| A method for isoelectric focusing of antibodies in agarose gels with ampholytes synthesized in the laboratory from pentaethylenehexamine is presented. The ampholytes are easy to prepare, give results comparable to those with commercial ampholytes, and are much less expensive.;In one family of outbred rabbits, several members demonstrated natural auto-anti-idiotypic (AAI) antibody responses which correlated with changes in anti-micrococcal antibody clonotype expression. These natural AAI responses were examined by radioimmunoassay techniques, and it was shown that, in each case, the natural AAI antibodies recognized identical idiotopes on F(ab')(,2) fragments prepared from the anti-micrococcal antibodies of each rabbit that made an AAI response. The cross reactive idiotopes were shown to be of paternal origin and were found in the anti-micrococcal antibodies of each offspring that synthesized AAI antibodies but not in anti-micrococcal antibodies of AAI-negative offspring.;The nature of the anti-micrococcal idiotopes which were recognized by the natural AAI antibodies was examined, and it was shown that the rabbit anti-micrococcal idiotopes were exquisitely sensitive to treatment with very low concentrations of sodium metabisulfite or 2-mercaptoethanol. Treatment with reducing agents selectively destroyed the anti-micrococcal idiotopes, while allotype epitopes and the antigen binding capacity of the anti-micrococcal F(ab')(,2) fragments were unaffected.;This isoelectric focusing method was used to study antibody clonotype patterns in outbred rabbits. Rabbits were immunized with the gram positive bacterium Micrococcus lysodeikticus and the anti-micrococcal carbohydrate antibody clonotype pattern of each rabbit was monitored throughout several immunization rounds. Examination of the antibody clonotype patterns in antisera from a number of outbred rabbits revealed that variations in clonotype expression often occurred within an individual during successive rounds of immunization. |
