Experimental methods(Laboratory applications)

Experimental Methods

For recognition and measurement of Abs and Ags in biological material

Experimental Methods:• Precipitation• Agglutination• Complement-binding reaction• Enzyme-linked immunosorbent assay (ELISA)• Radioimmunoassay• Immunoblotting (Western blot)• Immunfluorescence

Precipitation

When antibodies and soluble antigenare mixed in solution, the bi- or multivalent nature of immunoglobulins allows for a single antibody molecule to bind to more than one antigen.

If the antigen has more than oneepitopes, it may bind multiple different antibodies. Resulting cross-linked complex becomes so large that it falls out of solution as a precipitate.

Precipitation

Precipitation techniques are used to measure Ag or Ab concentrations.

Can performed in:Solid phase (radial immunodiffusion,

immunoelectrophoresis)Liquid phase (turbidimetry,

nephelometry)Precipitate formation requires that neither antigen (left panel in graph) nor antibody (right panel in graph) molecules are in excess.

Heidelberg curve

Precipitation: Radial immunodiffusion

Immunodiffusion in agar gels can be used to assay for the presence of antibodies and determine crossreactivity patterns between complex antigens and antibody samples.

Precipitation: Radial immunodiffusion

Both antigen and antibody diffuse radially from wells towardeach other, thereby establishing a concentration gradient.

At the relative antibody-antigen concentrations at which lattice formation is maximized, termed “equivalence,” a visible line of precipitation, or “precipitin line,” forms in the gel.

Аb Ab Ab Ab Ab

Ab Ab Ab Ab Ab

Ab Ab Ab Ab AbAg AgAgAgUp

Precipitation ringAg-Ab complexes

Gel AbsGlass

Agin different concentrations

Simple radial immunodiffusion method

according to Mancini

Precipitation: Turbidimetry

TurbidimetryAg sample placed in a cuvette and allowed to react with an

excess of antiserum with Abs.Soluble immune complexes form.The change of turbidity in the cuvette measured by

photometry.

Precipitation: Nephelometry

NephelometryAg sample placed in a cuvette and allowed to react with an

excess of antiserum with Abs.Soluble immune complexes form.The cuvette is irradiated with lazer light, which scattered

from the immune complexes.

Precipitation: Radial double diffusionRadial double diffusion (Ouchterlony)Ag and Ab diffuse radially toward each other in aqueous

agarose gel.Visible bands of precipitation form at the sites where the Ag

and Ab meet.Affective way to test the identity of unknown Ags.

Precipitation: electroimmunodiffusion

Ag and Ab migrate in opposite directions through the gel due to differences in their electric charges/

Immune complexes form (visible lines) during migration if the corresponding Ab is present in patient’s serum.

Precipitation: ImmunoelectrophoresisCombination of protein electrophoresis and

immunoprecipitation.Samples are first separated by electrophoresis.The antiserum diffuses perpendicularly to the direction of

separation.

Lines of precipitation form in the range of Ag and Ab equivalence.

Agglutination

Immune complexes form due to interaction between Abs and particulate Ags.

Direct agglutination (gemagglutination test for determination of blood type and Widal’s bacterial agglutination test)

Indirrect agglutination (latex agglutination test, Boyden’s passive gemagglutination test)

Agglutination

Agglutination: bacterial agglutination

Ab detection (Vidal method)Bacterial suspensions incubated in serial dilutions of the

patient’s serum serve as the Ag.Agglutination occurs if the Ab corresponding to Ag is

presents in patient’s serum.

Ag detection (Gruber method)Bacterial cultures are incubated with class-specific and

type-specific Abs.Used for bacterial type determination

HemagglutinationHemagglutination reactions can be used to detect any

antigen conjugated to the surface of red blood cells

Demonstration of hemagglutination using antibodies against sheep red blood cells (SRBCs).

The control tube (10) contains only SRBCs, which settle into a solid “button.” The experimental tubes 1 to 9 contain a constant number of SRBCs plus serial twofold dilutions of anti-SRBC serum. The spread pattern in the experimental series indicates positive hemagglutination through tube 3.

RBC

RBC

Hemagglutination

Hemagglutination inhibition reactions are also useful toolsin the clinic and in the laboratory for the detection of viruses and of antiviral antibodies.

Some viruses (most notably influenza) bear multivalent proteins or glycoproteins on their surfaces that interact with macromolecules on the RBC surface, and induce agglutination of the RBCs.

Complement-binding reaction

1 stageAg + Ab +

complement(complement binds if

Ab present in serum)

2 stage+ Test red blood cells

and test Ab“+” – no complement-

mediated hemolyses“-” – complement is still

available to induce lyses of the test system

Enzyme-linked analyses

Enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA)

Each assay can be used qualitatively or quantitatively by comparison with standard curves prepared with known concentrations of antibody or antigen.

Antibody can be determined with an indirect ELISA:

Enzyme-linked immunosorbent assay (ELISA)Antibody can be determined with a sandwich ELISA:A sample containing unknown amounts of Ag is allowed to

react with the immobilized Ab. After the well is washed, a second enzyme-linked Ab specific for a different epitope on the Ag is added and allowed to react with the bound Ag.

After any free second antibody is removed by washing, substrate is added, and the colored reaction product is measured.

Enzyme-linked immunosorbent assay (ELISA)Antibody can be determined with competitive ELISA.antibody is fi rst incubated in solution with a sample containing antigen. Th

e antigen-antibody mixture is then added to an antigen-coated microtiter well.

The more Ag present in the initial solution-phase sample, the less free Abwill be available to bind to the Ag-coated well.

After washing off the unbound antibody, an enzyme-conjugated Ab2 specific for the isotype of the Ab1 can be added to determine the amount of Ab1 bound to the well. In the competitive assay, the higher the concentration of Ag in the original sample, the lower the final signal, just as in the cytokine-specifi c RIA described above.

Radioimmunoassay (classical method)Based on principle of competitive binding.Ag corresponding to the target Ab immobilized. Unlabeled

Ab in the serum and radioactively labeled Ab compete for Ag binding sites.

Free Abs removed by washing.

Radioimmunoassay (classical method)The higher the number of unlabeled Abs in serum, the

lower the number of labeled Abs able to bind to Ag.The measured radioactivity will be low when the serum Ab

titer is high and vice versa.

Immunoblotting (Western blot)Proteins separated according to molecular weight by

means of gel electrophoresis.All proteins obtain a negative charge.The addition of 2-mercaptoethanol diminishes internal

disulphide bonds.Since they lose their characteristic charge and form during

electrophoretic separation, the proteins separated according to molecular weight.

The proteins are then “blotted” – transferred from the gel to an immobilizing nitrocellulose membrane, where they can be recognized by specific Abs in the membrane.

Immunoblotting (Western blot)One of the main advantages of Western blotting is its ability

to identify a specific Ab after protein separation.

ImmunfluorescenceDirect Immunfluorescence: Abs are already conjugated with a fluorescent dye.

Inirect Immunfluorescence:Fluorochrome-labeled secondary Ab added in a second

step after the Ag-specific primary Ab gas been bound.