Study Of Enzyme Immunoassay For Nitrofurazone Metabolite

Nitrofurans veterinary drugs are synthetic broad-spectrum antimicrobials,which are used for the prevention and treatment of gastrointestinal infections caused by e.coli and salmonella.These drugs can be soon become to the corresponding metabolites and are stable for long times when they enter into animal body.Nitrofurans and their metabolites are a class of compounds with carainogenicity and teratogenicity.It will have a great harm to human health when these 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.Nitrofurazone is one of nitrofurans.At present,various instrumental analysis methods with high analytical precision have been used to detect the residue of semicarbazide(SEM).However,these methods require expensive equipments and professional and technical personnel to operate them and also time-consuming and costly.Compared with these methods,enzyme immunoassay technical requirements is lower,can rapid screen large quantities of samples,and has high accuracy,precision and specificity.Thus,this study established three kinds of enzyme immunoassay to detect SEM residue,including chemiluminescence enzyme immunoassay(CLEIA),biotin-avidin enzyme linked immunosorbent assay(BA-ELISA)and enzyme linked immunosorbent assay(ELISA).The hapten CPSEM was synthesized by conjugating SEM with 4-CBA.Then made it conjugate with OVA by using N-hyduoxysuccinimide ester method to synthesize coating antigen.The target detection object NPSEM was synthesized by conjugating SEM with 2-NPA.The main operation conditions which impact on the enzyme immunoassay were optimized.The optimized chemiluminescence fluid's formula:Liquid A was 8 mm of iodine phenol solution and 10 mm luminol solution mixed by the same volume;Liquid B was lOmL Tris-HC1 buffer solution including 5?L 30%H2O2 solution;mix the same volume Liquid A and Liquid B before use.The optimized reaction conditions for CLEIA were as follows:800-fold dilution of monoclonal antibody,800-fold dilution of CPSEM-OVA,1%skimmed milk as blocking solution,30min of competitive reaction time,60min of incubation time for HRP-IgG.The optimized reaction conditions for BA-ELISA were as follows:12800-fold dilution of monoclonal antibody,800-fold dilution of CPSEM-OVA,8000-fold dilution of SA-HRP,1%skimmed milk as blocking solution,30min of competitive reaction time,30min of incubation time for Biotin-IgG,60min of incubation time for SA-HRP,15min of chromogenic reaction time.The optimized reaction conditions for ELISA were as follows:6400-fold dilution of monoclonal antibody,800-fold dilution of CPSEM-OVA,0.5%skimmed milk as blocking solution,30min of competitive reaction time,60min of incubation time for HRP-IgG,15min of chromogenic reaction time.The three optimized methods were compared and evaluated in this study.The cross-reactivity values were lower than 0.1%with other structural analogues and derivatives.The linear detection range of the developed CLEIA was 0.123?2.398ng/mL,and the 50%inhibitory concentration(IC50)value was 0.544ng/mL;the intra-assay and inter-assay coefficients of variation were 1.9%?4.1%and 2.8%?5.3%,respectively;the recoveries in negative samples ranged from 89.6%to 98.0%.The linear detection range of the developed BA-ELISA was 0.074?4.883ng/mL,and the IC50 was 0.601ng/mL;the intra-assay and inter-assay coefficients of variation were 1.2%?3.6%and 2.5%?5.1%,respectively;the recoveries in negative samples ranged from 88.8%to 98.5%.The linear detection range of the developed ELISA was 0.560?14.315ng/mL,and the IC50 was 2.831ng/mL;the intra-assay and inter-assay coefficients of variation were 1.8%?5.1%and 3.4%?6.3%,respectively;the recoveries in negative samples ranged from 87.9%to 94.1%.These results indicated that the three kinds of enzyme immunoassay to detect SEM residue had some differences in terms of sensitivity,precision and accuracy,and their accuracy were not as good as HPLC-MS/MS method.But they can meet the need of detecting SEM residues in actual samples,and can be used for rapid screening of SEM in animal tissues.This study had paved the way for the next step developing SEM fast detection kit.