| Amnesic Shellfish Poisoning (ASP) is a kind of toxin that produced bytoxic red tide diatoms. The main compound involved in this toxicity isdomoic acid (DA), an excitatory neurotransmitter amino acid of the kainicgroup that had been isolated from a marine sponge. Domoic acid can bebio-concentrated by shellfish through the transmission of food chain, and canalso be discharged into marine water directly by diatoms. People and othermarine mammals who take in the contaminated sea foods could suffer fromcentral nervous system dysfunction, notably loss of short-term memory, evencoma and death.Colloidal gold immunochromatographic assay (GICA) is a newimmunochromatographic technique in which a cellulose membrane is used asthe carrier, and a colloidal gold-labeled antigen or antibody is used as thetracer. It is simple, rapid and cheap, and the result is clear and easy to analyzewithout any equipment/facilities and skilled technicians. Since it is verysuitable for spot, rapid detection and batch detection on site, the GICAtechnique has become one of the fastest growing immunological detectionmethods and is widely used for rapid detection in marine toxins analysis.In this study, the colloidal gold immunochromatographic strip for rapiddetection of domoic acid (DA) was developed by using the principle ofmonoclonal antibody and immunochromatographic assay.The results were reported as follows:1. Preparation of DA complete immunogen and coating antigenDA was coupled to keyhole limpet hemocyanin (KLH) and bovine serumalbumin (BSA) using activation ester method to prepare complete immunogenand coating antigen. The conjugates were proved that it was successfullyconjugated and the conjugating ratios were 1:25 and 1:16 seperately.2. Preparation of Domoic acid monoclonal antibodyBALB/c mice were immunized with DA-KLH and two hybridoma cellstrains secreting McAb against DA were screened by hybridoma cell fusiontechnology. The isotypes of two strains were IgG1 and IgG2a. The ascites ofthe mice was collected and purified to obtain the monoclonal antibody. Thetiter of the McAb was 1:64000 with high specificity.3. Preparation of colloidal and immuno-colloidal goldAfter the preparing conditions were established, colloidal gold of 20nmwas prepared with reducing reaction using trisodium citrate. Studies wereprogressed on the optimal conditions about preparing the colloidal goldconjugates with DA McAb previously prepared. After the optimal conditionswere developed (the optimal pH is 9.0, the optimal mount of monoclonalantibody is 9.72μg/mL), colloidal gold-DA conjugate was preparedrespectively according to the method previously described.4. Development of DA colloidal gold immunochromatographic stripThe various factors and conditions of the immunochromatographiclateral-flow assay were explored, and the optimal reaction conditions of theassay were ascertained. The gold-labeled antibody (with the dilusion 4×) wascoated on SB06 glass fiber and dried as gold conjugate pad. The coatingantigen DA-BSA (0.867mg/mL) and Goat anti Mouse IgG (1mg/mL) werespotted respectively on a Millipore180 NC membrane as test line and controlline. SB06 glass fiber and SX18 absorbent paper were chosen as sample padand absorbent pad. Then gold conjugate pad, NC membrane, sample andabsorbent pad were assembled to immunochromatographic test strip.6. Application of DA colloidal gold immunochromatographic strip The detection sensitivity, specificity and stability of theimmunochromatographic strip were studied. The results showed that, thedetection limit of colloidal gold immunochromatographic test strip was20ng/ml and the whole analysis process could be completed within 15 min.The method established is sensitive and the procedure of determination issimple and quick without special equipment. The colloidal goldimmunochromatographic test strip could be widely used for batch detection ofdomoic acid in shellfish on site and has great prospect for commercialdevelopment. |
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