The Phenomenon That Antibodies Were Activated By Non-corresponding Antigen In Immune Response And The Correlations With H2-Eb Polymorphism

Objective:Natural Autoantibody (NAA) means that antibodies without active immunization by any antigen, and produced by the organisms to recognise one or more antigens of their own and (or) foreigners, exist widely in almost every vertebrate body.In this paper, four types of antibodies obtained by interval immunization of mice, including anti-BSA (Bovine Serum Albumin) antibodies, anti-HSA (Human Serum Albumin) antibodies, anti-ovalbumin antibodies and anti-protamine antibodies, were measured by enzyme-linked immunosorbent assay (ELISA) to observe the characteristic of non-corresponding excitation in immune responses. The above four antigens were used for the cross detection. The correlations with H2-Eb genetic polymorphism by Polymerase Chain Reaction-Sequence Specific Primer (PCR-SSP) technology were also analyzed. Through these experiments explored the source of NAA.Methods:1.First of all, standards of Bovine serum albumin (BSA) with a known concentration and the tested serum were prepared for detecting the relative contents of antibodies and to establish a method by which the contents of antibodies were detected based on ELISA, precisely.2.Then tested and verified the above method by recovery test method.3.We selected four albumins as antigens, that two has closer genetic relationship with mice (BSA and HSA) and the others distantly related to mice (Ovalbumin and Protamine). Interval immunization method was used to establish the animal model of non-corresponding excitation and prepare the tested serum.4.Microtiter plates were coated with these four antigens, which were used to measure the antibodies levels of each group, respectively. According to the types of antigen, mice serums which obtained through twice boost immunization were mixed together respectively as the standards.5.Then we took the liver of the boost immunization group mice and extracted genomic DNA. Both MudoEb5 and MudoEb7 alleles at muridae H2-Eb locus were detected by PCR-SSP technology in order to analyze the correlation between their genetic polymorphism and the phenomenon that antibodies in mice activated by non-corresponding antigens in immune responses.Results:1.The results showed that regression coefficient (R2) in the recovery test was 0.994, and regression equation was Y=1.097X-11.021. The linear regression curve was good, and the relative antibody contents were positively correlated with serum concentrations. The recovery test was successful, which proved that the ELISA test was accurate.2.We found that the method that prepared tested serum in interval immunization was valid. After the antibodies in mice were activated effectively by their specific antigens, relatively, there were significant differences between the antibody levels of booster immunization groups and basic immunization groups, respectively, P< 0.05. The differences were of statistical significances.3.It was shown that in the four kinds of antibodies, the contents of the anti-HSA (Human Serum Albumin) antibodies increased significantly after activated by protamine, P<0.05, and the difference was of statistical significance. There was no significant difference of the other three antibody levels between booster immunization groups and basic immunization groups, P>0.05. There was no statistically significant.4.The results of H2-Eb genetic polymorphism:Expressions of MudoEb7 and MudoEb5 were checked out in 40 and 36 of total 45 mice, and the positive rate were 88.89% and 80%, respectively. After stimulated by HAS, the contents of anti-ovalbumin antibodies, without MudoEb5 allele increased observably, P<0.05. The difference was of statistical significance. There was no significant difference of the antibody levels in the other immune responses, whether with MudoEb5 and MudoEb7 or no, between booster immunization groups and basic immunization groups, P> 0.05. There was no statistically significant.Conclusions:1.As was shown that the method which detected the content of antibodies by ELISA test can be used widely in a variety of antibodies for its preciseness and effectiveness. 2.Antibodies such as anti-HSA (Human Serum Albumin) antibodies may be stimulated by non-corresponding antigens (ovalbumin) in all probability.3.There were some correlations between activating antibodies by non-corresponding antigens, separately, and the H2-Eb genetic polymorphism in mice, probably. Such as the phenomenon that anti-ovalbumin antibodies were activated by HSA was related with MudoEb5.