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目的制备能够识别多种β-内酰胺酶结构的广谱特异性抗体,并初步建立用于检测牛奶中违法添加β-内酰胺酶的间接竞争酶联免疫分析方法(ELISA)。方法以混合β-内酰胺酶作为全抗原,免疫动物获得β-内酰胺酶广谱特异性多克隆抗体,分离纯化后进行详细表征,确定最佳使用条件,并开发适用于牛奶样品中β-内酰胺酶检测的免疫分析方法。结果获得了能够识别多种β-内酰胺酶的多克隆抗体,血清中抗体浓度达到17.9～18.9mg/ml,抗体重链和轻链分子量分别为55和25kD,总分子量为160kD,亲和常数为3.6×108L/mol,以该抗体为基础建立了检测β-内酰胺酶的间接竞争ELISA法,方法检出限为0.2ng/ml,线性范围0.2～200ng/ml,平均回收率在80%～115%之间,相对标准偏差小于20%。结论获得了β-内酰胺酶多克隆抗体,并建立了间接竞争ELISA方法测定牛奶中β-内酰胺酶含量。
OBJECTIVE: To prepare a broad-spectrum specific antibody capable of recognizing various β-lactamase structures and to establish an indirect competitive ELISA method for the detection of milk β-lactamase. Methods The β-lactamase-specific polyclonal antibody was obtained by immunization with mixed β-lactamase. The polyclonal antibody was isolated and purified. The optimal conditions were determined. Immunoassay for lactamase detection. Results A polyclonal antibody capable of recognizing various β-lactamases was obtained. The antibody concentration in the serum reached 17.9-18.9 mg / ml. The heavy and light chains of the antibodies were 55 and 25 kD, respectively, with a total molecular weight of 160 kD. The affinity constants Was 3.6 × 108 L / mol. The indirect competitive ELISA method was established for the detection of β-lactamase based on this antibody. The detection limit was 0.2 ng / ml and the linear range was 0.2 ~ 200 ng / ml with the average recovery of 80% ~ 115%, the relative standard deviation of less than 20%. Conclusion The β-lactamase polyclonal antibody was obtained, and indirect competitive ELISA was established to determine β-lactamase in milk.