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

制备了葡萄球菌肠毒素（SEs）和蜡状芽孢杆菌致泻毒素（DT）的小鼠单克隆抗体（mab），用于检测受SEs和DT污染的食品。测定这些单克隆抗体的免疫特异性，开发利用这些单克隆抗体的免疫学方法。我们成功地建立了分离se和DT的免疫亲和层析，以及检测se的夹心酶联免疫吸附试验（ELISA）。所获得的结果描述如下：1。针对SEs的单克隆抗体；以不同的SEs作为抗原，我们获得13个单抗（2个单抗仅与SEA反应，1个单抗仅与SEE反应，3个单抗同时与SEA和SEE反应，5个单抗仅与SED反应，1个单抗同时与SEA、SED和SEE反应）。所有这些单克隆抗体都属于IgG_1或IgG_2。ELISA检测到的se最低含量为1 ~ 100 ng/ml。其中一种单克隆抗体（AE-53）与SEA和SEE均有反应，用于制备免疫吸附剂。采用具有双重特异性的免疫吸附剂与AE-53偶联，可以很容易地从培养滤液中纯化SEA和SEE。约70- 75%的SEA和SEE通过一步免疫亲和层析得到。用单抗（KD-1）纯化SED也进行了类似的尝试。该方法对高收率地纯化SED也非常有用。建立夹心ELISA检测SEA的方法，将获得的单克隆抗体作为包被的第一抗体。在夹心ELISA法中，SEA的最低检出量为1 ng/ml。上述结果表明，采用单克隆抗体的夹心ELISA法检测培养滤液和食品中的硒具有较高的灵敏度和实用性。单克隆抗体对DT；对蜡样芽孢杆菌产生的一种DT获得了3个单抗。用其中一种单克隆抗体制备免疫吸附剂，从培养滤液中纯化DT。为获得较高的DT收率（16 - 32%），pH为7.2、6 M胍- hc1为较好的洗脱液。经SDS-PAGE分析，纯化后的DT是均匀的。因此，发现免疫亲和层析法在简单，纯度和回收率方面比传统方法更好地分离DT。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DT多克隆和单克隆抗体。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