The Synthesis Of Artificial Antigen And The Preparation Research Of Monoclonal Antibody Of Rosiglitazone

In the current enthusiasm for nutrition and health food, the phenomenon of abusive and indiscriminate using of various nutrients and other nutritional components is serious. Rosiglitazone is a thiazolidinedione and anti-diabetic drug which control blood sugar effectively through increasing sensitivity of insulin. There are many functions of it: to adjust the insulin-responsive gene transcription, to control generating, transporting and using of blood sugar, to improve high glucose toxicity and so on. Because of these, in recent years it is added into the health and losing weight food. This article launchs the immunoassay technique research of Rosiglitazone. Main findings are as follows:1.Rosiglitazone was not immunogenic in itself, it must be coupled with carrier protein before the induced antibodies. The hapten of rosiglitazone was prepared and identified by mass spectrometry in this study.2.Rosiglitazone was coupled with the carrier protein BSA and OVA by active ester method; analyzed by ultraviolet spectrum, both the artificial antigens changed significantly, the positions of max absorption shifted to far ultraviolet, indicating the successful synthesis of artificial antifens. Measured by BCA, rosiglitazone hapten-BSA concentration was 4.668mg/ml, and rosiglitazone hapten-OVA concentration was 3.312mg/ml. The coupling ratio of the two were 14:1, 8.2:1. All of the above artificial antigens preserve at -20℃after packing.3. Immunizing Balb/C mice with antigen (Rosiglitazone-BSA), we received Rosiglitazone antiserum, and then made the spleen cells and SP2 / 0 myeloma cells of the immune mice integrated,and the best coating antigen concentration was being diluted 10,000 times; optimal antibody dilution was 10,000 times; their ideal anti-dilution was 1:10000. At the same time, the spleen cells of mice immunized were fused with SP2/0 myeloma cells. The two cell lines that can secrete monoclonal antibodies against Rosiglitazone stablely were obtained after cell subclone and selection, named ED3 and CC4.4. It also conclude the preparation of rabbit polyclonal antibodies, while optimizing the reaction conditions of indirect competitive ELISA, in which rosiglitazone hapten-OVA was the best coating antigen; the best antibody dilution was the Y, the optimal dilution of coating antigen was 1/20000, the optimal dilution of antibody was 1/20000, the best two anti-dilution was 1/10000, the best one anti-competitive time was 50 min, while the best two anti-reaction time was 45min.5. Rosiglitazone standard curve was plotted. The rosiglitazone indirect competitive ELISA detection methods were established, the limit of detection was 2.18 ng/mL, the range of detection is 6.07-200.11 ng/mL. Between mice polyclonal antibody and rabbit polyclonal antibody, we found that the detection limit of rabbit polyclonal antibody was the lower one,which has higher potency.This paper lays the foundation to the developing of rapid diagnostic cassette for illicit drugs in the health-care foods, and provides basis for sample pretreatment of health-care foods. With present popularization of health-care foods, the detection of illicit drugs in the health-care foods would be the key of food safety testing.