Development Of Luminescent Immunoassay For Determination Of 3-methyl-quinoxaline-2-carboxylic Acid And Quinoxaline-2-carboxylic Acid In Animal Tissues

Olaquindox?OLA?and carbadox?CBX?are the member of quinoxaline antibiotics,which are used for hastening growth and anti-infectious activities on animals as feed additives.In animal tissues,OLAandCBXcanberapidlymetabolizedandconvertedinto3-methyl-quinoxaline-2-carboxylic acid?MQCA?and quinoxaline-2-carboxylic acid?QCA?,MQCA and QCA have carcinogenic,mutagenic and photoallergenic effects on people,threating the safety of food.Therefore,it is necessary to establish more sensitive and effective method for the monitoring of MQCA and QCA.Herein,the aim of the study is to establish a sensitive chemiluminescent competitive indirect enzyme-linked immunosorbent assay?CL-ciELISA?and background fluorescence quenching immunochromatographic assay?bFQICA?for simultaneous determination of MQCA and QCA with immunomagnetic beads for the simple and time-saving sample preparation,which was the technical support for screening purposes.The sensitive MQCA monoclonal antibody was produced.The hapten was MQCA-NH₂,coupling carrier protein with BSA,KLH and OVA,and obtained the immunogen MQCA-NH₂-BSA?MQCA-NH₂-KLH?and coating antigen MQCA-NH₂-OVA.The monoclonal antibody by the fusion of myeloma cell and spleen cell of mice for good immune effect,the concentration was 4 mg/mL after purifying with protein A affinity column,the half maximal inhibitory concentration(IC₅₀)value was 0.2?g/L,and the cross-reactivity with QCA was 40%.AsensitiveCL-ciELISAisproposedforsimultaneousdeterminationof3-methyl-quinoxaline-2-carboxylic acid?MQCA?and quinoxaline-2-carboxylic acid?QCA?with immunomagnetic beads for sample preparation.Half maximal inhibitory concentration(IC₅₀)value was 0.02?g/L for MQCA,linearity range was 0.008-1.00?g/L,the cross-reactivity with QCA was40%.1 mg streptavidin magnetic beads conjugating MQCA antibodies?30?g?formed immunomagnetic beads;2 g samples?fish,shrimp,pork and chicken?were extracted with 2 mL PBS after homogenizing,0.3 mg immunomagnetic beads were added to 800?L sample extraction for extracting MQCA and QCA?10 min?,separating from sample matrix with magnetic field.Immunomagnetic beads were eluted by heating?PBS as the elution fluid?at 85?for 5 min and formed 100?L reconstitution fluid.The total sample pretreatment would consume 30 min,and the concentration of MQCA and QCA could be 4 times of primary sample.The detection limit?LOD?for MQCA in fish,shrimp,pork and chicken were 0.050,0.043,0.048 and 0.050?g/kg and the LOD for QCA in fish,shrimp,pork and chicken were 0.090,0.10,0.13 and 0.18?g/kg.The recoveries ranged from 77.0%to 117.0%?0.05-3.0?g/kg?in fish,shrimp,pork and chicken tissue.Coefficients of variation ranged from 4.5%to 11.5%.Furthermore,40 field samples were analyzed with the developed CL-ciELISA,and the results correlated well with those obtained in liquid chromatography-tandem mass spectrometry?LC-MS/MS?,confirming the utility of CL-ciELISA for quantitation of MQCA and QCA in fish,shrimp,pork and chicken with a good accuracy and reliability.A strategy is proposed for determination of 3-methyl-quinoxaline-2-carboxylic acid?MQCA?and quinoxaline-2-carboxylic acid?QCA?simultaneously with immunomagnetic beads for the separation and enrichment of MQCA and QCA from chicken muscle,based on bFQICA.The IC₅₀ of MQCA was 0.34?g/L for MQCA with a cross-reactivity of 40%for QCA.The LOD for MQCA in fish,shrimp,pork and chicken were 0.35,0.40,0.45 and 0.40?g/kg and the LOD for QCA in fish,shrimp,pork and chicken were 0.85?1.10?1.20?1.00?g/kg.The mean recoveries ranged from 72.0%to 118.0%at the spiked levels?0.5,1 and 2?g/kg for MQCA;1.25,2.5 and 5?g/kg for QCA?in fish,shrimp,pork and chicken.Coefficients of variation varied from 5.2%to 11.5%.Furthermore,40field samples were analyzed with the developed bFQICA,and the results correlated well with those results obtained in a previously reported liquid chromatography-tandem mass spectrometry?LC-MS/MS?,confirming that the utility of bFQICA for quantitation of MQCA and QCA in fish,shrimp,pork and chicken with a good accuracy and reliability as the good consistency.The bFQICA is more sensitive and could quantify determination compared with traditional colloidal gold detection card,and could field test compared with ELISA.