Dideoxy mRNA Sequencing as a Method to Analyze Antibody Diversity
The antigenic specificity of an antibody (Ab) molecule is determined by the amino acid sequence of the light and heavy chain variable regions. We have analyzed the extent to which various possible mechanisms for generating Ab diversity contribute in an immune response to a simple antigen, the hapten 2-phenyloxazolone. In order to identify the relative contributions of the different mechanisms which can generate Ab diversity (e.g. the use of different germ-line V genes, different combinations of V, D and J gene segments and the role of somatic point mutations), nucleotide sequence information was required. We therefore analyzed the V region expression of a series of oxazolone-specific hybridomas by sequencing the mRNA isolated from these cells. The major advantage of this technique is the speed and ease with which the required sequence information can be generated.