Detection of Novel Amyloid-Associated Protein by Monoclonal Antibody and its Gene Analysis

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We have found that monoclonal antibody AM34 (IgGl) recognizes a novel antigen which exists in amyloid deposits in various organs of the rheumatoid arthritis.In addition, anti-beta-protein monoclonal antibodies (TB1, TB2, TB3, TB4 and TB5) were generated from the fusion of mouse myeloma X63-Ag8.653 cells with splenocytes obtained from a BALB/c mouse that had been immunized with a synthetic peptide of beta-protein consisting of 24 amino acids. All these antibodies bound specifically to the amyloid changes of senile plaque and amyloid angiopathy of the brain tissues of patients with Alzheimer's disease (AD) or with senile dementia Alzheimer type (SDAT) , but not at all to protein in the normal brain or other tissues by the immunoperoxidase method. Formic acid pretreatment of brain tissue enhanced this reactivity. Positive reactions were clearly visible even in small deposits of plaque, senile plaque crown amyloid and amyloid deposits of capillary vessels under this condition.Western blot analysis revealed that monoclonal antibodies TB2 and TB3 reacted mainly with 116 kD band when extract of SDAT brain was used. They failed to react, however, with 116kD band when extract of normal brain derived from patients with other diseases was used. Two dimensional electrophoresis revealed that the 116 kD band migrated at around pl8.5. MoAbs TB1, TB2 and TB4 strongly reacted with the 1st part (7 amino acids near to N-terminus) of the synthetic peptide (24 amino acids) of the beta-protein, which consists of hydrophilic amino acids.