| Objectives: To produce the McAbs against GTX2,3 and characterize the antibodies, to establish direct-competitive and indirect- competitive enzyme-linked immunoassay for the detection of paralytic shellfish poisoning toxins primarily , and make a basis for the ultimate establishment of immunological detection methods of GTX2,3.Methods: GTX2,3 was coupled with KLH by the method of formaldehyde in order to provide GTX2,3 the immunogenicity. Similarly, GTX2,3 was coupled with Glucose Oxidase(GOX) in the way of periodate oxidation. Several Balb/C mice were immunized with GTX2,3-KLH conjugates. The McAbs against GTX2,3 were produced through general hybridoma technology. The titration ,subtype and affinity of the antibodies were characterized in the way of indirect enzyme-linked immunosorbent assay, and the specificity were identified in the way of indirect competitive enzyme-linked immunosorbent assay. The antibodies were purified by the way of Protein A affinity chromatography. Direct and indirect competitive enzyme-linked immunsorbent assay were established primarily. The sensitivity, detection limits, working range of the two methods were compared .Results: Accord to the methods of formaldehyde and periodate oxidation, GTX2,3-KLH and GTX2,3-GOX conjugates were prepared successfully. After immunization and cell fusion, a hybridoma which can stably secrete McAbs against GTX2,3 was obtained. The subtype of the secreted antibodies is IgG1, the antibodies in ascites is 2.5×105, the relative affinity is 1.2×10-8, no cross-reactivity exists between the antibodies and the carrier protein BSA and GOX. The sensitivity of indirect and direct competitive enzyme-linked immunsorbent assay is 15μg/ml and 6μg/ml respectively, the detection limit is 6μg/ml and 0.6μg/ml, working range is 6-50μg/ml and 0.6-50μg/ml. All these research make a good basis for the deep research of fast detection of paralytic shellfish poisoning toxins.
|
PDF Full Text Request