Synthesis Of Methyl-3-quinoxaline-2-carrboxylic Acid Olaquindox Metabolite And Preparation Of Monoclonal Antibodies

The drug residues of Methyl-3-quinoxaline-2-carboxylic acid,which comes from the metabolite of olaquindox in livestock of China does threat the health of human,and influence the export of livestock products.Therefore,the development of rapid detection of Methyl-3-quinoxaline-2-carboxylic acid is with a certain significance.The rapid immunological determination is built up on antibody specifically reactive with the antigen,This method is specificity,high sensitivity,low cost required,sample preparation simplify,fast analysis time,commonly used for initial screening of samples.In this paper,5 methods are used to study the synthesis of Methyl-3-quinoxaline-2-carboxylic acid.Including four synthetic methods reported in the literature as well as one new method combining these four methods design,Synthetic products were identified by high performance liquid chromatography,high performance liquid chromatography-mass spectrometry,nuclear magnetic resonance spectroscopy,then compare the five kinds of synthetic methods.The result shows that combined with HPLC retention times,identification of HPLC-MS and appraisal of the results of MRI,the synthetic substances by 5 method are all Methyl-3-quinoxaline-2-carboxylic acid.Compare the five methods,the new Methyl-3-quinoxaline-2-carboxylic acid synthesis methods yields the highest yield rate of 51%,a purity greater than 99%,while the operation of this method is most convenient and safe.Use the active ester method,carbodiimide method and DIC catalytic approach to do the synthesis of Methyl-3-quinoxaline-2-carboxylic acid artificial immune antigen,use mixed anhydride method to synthesize artificial coating antigen,By animal immunization,detection of serum we find that using N,N-diisopropylcarbodiimide(DIC)catalyst which is different with Dicyclohexylcarbodiimide(DCC)can get the best effect for synthesis of MQCA immunogen.The MQCA we synthesize by ourselves has a better inhibitory effect on serum than that of bought from Sigma.Use synthetic artificial immunogen to immunize Balb/c female mice,obtain hybridoma cell lines obtained by cell fusion experiments,Detected by ELISA we find that the positive wells is rare,There has been a lot of holes cell colony.but little or no antibody-secreting.and lower rates of cell fusion.As for the phenomenon of a big quantity of cells and low OD value.Analysis may show that A of HAT may be invalid or the myeloma cell mutation There may be weakly immunogenic antigens,immune ineffective.For the original antibody-secreting hybridoma cells becomes negative,may be cells mycoplasma contamination may,or non-competitive antibody secreting cell clones grown,Thereby inhibiting the growth of antibody-secreting cells,may be the chromosome is missing.