| Objective:A kinetic ELISA method was established to improve the test accuracy and precision without requiring changing the original equipment basing on the routine ELISA. Also the method was used to measure the nature antibody of autoimmune disease and research the relationship between different carrying of HLA-DQB1 and natural antibody.Methods:1. A multivariate and muti- level quantitative analysis was choosed to explore the factors which may appear in any part of the experiment and to find out the larger experimental factor.2. The linearity, precision, and accuracy of K-ELISA were investigated with the established detection wavelength.3. Specific amounts of the four proteins including bovine serum albumin(BSA), starch polysaccharides, egg nucleus antibody, ovalbumin(OVA) were detected with the K-ELISA method in the serum selected randomly.(Blood samples were collected from the Second Affiliated Hospital of Dalian Medical University).The allelomorphic genes of HLA-DQB1 were determined by polymerase chain reaction(PCR) in the 50 patients to analyze the correlation between the natural antibody and the different carrying of HLA-DQB1.4. The difference among the people with autoimmune disease was deteced with K-ELISA with the specific amounts of the four proteins including BSA, starch polysaccharides, egg nucleus antibody, OVA.(Blood samples were collected from the Second Affiliated Hospital of Dalian Medical University).Results:1. The enzyme reacting time, serum concentration, microplate, reaction time of the substrate was found the four biggest factors of the routine ELISA by exploring the factors of the routine method in order. The other three which were not affected much in the experiment can be effectively controlled by strictly controlling the reaction conditions.2. The new method established with kinetic ELISA was shown a good stability and revealed a linear relationship between serum volume and assay results with the intraand inter-assay average coefficients variation(CV) were 8.91% and 8.02%. Compared with the commercial ELISA kit(10 mIU/ml), the sensitivity of k- ELISA for the detection of anti-HBs in human serum samples is much higher(0.625 mIU/ml) which show that K-ELISA an excellent sensitivity. The influence of microplate and reaction time of the substrate have been removed basically by K-ELISA method.3. In the correction analysis, we found that there is a significant correlation between the content of BSA in serum and HLA-DQB1*04 locus.4. There are differences among the people with autoimmune disease. Conclution:1. Kinetic ELISA could be suggested to widely used and proved more effective and sensitive.2. HLA-DQB1*04 locus have significant influences on natural antibody of BSA.3. There are differences among the people with autoimmune disease. |
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