Anti-oxazolone hybridomas and the structure of the oxazolone idiotype

Abstract

Antibodies raised in several mouse and rat strains against the hapten 2-phenyloxazolone (phOx, "oxazolone") regularly contain a fraction recognized by antiidiotypic reagents. We have studied this response in BALB/c and DBA/2 mice by generating over fifty anti-phOx antibody-secreting hybridoma clones. The hybridization was performed either 7 or 14 days after a primary immunization with phOx-protein conjugate. Most of the hybrids secreted IgG1. Whereas over 80% (17/21) of IgG-producing hybrids from day-7 fusions secreted oxazolone-idiotype positive immunoglobulin, all hybridomas originating from day-14 fusions were idiotype negative. The mRNA for heavy (H) and light (L) chains of three idiotype-positive and one idiotype-negative IgG1 hybridomas were sequenced by a modification of Sanger's dideoxynucleotide method of DNA sequencing, using crude mRNA as template, synthetic oligonucleotides as primers, and reverse transcriptase to incorporate both dideoxynucleotides and labeled deoxynucleotides. The sequence of the mRNA coding for the whole variable region of each chain was established using primers complementary to the constant region near the V-C boundary and another two that coded for a framework segment in either VH or VL. This method not only provided more information than protein sequencing but was also faster and simpler. The mRNA preparation did not need fractionation beyond the poly A-containing fraction. The sequences of the H and L chain mRNA of the three idiotype-positive anti-oxazolone antibodies were extremely similar or identical, and from them a tentative oxazolone-idiotype basic sequence was derived. Only three nucleotide differences were detected; these occurred in the D segment of one H chain mRNA, in the V-J boundary of one of the light chain mRNA, and in the first hypervariable region of another. The idiotype-negative antibody had a totally different H chain mRNA and a light chain mRNA that differed by 21 bases, almost all affecting the amino acid sequence.