| Objective:To measure of antibody affinity in clinical specimens,a new method was founded based on Enzyme linked immunosorbent assay(ELISA)and the traditional urea elution method,and then the role of antibody affinity in anti infection immunity was discussed based on the new method.The relationship between the polymorphism of HLA-DQB1 gene and the affinity of antibody to hepatitis B virus(HBV)antibody was discussed at the molecular level.Methods:1.Serum samples were collected from normal subjects who underwent a physical examination,based on the ELISA detection,multivariate and multi-level and single factor quantitative analysis were chosen respectively to study the influence factors which associated with antibody affinity,then the effect of the factors to the ELISA test results were explored to find out the significant influence factors2.Based on the traditional urea elution method,two factors which have significant influence on the affinity of antibody were selected to carry out the design of antibody affinity measurement method.Such as measurement of the affinity of anti-HBs,Serial dilutions of antiserum samples were prepared to find the threshold concentration of anti-serum that corresponded with the detection limit,and the new method(Terminal antibody method,TA method)was founded by the threshold concentration.Besides,to detect the applicability of the new TA method,the antiserum samples of rabbits with known antibody affinity were measured.3.Positive serum samples of anti-HBs were obtained and divided into two groups according to anti-HBe:anti-HBe positive group and anti-HBe negative group,the anti-HBe positive group consisted of 33 subjects and the anti-HBe negative group consisted of 92 subjects;meanwhile,fifty-three cases of serum samples with HCV antibody(including negative and positive HCV-RNA samples)were collected.And the TA method was used to measure the affinity of the anti-HBs and affinity of anti-HCV.Then the validity of the method in the clinical was verified.4.Sixty cases of serum samples with positive of anti-HBs and 1 case of serum sample with positive HBsAg were collected,and the TA method was used to measure the affinity of anti-HBs,then,HBsAg was neutralized by the anti-HBs with different level of antibody affinity,to explore the role of antibody affinity in anti infection immunity.5.Seventy cases of serum and blood samples were collected.The TA method was used to measure the antibody affinity to screen out the high-and low-affinity antibodies;What's more,PCR-SSP technology was used to detect the frequency distribution of HLA-DQB1 allele in the 70 cases whole blood samples,to analyze the association between different levels of antibody affinity and HLA-DQB1 gene polymorphism.Results:1.The key factors that affected the ELISA,in decreasing order,they were electrolyte strength,reaction time and incubation temperature,and the electrolyte strength was the most significant factor.2.The TA method could accurately distinguish of the high-affinity antiserum and the low-affinity antiserum,and had good stability.3.The study showed that the antibody affinity in anti-HBe positive group was significantly higher than that of anti-HBe negative group;the affinity of anti-HCV in HCV-RNA negative group was significantly higher than that of HCV-RNA positive group.4.Both antibody affinity and the protein content of the antibody were positively proportional to the neutralization results.5.The analysis of relationship between affinity and HLA-DQB1 polymorphism showed that there were significant differences between the high-and low-affinity groups for the positive rates of HLA-DQB1*02 and HLA-DQB1*06.Conclusions:1.A new method for the measurement of antibody affinity in clinical samples with good stability and accuracy was established,which named TA method.And the anti-infection effect of the high-affinity antibody might be stronger in virus infection immunity.2.The expression of HLA-DQBl*02and HLA-DQB1*06 genes might be related to the level of affinity to HBV antibody. |
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