【作者】 何丹；

【导师】 郑晓冬；

【作者基本信息】 浙江大学 ， 食品科学与工程， 2004， 硕士

【摘要】 氨苄青霉素(Ampicillin)是目前较常用的广谱半合成青霉素，抗菌谱比青霉素G广，毒性极低，具有较强的杀菌作用。对革兰氏阴性菌和革兰氏阳性菌均有作用。在兽药临床上，氨苄青霉素多以注射方式用于治疗各种炎症、感染。如果将停药期间内的动物屠宰或挤取尚在停药期间内奶牛的牛奶，就会造成动物性食品中氨苄青霉素的残留。人们食用残留有氨苄青霉素的食品后，会诱导耐药菌株的产生，敏感的人群会产生过敏反应。发酵乳生产中残留的氨苄青霉素会抑制生产菌的生长。因此，建立针对氨苄青霉素残留的快速检测方法对控制动物性食品的卫生质量、保障消费者健康、保证工业生产等方面都具有比较重要的社会经济意义。 本研究制备了氨苄青霉素的多克隆抗体，建立了氨苄青霉素免疫检测技术。通过比较两种合成方法偶联的氨苄青霉素和BSA免疫抗原刺激机体产生抗体的亲和性和效价，比较了两种合成方法的优劣。同时，对ELISA检测条件进行了优化，建立氨苄青霉素ELISA检测技术。其主要研究结果如下： 1 通过两种方法——物理直接偶联和以戊二醛为连接物合成了氨苄青霉素和大分子蛋白质的免疫抗原和包被抗原并用紫外光谱扫描和红外吸收光谱扫描的方法对合成物质进行了鉴定。 2 将合成的氨苄青霉素免疫抗原与等量佐剂混合后多途径多次免疫兔子，第七次免疫后心脏采血，分离获得血清，并将这些血清分成3部分进行处理：一部分直接冷冻于-20℃，一部分与等量甘油混合后保藏于-20℃冰箱，另一部分血清纯化分离IgG、冷冻干燥后保存于-20℃冰箱。 3 用琼扩实验对抗血清进行分析。发现抗血清能与免疫抗原产生沉淀线，而不能与以OVA作载体蛋白的检测抗原产生沉淀线。这说明琼扩实验是一种很粗略的检测方法，能检测出血清中大量存在的载体蛋白抗体，半抗原的抗体因为量少不能在琼扩实验中产生沉淀线而被检测出来。 4 对ELISA检测中所用的HRP酶标山羊抗兔IgG的使用浓度和包被抗原类型及其浓度进行确定。实验结果表明，酶标二抗的最佳使用浓度是1600倍稀释。由于HSA和BSA有很大同源性，以其为载体蛋白的合成抗原不能作为检测抗原。同时，抗血清中存在抗桥抗体，用于检测的包被抗原和免疫抗原最好由两种合成方法含成，以消除抗桥抗体的影响。 56只兔子产生的抗血清的效价分别为：1号抗血清和2号抗血清的效价均是64×10~3，3号抗血清的效价是1×10~3，4号抗血清的效价是8×10~3，5号抗血清的效价是128×10~3，6号抗血清的效价是4×10~3。其中1、2、5号兔子产生的抗浙江大学硕士论文血清的效价都远远高于3、4、6号兔子产生的抗血清。而1、2、5号兔子都是以Agl作为免疫抗原，3、4、6号兔子的免疫抗原都是Ag4。这说明不同合成方法合成的免疫抗原有不同的免疫原性。 ‘对间接ELISA检测条件进行优化，包括底物溶液的组成、包被液的选择、包被时间和温度的选择、封闭液的选择、兔子抗血清的温育时间、HR卫酶标羊抗兔lgG抗体的温育时间及底物加样后温育时间，并用L二(56)正交表对后6个因素进行正交实验。正交实验证明，底物溶液的最佳配方是10ml底物缓冲液+7 pl珑仇十200川TM[B。间接ELISA的最佳实验条件是以CBS作为包被液。加好包被液的酶标板37℃温育2h后转入4℃冰箱温育14h包被。然后以5%甘氨酸封闭醉标板。一抗的最佳温育时间是1 00min，二抗的最佳温育时间是120min，加底物显色1 smin产生的阴阳性抗血清的OD月5。差值最大。包被液的类型对实验结果的影响最大，其次是封闭液的类型和底物作用时间。由于二抗的温育时间对实验的结果影响不是很主要，而且二抗温育40min和温育1 00min产生的实验结果相差很少。从节约时间和成本的角度出发，在不降低实验灵敏度的前提下，二抗的握育时间定为40min。 7用间接竞争EUSA实验对抗血清的亲和性进行测定，实验结果表明以Agl为免疫抗原获得的抗血清的亲和性总体来说比免疫抗原为A妙的抗血清的亲和性差。虽然Agl免疫兔子后产生的抗血清的效价比Ag4的高，但亲和性远远不能达到犯LlsA检测抗体的要求。对于进行ELISA检测的抗血清，其亲和性的重婆性比效价高的多。这表明以物理方法直接合成的免疫抗原比戊二醛法合成的免痰抗原好，其免疫兔子产生的抗血清更适宜作为EUSA检侧抗体。6号抗血清是最佳血清。 忍用氨爷青霉素的结构类似物和几种常见的抗生素与6号抗血清进行间接竞争实脸，以测定抗血清的特异性。由于阿莫西林与氨节青霉素的结构只差一个翔签，扰血清与阿莫西林的交叉反应率高达78%。抗血清与青霉素钾的交叉反应率是1 .5机，与苯哇青霉素的交叉反应率是0涛%，而抗血清与硫酸链霉素、硫酸卡那舜李和礁酸庆大霉素的交叉反应率均小子0.0125%。这说明该抗体的特异性是好的，除了不能分辨样品中的氨节青霉素残留和阿莫西林残留，其他抗生素不会对检翎结果产生干扰。 96号抗血清的竞争抑制实验标准曲线方程是:卜25 .046X一5.5396，检测极限是4.1知留抽l，能达到动物性食品中氨节青霉素残留检测的要求。更多还原

【Abstract】 Ampicillin is one of the most common semi-synthesis antibiotics. It has strong sterilizing ability and low toxicity. Ampicillin has a wider sterilizing range than penicillin G, and it’s active to both G+ and G-. Ampicillin was often used to treat animals’ inflammation and infection. If the animal was slaughtered or milked before ampicillin metabolized completely, there would have ampicillin residue animal food. When people eat these food, ampicillin residue remain in the food would induce anti-antibiotics bacterium. And it also would induce anaphylaxis in hypersensitive people. In industry, ampicillin residue in fermenting milk would restrain the growth of the zymogen. It’s very meaningful to establish a fast detection method to detect ampicillin residue to control animal food safety, to safeguard consumers’ health and to protect industry’s production.In the present study, we have produced anti-ampicillin polyclonal antibody and have established ampicillin’s immunoassay method. We used two synthesis methods to synthesis immunogen. Then valued the synthesis methods by compare anti-sera produced from rabbits immunized with this two immunogen. We optimized and then established ELISA process. The main results were as following:1. Synthesis immunogen with two methods-Physiological directly synthesis ampicillin and carrier protein conjugate; Chemical indirectly synthesis ampicillin-glutaradehyde-carrier protein conjugate. The products were scanned by UV spectrum aad infra-red spectrum to judge whether hapten was linked to the protein.2. Synthetical antigen was mixed with same amount adjuvant and then immunized rabbits with it. After the seventh immunize, the rabbits were blooded from heart. Separated blood sera were divided into three parts for treatment: One part was directly stored at -20℃ Another part was mixed with glycerol and then stored at -20 ℃; IgG was separated from the third part serum then freeze dried and stored at -20℃.3. Agar diffuses analysis anti-sera. We found that anti-sera reacted with immunogen but didn’t react with antigen that different from immunogen only in protein carrier. This proved that agar diffuses was not a precision method in detecting an anti-hapten antibody because there was not so much antibody in the serum.4. Determination working concentration of HRP linked goat anti-rabbitlgG antibody and working concentration of coating antigen. Working concentration of HRP linked goat anti-rabbitlgG antibody was 1600X dilution. Because HSA and BSA was very similar in Amino Acid sequence, antigen synthesized with HSA carrier protein can’t be used as coating antigen. Besides, there were anti-bridge antibody in anti-sera. Coating antigen and immunogen should synthesize with different methods.5. Working concentrations of six rabbit anti-sera were: No.1 anti-serum-64 X 103 dilution, No.2 anti-serum-64 X103 dilution, No.3 anti-serum-1 X 103 dilution, No.4 anti-serum-8 X 103 dilution, No.5 anti-serum-128 X 103 dilution, No.6 anti-serum-4X103. No.l, No.2 and No.5 anti-sera were collected from rabbits immunized with Ag1. No.3, No.4 and No.6 anti-sera were collected from rabbits immunized with Ag4. Antigen synthesized with different method has different immune ability.6. Optimized indirect ELISA process. Our study showed that the best formula of reaction regent was 10ml buffer+7ml H2O2+200 ul TMB. The best reaction-conditions were use CBS as coating buffer, plate should coat at 37@ for 2h then coat at 4@ for 14h, block the plate with 5% Glysine, anti-sera should incubate 100min, anti-antibody should incubate 40min.7. Anti-sera’s affinities were determined using competitive inhibitory indirect ELISA. The result showed mat anti-sera separated from rabbits immunized with Ag4(No.1, No2, No.5 anti-serum) has better affinities than anti-sera separated from rabbits immunized with Agl(No.3, No.4, N0.6 anti-serum). Though the later three anti-sera has higher working dilution than the former three ones, Physiological directly synthesis更多还原