Hapten Synthesis And Establishment Of Hybidoma Cell Lines Secreting Specific Antibody Of Methyl-3-Quinoxaline-2-carboxylic Acid,the Metabolite Of Olaquindox

Olaquindox(OLA)is a kind of quinoxaline antimicrobial growth promoter synthesized by organic chemistry method.It can promote protein synthesis,increase the yield of lean meat,feed conversion rate,and promote animal growth,can also be used for bacterial infections prevention and treatment of diseases.However,a large number of studies have shown that excess olaquindox stranded in animals can produce a variety of side effects,such as cumulative toxicity and DNA damage.And it is banned in many countries,including the EU and China.Olaquindox itself is unstable,can be metabolized in vivo of animal.While its main metabolic product,Methyl-3-quinoxaline-2-carboxylic acid(MQCA),is relatively stable,is identified by WHO/FAO Joint Expert Committee on Food Additives(JECFA)as olaquindox residues marker.With the continuous progress of science and technology and development of equipment manufacturing industry,the detection methods olaquindox and its metabolite are constantly evolving and optimized,the sensitivity improving,which provides effective ways to monitor olaquindox and its metabolite.In order to establish a rapid and sensitive detection method of olaquindox metabolites,the study designed and synthesized a variety of haptens of Methyl-3-quinoxaline-2-carboxylic acid and prepared monoclonal antibodies against them respectively,using monoclonal antibody technology.The experiment successfully synthesized four kinds of MQCA haptens and the synthesis conditions were optimized,and ultimately found the optimum conditions:isobutyl chloroformate is in an amount of 120 ?L,the molar ratio of methyl-3-quinoxaline-2-carboxylic acid and amino acids is 1:2.5,the activation time is 1 h,the reaction time is 1 h.The immunogens were prepared by conjugating MQCA haptens to BSA respectively,and the conjugation ratio were 10.69:1,9.53:1,9.11:1,10.02:1.Coating antigens wre prepared by conjugating MQCA haptens to OVA respectively,and the conjugation ratio were 3.81:1,5.33:1,2.67:1,3.36:1.Balb/C mice were immunized with the four kinds of immunogens.After the third immunization,the 3-5 mouse serum titer was 100 000,2-1 mouse had the best inhibitory effect,2-3 and 1-4 followed.Choosing 2-1 mouse to prepare spleen cells,fusing with myeloma.Through screening,the 4D9 hole was the best and prepared hybridoma cell lines.