The Study On Rapid Detection Assays For Okadaic Acid From The Shellfish

Okadaic Acid(OA), an important marine DSP toxin, is mainly produced from some poisonous marine dinoflagellates, and usually accumulated in the bodies of Mussels, clams and snails by the food chain, then leads to food poisoning with the key feature of diarrhea. The dinoflagellates with toxin distribute throughout the whole world and the poisoning events due to eating poisonous marine products by accident also occurred quite often in China. This has a severe effect on the human health and the exploitation for the marine product. Thus strict inspection systems have been established in many countries, and the relevant detection methods and means have also been founded, such as mouse bioassay, chemical instrumental method, immunological detection and other specific detection methods according to the specialities of the different toxins. In China, the study on the Okadaic acid which is the key causative agent of DSP started quite late, especially the study on the detection methods drops behind. Therefore in this experiment the micromolecule OA was studied to develop for OA immunological detection and the high performance liquid chromatography with triple-quadrupole tandem mass spectrometry. At the same time, the application outlook of the ScFv and fusion ScFv during the broad spectrum detection for marine biotoxin was investigated. In order to offer the reliable basis for rapid detection of marine biotoxins for China's importing and exporting marine products. The preparation of OA complete antigen As OA is a low molecular weight substance, OA was coupled with human IgG and BSA as immunity antigen and detection antigen by active ester method. The man-made antigen was successfully coupled with carrier protein by identification of the agarose gel electophoresis and denaturalized polyacrylamide gel electrophoresis, and OA molecule conjugation ratio with human IgG and BSA were 24:1 and 8:1 by the indirect competitive ELISA which was set up in this experiment. It turns out that OA has been coupled with carrier protein, and OA immunity antigen and detection antigen have been successfully prepared.The preparation and specialities study of OA monoclonal antibody The spleen cells from the Balb/c mice hyperimmunized with a human IgG-OA conjugate were fused with SP2/0 myeloma cells. One hybridoma cell line was identified that secretes IgG1 monoclonal antibody against Okdacid acid, designated 3C5 after subcloned for 3 cycles by indirect ELISA. The average number of the hybridoma chromosome was 50±2. The molecule weight was 159 390, the affinity constant was 9.8×108L/mol and the titer of the monoclonal antibody was 2.56×106 ,and it had no cross reacting with TTX ,GTX1-4 and GTX2-3. The OA monoclonal antibody was largely manufactured by using the method of intraperitoneal injection Balb/c mice and purified by CA-AS and AS method. The result shows that the recovery of CA-AS method is 54.22% which is less than AS method (98.92%); but the purity for IgG by the CA-AS method is 83.21% which is higher than AS method (58.35%). That is to say, the CA-AS method is a sort of good mouse ascetic extracting method which is easy-operated, low-costing, excellent purified and recovered effects. The establishment of OA competitive inhibition ELISA detection method The one-step competitive inhibition ELISA for detecting OA (enzyme labeling monoclonal antibody) and the two-step competitive inhibition ELISA detection method (commercial enzyme labeling ant-antibody) were improved respectively using OA monoclonal antibody. The regression equations and correlation coefficients of the two detection methods are y=-38.678x+130.01, R2=0.988 6 and y=-34.212x+83.49, R2 =0.978 4; The linearity ranges are 1.56-75μg/L and 0.4-25μg/L; The lowest concentrations, which can effectively detect OA are 0.6μg/L and 0.18μg/L. The simulate shellfish sample (add some OA) was homogenated and dissolved by methanol/water(8/2). After ethane degreasing, chloroform extraction, evaporation fixation, the two kinds of ELISA were used to detect the sample. The recoveries are 84.72% and 98.38%. In addition, the OA competitive ELISA detection kit in plate were packed according to optimized condition of indirect ELISA.The establishment of OA high performance liquid chromatography with triple-quadrupole tandem mass spectrometry The shellfish extract was preseparated and gathered by AccuBondⅡSPE silica column extractor, a triple-quadrupole tandem mass spectrometer was used as a detector for HPLC to determine OA. As for the MS/MS, the multi-reactions monitoring (MRM) scan type and the positive ion Electrospray Ionization(ESI) mode were employed. Selected the precursor ion→product ion(m/z 827→m/z 723,m/z 827→m/z 809) as quantitate detection ion pair. The mobile phase of HPLC was the methyl cyanide:1%methanoic acid-water(vol/vol:70:30) and the chromatographic column was the Agilent Extend-C18 (2.1×150mm,Φ5.0μm). The standard OA, the shellfish being added OA and the shellfish sample were determined by the HPLC-MS/MS. The standard curve of OA shows good linearity over the concentration range of 10-800μg/L, the equation of linear regression is y=193.07x-780.6 ( Q1/Q3: m/z 827.4→723.5);y=83.021x-335.6(Q1/Q3: m/z 827.4→809.5),r2 are all 0.9991, The average recovery of sample being added OA is 83.99% and the average RSD is 4.34%. This HPLC-MS/MS is highly sensitive, fast, and very accurate, so it can be used for detecting limit of OA from shellfish.The compounding of SCFv against OA The VH and VL genes of monoclonal antibody against OA were amplified from the hybridoma cell line 3C5 by RT-PCR. They were linked using a flexible linker gene by SOE-PCR. The OA-ScFv gene was cloned into PMD18-T vector and was sequenced. The ScFv gene consists of 726bp which encoded 242 amino acids including 15 linker amino acids. The VH and VL accord with the feature of the variable region gene of the mice antibody by Igblast analysis. They have the typical structure domain (IGV) of the immunoglobulin and have very high homology with many ScFvs on record by amino acid homology comparing analysis. Both VH and VL genes are confirmed as functionally rearranged mouse immunoglobulin variable region genes.Clone and expression of the fusion ScFv against OA-STX The fusion ScFv against OA-STX were linked on gene level to construct the express carrier of pET-22b and transformed into DE3. The molecular weight of the expressed fusion protein is about 53ku by IPTG induction. The expressed fusion protein has a favorable reactionogenicity with OA by ELISA. This will established the study foundation for using the fusion ScFv to construct the rapid detection which can screen three kinds of biotoxin simultaneously. In brief, the monoclonal antibody against micromolecule OA was prepared in this experiment, and two sorts of competitive inhibition ELISA which were more rapid, sensitive and convenient were constructed. They had good correlation with the HPLC-MS/MS set up in this experiment for the same sample. At the same time, the VH and VL of the monoclonal antibody against OA were amplified and the OA-ScFv was packed by the SOE-PCR. Then OA-ScFv was linked to STX-ScFv by Gly4Ser. The OA-STX-ScFv was cloned into the pET-22b carrier and expressed by IPTG induction in DE3. The expression protein has a good reactionogenicity with the OA. This result will establish a study foundation for using the fusion ScFv to construct the rapid detection methods which can screen the three biotoxins simultaneously (National Natural Science Foundation of China (NO.30671762).