Detection of novel proteins associated with secondary amyloidosis and Alzheimer's disease by monoclonal antibody

Abstract We have established a monoclonal antibody (MoAb) AM34 (IgG1) which was prepared by a hybridoma constructed from fusion between murine myeloma cells and murine splenocytes. Crude amyloid proteins which were used as immunogen, were extracted from the kidney of a patient with rheumatoid arthritis by the distilled water method. This antibody strongly reacted with all 8 cases of secondary amyloidosis, but did not react or very weakly reacted with 17 tissue sections of primary or myeloma-associated amyloidosis. Other amyloid tissues did not give any positive reaction. Interestingly, 6 brain tissues of Alzheimer's disease clearly showed positive staining with this antibody, whereas two apparently normal brain tissues exhibited negative staining. Senile plaque cores, neurofibrillary tangles (weakly stained) and cerebrovascular amyloid in Alzheimer's disease were stained. Absorption of the MoAb AM34 with the crude amyloid proteins abolished the immunoreactivity of the MoAb AM34 not only with the kidney tissue section of the secondary amyloidosis, but also with the above mentioned portions of the brain in the case of Alzheimer's disease. Therefore, these immunohistological data suggest that the MoAb AM34 recognizes common epitope which exists in amyloid deposits of both secondary amyloidosis and Alzheimer's disease. An inhibition test on the kidney section showed that the reactivity of MoAb AM34 was not at all inhibited by the pretreatment of the section with 10 times higher concentration of anti-human amyloid A (AA) MoAb KM268 which was prepared against synthetic peptides of AA protein, suggesting that MoAb AM34 might react with amyloid-related protein other than AA protein. In addition, MoAb KM268 did not react with any lesions in Alzheimer's disease. MoAb AM34 reacted with two molecules having an M r of 45,000 and 42,000 Da from the crude amyloid extract obtained from the kidney under both reducing and non-reducing conditions in SDS-PAGE and Western blotting. MoAb KM268 to synthetic AA peptides did not recognize these two bands, although it reacted with 9000 Da component which was considered to be AA protein. Furthermore, anti-amyloid P antiserum, anti-apolipoprotein A, anti-immunoglobulin light chain,anti-α 1 -acid glycoprotein, or anti-prealbumin antiserum did not react with these 45,000 and 42,000 Da molecules in Western blotting. Therefore, these data strongly suggest that our MoAb AM34 does not recognize the conventional AA protein, but rather reacts with high-molecular-weight amyloid-related proteins which are different from known amyloid-associated substances.