Preparation Of Cadmium Artificial Antigen And Anti-Cadmium Polyclonal Antibody

Cadmium, one of the most common heavy metal elements in food and drinks pollution, can cause food and water pollution through environmental pollution, biological enrichment and the use of chemical fertilizer containing cadmium. The detection rate of cadmium in rice and vegetables is high, and the phenomenon is serious. It has a strong renal toxicity, liver toxicity, reproductive toxicity, immune toxicity, bones toxicity, cerebrovascular toxicity and carcinogenicity. At present, the detection method is mainly instrument method, which has high accuracy, good result reproducible and specificity, but always need large equipment and high cost. Besides, the pretreatment is trival, cannot meet living and on-line analysis. The immunological methods, like ELISA, are preferred for its simple, rapid, low cost and living detection adapted to environment and market.In the present research, bifunctional chelator, p-SCN-Bn-DTPA, was employed to prepare antigen of Cd2+, Ultraviolet-visible (UV-Vis) spectroscopy, Atomic absorption spectrometry (AAS),2,4,6-trinitrobenzene-l-sulfonic acid(TNBS), Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and X-ray absorption fine structure (XAFS) were adopted to identify and analysis the artificial antigens. Then the BABL/c mice were immuned, and studied the characteristics of antiserum by ELISA. In addition, the indirect competitive ELISA was established to detect water samples. The details are as follows:1 Preparation of artificial antigen of Cd2+Cadmium is a heavy metal element with the molecular weight 112.4.It has only reactiongenicity but no immunogenicity. It can stimulate body to produce antibody when coupled with bifunctional chelator and carrier proteins. In this study, in order to prepare immunogen, Cd-p-SCN-Bn-DTPA-BSA (Cd-BSA), and coating antigen, Cd-p-SCN-Bn-DTPA-OVA (Cd-OVA), cadmium ion was chelated by the DTPA group of bifunctional chelator, p-SCN-Bn-DTPA (2-(4-isothiocyanatobenzyl)-diethylenetriaminepen-taacetic acid), and then conjugated to free amino-group of BSA (Bovine serum albumin) and OVA (Ovalbumin) through SCN group. Meanwhile, p-SCN-Bn-DTPA-BSA (DTPA-BSA), p-SCN-Bn-DTPA-OVA (DTPA-OVA) and Cd-DTPA were constructed to analysis the conjugation state of Cd.2 Characterization of artificial antigen of Cd2+The UV-VIS spectra results of BSA, OVA, DTPA-BSA and DTPA-OVA showed that the maximum absorbing wavelength of DTPA-BSA, DTPA-OVA were slightly blue-shifted from 278 nm to 270 nm compared with BSA and from 280 nm to 268 nm compared with OVA, respectively.The free amino-group of Cd-BSA and Cd-OVA were determined by triniro-benzene-sulfonic-acid. The results showed that the free amino-group of Cd-BSA decrease to 48.8% with comparison to that of BSA, while for Cd-OVA, it was 41.4%.The results of SDS-PAGE implied that the molecular weight of BSA(OVA), DTPA-BSA (DTPA-OVA) and Cd-BSA (Cd-OVA) declined one by one. The EXAFS (extended X-ray absorption fine structure) results showed that the coordination type and number of Cd2+ in Cd-DTPA, Cd-BSA, and Cd-OVA) were basicly same. At the same time, the X-ray absorption near edge structure showed that the cadmium is bound to DTPA in the artificial antigens for the reason that the edge position of the antigens are at the same energy and possess the same edge shape as the Cd-DTPA compound. The XANES region for the three compounds was same, and this signified a same in the local structure of the three antigens.The cadmium is bound to DTPA in the artificial antigens for the reason that the edge position of the antigens are at the same energy and possess the same edge shape as the Cd-DTPA compound.In summary, the artificial antigen of Cd-BSA and Cd-OVA are successfully constructed.In view of free amino-group in Cd-BSA and Cd-OVA and the protein amount in conjugates, the conjugation ratio of Cd-BSA and Cd-OVA were 28.7:1 and 11.7:1, respectively.3 Anti-Cd2+ antibodies preparation and their characters analysisThree groups (three mice/group) of Balb/c mice (marked as No.1-9) were immunized by Cd-BSA. The immunization period of the first immunization was 21d, and 14d in the next immunization. The doses for each mouse in each group were 200μg,100μg and 50μg, respectively. It could be concluded form the antibody production process curves that mice in each group had immune response. Although it differed with different mouse, it was similar in total. The study on antiserum of all Balb/c mice after the fifth immunization implied that its titer had no significant variety, ranging from 2.6×104 to 4.2×104.Indirect compete ELIS A was established to analyze antiserum of all Balb/c mice after the best work conditions of the antigen-antibody reaction obtained by phalanx titration experiment. The sensitivity of No.1 mouse was 120ng/mL, and it was 200、70、59、58、155、150 and 167 ng/mL from No.3 to No.9. The antiserum of No.1, No.6 and No.8 Balb/c mice in each group were selected for specificity analysis, and the results showed that all antiserum had cross reaction with mercury in a certain extent, but no cross reaction with Pb, Fe, Cu and Zn.4 Studies on the establishment of ELISA method for Cd2+ detection and its application on detection of Cd2+ in a lakeBased on the antiserum of No.6 Balb/c mice, by indirect competitive ELISA method Analysis, The method for Cd2+ detection was established. In view of this method, the tap water was spiked with 10-200 ng/mL, and the results implied that the linear equation was y=-20.62 x+87.069 (R2=0.9919), the average recovery achieved 85.3%-90.3%. Some samples obtained from three Lakes in wuhan city were chose for testing, the results showed that the concentration of Cd2+ in A lake reached 6.08 ng/mL, and compared to AAS analysis results, the similarity is 105.7%.