| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1989: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1988: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
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
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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
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