| Okadaic Acid (OA) and Dinophytoxin-1（DTX-1) as the major disease-causingtoxin in DSP, is considered to pose the greatest risk to human health. It usuallyaccumulated in the bodies of Mussels, clams and snails by the food chain.Consuming contaminated shellfish can cause diarrhea, nausea, vomiting andabdominal pain, in addition to other characteristic DSP symptoms. when people eatthe contaminated seafood, it could cause food poisoning, appear the symptom suchas diarrhea, nausea, vomiting and stomach cramps then leads to food poisoning withthe key feature of diarrhea. Although OA groups poisoning has never resulted inhuman fatality, their carcinogenic, mutagenic and immunotoxic effects have beendemonstrated in animals. The toxins are stable at high temperatures and havelong-term carcinogenicity. Due to the potential toxicological risk of lipophilic toxins,previously known as DSP toxins, which are a serious threat to public health and tothe shellfish industry, thus, the development of practical, reliable and sensitivedetection methods for these toxins is necessary.To date, several assays for DSP in biological samples have been proposed,including mouse bioassays, liquid chromatography coupled to mass spectrometric(HPLC-MS/MS) detection, enzyme-linked immunosorbent assays (ELISA), lateralflow immunochromatographic (LFIC), and phosphatase inhibition assays. ELISAmethod is specific, sensitive and inexpensive. And HPLC-MS/MS can quantitativelydetect OA in seafood. In addition, lateral flow immunochromatographic (LFIC) issimple, suitiable to assay on sit.In this work, LFIC, Sensitive and reliable micro-plate chemiluminescenceenzyme immunoassay (CLEIA) and HPLC-MS/MS were established for detectionof OA in Commercially Available Seafood, which collected from six coastal citiesof four Inner China Seas. These data from LFIC, CLEIA and HPLC-MS/MS mightplay an important role on protecting consumer health and screening safe marine products. In addition to monoclonal antibody, aptamer as a novely assay tool hassome advantages, such as high affinity and specificity. Our group report aptamers asa novel non-toxin probe binding to monoclonal antibody against OA as well asbinding to OA with magnetic-bead SELEX. An non-toxic magnetic-bead ELISAwas established based on aptamer that mimic monoclonal antibody against OA.The establishment of LFIC for key DSP. The prepared colloidal gold wasscanned with TEM, and the images revealed that the size of colloidal gold particleswas homogeneously distributed with diameters around20nm. Theantibody-colloidal gold particles conjugate was prepared as the detector reagent. Ttest and C test were immobilizated in the NC membrane. The qualitative detectionlimit of the test strip was50ng/mL and visible decidable range was1.6-50ng/mL.The establishment of CLEIA for key DSP A CLEIA based on a McAb againstOA was developed and optimized. The optimized concentration of antigen was50ng/mL at4℃overnight. The optimal time of blocking time was120min. Theconcentration of the McAb in CLEIA was1:200000at37℃for60min. Theworking concentration and time of goat anti-mouse IgG-HRP were1:5000, at37℃f or60min. The standard curve equation of the CLEIA wasY=59.74+12.99Ln(X), R=0.99. The linear range of0.0098ng/mL-10ng/mL. The average recoveriesof the low, middle and high concentration samples were97.2%,111.2%and104.7%,respectively. The CVs of the intra-and inter-assay were all less than10%.The establishment of HPLC-MS/MS for key DSP. After extracted DSP with80%methanol and purified by HLB columns, the OA and DTX-1were detected byHPLC-MS/MS method. The detection ion pairs of OA was m/z827.7/723.7,m/z827.7/576.1; The detection ion pairs of DTX-1m/z841.7/737.9; m/z841.7/824.0. Themobile phase of HPLC was the methanol:1%methanoic acid-water (vol/vol:80:20with a gradient eluted method. The equation of linear regression of OA and DTX-1are:Y＝1.68e3X＋3.33e3(r＝0.9995),Y＝901X＋1.55e3(r＝0.9992), respectively.The matrix standard curve of OA and DTX-1are Y＝557X－287(r＝0.9997),Y288X－342(r＝0.9994). The limit detection of HPLC-MS/MS is2μg/kg:. Theaverage recoveis of10,20and50μg/kg: are63.1%,87.9%and93%. The CV oHPLC-MS/MS is0.719% Contamination of Commercially Available Seafood by Key Diarrhetic ShellfishPoisons Along the Coast of China.40species were collected from six coastal citiesof four inland seas in China. Above three assay for key DSP were used to analysethe samples, and the results were further confirmed using a commercially availableELISA kit. The monitoring results indicated that23of40species were positive forcontamination. In addition,14of the positive species were determined to be inediblebecause the content of OA and DTX-1was above the regulatory limit.Simultaneously, we verified that the digestive glands of shellfish tended toaccumulate toxin, in contrast to the flesh. The highest concentrations of OA andDTX-1were recorded in Scapharca broughtonii, which was collected from QingDao, in relation to the other analysed species. Moreover, the Arca family as well asMytilus galloprovincialis were severely contaminated by OA and its analogue. Theabove results indicate that some of the commercially available seafood from thecoastal cities in China may be inedible due to serious marine toxin contamination.Generation aptamer binding to OA via magnetic-bead SELEX. A randomssDNA library with81bpin length was synthesized. The fixed probe modified withamino was designed to bind to library. The complex was immobiliated on theDynal beads. OA toxin was added into beads, then some sequences binding to OAwere eluted. The PCR amplification was performed with ssDNA as tempreture. Thesecond libary were prepared by Lambda Exonuclease. After15rounds of selectionvia magnetic-bead SELEX, the four aptamers were obtained and then were clonedand sequenced. The affinity constants of four aptamers were1.71nm,0.54nm,3.96nm and2.56nm.Generation internal-image functional aptamers via magnetic-bead SELEX. Arandom ssDNA library with85bp length was synthesized. The monoclone antibodyagainst OA were coated on Dynal beads by amino bingding. The randomssDNApool was added and specific sequence binding to mAb were fixed on beads.After elution of ssDNA, PCR amplification was performed. The second libary wereprepared by Lambda Exonuclease. After12rounds of selection, the four aptamerswere obtained and then were cloned and sequenced. The affinity constants of fouraptamers were1.21nm,0.68nm,0.81nm and0.95nm.Non-toxin ic-ELOSA based on internal-image aptamers. Non-toxin ic-ELOSAbased on internal-image aptamers (ID31). Aptamer31was modified with amino and. The optimization of procedure are as follow: the concentration of aptamer is0.5ng/mL.45min at37℃. HRPlabeled goat anti-mouse IgG was1:400045min at37℃.The regression equation of the standard curve was Y=-0.488x+0.972R2=0.996.The limit detection of OA is0.44ng/mL The IC50is4.49ng/mL and the detectionrange is0.416-42.65ng/mL. There are no cross-action with other eight marine toxin.The recoveries are between88.3%-99.57%. The concentrations of OA in Scapharcabroughtonii, Mytilus galloprovincialis and Chlamys farreri were12.83μg/kg,5.49μg/kg and6.27μg/kg:. |
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