Studies On ELISA Detection Of Quinocetone Marker Residue And Hapten Design And Synthesis

Quinocetone is a new drug of Quinoxlines developed by China. Because of the lower toxicity and higher activity compare with other Quinoxlines, Quinocetone is considered as a safty and high efficiency feed additive. Used as an animal medicine, its side effect for person through food chain must be investigated.So an effective and sensitive method to detect the residue of this compound in animal edible tissue must be developed. In this thesis, we carried out a study of antigen synthesis, antibody production and hapten alteration to develop a quicky and sensitive detection method of Quinocetone's residue in animal edible tissues.In this study, we synthesized 3-methyl-quinoxaline-2-carboxylic acid (MQCA) with good purity from 2-nitroaniline via four step, and used the 2-carboxyl as function group and conjugated it with the amino- of protein in presence DCC.The conjugations (MQCA-BSA, MQCA-RSA) were identified by ultraviolet spectrum. Polyclonal antibodies were produced by immunization of New Zealand rabbits using different conjugations as immunogens and different time intervals. The titer test showed that the polyclonal antibodies immunonized by two immunogens all had high titer and the longer interval made for obtaining higher titer. The speciality test showed that the polyclonal antibodies immunonized by conjugations MQCA-RSA had the better speciality than the other one. Usage of RSA as the carrier protein could avoid producing antibody for carrier protein. But the result of the competitive indirect ELISA showed that the sensitivity of the antibody was too low to reach the demand of residue detection.According to the result, we believed that the reason resulted in the low sensitivity was that the marker residue compound MQCA structure has changed after conjugated with protein and the antibody induced by the conjugation could not recognize the free MQCA in the ci-ELISA. Therefore we designed some hapten which could reserve the marker residue structural speciality mostly. After synthesis route selection and optimum, the hapten 7-Hydroxymethyl-3-methyl-quinoxaline-2-carboxylic acid was synthesized, used 4-methyl-2-nitroaniline as the start material via six steps, such as oxidation cyclization, bromize reaction, hydrolysis, Beirut reaction, reduction, hydrolysis. This work established basis for producing the high affinity antibody and developing the sensitive ELISA residue detection method.