N-羟乙酰神经氨酸IgY抗体的制备及鉴定

Preparation and Identification of N-glycolylneuraminic Acid IgY Antibody

本试验旨在建立N-羟乙酰神经氨酸(Neu5Gc)IgY抗体的制备及鉴定方法。通过碳二亚胺法将Neu5Gc和载体蛋白进行偶联,制备Neu5Gc人工完全抗原,用制备的完全抗原免疫海兰褐蛋鸡制备Neu5Gc-IgY抗体,经ELISA检测分析鉴定抗体活性。经紫外光谱扫描、琼脂糖凝胶电泳法初步判断Neu5Gc人工完全抗原制备成功,获得的特异性Neu5Gc-IgY抗体经ELISA检测分析,首次免疫后第7天产生抗体,抗体效价呈动态变化,随着免疫次数的增多,血清效价和抗体效价逐步上升,至初次免疫后63d达到高峰,效价达1∶10 000,停止加强免疫后抗体效价可持续一个月左右,1号鸡产生的IgY抗体具有很好的抗原竞争活性,获得竞争回归方程y=30.28x+16.923,线性系数R2=0.9581。以上结果表明,本试验制备的Neu5Gc IgY抗体取得了理想效果,为红肉、牛奶及肿瘤组织中Neu5Gc的检测奠定了基础。

The experiment was carried out to establish a method for preparation and identification of N-glycolylneuramic acid IgY antibody.Neu5 Gc was linked to carrier proteins by carbodiimide（EDC）method.After the immunization of immunogen to laying hens,the IgY antibody was gained.Neu5Gc-IgY was analyzed and identified by ELISA.The results of UV and agarose gel electrophoresis showed that the artificial antigens of Neu5 Gc were successfully synthesized.The development of the IgY antibody titer was monitored by indirect ELISA.The result showed that the IgY antibody was generated at 7th day after the first immunization.The titer was gradually increased and reached its peak（1∶10 000）at 63 th day after the first immunization.The high titer of the IgY antibody maintained through the whole observation period.The sensitivity of antiNeu5 AGc IgY antibody was detected by the competitive blocking ELISA.The linearity about the first chicken of the standard curve showed good,the liner regression equation was y=30.28x＋16.923,R^2=0.9581.The IgY antibody gained in this study laid the foundation for the establishment of indirect ELISA immunoassay method for detecting Neu5 Gc in red meat,milk and tumor tissues.