Study On Rapid Detection Of Ochratoxins Contamination In Food By Immunological Methods

With the development of science and technology and the improvement of the people’s living situation,food safety has become a hotspot that people focused on.In recent years,food safety incidents,caused by various hazardous substances in food,came out one after another,which brought panic to the people.Therefore,in order to safeguard food safety and protect people’s health,the establishment of a quick and efficient detection technology for hazardous substances has great significance.Ochratoxins are toxic fungal metabolites,and have toxic effects of kidney toxicity,liver toxicity,teratogenicity,carcinogenesis,mutagenicity and immunosuppression to animals and humans.This study developed an enzyme-linked immunosorbent kit and gold nanopaticle based immunochromatography test strip for the rapid detection of ochratoxins contamination in agricultural products.The mainly results are listed as follows:1.Preparation,purification and identification of anti-ochratoxins monoclonal antibodies: Healthy female Balb/c mice of 6 weeks old were immuned with OTA-BSA as immunogen.Blood was collected from caudal vein after the fourth immunization to examine the titer and affinity of the serum.The mouse whose serum had the best affinity to ochratoxin was chosen for cells fusion.Liquid medium was used for hybridoma culturing in the experiment.Hybridoma clones were isolated by limiting dilution,and finally eight strains of clones that could secrete high-quality anti-ochratoxin monoclonal antibodies(McAbs)were screened out by the enzyme-linked immunosorbent method(ELISA).The eight strains were as follows: 11B9,11D2,11G6,11C5,1E2,11F5,11A9,11E10.The sensitivities(expressed by IC50)of these ascites antibodies were 0.52 ng/mL,0.68 ng/mL,1.30 ng/mL,1.08 ng/mL,45.79 ng/mL,1.25 ng/mL,1.50 ng/mL,1.27 ng/mL respectively,and they have no cross-reaction to other mycotoxins,showing good specificity.2.Development and characterization of enzyme-linked immunosorbent method(ELISA)kit for ochratoxin A detection.Based on the anti-ochratoxin monoclonal antibody prepared by our laboratory,the ELISA kit was developed for simple,sensitive and efficient detection of OTA.The best coating concentration determined by chessboard titration method for antigen of OTA-BSA was 2 μg/mL,antibody was diluted 8000 times,HRP-IgG was diluted 5000 times.Under that condition,the other experimental conditions of ELISA were optimized.Standard curve was drawn under optimal assay conditions and linear equation was obtained as: y = 0.3613 + 0.3411 x;R2 = 0.9576.The sensitivity(expressed by IC50)for OTA was 0.22 ng/mL.The accuracy of kit was determined by coefficient of variation(CV)which was 3.62% in batch and 3.74% between batch.The kit had cross reaction with OTB and the cross-reactivity rate was 42.22%,except that no cross-reactivity was found with other mycotoxins.Ochratoxin A spiked in soybean,peanut and corn samples was detected using the developed kit,the recovery rates were all over 90%,showing that the kit could be used for ochratoxin analysis in agricultural products.3.Preparation and characterization of immunochromatographic test strips for major ochratoxins detection: Immunochromatography assay(ICA)for simultaneous detection of three major ochratoxins in food was developed and the test strip was prepared in this section.Gold nanopaticles prepared by sodium citrate reduction method with a size of 15 nm were used as signal labels.After the optimization of experimental conditions,the limits of detection were 0.05 ng/mL,0.025 ng/mL,0.1 ng/mL for OTA,OTB,OTC and the cut-off levels were 0.5 ng/mL,0.25 ng/mL,0.5 ng/mL for OTA,OTB,OTC.The test strip had a good reproducibility and good specificity with no cross-reactivity with other mycotoxins.The as-prepared strips could be stored at 4 oC for at least six months in dry conditions.In the adding experiment,the detection limits in corn,peanuts and oats samples were 0.2 μg/kg,0.4 μg/kg,0.1 μg/kg for OTA,OTB and OTC,which were far lower than the maximum residue level of 5.0 μg/kg in the unprocessed grain regulated by European Union.Results showed that the prepared test strip could be used to detect ochratoxins on-site in agricultural products.