In order to evaluate the possibility of using IgY as the secondary antibody in immunoassay, specific IgY (1: 128,000) was generated by immunizing hens with mouse serum IgG purified by protein A column. IgY was extracted from egg yolk by polyethylene glycol 6000 (PEG-6000), and further purified using protein M affinity chromatography column. The purified IgY was conjugated with horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC), in that order. The reactivity of conjugated antibodies was evaluated by ELISA, Western blot and Immunofluorescence, demonstrating that the obtained IgY was able to conjugate with enzymes, react with mouse primary IgG antibody, and subsequently amplify the antigen-antibody signals in different immune reaction conditions, in a comparable secondary effect to conventional goat anti-mouse IgG antibody. The obtained conjugated antibodies showed high stability in broad pH ranges (4–10; >70%) and high thermostability at 37 °C for 84 h (>85%). Despite the need to further consider and evaluate the industrial standardization and production process, our data provided the primary evidence that conjugated IgY antibodies can be used as a secondary antibody for broad immunological analysis.

为了评估在免疫测定中使用IgY作为二抗的可能性，通过用蛋白A柱纯化的小鼠血清IgG免疫母鸡，产生了特异性IgY（1：128,000）。通过聚乙二醇6000（PEG-6000）从蛋黄中提取IgY，并使用蛋白M亲和色谱柱进一步纯化。依次将纯化的IgY与辣根过氧化物酶（HRP）和异硫氰酸荧光素（FITC）缀合。通过ELISA，Western印迹和免疫荧光对偶联抗体的反应性进行了评估，证明了所获得的IgY能够与酶偶联，与小鼠一抗IgG反应，并随后在不同的免疫反应条件下扩增抗原-抗体信号。具有与常规山羊抗小鼠IgG抗体相当的次级效果。获得的偶联抗体在宽pH范围（4-10;> 70％）中显示出高稳定性，并在37°C下持续84 h（> 85％）时具有高热稳定性。尽管有必要进一步考虑和评估工业标准化和生产过程，但我们的数据提供了初步的证据，证明结合的IgY抗体可用作广泛的免疫学分析的第二抗体。