抗水牛乳β-乳球蛋白多克隆抗体的制备与应用

发布时间：2018-09-17 07:45

【摘要】： 牛乳中β-乳球蛋白是一种主要的过敏原，大约有60％牛乳过敏人群对β-乳球蛋白过敏。牛乳β-乳球蛋白与水牛乳β-乳球蛋白存在免疫交叉反应，对牛乳β-乳球蛋白过敏的人群也可能对水牛乳β-乳球蛋白过敏。因此，从食物过敏的角度探索水牛乳β-乳球蛋白具有一定理论价值。另外，由于水牛乳消费人群逐渐增加，为了保护过敏患者的健康，开展水牛乳的过敏研究将具有重要现实意义。 本研究的主要内容包括水牛乳β-乳球蛋白的分离纯化、兔抗水牛乳β-乳球蛋白多克隆抗体的制备以及水牛乳β-乳球蛋白间接竞争ELISA检测方法的建立。 在水牛乳β-乳球蛋白的分离纯化过程中，建立了DEAE-Sepharose Fast Flow离子交换结合Sephadex G-75凝胶层析分离纯化水牛乳β-乳球蛋白的方法。SDS-PAGE和IEF-PAGE鉴定结果表明，两种方法结合可以分离得到纯度＞90％的水牛乳β-乳球蛋白。 在抗水牛乳β-乳球蛋白多克隆抗体的制备过程中，建立了间接ELISA检测水牛乳β-乳球蛋白抗血清效价的检测方法及制备抗水牛乳β-乳球蛋白多克隆抗体的程序。免疫2只日本大白兔分别获得了间接ELISA检测效价高达1：204800和1：102400及双向琼脂扩散检测效价均为1：16的抗血清。通过双向琼脂扩散试验证实了水牛乳β-乳球蛋白与牛乳β-乳球蛋白存在免疫交叉反应。 建立了检测水牛乳β-乳球蛋白的间接竞争ELISA方法，确定的工作条件如下：包被抗原浓度2.5μg／mL，抗体稀释倍数为1：60000；二抗稀释倍数为1：10000。包被条件为4℃过夜；37℃封阻1h；孵育条件为37℃，1h；加终止液5min后比色。该方法检测的范围为0.1ng／mL～100ng／mL，，灵敏度为5.75ng／mL，最低检出限＜0.01 ng／mL。用建立的方法对4份样品进行了检测，测得阳光酸酸乳、阳光核桃花生乳、皇氏水牛乳、壮牛水牛乳的β-乳球蛋白含量分别为0.36g／L、0.472g／L、1.097g／L、0.717g／L。
[Abstract]:尾-lactoglobulin is a major allergen in milk, and about 60% of milk allergens are allergic to 尾-lactoglobulin. Milk 尾 -lactoglobulin and buffalo 尾 -lactoglobulin have immune cross reaction, and the people who are allergic to milk 尾 -lactoglobulin may also be allergic to buffalo milk 尾 -lactoglobulin. Therefore, it has certain theoretical value to explore buffalo milk 尾-lactoglobulin from the point of food allergy. In addition, as the number of buffalo milk consumers increases gradually, in order to protect the health of allergic patients, the study of buffalo milk allergy will be of great practical significance. The main contents of this study included the isolation and purification of 尾 -lactoglobulin from buffalo milk, the preparation of rabbit anti-buffalo 尾 -lactoglobulin polyclonal antibody and the establishment of indirect competitive ELISA assay for buffalo milk 尾 -lactoglobulin. In the process of separation and purification of 尾 -lactoglobulin in buffalo milk, the method of DEAE-Sepharose Fast Flow ion exchange and Sephadex G-75 gel chromatography was established to separate and purify 尾 -lactoglobulin from buffalo milk. SDS-PAGE and IEF-PAGE showed that, 尾-lactoglobulin of buffalo milk with purity > 90% could be separated by two methods. In the course of preparation of anti-buffalo 尾 -lactoglobulin polyclonal antibody, a method for detecting the titer of buffalo milk 尾 -lactoglobulin antiserum by indirect ELISA and a procedure for preparing anti-buffalo milk 尾 -lactoglobulin polyclonal antibody were established. Two Japanese white rabbits were immunized with antiserum with indirect ELISA titers of 1: 204800 and 1: 102400 and double Agar diffusion titers of 1:16 respectively. The immune cross reaction between buffalo 尾-lactoglobulin and milk 尾-lactoglobulin was confirmed by two-way Agar diffusion test. An indirect competitive ELISA method for the detection of 尾 -lactoglobulin in buffalo milk was established. The working conditions were as follows: the concentration of coated antigen was 2.5 渭 g / mL, the dilution multiple of antibody was 1: 60000, and the dilution multiple of second antibody was 1: 10000. The coating conditions were as follows: blocking at 37 鈩

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