Determination Of Cadmium Ion By Nano-SiO₂ Preconcentration With Heterologous IcELISA

The heavy metal cadmium（Cd）has different degrees of cumulative toxicity to human beings,animals and plants.Cadmium can pollute the air,water,soil and other natural environment,also contaminate the agricultural byproduct via the food chain,and ultimately endanger human health and biological safety.Many countries and international organizations have adopted Maximum residue limits（MRL）standards and established corresponding detection methods of the heavy metal cadmium in food.Therefore,it is of great significance to establish an accurate,simple,rapid and no secondary pollution detection method of the heavy metal cadmium.Currently,physicochemical and immunological techniques which based on chromatography,spectrum,mass spectrometry,electrochemical signals and antigen-antibody reaction are often used in heavy metal detection.Physicochemical analysis has the advantages of high accuracy and low detection limit.However,this method has highly-detection cost and strict requirements for sample pretreatment.Compared with the physicochemical analysis,immunological detection method has high sensitivity,strong specificity and simple detection process,which can provide technical support for rapid detection of heavy metal cadmium on site.In this paper,monoclonal antibodies were prepared based on the successful synthesis of cadmium artificial antigen,With this antibody,a heterologous indirect competitive ELISA method for the detection of cadmium chelate was established right after.At the same time,nano-SiO₂ was used as adsorbent to preconcentrate cadmium in simulated samples,the amount of cadmium was quantitatively analyzed by hicELISA,which provided a reliable technical support for the rapid detection of heavy metal cadmium residues.The main contents and conclusions of this study were as follows:1.ITCBE,DTPA and EDTA were used as bifunctional chelating agents to chelate ion with carrier protein to prepare immunogen and heterologous coating antigen.The protein content of artificial antigen was determined by nucleic acid protein analysis,the coupling characteristics were analyzed by UV scanning,and the content of heavy metal ions in the antigen was determined by ICP.The above detection proved that the artificial antigen was synthesized successfully.2.Three groups of Balb/C mice were immunized with high（60μg/mouse）,medium（30μg/mouse）and low（10μg/mouse）dose of Cd²⁺-ITCBE-BSA respectively,then its immune effect was identified by ELISA.The results showed that the serum antibody titer of group M mice was high and stable.It is worth noting that the serum titer of M3 was as high as 1:51200.Hence,M3 was chosen as the splenocytes donor of cell fusion.3.The results of ELISA and statistics showed that the fusion rate was 92.97%and the positive rate was 18.49%.Five highly sensitive monoclonal cell lines 1D8B10,2F4E3,3B10B1,1C3F10 and1C3G6 were obtained after three subclones.The monoclonal antibody was prepared by inducing ascites,and its immunological characteristics were analyzed.The titer of monoclonal antibody 1D8B10 was the best,its indirect determination value was 1:1.28×10⁵,the antibody subtype was IgG₁/κ,the affinity constant Ka=8.1×10⁹ L/mol;the sensitivity test showed that the 50%inhibitory concentration was 86.5μg/L;the specific determination showed that the cross rate of the antibody with Hg²⁺-EDTA was 83.5%,but little else.4.Monoclonal antibodies 1D8B10 were used to establish hicELISA method for the detection of Cd²⁺-EDTA.The square array method was used to optimize the detection conditions,screen the best detection antigen,determine the coating concentration and the working concentration of 1D8B10 m Ab.Meanwhile,the sulfhydryl modified nano-SiO₂ was used to preconcentrate the trace cadmium,and the experiment of adding and recovering was carried out by hicELISA.The results indicated that Hg²⁺-ITCBE-OVA was the best heterologous antigen.The sensitivity of the established hicELISA method was 1.2mg/L,its linear detection range was 2.9～672.6mg/L.No cross reaction was found between the detection with Hg²⁺,Cu²⁺,Zn²⁺,Pb²⁺,Cr³⁺,Mo⁶⁺,Fe³⁺,Co²⁺-EDTA chelates.The adsorption capacity of Nano-SiO₂ for Cd²⁺in simulated water samples was 13.28%,the enrichment factor was 20 times,and the optimal concentration was 5～50 mg/L.The recoveries of ultra pure water,tap water and lake water samples were 92.47%～102.86%,and its relative standard deviation was 0.91%～3.41%.The results fully proved that the method was feasible.