ESTABLISHMENT BY THE RAT LYMPH-NODE METHOD OF EPITOPE-DEFINED MONOCLONAL-ANTIBODIES RECOGNIZING THE 6 DIFFERENT ALPHA-CHAINS OF HUMAN TYPE-IV COLLAGEN

A group of rat monoclonal antibodies recognizing the six different alp ha chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which e nlarged medial iliac lymph nodes of a rat injected with an antigen emu lsion via hind footpads are used as a source of B cells for cell fusio n to produce hybridomas. The immunogens used were synthetic peptides h aving non-consensus amino acid sequences near the carboxyl termini of type IV collagen alpha chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryost at sections of human kidneys. Because the epitopes of all antibodies w ere determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of alpha chains of type IV collagen at the protein level in normal and abnormal condit ions. The combined use of synthetic peptides as immunogens, the rat ly mph node method as making monoclonal antibodies, and the multipin-pept ide scanning as epitope mapping is found to be a strong tool for ident ification of peptides and proteins whose amino acid sequences are know n or have been deduced.