Preparation Of Monoclonal Antibody Against Orchratoxin A And Its Primary Application

Objective:The study aims at preparing monoclonal antibody (McAb) against ochratoxin A (OA) and conducting preliminary evaluation on the antibody. Based on this work, competitive inhibition ELISA (competitive inhibition enzyme linked immunosorbent assay,ci-ELISA) was devised to detect the OA. The extraction of OA from millet and maize was optimized. To determine the degree of pollution, the OA content in millet and maize commercially available in the market of the 5 districts of Jinan City were detected by the proposed ci-ELISA.Methods:In the proposed low-dose and long-cycle immunization scheme, female BALB/c mice were immunized with OA coupled to bovine serum albumin (OA-BSA). To detect the titre of anti-OA antibody in the serum from immunized mice and observe the cross reaction between the prepared McAb and BSA, OA-BSA and BSA were used as coating antigen in the enzyme linked immunosorbent assay (ELISA), respectively. Using the cell-fusion method, the McAb contained in supernatant of hybridoma cells was detected by ELISA. In the detection, OA-BSA was used as coating antigen, and a comparison detection using BSA as coating antigen was conducted as well. Thus, the hybridoma cell strain secreting McAbs against OA-BSA was obtained. The specificity of McAb was evaluated based on the observations of cross reaction between the McAb and BSA. Using limited dilution, the hybridoma cells capable of secreting specific McAbs were cloned, of which the specificity was determined with ci-ELISA. A large volume of McAbs was prepared by ascites in liquid paraffin-primed mice. Purify McAb in ascites with octanoic acid and saturation sulphuric acid ammonium and then determine the titre of anti-OA antibody in ascites before and after the purifying treatment by ELISA. Meanwhile, a contrast test using BSA as the coating antigen was conducted to observe the cross reaction. Using OA as competitive antigens, the ci-ELISA method for detecting OA was proposed. Prepare mixed liquors of methanol and sodium chloride with different concentrations and use the mixture as the solvent to extract OA from samples. Compare the recovery rates obtained with different extracting solvents. Determine the effect of the concentration of methanol and sodium chloride in the solvents on OA extraction and optimize the OA extraction process. Sample millet and maize that were commercially available in markets and supermarkets distributed in Shizhong, Lixia, Huaiyin, Tianqiao, and Licheng districts of Jinan City. Fifty portions of millet and maize were sampled respectively. Detect the OA content in the sampled millet and maize with the proposed ci-ELISA method and standard addition method before calculating the detectable rate. Based on the limit of OA contained in corns and beans stipulated in the Hygienic Standard for Grains (China National Standard GB2715-2005), calculate the unqualified rate. The data were analyzed with SPSS software package.Results:The titer of McAbs in sera from BALB/c mice immunized by OA-BSA was 1:512000 and strong cross reaction with BSA was observed. Hybridoma cell strains secreting McAbs against OA-BSA were screened with ELISA after cell fusion. The cross reactivity of McAbs against OA and BSA was merely 3.50%. The hybridoma cell strains secreting McAbs against OA-BSA were established by 3 rounds of cell sub-cloning and the antibody secreting positive rate reached 100%. One hybridoma cell strain capable of steadily secreting anti-OA-BSA McAb was obtained. The specificity of McAbs for OA was thus verified by ci-ELISA. A large volume of McAbs were prepared by ascites production. The McAb titer of purified ascites was 1:512000 and no cross reaction with BSA was observed. For ci-ELISA, the linear range was from 0.04ng/ml to 1250ng/ml; the linear equation y=-0.1368x+0.6796; the correlation coefficient r=0.9965; and the lower detection limit 0.04ng/ml. Optimal OA extraction was obtained when 5% sodium chloride and 60% methanol were contained in the extracting agent. Repeated tests were conducted using the extracting agents when the OA recovery rates with standard additions was 50ng/g,25ng/g, and 12.5ng/g. The millet recovery rate with each addition averaged 93.52%,96.86%, and 95.66% respectively. The coefficient of variation (CV%) was 4.59%,8.42%, and 8.94%, averaging 7.32%. The maize recovery rate with each addition averaged 96.30%,98.67% and 98.13%. The coefficient of variation (CV%) was 5.74%,5.59% and 9.56%, averaging 6.96%. For millet OA, the ci-ELISA linear equation of working curve was y= 0.3249x+1.079, the coefficient of correlation r= 0.9959. For maize OA, the ci-ELISA linear equation of working curve was y=-0.2554x+0.9561, the coefficient of correlation r= 0.9971. The lower detection limits of millet and maize OA were 0.04 ng/ml, and the lower output limits were 0.2 ng/ml. For all the samples, no marked difference of OA detectable rate between millet and maize was found (P> 0.05):the OA detectable rate of millet was 41% (41/100) while the rate of maize was 38% (38/100). In particular, the OA detectable rate of millet in market was 26% (13/50).The OA detectable rate of maize in market was 32% (16/50). No significant difference between millet and maize sampled from markets was found (P>0.05). The detectable rate of millet sampled from the supermarket was 56% (28/50). The detectable rate of maize sampled from the supermarket was 44% (22/50).No significant difference between millet and maize sampled from supermarket was found (P> 0.05). For all detected samples, the OA detectable rate of millet from supermarkets was 56%, which was significantly high above that of the millet sampled from markets (26%) (P<0.05). For all detected samples, the OA detectable rate of maize from supermarkets was 44%, with no difference from that sampled from markets (32%) (P>0.05). The OA contents in all the detected samples of millet ranged from 0.40 to 4.45ug/kg, the median was 1.73ug/kg; and the OA contents in all the detected samples of maize ranged from 0.48 to 4.02 ug/kg; the median was 1.61 ug/kg. There was no significant difference between the OA contents of the millet and maize (P>0.05). In particular, there was no significant difference between the OA contents contained in the millet (0.41-3.94 ug/kg, the median was 1.63 ug/kg) and maize (0.53-3.99 ug/kg, the median was 1.52 ug/kg) sampled in markets(P>0.05), and there was no significant difference between the OA contents contained in the millet (0.40-4.45 ug/kg, the median was 1.78 ug/kg) and maize (048-4.02 ug/kg, the median was 1.74 ug/kg) sampled in supermarkets (P>0.05). For all the millet samples, there was no significant difference between the OA contents of the millet sampled from supermarkets (the median was 1.78 ug/kg) and markets (the median was 1.63 ug/kg) (P>0.05). For all the maize samples, there was no significant difference between the OA contents of the maize sampled from supermarkets (the median was 1.74 ug/kg) and from markets (the median was 1.52 ug/kg) (P>0.05). Thus, the OA contents of all samples under this study ranged between 0.40 and 4.45ug/kg, the median was 1.67 ug/kg. According to the OA limit (≤5ug/kg) for corns and beans stipulated in Hygienic Standard for Grains (China National Standard GB2715-2005), the OA contents in all samples were below the limit.Conclusions:Hybridoma cell strains secreting McAbs against ochratoxin A were obtained. The anti-OA-BSA McAb secreted from the cell strain was OA-specific and features strong specificity. During the preparation of monoclonal antibody by hapten, the immune and detection antigens may share the same carrier so that the preparation and consequent detection of complete antigens were greatly simplified. Ci-ELISA method on the anti-OA-McAb basis was proposed for the detection of OA. The detection method under study had a high sensitivity and satisfied the limit of 5ug/kg OA contained in grains and beans stipulated in the Hygienic Standard for Grains (China National Standard GB2715-2005). It has proven that 60% methanol aqueous solution with 5% sodium chloride was the optimal solvent for extracting OA from millet and maize. The method boasts good repeatability. The results indicate that the millet and maize commercially available in markets and supermarkets of Jinan were to some extent polluted by OA. The OA detectable rate of millet sampled from supermarkets was higher above that of millet sampled from the markets. There was no significant difference between the OA contents of millet and maize. According to the 5ug/kg limit of OA contained in grains and beans that was stipulated in the Hygienic Standard for Grains (China National Standard GB2715-2005), the OA contents in the detected samples were well below the limit of OA; the median was 1.67 ug/kg.