Immunoassay Development For The Determination Of Benzodiazepines In Animal-derived Foods

Benzodiazepines(BZD),which include more than 20 derivatives,are prescription medications used in clinic for sedation and hypnosis.However,in recent years,benzodiazepines have been illegally used to feed livestock and poultry or added in the forage as sedatives and antistress drugs.The residues of BZD in animal-derived foods will cause serious harm to human health.Therefore,China’s Ministry of Agriculture has banned the use of benzodiazepines in feed or drinking water for animal rearing,including analogs of diazepam,nitrazepam and oxazepam,etc.At present,the detection of BZD mainly relies on instrumental based methods.These methods offer high accuracy but require professional personel and expensive equipment.So they are not suitable for rapid on-site detection of BZD.Therefore,it is necessary to develop a simple,rapid,low-cost,high-throughput immunoassay for on-site detection,and as a supplement to instrumental based methodology.In this study,two novel haptens were designed and synthesized to obtain broad-recognition monoclonal antibody against BZD(BZD-m Ab)with high affinity through animal immunization.Based on the prepared m Ab,a quantum dot nanobeads based immunochromatographic assay(QBs-ICA)was developed for the determination of BZD.The main results are as follows:(1)Haptens synthesis and artificial antigens conjugation:Two different BZD haptens were designed and successfully synthesised,which characterized by MS and~1H NMR.Then artificial antigens were prepared by coupling hapten with keyhole limpet hemocyanin(KLH)and ovalbumin(OVA),respectively.The antigens were identified by UV-vis spectra.(2)Generation of monoclonal antibody against BZD:After mouse immunization,cell fusion and subclonal screening,two hybridoma cell lines No.2-3D-7E-6B and 6-11A-7D-10G were obtained,which can stably secrete monoclonal antibody to broadly recognize BZD.Thereafter,ascitic fluid was obtained by 6-11A-7D-10G and and BZD-m Ab was purified.The results of indirect competitive enzyme linked immunosorbent assay(ic-ELISA)showed that the broad-recognition monoclonal antibody could simultaneously recognize oxazepam,nitrazepam,lorazepam,chlordiazepoxide and clonazepam.The half maximal inhibitory concentration(IC50)ranged from 103.64ng/m L to 761.74 ng/m L,and the limit of detection(LOD)ranged from 11.01 ng/m L to61.28 ng/m L.The cross-reactivity rate ranged from 13.61%to 98.82%.(3)Development of quantum dot nanobeads based immunochromatographic assay for the detection of BZD:Firstly,the QBs-m Ab conjugate was achieved after optimizing the p H value and volume of BZD-m Ab.Then,a series of condition parameters were optimized,including the concentration of coating-antigen on T-line,corresponding dosage of QBs-m Ab probe,time of reaction,p H value and content of methanol.Under the optimal conditions,the IC50 of QBs-ICA for the determination of five benzodiazepines ranged from 28.81 ng/m L to 179.04 ng/m L,and the LOD ranged from 2.27 to 12.10 ng/m L.Compared with ic-ELISA,the sensitivity of QBs-ICA was promoted to 3.3-4.3 times higher than the previous one.Under the excitation of UV light,the LOD of QBs-ICA for the determination of oxazepam,nitrazepam,lorazepam,chlordiazepoxide and clonazepam with naked eyes observing reached 10,10,10,39and 78 ng/m L,respectively.It indicated that QBs-ICA could achieve sensitive qualitative and semi-quantitative rapid on-site detection for BZD.The average recovery rate was 78.2-112.3%in the spiked pork sample and the coefficient of variation(CV)was 4.9-9.7%.Method validation was conducted in comparison with LC-MS/MS.The consistent results indicated that an accurate and reliable QBs-ICA was developed.