Establishment Of Enzyme-linked Immunosorbent Assays(ELISAs) For Two Kinds Of Drug Residues:Brombuterol And Diclofenac

In recent years,concerns about drug residues are increasing due to frequent incidents of residual drugs in the food and environment endangering human health.Brombuterol and diclofenac sodium is two kinds of common residual drugs.Brombuterol is a new?-agonist that can greatly promote muscle growth and improve feed conversion rate.However,meat products containing?-agonist residues can be harmful to human health.In China,brombuterol has been banned from animal feed and drinking water.Diclofenac sodium is a nonsteroidal anti-inflammatory drug?NSAID?,which has a strong antipyretic and analgesic effet.It's commonly used in the treatment and prevention of various animal diseases in human and animal husbandry.Diclofenac sodium and its metabolites will be discharged into the water environment through various ways,and are often detected worldwide in water environment.In the south Asian subcontinent subcontinent,according to the literature,vultures ate animal tissues and viscera containing diclofenac sodium,causing a sharp decline in the number of vultures and almost an ecological disaster.At home and abroad,methods for the determination of these two kinds of drug residues are limited.So it's necessary to establish rapid detection method of brombuterol and diclofenac sodium.In this study,we successfully prepared highly sensitive,highly specific monoclonal antibodies?mAb?of brombuterol and diclofenac sodium,and polyclonal antibody?pAb?of diclofenac sodium.We also established highly sensitive,highly specific enzyme linked immunosorbent assays?ELISAs?of brombuterol and diclofenac based on corresponding antibodies.Brombuterol-protein conjugates were prepared by coupling brombuterol to proteins using the diazotization method while diclofenac was conjugated to protein by the EDC/NHS ester method.As for brombuterol,two hybridoma clones(13D₁₂C₁ and 4D₁₁C₁)specifically secreting antibodies against brombuterol were successfully isolated.Under optimal conditions,the IC₅₀and LOD values of the ELISA for brombuterol based on the mAb secreted by the 13D₁₂C₁ clone were found to be 0.56 ng mL^-11 and 0.047 ng mL^-1,respectively.The ELISA displayed no cross-reactivity with structurally related?-agonist?ractopamine,salbutamol,phenylethanolamine A,phenylephrine and isoproterenol?and five other veterinary drugs,but showed 52.8%cross-reactivity with clenbuterol.Swine meat,liver and feed samples were spiked with different content of brombuterol and detected by ELISA.The recovery rates and the coefficients of variation of the intra-assay and inter-assay were found to be in the range of 87.3-107.5%and 3.5-11.6%?n=3?,and 85.7-116.0%and 4.1-12.7%?n=3?,respectively.Spiked samples were analyzed by ELISA and HPLC simultaneously.A good correlation between the two methods was obtained.The results demonstrated that the proposed ELISA was a feasible quantitative/screening method for brombuterol analysis in swine meat,liver and feed samples with the properties of high sensitivity.As for diclofenac,under optimal conditions,the IC₅₀and LOD values of ELISAs based on mAb and pAb were found to be 0.06 ng mL^-1and 0.01 ng mL^-1,0.13 ng mL^-1and 7.88 pg mL^-1,respectively.The two ELISAs both displayed no cross-reactivity with selected 11 compounds including structurally related compounds and anti-inflammatory drugs.Spiked water samples were detected by two ELISAs.The recovery rates and the coefficients of variation of the intra-assay and inter-assay of ELISA based on mAb were found to be in the range of 74.0-114.0%and 4.1-15.0%?n=3?,and 83.5-121.1%and6.7-13.8%?n=3?,respectively.And the recovery rates and the coefficients of variation of the intra-assay and inter-assay of ELISA based on pAb were found to be in the range of 84.0-115.2%and 2.4-12.0%?n=3?,and 82.9-117.8%and 5.3-18.2%?n=3?,respectively.Spiked samples were aslo analyzed by ELISAs and LC-MS/MS simultaneously.A good correlation between the two methods was obtained?for mAb:R=0.9903;for pAb:R=0.9864?,respectively.The results demonstrated that the proposed ELISA was a feasible quantitative/screening method for diclofenac analysis in water enviroment with the properties of high sensitivity,high specificity.In summary,three kinds of established ELISAs based on three kinds of antibodies of brombuterol and diclofenac had high sensitivity and high specificity and high throughput and low cost.These methods can be used for rapid detection of the real samples and provides feasible methods for rapid detection of brombuterol and diclofenac.