The Study On Development Of An Enzyme-Linked Immunosorbent Assay And A Gold Immunochromatography Assay For Detecting Okadaic Acid In Shellfish From China Coast

Diarrhetic shellfish poisoning (DSP) is a kind of lipophylic natural biotoxin produced by the Dinophysis and Prorocentrum.Okadaic acid (OA) and its derivatives have been reported to be the principal components of DSP toxin which may cause eaters to suffer from diarrhea, nausea, vomit, and gastrointestinal cramping pain and are a strong tumor promoter . At present, DSP are mainly analyzed by mouse bioassay, high-performance liquid chromatography(HPLC) and some immunological methods.OA were conjugated to KLH, BSA and OVA using carbodiimide reaction. The Balb/C mice were immunized by ip injection with OA-KLH and OA-BSA. Monoclonal antibody were prepared throughout fusing the spleen cells of immunized mice with Sp2/O cells. The polyclonal antibodies and monoclonal antibodies against OA were producted.An indirect competitive enzyme-linked immunosorbent assay for quantitative analysis of okadaic acid in the shellfish and seawater were developed using 3# polyclonal antibody against OA. Under optimal condition, the detection limit of OA was 0.1ng/ml. The quantitative limit for OA was 10ng/ml for sea water, 50ng/g for shellfish, respectively. The recovery of OA added to sea water was 62%～114%, with a coefficient of variation of 22%～24%;the recovery of OA added to shellfish was 51%～78%, with a coefficient of variation of 1%～10%.The Monoclonal antibody prepared using OA-KLH as the immunogen were applied in development the idc-ELISA(indirect competitive enzyme-linked immunosorbent assay) for detection OA. The idc-ELISA method with detection limit of 0.8ng/ ml was developed using 1# monoclonal antibody with the highest specificity against OA. An indirect competitive enzyme-linked immunosorbent assay for quantitative analysis of okadaic acid in the shellfish and seawater were developed using monoclonal antibody against OA prepared using OA-BSA conjugate as immunogen, the detection limit of OA was 31.2ng/ml, the mean recovery was 99.8%, with a mean coefficient of variation of 8.7%. An immunochromatographic strip test was developed for detection of okadaic acid by using colloidal gold-labeled monoclonal antibody(OA-BSA) of the okadaic acid molecule as the detection antibody.The lower detection limit for OA was 50ng/strip, 500ng/ ml.A Gold Immunochromatography Assay(GICA) was developed for detection of okadaic acid by using an colloidal gold-labeled monoclonal antibody(OA-KLH) of the okadaic acid molecule as the detection antibody. The test works on the principle of lateral flow immunochromatography using a strip format,providing a qualitative (yes/no) indication of the presence of OA within 15 min. At 12ng/ ml OA levels, the test is applicable only as a qualitative assay. The major advantages of the step strip test are that results can be obtained within 15 min and that all reagents are included in the test device.