吴鹏等

[摘要]目的 通过免疫母鸡制备抗人Ⅳ型胶原多克隆抗体（IgY），为检测人血清Ⅳ型胶原含量提供IgY。 方法 采用人胎盘来源的Ⅳ型胶原蛋白作为抗原，免疫Leghorn母鸡，采用PEG方法分离纯化鸡卵黄中的抗体，获得抗体粗提物。经Sephadex G100凝胶过滤制备多克隆抗体纯品。使用SDS-PAGE、免疫琼脂双向扩散等方法对提取物的含量、纯度及特异性进行鉴定。 结果 电泳结果显示，抗体粗提物纯度为81%。抗体纯品仅在70kd和25kd处各有一条带，纯度达到96%；免疫琼脂双向扩散实验结果显示，抗体纯品、粗品及免疫三周后鸡血清均与中心孔的抗原形成抗原抗体复合物的乳白色沉淀线。 结论 本研究分离纯化方法制备的抗Ⅳ型胶原IgY，具有抗体纯度高、特异性强等特点，为多克隆抗体广泛应用奠定了基础。

[关键词] IgY；Ⅳ型胶原；多克隆抗体

[中图分类号] R392 [文献标识码] A [文章编号] 2095-0616（2014）10-27-04

[Abstract] Objective To detect the content of collagen type Ⅳ in human serum， and prepare polyclonal antibody （IgY） against it by immunized hens. Methods The collagen typeⅣ from human placenta was used as antigen to immunize Leghorn hens. It was used the PEG method to prepare antibody-IgY from the chicken egg yolk and obtain its crude extraction. The purity of the polyclonal antibody（IgY） was obtained by Sephadex G-100 gel filtration column. The content， purity and specificity of extract were measured with SDS-PAGE and double agar gel immune-diffusion test. Results The result of electrophoresis showed that the purity of antibody crude extraction was 81%， The purified IgY were shown two straps and the molecular weight（MW） of them were estimated to be approximately 70kD and 25kD respectively， its purity was 96%； the result of double agar gel immune-diffusion test showed that such as the purification and crude extraction of antibody and chicken sera after immunization three weeks， all of product were formed milky white precipitation lines of antigen-antibody complexes with antigen in the center hole. Conclusion In this study， the IgY against human collagen type Ⅳ with high purity and strong specificity was produced by separation and purification methods， which afford a theoretical foundation for polyclonal antibodies widely application.

[Key words] IgY； Collagen type Ⅳ； Polyclonal antibody

近年来，用于疾病研究、诊断的抗体主要来源于哺乳动物的单克隆抗体和多克隆抗体[1]。由于多克隆抗体在制备和纯化方面较单克隆抗体简单，且可在完成一次免疫后连续获得大量抗体。因此，多克隆抗体在疾病的诊断方面已经广泛应用[2]。IgY是禽类血清中的主要免疫球蛋白，鸡经特异性抗原免疫后，在卵黄中可富集特异性抗体（IgY）[3]。卵黄中的IgY纯度高，有较强的特异性。

1.8 IgY特异性鉴定

采用免疫琼脂双向扩散方法[7]。1.2%盐水琼脂糖制备琼脂板；待琼脂凝固后，打孔器打孔，孔径 4mm，孔距 15mm；加样，1孔加生理盐水对照、2孔加入抗体纯品、3孔加抗体粗品、4孔加入血清（鸡免疫后3周），每孔加样品20μL；……