| The veterinary drug residues in animal-origin foods caused widespread concern at home and abroad in recent years. Nowadays, most of the immunoassays focus on detecting one analyte or one class of structural-related analytes. In order to improve dtermination efficiency, decrease detecting time and decrease expenses, it is urgent to develop multi-analyte immunoassays. In this paper, we developed immunoassays for rapid detecting two marker residues of quinoxaline and immunoassays for detecting two class of veterinary drugs (SAs and QNs)In this paper,4-(3-methylquinoxaline-2-carboxamido)butanoic acid was used as hapten for monoclonal antibody production. The produced MAb showed good cross reactivities (50%-2400%) with the metabolites of quinoxaline, and the IC50values of QCA and MQCA were4.8and9.6ng/mL. Based on this MAb, a broad specificity ELISA was developed for simultaneously detecting MQCA and QCA in animal edible tissues (fish, shrimp, pork and chicken). The limit of detection was1.54μg/kg for MQCA and QCA in animal edible tissues. Satisfying recovery ranged from76%-108%was obtainded with the coefficients of variance ranged from4.2%to13.3%, when the samples were spikded with MQCA (2,4,8μg/kg) and QCA (4,10,20μg/kg). All these results meet the requirement of residue analysis.To improve the sensitivity of immunoassay, BA-ELISA and CLIA were debeloped for MQCA and QCA residues analysis. The IC50of BA-ELISA was1.6and3.6ng/mL for MQCA and QCA in buffer, and the LOD was1.04μg/kg in animal edible tissues (fish, shrimp, pork and chicken).When the blank samples were spiked with MQCA at2,4,8μg/kg, the intra-assay and inter-assay recoveries were in the range of72%-106%, and the corresponding CV values ranged from6.7%to12.6%. The IC50of CLIA was0.42and1.05ng/mL for MQCA and QCA in buffer, and the LOD was0.76μg/kg. When the fish sample were spiked at1,2,4μg/kg, the intra-assay and inter-assay recoveries were in the range of71%-108%, with the CV values ranged from7.0%to14.4%. The detection limit and sensitivity of the immunoassays meet the requirements of the legislation of EU, US and China.In this study, a novel DC-ELISA was developed for simultaneously detecting22SAs and13QNs in milk. The DC-ELISA using ALP labeled goat anti-mouse IgG to recognize anti-QN MAb, and using HRP labeled goat anti-rabbit IgG to recognize anti-SA PAb. In this way, the DC-ELISA could be used for simultaneous detecting SAs and QNs in one well. The method for SAs and QNs detection limits were5.8and2.4ng/mL. When the milk samples were sipked with SAs and QNs (10,50,100ng/mL), the mean recoveries were in the range of67%-101%for detecting SAs residues in milk, and the corresponding CV values ragned form8.1%to16.4%. The mean recoveries were in the range of72%-105%for detecting QNs residues in milk, and the corresponding CV values ranged from4.8%to9.3%. All these results meet the requirement of residue analysis.In this paper, we used agrose as the carrier of the immune-reaction and developed a novel FTIACT method for simultaneously detecting14SAs and13QNs. In this paper, we tested four different labels (horseradish peroxidase, fluorescent microspheres, quantum dots, lipsome encapsulated quantum dots) and their application in FTIACT. The visual detection limits of the LQDs-FTIACT method were1and0.5ng/mL. The OD values were processed and analyzed from the image of the FTIACT results using Image Pro Plus6.0software, and the OD values were used to develop a standard curve for FTIACT quantitative analysis. When using LQDs as labels, the detection limit of the FTIACT method were0.27ng/mL (SAs) and0.12ng/mL (QNs). The mean recovery ranged from89%to114%, and the CV values ranged from9.2-12.3%when the blank milk samples were spikded at0.25,0.15μg/kg for SM2and0.1,0.25μg/kg for CIP. All these results meet the requirement of the legislation and indicated that the FTIACT method could be used for simultaneously screening SAs and QNs in milk. |
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