Preparatory Electroimmunodiffusion for Making Prepitins to Selected Native Antigens

Abstract

Miniaturized two-dimensional single electroimmunodiffusion (2-D EID) was used to produce directly and simply minute quantities of purified human serum antigens complexed with their precipitins. These complexes injected in incomplete Freund adjuvant readily primed rabbits for vigorous selective production of precipitins to the complexed antigens when the animals later were boosted with whole human serum. This procedure of priming animals with portions of electroimmunodiffusion-produced arcs of antigen-antibody precipitate and later boosting them with crude antigen mixture thus provides a simple, effective means for preparing antisera with chosen restricted specificities without preliminary, frequently difficult conventional biochemical purifications of antigens. The technique was unexpectedly sensitive, readily detecting native associations between certain serum antigens (e.g., between albumin and both β-lipoprotein and α₂-macroglobulin). Thus rabbits primed with nearly 100,000-fold differences in quantities of antigen nevertheless were able to react to similar boostings with no more than 2.5-fold differences in precipitin production. The preparatory-immunization technique described therefore will be useful not only for easier production of monospecific antisera but also for sensitive analyses of native antigen-antigen associations in such natural mixtures as human serum.