Synthesis of Bacterial Urease Flap Region Peptide Equivalents and Detection of Rheumatoid Arthritis Antibodies Using Two Methods

Abstract

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that leads to cartilage damage, joint destruction and bone erosions. Serological analysis is one of the most important tools for diagnosis of RA. The aims of studies were the synthesis of an amino-acid library of epitope CHHLDKSIKEDVQFADSRI corresponding to the flap region of H. pylori urease and investigation recognition by serum antibodies from one rheumatoid arthritis patient (RAP) and one volunteer blood donor (VBD) tested by two semi-quantitative methods. In this study we compared two immunoblot variants for estimation of antibodies recognizing five synthetic peptides corresponding to the urease flap region sequence from different organisms. One immunoblot variant was a classic dot-blot using HRP-conjugated anti-human antibodies, where the level of bound immunoglobulins was estimated by digitization of color formed by reaction with secondary antibody. The second immunoblot variant was based on fluorescein-conjugated anti human antibodies. Both semi-quantitative methods were effective for evaluation of antibodies, and their advantages and disadvantages are discussed. To identify the amino-acid residues critical for reaction with antibodies, an amino-acid scan of the complete sequence of the flap region from Helicobacter pylori urease (epitope BK-61B) was conducted. Each sub-library (1–19) contained 19 peptides, each with different amino acids (a–w) at defined positions. All components of the library were synthesized using a divergent strategy. Patterns of serological reaction with the peptide library were unique for each serum sample from an RA patient or control blood donor. The amino-acid residues in epitope BK-61B necessary for strong reaction with antibodies and preventing reaction with antibodies were identified.