Preparation Of Monoclonal Antibody Against Cadmium Ion And Its Application In The Immunological Detection

As is known to all,the concentration of cadmium（Cd）is very small in the environment.However,heavy metals were enriched and polluted the environment more and more seriously with the rapid development of industry in recent years.Previous study showed that cadmium has a long half-life in the environment and could accumulate in the human body through the food chain,which results in irreparable damage to the kidneys and liver with high concentration of it.Thereforce,people paid more and more attention to the problem of cadmium pollution.Due to the threat of cadmium to the health of humanbeings,it is urgent need to develop the effective,sensitive and rapid method for the detection of cadmium.The purpose of this study was to obtain the antibody against cadmium ion,and then establish the immunoassay for rapid detection of cadmium ion based on the antibody.For preparation of the complete antigens,1-（4-Isothiocyanatobenzyl）ethylenediamine-N,N,N’,N’-tetraaceti c acid was selected as the bifunctional chelating agent,and KLH and BSA were chosen as the carrier proteins.Then,detection antigen Cd-ITCBE-BSA and immune antigen Cd-ITCBE-KLH were successfully obtained,which were further identified by agarose gel electrophoresis.Subsequently,6-week-old BALB/c mice were immunized with immune antigens.After five immunizations,the spleen cells of the immuned mouse were fused with myloma cells.In our experiment,9A12 and 3B3cell lines stablely secreted antibody against cadmium ion were obtained after four subcloning.The antibody secreted by 9A12 cell line displyed higher titer than that of 3B3 cell line,hence 9A12 cell line was selected to do further study.Moreover,the ascites of 9A12 hybridoma cells was purified and the characterization of antibody was determined.The results showed that the subtype of antibody belongs to Ig G1,the titer reached up to 6.4×10⁴,and the affinity constant was Kaff=3.0×10⁹L/mol,indicating that high titer and affinity antibody against cadmium ion was successfully obtained in our study.Indirect competitive enzyme-linked immunosorbent assay（ic-ELISA）and immunochromatographic test strips（ICTS）were established for Cd determination based on the obtained antibody.The standard curve equation of ic-ELISA for Cd detection was y=0.03143+[0.96751/（1+x/0.1476）^1.15721]with R²=0.99935,and the linear range of ic-ELISA detection was 0.032-1.111 ng/m L（IC50 was0.15 ng/m L）.In order to shoten the detection time,conventional gold nanospheres（Au NS）with the average diameter of 20 nm that could be observed by eyes were synthetized,and Au NS was conjugated with antibody to form gold-labelled antibody.Finally,Au NS based strip was developed for rapidly detection of cadmium ion.The linear range of Au NS based strip was 12-0.375 ng/m L,and the limit of detection was0.375 ng/m L.The results could be quickly observed within 10 min.In sumary,monoclonal antibody against cadmium ion was successfully prepared in the study.Competitive ELISA and colloidal gold strip test were established based on the obtained antibody,and both methods could be used for rapid detection of cadmium ion.