抗MRSA-PBP2a鸡卵黄抗体的制备及乳胶凝集检测法的建立

制备抗耐甲氧西林金黄色葡萄球菌青霉素结合蛋白2a( MRSA- PBP2a)抗原的鸡卵黄免疫球蛋白(IgY),建立检测MRSA的乳胶凝集方法.采用体外诱导的方法制备PBP2a蛋白,胸部肌肉多点注射方式免疫6只海蓝蛋鸡,水稀释法提取IgY,BCA法测定蛋白含量,Western blotting进行特异性分析,用提取的IgY抗体致敏聚苯乙烯乳胶,建立检测PBP2a的乳胶凝集方法.成功诱导并制备获得纯化的PBP2a蛋白,首次免疫后1月每枚鸡蛋提纯后可获得约48 mg IgY抗体,Western blotting结果显示IgY抗体能有效识别纯化的PBP2a蛋白；成功建立检测PBP2a的乳胶凝集法,敏感性达1 mg/L.抗MRSA- PBP2a鸡卵黄抗体具有较高的敏感性和特异性,基于其建立的乳胶凝集检测方法具有较好的灵敏性.

Preparation of Chicken Egg Yolk Antibodies against MRSA-PBP2a and the Establishment of a Emulsion Agglutination Test Method

In order to prepare chicken egg yolk immunoglobulin （IgY） against methicillin resistant Staphylococcus aureus penicillin binding protein 2a （MRSA-PBP2a） and to establish a emulsion agglutination test （EAT） method to detect MRSA. The PBP2a was prepared adopting induction in vitro by immunizing six sea blue layers intramuscular multi-pointed injection on their breasts. And the IgY was extracted with water-dilution method; the protein content was measured by BCA method, and analyzed by Western blotting to evaluate the specificity： Polystyrene emulsion was sensitized with the extracted IgY antibody to establish EAT method to detect PBP2a. The results showed that the PBP2a was induced successfully. Approximately 48 mg IgY was extracted, obtained, and purified from each egg one month after the first immunization. The results of Western blotting demonstrated that the IgY antibody could be effectively recognized PBP2a. The sensitivity of the successfully established PBP2a EAT method was as high as 1 mg/L. Therefore, chicken egg yolk immunoglobulin IgY against MRSA-PBP2a possessed fairly high specificity and sensitivity, and EAT method based on it possessed fine sensitivity.