UNIT 11.17 Determination of the Specific Antibody Titer
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Cooper, H. M. and Paterson, Y. 2001. Determination of the Specific Antibody Titer. Current Protocols in Molecular Biology. 50:V:11.17:11.17.1–11.17.13.
- Published Online: 1 MAY 2001
- Published Print: APR 2000
The amount of specific antibody present in polyclonal antiserum, ascites fluid, or hybridoma supernatant can be quantitated by either solid-phase radioimmunoassay (RIA) or by direct ELISA. In the solid-phase assay described here, serially diluted antiserum is incubated in microtiter wells previously coated with the relevant antigen. Bound antibody is detected by employing 125I-labeled anti-immunoglobulin antibodies. The amount of specific antibody in the antiserum is then determined from a standard curve generated with a specific antibody of known concentration. The unknown antiserum and the standard antibody are assayed in parallel. The support protocols describe the chloramine T and IODO-GEN procedures for radioiodination of the anti-immunoglobulin reagent. The use of the solid-phase RIA procedure to determine the light-chain and heavy-chain isotypes present in polyclonal antisera and fluids containing monoclonal antibodies is also described.