A novel protein immunoassay with predetermined specificity using monoclonal antibodies against tryptic fragments:...

We have developed a new protein immunoassay method which uses antibodies raised against synthetic peptides. These synthetic peptides are selected to correspond to fragments of the protein that can be obtained by proteolytic treatment of the protein by trypsin. Just before assay, biological samples are treated with trypsin to liberate the fragments which bind to the anti-peptide antibodies with high affinity. The exact specificity of the assay is predetermined by the amino acid sequence of the fragment which may be either conserved within a family of antigens or, conversely, entirely specific for a particular protein. This method has been successfully employed in the development of an immunoassay for HIV P24 antigen. In that case, peptides were selected that were strongly conserved among the different HIV-1 and HIV-2 strains. This methodology has permitted the development of a sensitive immunoassay with a broad specificity despite many amino acid variations between HIV strains. The methodology could be extended to other protein antigens.