A new approach to the preparation of ACTH antibody by immunization of the rabbit with an antigen-antibody complex.

Immunization with antigen-antibody complex of ACTH produced sensitive ACTH antibody applicable to the radioimmunoassay. The antibody prepared by immunization with the synthetic 1-24 ACTH was mixed with the antigen porcine ACTH, and the mixture was incubated to form the antigen and antibody complex. Two rabbits were initially immunized with this complex, then further immunized with only the porcine ACTH. Both rabbits produced the ACTH antibody with a titer of 1:25,000, which could detect ACTH of 5 pg per tube when applied to the radioimmunoassay of the human ACTH. This antibody showed a strong crossreaction with synthetic 1-24 ACTH, which was confirmed by radioimmunoassay method and immunostaining technique. On the other hand, control animals immunized with only procine ACTH from the beginning showed no production of ACTH antibody. It was concluded that addition of ACTH antibody to the antigen for the first immunization took an important role in eliciting production of the antibody. Possible roles of the antigen-antibody complex for immunization were discussed.