Establishment Of ELISA Method For Determination Of Total Arsenic In Aquatic Products

In recent years,with the pollution of environment and water sources,heavy metal pollution in aquatic products has become increasingly serious.The quality of aquatic products is related to the safety of every consumer.The rapid detection of heavy metal content has become one of the hot spots of food safety testing.At present,the detection of heavy metal elements such as lead,merc-ury,chromium and cadmium in food by enzyme-linked immunosorbent assay has been reported,but no ELISA kit for arsenic has been found.In this study,a chelating agent was used to couple arsenic with a carrier protein to form an immunogenic macromolecular artificial antigen,and an immunized an animal obtained a polyclonal antibody,and an indirect competitive ELISA assay for detecting arsenic was established.And then used it for the detection of total arsenic in aquatic products?fish,fish skin,red shrimp,green shrimp?,achieved satisfactory results.The main research contents and results are as follows:1.Synthesis and identification of arsenic artificial antigenSynthetic arsenic artificial immunization antigen and artificial coating antigen were used to prepare polyclonal antibody against arsenic,which was used to provide antigen and antibody for establishing rapid detection method of ELISA.The arsenic was coupled with the carrier proteins BSA,OVA and KLH by using isoth-iocyanate benzyl ethylenediaminetetraacetic acid?ITCBE?as a chelating agent and the artificial antigens As³⁺-ITCBE-BSA,As³⁺-ITCBE-OVA and As³⁺-ITCBE-KLH were prepared.Qualitative identification by nucleic acid protein concentration,UV spectr-ophotometer scanning and SDS-PAGE electrophoresis,and the content of As³⁺ in artificial anti-gen was detected by atomic fluo-rescence spectrometry.Results:The concentrations of As³⁺-ITCBE-BSA,As³⁺-ITCBE-OVA and As³⁺-ITCBE-KLH were 4.692 mg/mL,5.726 mg/mL and4.556 mg/mL,respectively.The maxi-mum absorption peak of UV scanning shifted;The elec-trophoresis bands were slightly delayed for the respective carrier proteins;The contents of As³⁺ were 0.254 mg/mL,0.190 mg/mLand 0.243 mg/mL,respectively,and the coupling rates reached70.67%,52.81%,and 67.61%.The results showed that the above three artificial antigens were successfully synthesized and could be used for the preparation of elemental arsenic polyclonal antibodies.2.Preparation and identification of polyclonal antibody against arsenicIn order to establish an ELISA method for arsenic,the artificial immune antigen AS³⁺-ITCBE-BSA was implanted with Freund's complete adjuvant and Freund's incomplete adjuvant,and Balb/c mice and New Zealand white rabbits were injected subcutaneously into the back of the neck.The immunization was performed once every 2 weeks,and the serum was collected after the four exemptions.Then,the polyclonal antibody was prepared and the polyclonal antibody was pu-rified by Protein A column.Finally,the concentration was determined.The best coated antigen was screened by indirect competitive ELISA and the potency,sensitivity and specificity of the pol-yclonal antibody were identified.The As³⁺-EDTA standard chelate solution was used as an inhi-bitor,and the competitive inhibition curve of As³⁺-EDTA against polyclonal antibody was dete-rmined by indirect competitive ELISA method.The detection limit and sensitivity were dete-rmined.Through the cross-reaction test of As³⁺ with Pb²⁺,Hg²⁺,Cd²⁺ heavy metal ions,the cross reaction rate was measured.Results:The OD₂₈₀nm value of the polyclonal antibody was 1.973;The coating antigen As³⁺-ITCBE-KLH was more ideal than the AS³⁺-ITCBE-OVA coating;The polyclonal antibody titer of the 2nd and 4th mice was 1:6400;New Zealand white rabbit No.2 had a serum titer of 1:4×10⁴;The regression equation of the competition inhibition curve was y=-0.2657x+1.1618,R²=0.9859,and the IC₅₀ value reached 2.4907?g/L.The arsenic element did not cross-react with other metal elements,and the prepared As³⁺ polyclonal antibody had strong spe-cificity.Therefore,the polyclonal antibody of New Zealand White Rabbit No.2 could be used for the esta-blishment of arsenic ELISA assay.3.Establishment and preliminary application of ELISA method for arsenicThrough the determination of optimal working concentration of coated antigen and antibody by indirect competitive ELISA,the ELISA method for detecting arsenic had been established.Results:The optimal coating antigen As³⁺-ITCBE-KLH concentration was 1.5?g/mL,and the antibody dilution factor was 1:1500.The standard curve regression equation was y=-0.2672x+1.158,R²=0.9913,the IC₅₀ value was 2.4626?g/Land the IC₁₅ value was 3.772?g/L.Sample pretreatment was carried out by dry ashing and wet digestion,respectively.The total arsenic content in the samples was detected by silver salt method,ELISA method and HG-AFS method,and the recovery test was carried out.Results:The content of total arsenic in the samples was significantly different by ash method and silver salt method?P<0.01?,but not significantly different from HG-AFS method?P>0.05?.When the samples were treated by wet digestion,the content of total arsenic in the samples detected by ELISA and silver salt method was significantly different?0.05>P>0.01?,and the difference was not significant with HG-AFS method?P>0.05?.Moreover,the dry ashing method was more better than the wet digestion method,and the preferred dry ash method was preferred as the treatment method for aquatic products.When the same kind of sample was detected by different methods,the detection effect was HG-AFS method>ELISA method>silver salt method.The recovery rate of the test sample was HG-AFS method silver salt method>ELISA method>silver salt method.The total arsenic content in fish skin and fish samples was lower than the national standard limit of 0.05mg/kg,but the total arsenic content in red shrimp and green shrimp exceeded the national standard limit.