatable".
In fact, there is some In fact, there is some evidence to suggest that evidence to suggest that antibodies may lead to a antibodies may lead to a cure for AIDS (P/S/L, 1997). cure for AIDS (P/S/L, 1997).
Identify and to trace specific cells or Identify and to trace specific cells or molecules in an organism. molecules in an organism.
e.g. developmental biologists at the e.g. developmental biologists at the University of Oregon use monoclonaUniversity of Oregon use monoclonal antibodies to find out which proteil antibodies to find out which proteins are responsible for cell differentians are responsible for cell differentiation in the respiratory system (Frateltion in the respiratory system (Fratella, 1998). la, 1998).
AlleviateAlleviate （（减轻减轻） ） the problem othe problem of organ rejection in patients who f organ rejection in patients who have had organ transplants (Trahave had organ transplants (Transweb, 1996). nsweb, 1996).
Problems with monoclonal therapy
This is not a happy prospect for the This is not a happy prospect for the
pharmaceutical industry. The very pharmaceutical industry. The very specificity of antibody therapy specificity of antibody therapy guarantees that the market is small, guarantees that the market is small, because every different type of because every different type of cancer requires a different product. cancer requires a different product. Nevertheless the investment Nevertheless the investment required to carry out clinical trials is required to carry out clinical trials is enormous. enormous. Continue to see
next slides
Why are there so few monoclonals being used in human therapy a quarter century after their discovery? The main difficulty is that mouse antibodies are "seen" by the human immune system as foreign, and the human patient mounts an immune response against them, producing HAMA ("human anti-mouse antibodies"). These not only cause the therapeutic antibodies to be quickly eliminated from the host, but also form immune complexes that cause damage to the kidneys.
Using genetic engineering it is possible to make mouse-human hybrid antibodies in an attempt to reduce the problem of HAMA. •Chimeric antibodies. The antibody combines the antigen-binding parts (variable regions) of the mouse antibody with the effector parts (constant region) of a human antibody. Infliximab, rituximab, and abciximab are examples.
•Humanized antibodies. The antibody combines only the amino acids responsible for making the antigen binding site (the hypervariable regions) of a mouse (or rat) antibody with the rest of a human antibody molecule thus replacing its own hypervariable regions. Daclizumab, Vitaxin, Mylotarg®, Herceptin, and Xolair® are examples.
The selection of productThe selection of product
It is difficult and criticalIt is difficult and critical
1)1) No sufficient volunteers or No sufficient volunteers or patients to do test. patients to do test.
2)2) Patent rights. Patent rights.
Combinations of antibodiesCombinations of antibodies
It may be significantly more It may be significantly more effective,but these combinations effective,but these combinations need to be tested in small pilot need to be tested in small pilot studies using new methods for studies using new methods for detecting and measuring small detecting and measuring small numbers of tumor cells to get an numbers of tumor cells to get an early indication of the likely early indication of the likely outcome.outcome.
Summary for this Chapter
Main categories of techniques introduced in this chapter
1) Imagery
2) Detection
3) Isolation/Fractionation
4) DNA manipulation
5) Cell/Tissue culture
6) Uses of antibodies
