Preparation Of Anti-vitamin B₁₂-anti-acridinium Ester Bispecific Antibody And Study Of Immunochemiluminescence Method

This research adopted the method of cell fusion to prepare a bispecific antibody which based on the acquired the monoclonal antibody against vitamin B₁₂(VB₁₂).On this basis,the immunochemical luminescence detection method were established with the bispecific antibody,and the applied research was carried out.The main research contents are as follows:1.The preparation of acridine artificial antigenThe acridine ester（AE）was conjugated to carrier protein of keyhole limpet hemocyanin（KLH）and bovine serum albumin（BSA）by active ester method to preparaed the immune antigen AE-KLH and the detection antigen AE-BSA.The UV spectra and immunogenicity titration indicated that two kinds of antigens were synthesized successfully.2.Preparation of anti-vitamin B₁₂-anti-acridine ester bispecific antibody by electric fusionThe monoclone hybridoma cell of anti-VB₁₂2 was induced to obtained the hybridoma cell lines with a defectived the hypoxanthine guanine phosphate ribozyme which was sensitive to the HAT medium.The hybrid-hybridoma cell that produced the bispecific antibody of anti-VB₁₂-anti-AE was obtained by electric fusion between the defective hybridoma cell of anti-VB₁₂2 monoclone hybridoma and the spleen cells of the mice immunized with the antigen AE-KLH.The supernatant secretion of hybridoma cell were detected by the indirect ELISA,which indicated that the half inhibition(IC₅₀)of the VB₁₂2 and AE separately were 5.90 ng/mL and 1.90 ng/mL,and the chromosomes number of 5strains hybridoma cell were 147±5 averagely by the chromosome analysis,which were confored to the number of the three hybridoma chromosome number.It was proved that the hybridoma cells were obtained which secreted the anti VB₁₂-anti AE bispecific antibody.3.Establishment of Chemiluminescence immunoassay method（CLIA）based on the specific antibodyThe CLIA method that based on the specific antibody was established.The effects of the concentration of antigen,dilution ratio of acridine ester coupling of horseradish peroxidase,pH and salt ions（Na⁺）concentration on the chemiluminescence intensity（RLU）and half inhibitory concentration(IC₅₀)were optimized.The optimal reaction conditions as follows:the concentration of antigen coating was 8μg/mL,the dilution ratio of acridine ester coupling horseradish peroxidase was 1:400,antibody dilution buffer was pH 7.5 0.01 mol/L PBS.The competitive inhibition curve was established,which the linear range was 1-50 ng/mL,IC₅₀0 was 6.18 ng/mL,and the IC₁₀0 was 0.93 ng/mL.Recorvery experiment was performed on three levels,and the average recovery rate of care tablets and functional drinks were 94.98%-101.50%and 86.11%-102.12%,the coefficient of variation were2.37%-4.07%and 2.71%-7.75%,respectively.Three kinds of actual samples were tested respectively,and the detection value was basically consistent with the label value.