山羊IFN-γ免疫血清制备及抗体ELISA检测方法的建立

Preparation of Goat IFN-γImmune Serum and Establishment of an ELISA for Detecting Antibodies

摘要：为制备山羊干扰素-γ（IFN-γ）免疫血清并建立山羊IFN-γ抗体ELISA检测方法，以原核表达的山羊γIFN重组蛋白（rIFN-γ）为材料，免疫小鼠制备免疫血清；通过对测定不同的抗原包被浓度及包被时间，酶标抗体稀释度，免疫血清的孵育时间等条件的优化建立检测抗体效价的ELISA检测方法，并采用建立的方法测定免疫血清的抗体效价。结果显示，rIFN-γ抗原包被浓度为10μg／mL，4℃包被12h；酶标抗体稀释度为1：5000；免疫血清孵育时间为60min，可以得到最佳ELISA的检测结果。重复试验显示该方法变异系数（CV）在1．5％～5％之间。采用建立的方法测得免疫小鼠免疫血清效价为10^7。该ELISA检测方法灵敏高，稳定性好，为山羊IFN-γ抗体检测提供了技术支持。

In order to prepare immune serum against goat IFN-γ and develop a method of ELISA, Balb/c mice were immunized with recombinant goat IFN-γ （rIFN-γ） expressed prokaryotically to produce immune serum, and then the coating concentration and time of rIFN-γ, the HRP-IgG dilution, and incubation time of the immune serum were all determined to optimize the ELISA method. And the potency of immune serum was also detected. The results suggested that an optimized ELISA result could be acquired when rlFN-γ was coated at 4℃ for 12 h in a concentration of 10 μg/mL, HRP-IgG was diluted at 1 ： 5 000, and the immune serum was incubated for 60 min. The repeated experiments suggested that the coefficient of variation （CV） varied between 1.5% and 5%. The immune serum potency was proved to be 10^7. This ELISA method with high sensitivity and stability could provide a strong requirement for goat IFN-γ detection.