Application of Antibody

Application of Antibody -Antibody -Western blot -Immunoprecipitation -Immunohistochemical staining -Elisa -Elispot -Flow cytometry -Fluorescenct microscope -EMSA -Affinity purification -Immunotoxin -Mimickry of ligand (activatory, inhibitory) --neutralizing Ab as therapeutics 2013/10/2 Antibodies 1

References 1. Antibodies laboratory manual David Lane et al chapter 5,6,14 2. Immunology Kuby et al. chapter 3, 6 2013/10/2 Antibodies 2

Antibody - Classification -Antibody production and diversity -Structure and function -Antibody/antigen interaction -Reagents able to affect Ab 2013/10/2 Antibodies 3

Antibody Class Immunoglobulins--- Ig A, IgD, IgE, IgG, IgM. IgG predominates. The light chains have a variable portion that is different in each type of antibodies and is the active portion of the molecule that bind with the antigens. The light chains and the heavy chains are connected by S-S bond. Soluble in water or dilute salt solution. Antibodies can act independently of the rest of the immune sys. 2013/10/2 Antibodies 4

Classes of Antibodies Five classes of antibodies (IgA, IgD, IgE, IgG, and IgM) can be distinguished by differences in the heavy chain Differences in the heavy chains (the tail regions) impart distinctive functional properties to the classes of antibodies 2013/10/2 Antibodies 5

Classes of Antibodies IgG: Produced during the secondary immune response. Major class secreted into the blood. Activates the complement system. Binds to macrophages and neutrophils inducing phagocytosis. Only antibody to pass from mother to fetus via the placenta. Secreted into the mother s milk. 2013/10/2 Antibodies 6

Classes of Antibodies IgM: First antibody produced by B cells. Membrane bound then secreted into the blood in the primary response. Activates the complement system IgD: Produced by activation of resting B cells. Contains same antigen binding sites as IgM. Membrane bound. Not secreted. Function unknown 2013/10/2 Antibodies 7

Classes of Antibodies IgA: Principle class of antibodies secreted in saliva, tears, milk, respiratory track, and intestines. IgE: The Fc region binds with high affinity to the surface of mast cells in tissues and basophils in blood. Binding of antigen produces allergic reactions 2013/10/2 Antibodies 8

Antibody Production The antigen receptor of a membrane bound antibody molecule is presented on the surface of a resting B cell. Upon activation, B cells proliferate and produce millions of copies of a secreted antibody with the same antigen binding sites. 2013/10/2 Antibodies 9

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Antibody Diversity A human can make at least 10 15 antibody molecules. Antibodies are proteins encoded by genes. The human genome is thought to contain less than 10 5 genes. How can a human make more antibodies than there are genes in its genome? 2013/10/2 Antibodies 11

Antibody Diversity During B cell development, separate gene segments are assembled in a genetic recombination event before the gene is transcribed. Experiments done in 1976 showed that the C-coding and V-coding regions on different parts of the chromosome were spliced together. 2013/10/2 Antibodies 12

Combinatorial Diversification In the mouse variable light chain, about 300 different V-regions can be joined to 4 different J segments which are then joined to one C-region. In the mouse variable heavy chain, 500 different V-regions can be joined to 4 J segments and 12 D segments. What is the total number of possible combinations? 2013/10/2 Antibodies 13

Combinatorial Diversification Light Variable Chain: 300 x 4 = 1200 Heavy Variable Chain: 500 x 4 x 12 = 24,000 Total possible combinations: 1200 x 24,000 = 28,800,000 2013/10/2 Antibodies 14

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Antibody Structure Consists of four interconnected polypeptide chains. Two heavy chains (H-chains) and joined to two shorter chains (L-chains). These four chains are arranged in the form of a Y ; with the stalk of the Y is called the crystallizable fragment and the top of the Y is known as the antigen-binding fragment. 2013/10/2 Antibodies 18

Hv Lv Lc Hc The amino acid sequence and configuration of an antibody were determined in the 1960s by the biochemists Gerald Edelman, an American, and R.R. porter, An English man; for this achievement they shared the 1972 Nobel Prize for physiology or Medicine. http://www.accessexcellence.org/ab/gg/antibodies.html 2013/10/2 Antibodies 19

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A Fab is the one copy of the variable region of an antibody. 2013/10/2 Antibodies 21

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Antibody Fine Structure Light and heavy chains have variable regions and constant regions (relative to amino acid sequence) The 110 amino acid variable regions are located at the amino-terminal ends and form the antigen binding site Within the variable regions are three hypervariable regions (5 to 10 amino acids) 2013/10/2 Antibodies 23

Constant and Variable Regions 2013/10/2 Antibodies 24

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Antibody Domains Both chains are composed of 110 amino acid repeating segments The repeating segments fold independently to form functional domains Each domains is held together by one intrachain disulfide bond The domains evolved by gene duplications 2013/10/2 Antibodies 26

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Three-Dimensional Structure Each domain folds into a very similar three-dimensional structure In the variable domain are three hypervariable loops that form the antigen binding site Changing the length and amino acid sequence of the hypervariable loops does not disturb the threedimensional structure 2013/10/2 Antibodies 28

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Structure of Light & Heavy Chains Heavy Chains Genes encoding the constant regions are similarly designated (Cm, Cd, Cg, Ca, Ce) Any individual of a species makes all 5 types of H chains In any one Ab molecule both H chains are identical Several subclasses IgG1, IgG2, IgG3 and IgG4 IgA1 and IgA2 2013/10/2 Antibodies 31

Ig Structure Variable Region Part that binds to the epitope Determines the numerous individual specificities Greatest variability in sequence in the N-terminal 110 aa of both the L and H chains Kabat and Wu showed there are three regions of the L and H chains that show the greatest amount of variability hypervariable regions Participate in binding with Ag Form the complementarity-determining regions (CDRs) of the L and H chains Folding brings the the CDR s together Variability in the CDRs provides the diversity in shape and size of the combining site site often takes the form of a cleft or depression thus fit affects affinity and cross-reactivity 2013/10/2 Antibodies 32

Variable Region A particular Ab combining site may have the ability to combine with two or more Ags redundancy Cross-reactivity may reduce the number of different Abs needed to defend an individual against the range of Antigenic challenge 2013/10/2 Antibodies 33

Immunoglobulin Variants Isotypes 5 different classes of Heavy chains Allotypes Genetic differences between individuals Allelic forms of the same protein Allotypic differences at known loci usually involve changes in only one or two aa in the constant region of a chain Idiotypes The combined antigenic determinants (idiotypes) expressed in the variable region of Ab of an individual that are directed at a particular Ag 2013/10/2 Antibodies 34

Immunoglobulin Isotypes Biological Properties of IgG classical Ab Largest % of total Ig and 15% of total protein in serum Equally distributed b/w intra and extra-vascular spaces Four subclasses IgG1, IgG2 and IgG 4 ½ life of approx 23 days longest of all Ig isotypes (IgG3 about 7 days) Most suitable for passive immunization by transfer of ab 2013/10/2 Antibodies 35

Antigenic Determinants (Epitopes) The specific site of an antigen that binds to an antibody is called an antigenic determinant or epitope. Most antigens have a variety of epitopes that generate a number of different antibodies that are called polyclonal. A single immune response to an antigen is termed monoclonal. 2013/10/2 Antibodies 36

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Antibody-Antigen Interactions Antibodies have two antigen binding sites that are connected by a hinge region to a common tail region. The bivalent nature of antibodies allows for cross-linking of antigens. The hinge region can expand or contract for flexibility in antigen binding. 2013/10/2 Antibodies 38

NATURE OF ANTIGEN-ANTIBODY REACTIONS Lock and Key Concept ---There must be a pretty good fit between the shape of the antigen and shape of the antibody receptorsite. Non-covalent Bonds --- Chemical Bonding( Hydrogen Bonds H-N, H-O) --- Physical Bonding Antibodies are negatively charged whereas antigens are frequently positively charged.( electrostatic) Van der Waals force Hydrophobic Bonding Reversible 2013/10/2 Antibodies 39

AFFINITY AND AVIDITY Affinity - Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. Affinity is the equilibrium constant that describes the Ag- Ab reaction Avidity - Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. 2013/10/2 Antibodies 40

SPECIFICITY AND CROSS REACTIVITY Specificity - Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. Cross reactivity - Cross reactivity refers to the ability of an individual antibody combining site to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one antigen. 2013/10/2 Antibodies 41

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Production of monoclonal antibodies -Immunising an animal-usually a mouse -Obtaining immune cells from its spleen -Fusing cells with cancer cells (myeloma) -Tumour of fused cells - hybridoma secretes mab -Cells must grow and form a clone to produce mab Injecting cells into peritoneal cavity of mouse, hybridoma cells multiple and produce fluid (ascites) In-vitro cell culture techniques http://users.rcn.com/jkimball.ma.ultranet/biologyp ages/m/monoclonals.html 2013/10/2 Antibodies 44

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How to choose a region as antigen to immunize animals 2013/10/2 Antibodies 46

Deciding which antibody Antibody raised against which portion of the Protein Monoclonal or polyclonal Species cross reactivity Host animal 2013/10/2 Antibodies 47

Antibody purification -Most common technique absorption and elution from beads coated with protein A -Protein A binds strongly to sites in 2nd and 3rd constant region of the Fc portion of the immunoglobulin heavy chain -Antibodies bind to protein A chiefly by hydrophobic interactions and are disrupted by transient exposure to low PH -Different classes of IgG vary in sequence in their Fc regions {eg: human IgG1,2,4 high affinity, IgG3, weak} -Unimportant for polyclonal sera, where antibodies against target antigen are distributed throught IgG subclasses -Monoclonal antibodies only one subclass of IgG must be identified prior to purification 2013/10/2 Antibodies 48

Terminology Antigen/Immunogen/Antigenic determinants (epitopes) Paratope Priming, boosting, immunization and vaccination Polyclonal antibodies Monoclonal antibodies Memory cells Fab and Fc fragments 2013/10/2 Antibodies 49

Western blot/immunoblot : Using Ab to recognize a specific protein 1 0 Ab Ag Enzyme/ Fluorochrome 2 0 Ab 2013/10/2 Antibodies 50

I : Western Blotting prepare protein/ag (1) Protein extraction -Total protein standard lysis buffer -Nuclear cytoplasmic extraction (NER-PER, extraction kit, P ierce) To prevent degradation -always centrifuge samples at 4 0 C and -include a protease inhibitor eg Pefabloc (Roche diagnostics) Protein Qantification : Bradford reagent Standard BCA assay (Pierce) 2013/10/2 Antibodies 51

I : Western Blotting Electrophoresis (2) Polyacrylamide gels Proteins dissociated: combination of anionic detergent (SDS), reducing agent and heat Polypeptides bind to SDS: ve charge SDS-polypeptide complexes migrate through gel according to size Modifications to polypeptide backbone eg; phosphorylation and glycosylations may have impact 2013/10/2 Antibodies 52

I : Western Blotting Electrophoresis (3) SDS-PAGE carried out with discontinuous buffer system Sample and stacking gel Tris-Cl (ph6.8) Resolving gel Tris-Cl (ph8.8) Buffer reservoirs Tris-glycine (ph8.3) - cathode 2013/10/2 Antibodies 53

I : Western Blotting Electrophoresis (4) Stacking gel (5%) PH=6.8 Separation gel (>5%) PH=8.8 Constituents : Bis : crosslink monomer of acrylamide SDS : denaturing reagent, TrisCl : Chloride ion front to drive protein into positive electrode. Amonium persulfate : free radical provider TEMED : polymerization stabilizer 2013/10/2 Antibodies 54

I : Western Blotting Protein transfer (5) -Wet transfer -Semidry transfer + 3MM paper membrane Gel 3MM paper - 2013/10/2 Antibodies 55

I : Western Blotting Types of Membranes (6) Nitrocellulose (pore size 0.45 µm) standard membrane Nylon membranes Binds protein with greater affinity, high background Polyvinylidene fluoride (PVDF) high affinity binidiny, pre-wash with methanol Immobilised protein can be visualised with ponceau S (not nylon membranes) 2013/10/2 Antibodies 56

I : Western Blotting blocking reagents (7) Western Blotting Horseradish peroxidase 5% non-fat dry milk (Marvel), bovine serum albumin, BLOTTO Alkaline phosphatase 6% casein, 1% polyvinylpyrrolidone, 10mM EDTA in phoshpate-buffered saline, heated to 65 0 C for 1 hour and stored at 4 0 C Immunohistochemistry Sera of animal in which secondary antibody was raised 2013/10/2 Antibodies 57

I : Western Blotting Signal Detection (8) Radioiodinated antibodies Antibodies conjugated to enzymes Horseradish peroxidase Alkaline phosphatase Antibodies coupled to biotin Fluorochrome-labelled antibodies : FITC, PE, EC5, PC5 2013/10/2 Antibodies 58

I : Western Blotting conjugated enzymes (9) :Hosrseradish peroxidase/alkaline phosphatase 1. Chromogenic Detection: HRP: Diaminobenzidine (DAB)-brown precipitate AP: BCIP/NTP- purple/blue precipitate 2. Chemiluminescent detection HRP: Luminol- oxidised luminol emits blue light AP: AMPPD dephosphorylated product emits light 2013/10/2 Antibodies 59

I : Western Blotting Immunodetection (10) 2013/10/2 Antibodies 60

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I : Western Blotting Immunoblotting Variables (9) Blocking membranes -5% Blocking buffer (RT or 4 0 C) -incubation time : 60 min ~ O/N Wash 15 min (RT) Primary antibody -concentration :1 mg/ml -time: 1 hour (RT) Wash 15 min (RT) Secondary antibody -concentration :1/10,000 dilution (Sigma) -time: 1 hour (RT) Wash 15 min (RT)+0.5 M NaCl Wash 15 min (RT) Detection: Chromogenic or chemiluminescent 2013/10/2 Antibodies 63

Trouble shooting :Non-specific binding -Hyperimmune antisera will contain IgG directed against target antigens and also antibodies directed against other antigens -Antisera may also bind other non-target molecules with low afinity -Antisera can manifest levels of background reactivity that is unacceptably high 2013/10/2 Antibodies 64

II. ELISA Enzyme-Linked ImmunoSorbent (ELISA) detecting and quantitating substances such as peptides, proteins, antibodies and hormones. Usually performed on a 96-well plate (Corning s/nunc) Plate coated with capture protein (2-10µg/ml) in buffer Carbonate-Bicarbonate Incubated overnight at 4 0 C Non-specific sites blocked (1% BSA or Super block) Standards/samples added (50-100 µl/well) Antibody directed against antigen of interest conjugated to enzyme HRP/AP Add substrate and detect 2013/10/2 Antibodies 65

conjugate bovine serum albumin Second antibodies TNB ntri.tamuk.edu/protocols/ elisa.html 2013/10/2 Antibodies 66

Enzyme Linked Immunosorbent Assay (ELISA) based on the measurement of an enzymatic reaction associated Microwell Products with immune complexes The method and principle :By using known amounts of a standard unlabeled antigen one can generate a standard curve relating cpm (Enzyme) bound vs amount of antigen. From this standard curve one can determine the amount of an antigen in an unknown sample. 2013/10/2 Antibodies 67

III. Immunohistochemistry Localisation of antigen within Cellular and intracellular localisation Qualitative expression in patient populations; correlations with clinicopathological data 2013/10/2 Antibodies 68

Immunohistochemistry variables Cryosections (~7µm) from frozen sections or paraffin embedded sections (~ 5µm) Blocking: endogenous peroxidase H202 ; nonspecific binding with sera from animal in which 20 antibody raised in Concentration of primary antibody (1-5µg) and incubation time 20 antibody, peroxidase conjugated/biotinylated detection with DAB (brown) Counter-stain with haematoxylin (blue) 2013/10/2 Antibodies 69

Immunohistochemical localization 2013/10/2 Antibodies 70

Tissue microarray 2013/10/2 Antibodies 71

IV. Immunofluorescence Co-localisation of two different antigens within tissue Intracellular localisation of antigens 2013/10/2 Antibodies 72

Immunofluorescence variables Optimise staining first with immunohistochemistry Primary antibodies 10X concentration Different secondary antibodies with different fluoroscene label Sections mounted in DAKO mounting media, viewed under UV Sections can be mounted with DAPI, stains nuclei blue 2013/10/2 Antibodies 73

Flurochrome labelled antibodies When possible use fluorochrome-labeled, affinity purified antibodies Excitation (nm) Emission (nm) Fluorescein 495 524(green/yellow) Texas Red 595 620(red) R-phycoerythrin 565 574(orange/red) 2013/10/2 Antibodies 74

Co-localization 2013/10/2 Antibodies 75

IV. Immunoprecipitation Antibody directed against a particular antigen is added to cell lysate forming an antibody-antigen complex along with lysis buffer Protein A/G agarose complex is added to the lysate, binding the antigen-antibody complex and precipitating the complex out of solution. After centrifugation, non-specific protein will be removed in the supernatant. The complex is then washed to further remove any non-specific protein binding 2013/10/2 Antibodies 76

Application of immunoprecipitation Co-immunoprecipitation is a process that allows the examination of a protein-protein interaction - precipitated out in the complex and detected by Western blotting Activity of immunoprecipitated proteins eg; acetylation assays, DNA footprinting 2013/10/2 Antibodies 77

V. EMSA/Gel retardation/(super)band shift assay 2013/10/2 Antibodies 78

- +50X Anti-ERa Anti-ERb Anti-SRC1 Anti-SMRT Supershift Estrogen Response Element 2013/10/2 Antibodies 79

VI. Antibody (Ab) Microarray A complete microarray-based system for profiling protein expression in biological samples; used to compare two biological samples to measure the relative differences in protein expression. The microarray consists of hundreds of monoclonal antibodies covalently bound in an ordered layout to a glass slide. A protein which can be synthesized in pure form by a single clone (population) of cells. These antibodies can be made in large quantities and have a specific affinity for certain target molecules called antigens which can be found on the surface of cells and those that are malignant. The array can be used as a means to correlate specific proteins with physiological or pathological process of interest, by comparing hundreds of proteins at a time. It is used for toxicity testing, disease investigation, and drug discovery. 2013/10/2 Antibodies 80

Factors affecting measurement of Ag/Ab reactions Affinity Avidity Ag:Ab ratio Physical form of the antigen http://www.med.sc.edu:85/mayer/ab-ag-rx.htm 2013/10/2 Antibodies 81

Ab Array Procedure Extraction of total cellular protein from biological samples of interest (eg. Serum samples). Labeling of extracted protein with fluorescent dyes Cy5 and Cy3 (direct labeling, direct labeling with hapten tag, paired Ab sandwich assay). Removal of unbound dye. Incubation of labeled protein with the array. Scanning of the array and the analysis of the results. 2013/10/2 Antibodies 82

This procedure is a fluorescence-based analysis; covalently immobilized antibodies are used to capture fluorescently labeled antigens. They do not measure absolute concentrations- instead they provide a relative measure of protein abundance [i.e. the abundance of protein in one sample as compared to another sample]. As part of array development, all antibodies are printed and tested against their specific purified antigen (when available) and against cell lines and tissues samples (for quality control). A reference pool is also used, and similar to the gene expression microarrays, a pool of equal aliquots from each sample to be measured is used, thus ensuring that all proteins from the samples are represented in the reference. 2013/10/2 Antibodies 83

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Enzyme linked immusorbent assay (ELISA) 2013/10/2 Antibodies 85

Direct Labeling (w/ hapten tag) A convenient method to measure multiple proteins in a complex mixture. All proteins are labeled with either a fluorophore or a hapten tag such as biotin. Advantages: Only one captured antibody per target is required, as compared to the next method- easier to expand detection to new targets for which matched antibody pairs may not be available. Can label different samples with different tags and to co-incubate the samples on the same arrays. Disadvantages: Potential for a high background: all proteins are labeled from the sample, including high concentration proteins such as albumin in serum; nonspecific binding or adsorption of these proteins to Ab could cause interference reduce detection sensitivity or data accuracy. Potential for disruption of antibody-antigen interactions if the labeling reaction severely alters an antigen s binding site. 2013/10/2 Antibodies 86

A. Direct Labeling B. Direct Labeling with a hapten tag. C. Paired Ab sandwich assays. 2013/10/2 Antibodies 87

Dual Antibody Sandwich Antibodies spotted onto microarray substrates capture specific antigens, and a cocktail of detection antibodies, each antibody matched to one of the spotted antibodies, is incubated on the arrays. Advantages: Quantification of the bound detection antibodies provides a measure of each antigens abundance. Sandwich assays are more sensitive than the direct labeling method because background is reduced through the specific detection of two antibodies instead of one. Disadvantages: The development and validation of assays measuring many targets in parallel is difficult because of the cross reactivity and precipitation when using many detection antibodies. 2013/10/2 Antibodies 88

VII. Flow cytometer /FACS 2013/10/2 Antibodies 89

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Flow Cell Injector Tip Sheath fluid Fluorescence signals Focused laser beam 2013/10/2 Antibodies 91

Flow Cytometry Optics PMT 4 Flow cell Dichroic Filters PMT 1 Bandpass Filters PMT 2 Laser PMT 3 2013/10/2 Antibodies 92

Optical Filters Dichroic Filter/Mirror at 45 deg Light Source Transmitted Light Reflected light 2013/10/2 Antibodies 93

Standard Band Pass Filters White Light Source 630 nm BandPass Filter Transmitted Light 620-640 nm Light 2013/10/2 Antibodies 94

Standard Long Pass Filters 520 nm Long Pass Filter Light Source Transmitted Light >520 nm Light Standard Short Pass Filters 575 nm Short Pass Filter Light Source Transmitted Light <575 nm Light 2013/10/2 Antibodies 95

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THEN THERE WAS FSC AND SSC N U M B E R VOLUME 2013/10/2 Antibodies 97

Anti-TCR-gamma-delta-1 PE --> THIS PRODUCES PATTERNS OF CELL CLUSTERS C D 4 10 1 10 2 10 3 10 4 CD3+CD4+ CD3-CD4+ CD3+CD4- CD3-CD4- green cyan cyan black CD3 10 1 10 2 10 3 10 4 CD8 TC --> RPCI 2013/10/2 Antibodies 98 LFC

VIII. Elispot -Detection of individual cytokine-secreting cells using two high-affinity cytokinespecific antibodies -Capture of secreted cytokines directly at the site of secreting cells -Generation of Spots based on a colorimetric reaction: Spot = Footprint of cells -Spots are permanent and can be quantitated 2013/10/2 Antibodies 99

Single cell resolution Advantages High sensitivity(one per million cells) ImmunoSpot plates : synthetic high-proteinbinding membrane No dilution, degradation or absorbtion of cytokines by bystander cells 20-200-fold more sensitive than ELISA or intracellular cytokine staining True frequency of cells in vivo Easy to set up Rapid and objective analysis of large number of samples using computer-assisted image analysis 2013/10/2 Antibodies 100

Technical Aspects ELISPOT Assay 1. Coating the ImmunoSpot plates with Capture antobody 2. Block the Plates Scannnig the plates ImmunoSpot plate scanner Analysis of data ImmunoSpot Software 3. Incubation of cells with stimulating antigens/mitogens usually 24 hours 4. Lysis of Cells Add secondary Detection antobody 5. Detection of antibody-cytokine complex using a chromogen 2013/10/2 Antibodies 101

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HUMAN ELISPOT ASSAY 2013/10/2 Antibodies 103

VIII. Enzyme Linked Immunosorbent Assay (ELISA) 1. Based on the measurement of an enzymatic reaction associated with immune complexes. 2. The method and principle :By using known amounts of a standard unlabeled antigen one can generate a standard curve relating cpm (Enzyme) bound vs amount of antigen. From this standard curve one can determine the amount of an antigen in an unknown sample. Coat 1st Ab Ag Wash Coat 2nd Ab* Colorimetry 2013/10/2 Antibodies 104

Elisa microplate reader 2013/10/2 Antibodies 105