Antigen Antibody reaction

2014

Antigen & Antibody Reactions

Affinity vs. Avidity• Affinity

– Measure of the binding strength between an antigenic determinant (epitope) and an antibody combining site.

• Avidity– The cumulative binding strength of all

antibody-epitope pairs which results from multivalent antigen and antibody.

Antibody generation

HAT medium selection

Antigenic determinant or epitopes• The specific part of an antigen where an

antibody becomes attached.

CH2 CH3

CH2 CH3

IgG has a Valence of 2

TWO Identical ANTIGEN BINDING SITES

Non-Covalent Interactions Ball in glove fit

Antigenic Determinant

VL

VH

Antigenic determinant 1

Antigenic determinant 2

Antigenic determinant 3

Antigenic determinant 4

PROTEINANTIGEN

YY

Y

POLYCLONAL ANTIBODYand at least two different antigenic determinants

TO CROSS-LINK PROTEIN ANTIGENS

Immune Complexes

Immunoprecipitation reaction

• When antibodies and soluble antigen are mixed in solution the bi-or multivalent antibody nature of immunoglobulins allows for a single Ab molecule to bind to more than one Ag. Eventually the resulting cross-linked component becomes so large that it falls out of the solution as a precipitate.

YYY

YANTIBODYEXCESS

NO CROSSLINKSNO Precipitate

YYY

Y

Y YY

Y

Excess Antibody

Y

Excess Antigen = Not enough Cross-links to cause a Precipitation

Immunodiffusion Techniques

• Radial Immunodiffusion– A single diffusion technique where Ab is put into a

gel and Ag is measured by the size of a precipitin ring formed when it diffused out in all directions from a well cut into the gel

• Ouchterlony Double Diffusion– Both Ab and Ag diffuse from wells into a gel

medium

Radial Immunodiffusion

• Ag is added to an antibody rich media. The two continue to react until the zone of equivalence is reached. The area of the ring is a measure of the Ag concentration.

• Both antigen and antibody can diffuse independently.

Ouchterlony

Non identity- no shared antigenic determinant

Radioimmuonoassay (RIA)• It is an in vitro assay that measures the presence of an antigen and has

very high sensitivity

• RIA has been the first immunoassay technique developed to analyze nanomolar and picomolar concentrations of hormones in biological fluids.

• All the variations of this assay developed so far depend on the availability of radioactively labeled antibody or antigen.

• The radioactive label that is most commonly used is 125I because of the ease of introduction of iodine atoms to tyrosine residues of proteins

ELISA

• ELISA assays are similar in principle to RIA but instead of using antibodies or antigens conjugated to radioisotopes they use antigen or antibodies covalently bound to enzymes.

• The conjugated enzymes are chosen based on their ability to catalyze the conversion of a substrate into a colored, fluorescent or chemiluminescent product.

• Safer and cost effective

Indirect ELISA

• Antibody can be detected or its concentration can be determined with an indirect ELISA assay

• This version of ELISA is the method of choice to detect the presence of serum antibodies against HIV. In this assay, recombinant envelope and core proteins of HIV are adsorbed as solid-phase antigens to microtiter wells. Individuals infected with HIV will produce antibodies to epitopes on these viral proteins.

Sandwich ELISA• Antigen can be detected or measured by the sandwich ELISA.

• In this technique the antibody is immobilized on a microtiter plate. A sample containing unknown amounts of antigen is allowed to react with the immobilized antibody.

• They are useful for the measurement of soluble cytokine concentrations in tissue culture supernatants, as well as in serum and body fluids.

• For this assay to work, the two antibodies use for the antigen immobilization and detection phases respectively must bind to different antigenic determinants or epitopes. Monoclonal antibodies are therefore often used for this purpose.

Competitive ELISA• It provides another extremely sensitive variation for

measuring the amounts of antigen.

• In this technique antibody is first incubated in solution with a sample containing antigen. This mixture is then added to an antigen coated well.

• The more Ag is bound to the Ab in the solution phase sample, less free Ab will be available to bind to the Ag-coated well. Higher the conc. of Ag in the final sample, lower will be the signal.

ELISPOT assay• It allows for the quantitative determination of the number

of cells in a population that are producing a particular type of molecule.

• Frequently used to detect and quantify the number of cells in a population that are producing a particular cytokine.

• Substrates for ELISPOT assays are usually colorless but when acted upon by their cognate enzyme, they precipitate out of solution and produce a visible ‘spot’ which can be counted under a dissecting microscope.

Western Blot or Immunoblot

• Allows for the preliminary quantification of a specific protein in a complex mixture of proteins

Electrophoresis Techniques

• Electrophoresis separates molecules according to differences in their electrical charge.– Rocket Immunoelectrophoresis– Countercurrent Immunoelectrophoresis– Immunoelectrophoresis– Immunofixation Electrophoresis

Immunoelectrophoresis

• It is used to check the presence, specificity and homogeneity of antibodies

• It can also determine relative antibody concentrations

1. The Ag mixture s first added to the wells to separate its constituents by charge2. The antiserum containing the antibodies is added to the troughs3. At specific Ag/Ab ratio, huge macromolecules are formed. They for a visible while Complex which precipitates as arcs within the gel

Immunoeletrophoresis

Antigens on Cells or on Tissue Sections

UV Light

Fluorescence

Fluorescence Double layer Sandwich

UV Light

Antigens

Fluorescence microscope

Confocal microscope

Measurement of affinity of Ag-Ab interaction

Surface Plasmon resonance

Agglutination

When a particulate Ag reacts with an Ab, a visible precipitate is formed.

Hemagglutination: When Ab bind Ags on the surface of RBCsThey are routinely performed to type RBCs

Coombs testDirect Coomb's Test

When antibodies bind to erythrocytes, they do not always result in agglutination. This can result from the antigen/antibody ratio being in antigen excess or antibody excess or in some cases electrical charges on the red blood cells preventing the effective cross linking of the cells. These antibodies that bind to but do not cause agglutination of red blood cells are sometimes referred to as incomplete antibodies. In order to detect the presence of non-agglutinating antibodies on red blood cells, one simply adds a second antibody directed against the immunoglobulin (antibody) coating the red cells. This anti-immunoglobulin can now cross link the red blood cells and result in agglutination.

Indirect Coomb's Test

If it is necessary to know whether a serum sample has antibodies directed against a particular red blood cell and you want to be sure that you also detect potential non- agglutinating antibodies in the sample, an Indirect Coomb's test is performed (Figure 11). This test is done by incubating the red blood cells with the serum sample, washing out any unbound antibodies and then adding a second anti-immunoglobulin reagent to cross link the cells.

Applications

These include detection of anti-rhesus factor (Rh) antibodies. Antibodies to the Rh factor generally do not agglutinate red blood cells. Thus, red cells from Rh+ children born to Rh- mothers, who have anti-Rh antibodies, may be coated with these antibodies. To check for this, a direct Coombs test is performed. To see if the mother has anti-Rh antibodies in her serum an Indirect Coombs test is performed.