Production of monoclonal antibody against amyloid fibril protein and its immunohistochemical application.

An autopsy case of multiple myeloma (IgA-lambda) with extensive amyloid arthropathy of the systemic joints was described. Heavy deposits of amyloid (amyloidoma) were observed in the articular cavities of the joints. Furthermore, numerous amyloid deposits were found in the walls of small blood vessels in the general organs. Incubation of the paraffin sections of amyloid with potassium permanganate produced little loss of Congo red affinity and apple-green birefringence under polarized light. A crude preparation of amyloid protein was isolated from a frozen intra-articular mass, which had been obtained at necropsy, and injected into the footpads of BALB/c mice with complete Freund's adjuvant. The immune spleen cells were fused with myeloma cells (P3 X 63-Ag8.653) under the presence of polyethylene glycol. Hybridoma cell lines, producing supernatants which reacted not only with amyloid substances but also with normal human tissues, were omitted from the subjects of recloning, and one hybridoma cell line (Am-1) producing a specific monoclonal antibody (MAb) against the immunized amyloid substance was finally obtained. Using the indirect immunoperoxidase method, the specificity of MAb Am-1 was confirmed in the cryostat sections as well as in the formalin-fixed paraffin sections of various organs of the case from which the crude amyloid protein was obtained and used for immunization. Amyloid deposits in 25 cases with amyloidosis or amyloid deposits were examined with MAb Am-1, and 2 cases showed positive reactivity with Am-1. These 2 cases presented primary amyloidosis and focal amyloid deposits in the oral cavity, respectively; Congo red staining of amyloid in these cases showed resistance to treatment with potassium permanganate, as observed in the amyloid of the original case used for immunization. Trypsin treatment of the sections resulted in a loss of positive reactivity with MAb Am-1 in all these cases. This result indicates that Am-1 recognizes a substance composed of protein molecules. Furthermore, the immunohistochemical distribution of Am-1 positive reaction was consistent with the histological distribution of substance which could be stained by Congo red and showed apple-green birefringence under polarized light. These results have suggested that Am-1 reacts with a portion of amyloid protein which is resistant to treatment with potassium permanganate followed by Congo red staining.