渔药恩诺沙星完全抗原合成方法及效果的研究

Studies on synthesis and characteristics of complete antigens of Enrofloxacin

采用牛血清白蛋白(BSA)和鸡血清白蛋白(OVA)为免疫用载体蛋白,匙孔血蓝蛋白(KLH)为包被抗原用蛋白,采用不同的方法合成完全抗原,对BALB/C小鼠进行免疫,并通过紫外光谱特征、抗血清检测等手段对完全抗原的合成效果进行了分析。实验表明蛋白-恩诺沙星偶联物的紫外光谱特征可以较为准确、简便地提示完全抗原的合成效果,并能够与抗血清的酶联免疫吸附试验(ELISA)分析结果吻合。用乙二胺(EDA)对载体蛋白进行处理,可以明显提高完全抗原的合成效果;而偶联方法对于不同载体蛋白的影响也存在差异,OVA采用活化酯法效果较好,而BSA采用碳二亚胺法得到的血清效价较高。

Fluoroquinolones （FQs）are one of the commonest antibiotic groups that have been used for the treatment of bacterial infection. Enrofloxacin（EF） belongs to this group and has been increasingly used in veterinary medicine . A simple and reliable analytical method for this drug is required. The current determination by high performance liquid chromatography （HPLC） is sensitive but labor-intensive, lmmunoassay methods have been successfully developed to rapidly screen EF residues. The synthesis and characteristics of complete antigens of EF were experimented. The results of this study indicated that the antibodies against EF may be used as the basis for an immunoassay to rapidly screen samples for the presence of FQs in tissues or body fluids. EF is a small molecule （MW 〈 1000） that must be conjugated to a cartier to elicit an immune response. Proteins are commonly used carriers, such as bovine serum albumin （BSA）, ovalbumin （OVA）, keyhole limpet hemocyanin （KLH） and Albumin Human （HSA）. Using the free carboxylic acid on EF, it could be linked to protein carriers through the amide linkage. BSA and OVA were used as protein carriers to synthesize EF complete antigens respectively by both carbodiirnide method and active ester method, which were then immunized to BALB/C rats. KLH was coupled to EF by carbodiimide method , the conjugation was used as coating antigen. The effects of coupling between haptens and carriers were investigated by UV spectrum, anti-serum, etc. The UV spectrum showed that the major absorbances of EF were in 208 nm, 271 nm and 334 nm, while protein had its major absorbances at 229 nm and 278 nm ; the EF-protein conjugates had a significant peak at 330 - 335 nm which were the main characteristic of EF but could not be seen in protein, so it could be concluded that the linkage between EF and protein were successfully set up. An indirect enzyme linked immunosorbant assay （ ELISA ） was used to determine the titers of the antiserum, the titers of all the resultant polyclonal antibodies could reach 1：50000. In ELISA procedure, the optimal dilution of coating antigen was determined by the checkerboard test and the optimum condition selected was coating antigen concentration in 10μg·mL^-1 and secondary antibody diluted 1 ： 10000. The haptenprotein ratio was calculated by formula at the range of 1 - 6. It used to believe that the amount of haptens coupled to the protein should be as much as possible. But in theory, the carder protein linked even to one hapten may be enough to produce specific antibody, and in fact many researchers have proved that in some cases overabundance of the haptens with the cartier cannot result in desired responses, because it may prevent the protein from being present at the surface of immunoglobulins on B cells, and then interfere with the immune reaction. The results showed that the UV spectrum of protein-EF could coincide with the results of enzyme immunoassay of anti,rums, so it could be used to indicate the characteristics of the complete antigens simply. During the reaction, amino group on the protein can also be linked with the carboxyl group on itself. To reduce the self-coupling, we added excess ethylenediamine （EDA） first to block the proteins and it could be seen that the characters of the modified proteins did not change from its UV spectrum. Now no relative reports about modified protein could be obtained in China. Also it was found that coupling effects could be obviously improved after the carrier proteins were treated with EDA. But to various carrier proteins, the coupling effects were greatly influenced by different coupling methods. It was better to use active ester method than carbodiimide method when carrier protein was OVA. But when BSA was used as carrier protein, the coupling effects of the carbodiimide method were better.