UC Irvine

Various methods were employed to induce antibodies in rabbits that were capable of neutralizing different families of lymphotoxins (LT). Both stable (alpha-LT) and unstable (beta-LT) molecules, released by activated human lymphocytes in vitro, were neutralized. The different LT families were first separated into their respective groups by physical-chemical methods. Immunization with small quantities of antigen yielded a high percentage of responder animals. Techniques were developed for eliciting alpha-LT antibodies using as little as 2--3 ml of a cell-free supernatant. The situation was more difficult, however, when the unstable beta-LT molecules were employed as antigens. We found that because of the low concentration and lability of beta-LT in supernatants, the immunizing dose had to be: a) handled rapidly, b) larger than that used with the alpha-LT, and c) injected at closer intervals and over a longer immunization protocol. Physical-chemical studies supperted the concept that the LT-neutralizing activity in the immune serum was immunoglobulin.