Study Of Enzyme Immunoassay For Detection Of Nitrofurantion Metabolite

Nitrofurans and their metabolites are a class of compounds with strong toxicity, carainogenicity and teratogenicity. It will have a great harm to human health when those compounds remained in anaimal-derived food. As a consequence, many countries have classified nitrofurans as banned drugs. But due to its low cost and efficacy significantly, there are still lawless elements to reap huge profits to continue to use them. Nitrofurantoin is one of nitrofurans. At present, various instrumental analysis methods with high analytical precision have been used to detect the residue of AHD. However, these methods require expensive equipments and professional and technical personnel to operate and also time-consuming and costly. Thus, the establishment of a convenient, rapid and sensitive method for detecting residues of AHD in anaimal-derived food will have a great significance to the effective control of food safety risk and ensure the safety of people’s diet. In this study, CLEIA and ELISA methods for AHD were developed.The hapten CPAHD was synthesized by conjugating AHD with 4-CBA. Then made it conjugate with OVA by using N-hyduoxysuccinimide ester method to synthesize coating antigen. The enzyme-labeled antigen was synthesized by N-hyduoxysuccinimide ester method and mixed anhydrides method respectively. Then this two kinds of enzyme-labeled antigen were identified by dc-ELISA. The results indicated that the one synthesized by N-hyduoxysuccinimide ester method has a higher sensitivity.The indirect competitive CLEIA and direct competitive CLEIA for AHD were established. Then series of test parameters, such as chemiluminescent solution, coating condition, blocking liquid and competitive reaction time were optimized. Besides, coating antigen concentration and antibody concentration were optimized in indirect competitive CLEIA. Antibody concentration and enzyme-labeled antigen concentration were optimized in direct competitive CLEIA. The results indicated that the IC50 of indirect competitive GLEIA and direct competitive CLEIA were 0.753ng/mL and 0.539ng/mL. The cross reactivity studies showed that the CLEIA had good specificity for AHD and there was negligible cross reactivity with other structurally related compounds except nitrofurantoin. Recoveries rates of AHD ranged from 84.9% to 103.4% in fortified pork, chicken and fish samples. The intra and inter assay coefficient of variation were less than 12%. The detection limit of indirect competitive CLEIA for pork, chicken and fish samples were 0.020,0.021 and 0.019μg/kg respectively. While the detection limit of direct competitive CLEIA for pork, chicken and fish samples were 0.013,0.013 and 0.012μg/kg respectively.The indirect competitive ELISA and direct competitive ELISA for AHD were also established in this study. The developing time was optimized. And the rest of optimization conditions were set the same as the method of CLEIA. The results indicated that the IC50 of indirect competitive ELISA and direct competitive ELISA were 2.186ng/mL and 1.291ng/mL. The ELISA had good specificity for AHD and there was negligible cross reactivity with other structurally related compounds except nitrofurantoin. Recoveries rates of AHD ranged from 82.6% to 104.7% in fortified pork, chicken and fish samples.The intra and inter assay coefficient of variation were less than 13%. The detection limit of indirect competitive ELISA for pork, chicken and fish samples were 0.219,0.214 and 0.208μg/kg respectively. While the detection limit of direct competitive ELISA for pork, chicken and fish samples were 0.115,0.113 and 0.109μg/kg respectively.These results indicated that the CLEIA and ELISA methods with good sensitivity, precision, accuracy and detection limit can meet the need of detecting AHD residues in actual samples. The sensitivity and detection limit of CLEIA methods were better than ELISA methods. Meanwhile, compared with indirect competitive CLEIA method, direct competitive CLEIA has higher sensitivity and lower detection limit. It can provide an accurate, reliable and convenient way for grassroots food safety regulators to detect AHD residues. Combined with the gray evaluation method for a small sample of food, which was established early, it can be more fully to determine the risk extent of food from different dimensions. This initiative has a strong practical value for effective and positive prevention and control of major food safety risks.