Preparation Of Anti-IFN-α Monoclonal ELISA Kit And The Clinical Usage

[Objective] We have proliferated monoclonal antibodies from established three hybridoma cell lines secreting immunoglobulin G (IgG) antibodies specific for human alpha-interferon. The high specific McAb have been used to preparing biotinylated antibody for ELISA kit. The proposal of this kit is analyzing clinical samples, which come from HBV infection patients. Discuss the possibility of the clinical usage of this ELISA kit based on the results of the analysis. [Method]The harvested monoclonal antibodies have been purified by saturated ammonium sulfate and SPA-sepharose CL-4B. We added 100μg of the BNHS reagents per 1 mg of the McAb, incubated them at room temperature for 4 hours. Then add 20μl 1M NH₄Cl per 250ug BNHS. 10 minutes later, we applied the McAb sample to dialyze and got the purified biotinylated McAb. The Avidin-biotin ELISA tested the activity of antibody.Comparing different coating system, immobilized monoclonal antibody and reaction time to find out the most suitable reaction system. Made up all experiment data to set up Sandwich ELISA kit and draw the standard curve and stabilization curve.Detected IFN- α in patients' serum who have been infected by hepatitis B virus by the ELISA kit constructed in the primary part. Compared the IFN- α level between HBV negative and positive people based on the information got from the ELISA analysis. [Results]We harvested three strains anti-IFN α McAbs After labeling with BNHS,the antibodies keep high biological activities and sensitivities in detecting the sampleofrhlFN-a by ELISA.The Sandwich ELISA kit is based on two strain McAbs, 3D9B10 and IA11C3. 3D9B10 was biotinylated and act as labeling antibody. IA11C3 was immobilized by 0.01 M, pH7.4 PBS. The sensibility of the ELISA kit is 460 pg/ml.The IFN- a level in samples of HBV infection patients are higher than HBV infection negatives. The information based on the ELISA analysis could be used in clinical field, which can help to evaluate the celluar immunity ability of the people. [Conclusion]The method of preparing McAb and biotilyzation McAb is simple and stable.The Sandwich ELISA kit based on the solid McAb, 1A11C3, and labeled McAb, 3D9B10, has a comprehensive sensitivity, accuracy and stability.After applying this kit for serum from HBV infection negative and positive people, we have observed that the IFN- a level were difference between them.