Anti-staphylococcus Aureus Intestinal Toxins C2 And Anti-mannose Glycans Combination The Establishment And Application Of Lectin Monoclonal Antibody Preparation, Identification And Elisa Method For Detection

Staphylococcal enterotoxins(SEs) is a series of extracellular water-soluble proteins secreted by Staphylococcus aureus, which can be divided by their serological activity into five distinct types designated staphylococcal enterotoxin A(SEA), B(SEB), C(SEC), D(SED) and E(SEE). The SEC is also divided into three subtypes: SEC1, SEC2 and SEC3. Compared with common antigens, SEs can activate much more T lymphocyte at lower concentration and induce more powerful immune response, so they are considered to be superantigens. Among the members of SEs, SEC is poorly studied.Before this study, SE was purified from the bulk material of the biological product "Injection Jinpusu" that prepared by the culture of Staphylococcus aurues. The N-terminal amino acid sequencing confirmed this SE was SEC2. Further pharmacological tests indicated that SEC2 was one of the active components in the "Injection Jinpusu". Based on all those previous data, it can be concluded that the amount of SEC2 was a reliable indicator for the quality control of the "Injection Jinpusu". Thus, a reliable and simple method for quantitatively detecting SEC2 became necessary.In this study, the monoclonal antibodies against SEC2 were prepared and the diagnostic sandwich ELISA was established by using these McAbs. The hybridoma technique was used to prepare McAbs and the ELISA was used to screen the culture supematant of hybridoma. Four hybridoma cell strains(3B7, 4F7, 4D7 and 4F6) stably secreting anti-SEC2 McAbs were obtained, whose Ig isotype was IgG₁(κ) and ascites titer was between 1: 10⁵ and 1: 10⁷. Those ascites were purified by caprylic acid-ammonium sulfate precipitation. The result of SDS-PAGE showed that the purity of McAbs were above 80％. These McAbs can specifically recognize SEC2 without cross-reaction to bovine serum albumin, human serum albumin, SEA, SEB and SED. The McAb-3B7 had cross-reaction to SEC1 when the concentration of SEC1 was more than 1μg/ml, and the others had no cross-reaction to SEC1. The affinity of these McAbs measured by non-competitive enzyme immunoassay was from 1.1×10⁹M^-1 to 2.46×10¹⁰M^-1. The purified McAbs were conjugate with HRP by conventional method. The McAbs recognize different antigenic binding site except McAb-4F7 and McAb-4D7 sharing the same binding site. Two anti-SEC2 McAbs that recognize different antigenic binding site (McAb-4D7 and HRP-McAb-3B7) were used as the coating antibody and detecting antibody respectively and a quantitative sandwich ELISA for measurement of SEC2 was established. This method had good specificity, sensitivity and reproducibility, which can reliably detect SEC2 at the concentration from 0.5 to 20 ng/ml. The inter-assay coefficients of variation(CV) ranged between 3.71％and 6.93％, and the intra-assay CV of mean was 1.02％. The recovery rate ranged between 97.8％and 101％, and the CV value of recovery test ranged between 2.2％to 5.2％. This monoclonal ELISA method and polyclonal ELISA method were applied in parallel for quantitative analysis of the content of SEC2 in the 40 lots sample of "Injection Jinpusu". Although the value tested by monoclonal method was lower, but there was no remarkable difference in results of two methods. The content of SEC2 in the 50 tested lots sample was 5 ng/ml to 16 ng/ml and the CV was 16.8％.The successful preparation of McAbs against SEC2 and establishment of a qualitative ELISA method with good specificity and sensitivity will provide a useful tool for the research of SEs and also for the quality control of "Injection Jinpusu". Preparation, characterization and preliminary application of monocional antibody against human Mannan-Binding LectinMannan-Binding Lectin(MBL) secreted by liver is a plasma C-type lectin with a collagen-like domain and Ca²⁺-dependent, which is considered as an essential element of innate immunity. Via 'carbohydrate recognition domains'(CRD) MBL can recognize and bind to the surface of a large number of pathogenic micro organisms and sequentially neutralize and/or opsonize them by activating complement system. As an acute phase protein and innate immune factor, MBL is important for human especially in the period before the maturation of adaptive immunity and before the occurrence of IgM in the situation of infection. The circulating MBL level is primarily affected by genotype, but it is also influenced significantly by some nongenetic factors. It has been reported that MBL deficiency or insufficiency is associated with many diseases, including repeated infection, autoimmune disease, repeated spontaneous abortions, some cancers and so on. A diagnostic kit for MBL is necessary for the research of MBL. But, till now, there's no this kind of kit in our country and the most of work releated to MBL depended on imported kit which is very expensive, and it is a block to our research of MBL.In this study, we made a diagnistic kit for MBL by preparing two monoclonal antibodies and establishing quantitative ELISA method. By using this kit, we detected MBL level in some different people. The MBL purified from serum of health people was used as antigen to immune Balb/c mice. Two specific McAbs(B3 and B10) against MBL were obtained by normal hybridoma technology, whose Ig isotype was IgG1, ascites titer were from 1: 10⁶ to 1: 10⁷ and antigenic binding site was different. Those ascites were purified by caprylic acid-ammonium sulfate precipitation and affinity chromatography of protein A. The result of SDS-PAGE showed that the purity of McAbs were above 90％. The two anti-MBL McAbs can specifically recognize MBL without cross-reaction to bovine serum albumin, human serum albumin, and the serum of deficiency of MBL. The purified McAb-B3 was conjugated with HRP by conventional method. The optimum concentration of coating McAb-B 10 was 8μg/ml and the dilution of HRP-McAb-B3 was 1:500. The sandwich ELISA method had good specificity, sensitivity and reproducibility, which can reliably detect MBL at the concentration from 1 ng/ml to 100 ng/ml. The inter-assay coefficients of variation (CV) ranged between 3.9% and 5.2% and intra-assay CV of means was 1.6%. The recovery rate was between 98.0% and 105.3%, and CV value of recovery test was between 2.1% to 4.3%. Compared with Denmark's method, this method had a wider detective range. The results of two methods had good consistency.The established sandwich ELISA was used for measuring the serum level of MBL in 277 healthy Chinese Dong and Zhuang people, and the results were analyzed statistically. The concentration of MBL in Dong was significantly lower than that in Zhuang (p＜0.01), and there was no difference between male and female. For different age, the concentration of MBL in adolescence was significantly higher (p＜0.01) and the deficiency or insufficiency ratio of MBL was significantly lower than that in other period. In this study, the concentration of MBL had a negative correlation with the deficiency or insufficiency ratio of MBL. The difference of concentration and deficiency or insufficiency ratio of MBL between Dong and Zhuang people suggested they had different genetic background. The serum level of MBL may be in positive correlation with the level of growth hormone. The PCR technology was used to analyze the point mutation of MBL Exon I in all these serum samples. The samples of point mutation in codon 54, 57 and 52 was 51, 1 and 0 respectively. All samples with point mutation had low MBL level(＜1μg/ml). Low MBL level was associated with the point mutation in MBL Exon I. Another investigation about serum level of MBL in 204 children showed that MBL level in 103 infectious children (pneumonia, cephalitis, diarrhea and sepraemia) was higher than that in 101 healthy children. This result suggested that infection can lead to the increase of MBL level and low MBL level may correlate with the susceptibility to infection.