Development And Preliminary Application Of Okadaic Acid Immunoaffinity Column

Okadaic acid(OA)is produced by Red tide algae armor, this toxin has no toxiceffect on the shellfish itself, however, when humans consume shellfish contaminatedby OA, Diarrhea, nausea, vomiting diarrhea symptoms will occure.It has no strongacute toxicity, but has mutagenic and immunotoxic effects. Human health is seriouslythreatened, therefore, the prevention and control of OA has important public healthsignificance. In liquid chromatography and immunology detection methods, effectiveextraction of the toxin is necessary for evaluating content of toxins in seafood. Toensure the safety of consumers, it is necessary to establish efficient, convenient andsimple pretreatment method.Imumunoaffinity column has many advantages, such as efficient, convenient andspecific as a commonly used pre-treatment methods in the methods of detection andanalysis, while ELISA is simple, relatively stable, low cost and widely used in rapiddetection of various food contaminants, in this study,immunoaffinity column wasprepared for pre-treatment of seafood samples spiked based on the obtained of OAmonoclonal antibody hybridoma cell lines, we can analyse the recovery and effect ofapplication quantitatively by indirect competitive ELISA.In this paper, we revived specific hybridoma cell lines secreting anti-OA andproduced a large number of mouse ascites by producing in vivo. Due to ascites ispurified by affinity chromatography of protein G, high purity OA monoclonalantibody is prepared successfully and titer is2.56×106. The IAC column wasconstructed by CNBr-activated Sepharose4B covalently coupling the monoclonalantibodies and coupling efficiency can reach93.4%, which indicate the success ofpreparation of immunoaffinity column, the extraction conditions is optimizedconsequently, loading buffer is PBS containing0.2mol/L NaCl (pH=7.4), the volumeof loading buffer is1.5mL, lotion liquid is methanol: water (v/v:1/20), Eluent ismethanol: water (v/v:9/10), volume of eluent is1mL.To apply the IAC column forextraction recovery of shellfish samples, two detection methods IC-ELISA and HPLC-MS/MS were used for contrast detection, which obtained similar results andIC-ELISA was90.3%,10kinds of shellfish samples were preliminary determinedcontent of OA toxin further, the results showed seven kinds of samples were positive,Chlamys Farreri was most containing33.91ng/g, Scapharca subcrenata was secondcontaining11.45ng/g, the others was less than10ng/g. Successfully preparedimmunoaffinity column can effectively gather OA content in the samples, and reduce theinfluence of matrix and thus improve the detection accuracy, it provided an efficientpre-treatment tool to monitor pollution and establish detection method of OA.