Ways of Determining the Magnitude of an Antibody Response

The responses of B cells to an injected foreign particle is measured mainly by analyzing the specific antibody production in a humoral immune response. This can be done most efficiently by assaying the antibody that accumulates in the plasma. The amount of antibody is a direct function of the number of responding B cells, their rate of antibody synthesis and the persistence of antibody after secretion (1).

Various assays can be used to detect and analyze specific antibodies found in a variety of fluids through the formation of precipitates, antigen-antibody complexes, with their corresponding antigens. These assays include, but are not limited to, a) a simple ring precipitation, b) a sinlge radial immunodiffusion (the Mancini version), c) a double diffusion test within gels (the Ouchther Test) and d) immunoelectrophoresis.

Radioimmunoassays (RIA) and Enzyme-linked Immunosorbent Assays (ELISA)

From serum, the use of serological assays can be used, such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). In a radioimmunoassay, antigen is radio-labelled (vice-versa) and unlabelled antibody is attached to a solid support and the fraction of the labeled antigen retained on the surface is determined in order to measure binding. An ELISA detects bound antibody to antigen by a linked enzyme that converts a colorless substrate into a colored product. For example, antigen can be coated on the surface of a well, add the serum containing antibodies specific to the antigen, wash and then add an antibody, linked to anenzyme that cleaves a colorless product, specific to the Fc portion of antibodies. Measuring the absorbance of light by the colored product (e.g.) can determine the magnitude of the antibody response. Moreover, affinity chromatography can isolate specific antibody, by allowing the spectrometer and the magnitude of antibodies produced viewed. The amount of precipitate formed can detect amount of antibodies present, as in the precipitin reaction, which depends on the large aggregates of antigen crosslinked by antibody molecules to form. Moreover, isoelectric focusing of antigens, can create a pH gradient that can be transferred to a solid support such as nitrocellulose paper and the anti-antigen antibodies added, with subsequent anti-Fc antibodies linked to a luorescence. The level of fluorescence for a given protein can then be assessed (like immunoblotting). Competitive binding assays can also be used, for example by inhibiting the binding of labeled ligand (1).

References:

1. Janeway, C.A., Travers P., Walport M., and Capra J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA, pages 1-40, 2.5-2.22, 3.1-3.12

2. Kissinger R, and Myl A.D. 1984. Improvements to the Plaque Assay for Antibody Secreting Cells. J. of Immun. Meth. 66:377-382

3. Wilson S., Munson A., and Meade J. 1999. Assessment of the Functional Integrity of the Humoral Immune Response: The Plaque-Forming Cell Assay and the Enzyme-Linked Immunosorbent Assay. Methods 19:2-7

4. Cunningham, A.J. 1965. Nature (London) 207, 1106

5. Cunningham A.J. and Szenberg A. 1968. Immuniligy 14: 599

6. Jerne N. and Nordin A. 1963. Science 140: 405

7. Delves P. and Roitt I. 1999. Encyclopedia of Immunology, Academic Press Inc., 2nd ed., San Diego, USA page 238.

8. Cruse, J and Lewis, R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA pages 1960-1965