【摘要】： 牛乳中β-乳球蛋白是一種主要的過敏原，大約有60％牛乳過敏人群對β-乳球蛋白過敏。牛乳β-乳球蛋白與水牛乳β-乳球蛋白存在免疫交叉反應，對牛乳β-乳球蛋白過敏的人群也可能對水牛乳β-乳球蛋白過敏。因此，從食物過敏的角度探索水牛乳β-乳球蛋白具有一定理論價值。另外，由于水牛乳消費人群逐漸增加，為了保護過敏患者的健康，開展水牛乳的過敏研究將具有重要現(xiàn)實意義。 本研究的主要內(nèi)容包括水牛乳β-乳球蛋白的分離純化、兔抗水牛乳β-乳球蛋白多克隆抗體的制備以及水牛乳β-乳球蛋白間接競爭ELISA檢測方法的建立。 在水牛乳β-乳球蛋白的分離純化過程中，建立了DEAE-Sepharose Fast Flow離子交換結(jié)合Sephadex G-75凝膠層析分離純化水牛乳β-乳球蛋白的方法。SDS-PAGE和IEF-PAGE鑒定結(jié)果表明，兩種方法結(jié)合可以分離得到純度＞90％的水牛乳β-乳球蛋白。 在抗水牛乳β-乳球蛋白多克隆抗體的制備過程中，建立了間接ELISA檢測水牛乳β-乳球蛋白抗血清效價的檢測方法及制備抗水牛乳β-乳球蛋白多克隆抗體的程序。免疫2只日本大白兔分別獲得了間接ELISA檢測效價高達1：204800和1：102400及雙向瓊脂擴散檢測效價均為1：16的抗血清。通過雙向瓊脂擴散試驗證實了水牛乳β-乳球蛋白與牛乳β-乳球蛋白存在免疫交叉反應。 建立了檢測水牛乳β-乳球蛋白的間接競爭ELISA方法，確定的工作條件如下：包被抗原濃度2.5μg／mL，抗體稀釋倍數(shù)為1：60000；二抗稀釋倍數(shù)為1：10000。包被條件為4℃過夜；37℃封阻1h；孵育條件為37℃，1h；加終止液5min后比色。該方法檢測的范圍為0.1ng／mL～100ng／mL，，靈敏度為5.75ng／mL，最低檢出限＜0.01 ng／mL。用建立的方法對4份樣品進行了檢測，測得陽光酸酸乳、陽光核桃花生乳、皇氏水牛乳、壯牛水牛乳的β-乳球蛋白含量分別為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

本文編號：2245202