| Because okadaic acid (OA), saxitoxin (STX) and tetrodotoxin (TTX) are highly harmful to people, they are concernful marine toxins to cause outstanding safty problems of food and environment. It is important to research rapid detection methods of these toxins. In this study, the immunological methods of detecting TTX including a indirect competitive enzyme-linke immunosorbent assay (icELISA) and colloidal gold immune test paper were established, and then, fusion single chain antibody which was used to decteting OA, STX and TTX was developed. This would open up a new way for detecting marine toxins and other food pollutants .Man-made complete antigens (TTX-OVA and TTX-BSA) were successfully prepared by methanal couple, and the conjugation ratios of TTX to carrier proteins BSA or OVA respectively were 14:1 and 6:1. BALB/c mice immuned by complete antigen(TTX-OVA)made immune response to produce antibodies against TTX. The hybridoma cells (3D4 and 4C5) which secreted monoclonal antibody (McAb) against TTX were established by monoclonal antibody technique. The characteristics of TTX-McAb were determined, such as titer, subtype, molecular weight, affinity, sensitivity and specificity. Two hybridoma cell lines were identified that secretes IgG1 monoclonal antibody against TTX, designated 3D4 and 4C5 after subcloned for 3 cycles by indirect ELISA. The average number of the hybridoma chromosomes were 50±2 and 50±4 and exceeded parent cells respectively. ELISA result showed that the titer of the cell culture supernatant were 1:640 and 1:320, antibody titer 5.12×105 and 3.2×105, and the affinity constant 6.2×106 L/mol and 5.9×106 L/mol respectively.The TTX-McAb of 3D4 was manufactured by means of intraperitoneally injecting BALB/c mice and purified by CA-AS method. The result showed that purification ratio was up to 61.24 %, the recovery was 23.48%, and the purity of IgG was up to 90.99%. Experiments of specificity detection to STX, OA, MC and NOD by indirect ELISA was carried out, showing that TTX-McAb performed no cross reaction with OA, MC and NOD except STX. Preparation of TTX-McAb had successfully established to facilitate immunological detection and preparation of single-chain antibody.An icELISA based on TTX-McAb was set up, detecting condition were optimized, TTX standard and mock samples were detected. Optimized ELISA reaction condition as follow: antigen covering concentration was 1.0μg/mL, antibody dilute strength 1:40 000,covering condition to stay overnight at 4℃, competition action at 37 OC for 30 min, action time of antibody enzyme marked 30 min, action time of substrate 10 min. Equation of linear regression was y=25.681x-4.2725, and coefficient correlation R2=0.989, concentration limit 0.05 ng/mL, linear range 0.05～50 ng/mL. TTX standard and mock samples of 216 portions, including crab, shellfish as well as spiral vagina muscle and egg tissue, were detected. Results showed that average recovery were respectively TTX standard samples 88.89%, crab spawn 87.05%, crab muscle 80.96%, snail 83.26%, shellfish 85.59%. Inner and interassay coefficient of variation were smaller than 7% and 10% respectively. ELISA kit of within plate was packaged, and its performance index reached to the fast,specific and sensitive detection requirement. Specificity detection to STX, OA, MC and NOD by indirect ELISA was done, and results indicated that the monoclonal antibody had no cross reaction with OA, MC and NOD except STX.Colloidal gold immune test paper based on monoclonal antibody was developed, and TTX standard samples were detected. Colloidal gold particles of 20 nm were prepared using trisodium citrate as reducer, and colloidal gold probe was prepared, and condition of paper test paper slip was optimized. Slip of test paper was packaged. Results showed that the best coating density for antibody and colloid gold was 8.0μg/mL, the best pH value of coating the colloid gold was 8.5, the lowest detection concentration limit 50 ng/mL. Specificity detection with STX, OA, MC, NOD by indirect ELISA was done, and result indicated that the monoclonal antibody had no cross reaction with OA, Mc and NOD other than STX. The time for guarantee the quality was 8 weeks at 4℃.VH and VL genes of TTX-McAb were amplified from the RNA of hybridoma cell by RT-PCR using VL and VH genes primers designed according to the monoclonal antibody of mouse source, and were ligated with linkers. The single chain antibody gene TTX-ScFv (VH -Linker-VL) was constructed by SOE-PCR. TTX-ScFv gene was 744 bp including VH of 366 bp, VL of 333 bp and linker of 45 bp. Result by NCBI blast analysis showed that VLand VH genes not only consisted with variable region characteristic of the monoclonal antibody of mouse, and but also had high homology with many single-chain antibody registered.Prokaryotic expression vector TTX-ScFv-22b and eukaryotic expression vector TTX-ScFv-PICZαwere constructed and expressed proteins with reactive activity with TTX were obtained. Recombinant plasmid TTX-ScFv-22b was transferred into E.coli BL21(DE3)and gained high performance expression. Molecular wieght of expressed protein was 26.045 ku. Fusion proteins were induced to express by IPTG at 37℃for 6 h with amounting to 51.38% in inclusion, but amounting to 42.14% at 25℃for 12 h in inclusion and solubile. Two types of expressed proteins were concentrated and purified by urea dissolving, gradient dialyzing to and ultrafiltrating. Activity was analysed by indirect competition ELISA(ic ELISA).TTX-ScFv gene was cloned into expression vector pPICZαA, and recombinant expression vector TTX-ScFv-PICZαwas constructed. After linearrization, recombinant plasmid was transferred into GS115 of Pichia pastoris, and then were induced to express by methanol at 30℃for 72h with amounting to 15.10%. Expressed fusion protein were concentrated and purified by dialyzing and ultrafiltration, and determined by SDS-PAGE. Activity was analysed by icELISA. ELISA results indicated that expressed fusion proteins in prokaryotic and eukaryotic systems performed reactivity with TTX antigens, and activity of the eukaryotic expressed protein was a little higher than the prokaryotic, but both of them were lower significantly than TTX-McAb.At the same time, three ScFv genes, including OA-ScFv, STX-ScFv and TTX-ScFv, were linked to OA-STX-TTX-ScFv by SOE-PCR. OA-STX-TTX-ScFv gene was 2 256 bp, encoding 752 amino acids. Fused single-chain antibody against three toxins were obtained. Molecular weight of expressed protein was about 79.000 ku and consisted with predicted value. Expressed fusion protein induced by IPTG at 37℃for 6 h amounted to 12.70% in inclusion, but expressed fusion protein induced by IPTG at 25℃for 12 h amounted to 10.90% in inclusion and solubile. Two types of expressed proteins were concentrated and purified by urea dissolving, gradient dialyzing and ultrafiltrating. ELISA results indicated that the expressed fusion protein had weak reactive activity with three toxins respectively. Fusion single-chain antibodies were expressed with bioactivity. How to improve the bioactivity of fusion single-chain antibodies to estiblish broad-spectrum detection methods need to study in the future. |
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