Preparation Of Antibodies Against Citrulline/Carbamylation And Development Of ELISA Method

Post-translational modification is a covalent process which changes the properties of a protein by proteolytic cleavage or by addition of a modifying group to one or more amino acids, such as phosphorylation, methylation, glycosylation, citrullination, saccharification, acetylation, hydroxyproline and ubiquitination. Different amino acids involved in different modifications were found in different diseases, they are often associated with autoimmune diseases (AID).Citrullination is one of the PTMs, which converts arginine residue to citrulline residue in many proteins. It may be an inflammation-associated phenomenon, and is thought to exist in many diseases, such as rheumatoid arthritis, psoriasis, multiple sclerosis.Carbamylation is also one of the PTMs, which converts protein lysine residue to homocitrulline (Hcit) residue by urea derivative-cyanate ester. It exists in uremia, renal failure (CRF), atherosclerosis and coronary artery disease and so on.Citrullination and carbamylation are studied in this issue. Hapten design, carrier coupling, antibody production, and establishment of ELISA detection method were done. The main research results are as follows:(1) Hapten designs of semicarbazide (SEM), furazolidone metabolites (AOZ), Thiram and phosphotyrosine were referred in the hapten design of citrulline artificial antigen. Carbamylation artificial antigens were synthesized. Artificial antigens were identified by BCA method and SDS-PAGE.(2) Rabbit and mice were immuned. Anti-Citrulline rabbit antiserum and anti-carbamylation mouse antiserums were acquired. Titers of anti-Cit-G-BSA antiserum were higer than3.2×104. Titers of anti-cBSA/cKLH were higer than6.4×104.(3) After fusion and screening, cell lines of anti-carbamylation were developed, named as13C11-F12-H2-F4,14E5-E8-G3-D8,14E10-F6-F7-E5and10E3-F9-G5-D8.(4) Two monoclonal antibodys were obtained by production of ascites fluid and protein A affinity chromatography, called as mAb-13C11and mAb-10E3.Titers were both above3.14×106. Isotypes were both IgG2b. They both recognize carbamylation proteins specifically.(5) ELISA detection method was established by sandwich ELISA and biotin- avidin system (BAS).The optimum conditions was mAb-13C1120μg/mL,1%BSA,Bio-13C113μg/mL,SA-HRP12.5ng/mL and TMB20min.The detection has reached the level of ng. Significant differences were successfully detected in coronary atherosclerotic heart disease, atherosclerosis myocardial infarction and human synovial fluid.This paper preliminary explored the hapten design of citrlline, at the same time, laid the foundation to developing of carbamylation detection kit (Sandwich ELISA) and specific disease detection kit (Sandwich ELISA).