Immunological application of monoclonal antibodies to detect bacterial toxins produced in foods

制备了抗葡萄球菌肠毒素(SES)和蜡样芽孢杆菌致泻毒素(DT)的小鼠单抗，用于检测食品中的SES和DT。确定了这些单抗的免疫学特异性，以开发使用这些单抗的免疫学方法。我们成功地建立了免疫亲和层析分离SES和DT，并成功地建立了夹心酶联免疫吸附试验(EL ISA)检测SES。所获得的结果如下所述。用不同的SEA作为抗原，得到13株单抗(2株仅与SEA反应，1株仅与SEE反应，3株同时与SEA和SEE反应，5株仅与SED反应，1株FLAB与SEA、SED和SEE反应)。所有单抗均属于Ig G_1或Ig G_2。用这些单抗可检测到的最低Se含量为1-100 ng/ml。其中一种单抗(AE-53)同时与SEA和SEE反应，用于制备免疫吸附剂。通过使用…更多相同的免疫吸附剂与具有双重特异性的AE-53结合，SEA和SEE都很容易从培养滤液中得到纯化。一步免疫亲和层析可得到约70-75%的SEA和SEE。使用单抗(KD-1)也进行了类似的纯化SED的尝试。为了建立检测SEA的夹心ELISA法，以获得的单抗为包被抗体，建立了检测SEA的双抗体夹心EL ISA方法。结论：1.在使用这些单抗的夹心ELISA方法中，SEA的最低检测浓度为1 ng/ml。这些结果表明，使用单抗的夹心ELISA法对于检测培养滤液和食品中的SES是非常敏感和有用的。获得3株抗蜡样芽孢杆菌产生的DT的单抗。用其中一种单抗制备了一种免疫吸附剂，用于从培养滤液中纯化DT。为了获得高得率(16-32%)的DT，6M盐酸胍，pH 7.2是一个很好的洗脱剂。经SDS-PAGE分析，纯化的DT为均一组分。因此，免疫亲和层析法在分离DT的简便性、纯度和回收率方面明显优于传统方法。通过免疫亲和层析获得的DT制剂也可用于制备高特异性的抗DT的多克隆和单抗。较少

Mouse monoclonal antibodies (MAbs) against staphylococcal enterotoxins (SEs) and a diarrheagenic toxin (DT) produced by B. cereus were prepared to detect foods contaminated with SEs, and DT. Immunological specificities of these MAbs were determined to develop immunological methods using these MAbs. We succeeded in developing immunoaffinity chromatography to isolate SEs and DT, and the sandwich enzyme-linked immunosorbent assay (ELISA) to detect SEs. The results obtained were described below.1. MAbs to SEs; With different SEs as antigens, we obtained 13 MAbs (2 MAbs reactive with only SEA, one MAb reactive with only SEE, 3 MAbs reactive with both SEA and SEE, 5 MAbs reactive with only SED, and one flab reactive with SEA, SED, and SEE). All of these MAbs were found to belong to either IgG_1 or IgG_2. The minimum amounts of SEs detectable by ELISA using these NAbs were 1-100 ng/ml.One of these MAbs (AE-53) reactive with both SEA and SEE was used to prepare an immunosorbent. By use of the … More same immunosorbent coupled with AE-53 with dual specificity, both SEA and SEE were easily purified from culture filtrate. Approximately 70-75 % of SEA and SEE were yielded by one-step immunoaffinity chromatography. Similar attempts were also made to purify SED using a MAb (KD-1). This method was found to be also very useful to purify SED in high yield.To develop the sandwich ELISA method to detect SEA, MAbs obtained were used as the first antibodies for coating. In sandwich ELISA method using these MAbs, the minimum amount of SEA detectable was found to be 1 ng/ml. These findings suggest that the sandwich ELISA method using MAbs is very sensitive and useful to detect SEs in culture filtrates and foods.2. MAbs to DT; Three MAbs were obtained against a DT produced by B. cereus. An immunosorbent using one of MAbs was also prepared to purify the DT from culture filtrate. To obtain high yield (16 -32 %) of DT, 6 M guanidine-HC1, pH 7.2 was found to be a good eluant. Purified DT was found to be homogeneous as analyzed by SDS-PAGE. Thus, immunoaffinity chromatography was found to be much better to isolate DT than conventional methods in terms of easiness, purity, and recovery. The DT preparation obtained by immunoaffinity chromatography will be also useful to prepare highly specific polyclonal and monoclonal antibodies against DT. Less