| Summary: | 碩士 === 中山醫學大學 === 生物醫學科學學系碩士班 === 95 === Ochratoxin A (OTA) is a widespread mycotoxin contaminating feed and food, and is suspected of being the etiological agent of Balkan endemic nephropathy (BEN) and its associated urinary tract cancers. To develop more rapid and sensitive methods for detection of OTA, monoclonal antibodies (mAb) against OTA were produced from a stable hybridoma cell line, H9, generated by the fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from BALB/c mouse immunized with OTA-keyhole limpet hemocyanin. The OTA-specific mAb H9 was used for developed a sensitive direct competitive enzyme-linked immunosorbent assay (cdELISA) and a rapid mAb based colloidal gold immunostrip assay. The concentration causing 50% inhibition (IC50) of binding of OTA–horseradish peroxidase with the antibodies by OTA in the cdELISA were found to be 0.27 ng/mL. The detection limit of OTA-specific immunostrip was 5 ng/mL for OTA. The results of immunostrip analysis of OTA in coffee samples were in good agreement with those obtained from cdELISA and the result can be observed directly by the nake eyes, and the detection time could be completed within 10 min. The OTA mAb-based cdELISA and immunostrip assay development in this study is suitable for the simple and rapid screening of OTA in coffee samples without sample cleanup.
Domoic acid (DA) is a naturally occurring neuroexcitatory toxin produced primarily by the marine diatom pseudonitzschia pungens. Human ingested DA contaminated shellfish leads to amnesic shellfish poisoning (ASP), which even cause death at high dose of DA. To develop more rapid and sensitive methods for detection of DA, monoclonal antibodies (mAb) against DA were also development, which was produced from a stable hybridoma cell line, 9F1F11, generated by the fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from BALB/c mouse immunized with DA-keyhole limpet hemocyanin. The DA-specific mAb 9F1F11 were used for developed a sensitive cdELISA and a rapid mAb based colloidal gold immunostrip assay. The concentration causing 50% inhibition (IC50) of binding of DA–horseradish peroxidase with the antibodies by DA in the cdELISA were found to be 0.58 ng/mL. The detection limit of the DA-specific immunostrip was 5 ng/mL for DA. The results of immunostrip analysis of DA in blue mussel samples were in good agreement with those obtained from cdELISA and the result can be observed directly by the nake eyes, and the detection time could be completed within 10 min.
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