General methods for the study of cells and serum during the immune response: the response to dinitrophenyl in mice.
Two new methods for antibody assay are reported: one for antibody in solution, the other for individual cells releasing antibody. They depend on the use of an immunoabsorbent-bound antigen to absorb the antibody, and radioactively-labelled anti-immunoglobulin antibody (anti-Ig) for its detection. In the first method immunoabsorbent was added to the solution containing antibody, washed free of serum components other than the bound antibody, treated with [131I]anti-Ig, and the uptake of radioactivity assessed by γ-ray counting. This method was standardized for anti-DNP antibody by comparison with results of equilibrium dialysis. It was shown to be independent of affinity, down to K0 = 106 1/M. It would detect 0·1 ng antibody. For the study of lymphoid cells releasing antibody the cells and immunoabsorbent were dispersed in agarose gel on microscope slides, incubated at 37°C in the presence of [125I]anti-Ig, washed, and autoradiographed. Antibody released by a cell could then be visualized as a circular area (`spot') of silver grains. When sheep red blood cells were used as the antigen, the number of spots approximated to the number of lytic plaques obtained by the addition of complement. The methods were shown to be generally useful for a number of antigens. Both methods were used in a study of the course of the primary immune response to dinitrophenyl in mice. Cells releasing anti-DNP antibody were detected from 2 days after immunization and rose to a peak number at 9 days of 2 × 105 in the lymph nodes local to the site of injection.