Preparation Of Monoclonal Antibody Against Bovine Resistin And Development Of ELISA For Resistin

Resistin was a newly discovered hormone secreted by adipocyte, it was thought to be a factor which induced the insulin resistance, regulated glucose metabolism and inhibited fat forming. The discovery of resistin provided a new hotspot for studying the animal diseases of energy metabolism disorder, especially for cow adiposis hepatica disease which had a relationship with the insulin-resistance (IR). IR commonly existed in metabolic syndromes such as adipositas, diabetes and hyperlipemia et al, and resistin could be the point linking obesity and diabetes, the level of fasting serum resistin could be an important sign in the occurrence and developing of diabetic, which was the evidence of insulin resistance mediated by adipose tissue. Serum resistin expressing in white adipose tissue decreased the sensitivity of adipose cells, skeletal cells and hepatocytes to resist insulin, and increased the blood sugar level and hyperplased adipose cells, and then lead to obesity. But in the perinatal period, when ketoacidosis, fatty liver, especially grave fatty liver happened, whether insulin resistance was present and what effect insulin resistance played on diseases need to be certified. The variation of serum resistin in ketoacidosis and fatty liver was not clear, thus detecting the concentration of serum resistin was very important to research resistin physiological action and insulin resistance and disease of energy metabolism disorder.Immunology test method based on monoclonal antibody, was widely used in detecting various kinds of materials. In this experiment, monoclonal antibodies against resistin were achieved by immuning resistin-maltose into BALB/c mice,fusing splenocytes from immunized mice with Sp2/0 myeloma cells, screening with resistin-GST across three continuous clones. Three strains of hybridoma cells were selected and named 1B2, 3E3 and 5G9 respectively, the subclass of which were all IgG1. These three McAbs were characterized for the cross reactivities, specificities and relative affinity. The titer degree of supernatant of these three strains of hybridoma cells was above 1:500, that of ascites fluid was above 105. Relative affinity showed 3E3>1B2>5G9, which of 3E3 was 4.2×108 L/mol. There were no cross reactivities with insulin, adiponectin and bovine serum-albumin. Compared to specificity and stability of monoclonal antibody, polyclonal antibody had its own advantages, for example, the shorter period of production, more antigen recognition sites, more powerfully bonding force. In this experiment, PcAb against resistin was achieved by injecting of resistin-maltose into rabbits, then collecting blood from carotid arteries, mixing the dissociated serum. The antiserum was purified by saturated ammonium sulfate method. The titer degree of PcAb was above 1:32 000, and the immunocompetence didn't change after purification. There were no cross reactivities with insulin, adiponectin and bovine serum-albumin. PcAb was successfully labeled with HRP, with the rate of labelling was above 90%. The active of HRP didn't change after labelling, and the PcAb labelled with HRP could bind with resistin specially.Immunodetection was one of the developing faster test methods. Immune analytical technique consists of enzyme-linked immunoassay, colloidal gold immunoassay, immunosensor. ELISA had the advantages of high sensitivity, low minimum detectability. Double antibodies sandwich ELISA was a sensitive method of detecting antigen, and the experiment was operated by elementary procedure to optimize. With purified mouse anti-bovine McAb against resistin as a coated antibody, and rabbit HRP-labeled PcAb as a second detection antibody, double antibody sandwich ELISA for resistin was established. The concentration of coated antibody was 15.0μg/mL, overnight at 4℃. The confining liquid was 1% gelatinum, incubated for 1.5 h at 37℃. And dilution of HRP-labeled PcAb was 1:1000, incubated for 1h at 37℃. OPD was chosen to be enzyme reaction substrate. The stopping solution was added after incubating in the dark at 37℃. The standard curve was obtained. Regression equation was got by a regression analysis. It was Y=0.927X+0.523, with the correlation coeffecient R2=0.9998, the lowest detectable limit of ELISA 4.0 ng/mL. This immunoassay provided an analytical method for the detection of resistin, established experiment foundation for bovine resistin immunodetection kit and test paper.