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目的:利用小鼠神经生长因子(mNGF)和纯化的兔抗mNGF IgG,建立mNGF直接竞争酶联免疫检测方法。方法:以亲和纯化兔抗mNGF IgG作为包被抗体,封闭、洗涤后加入用稀释液稀释的不同浓度的标准mNGF或样品液和生物素标记的mNGF混合液,于37℃孵育80 min或室温孵育120 min,洗涤后加入辣根过氧化物酶标记的链酶亲和素,洗涤后加入显色液显色,测定D450nm值,通过标准曲线计算待测样品中mNGF含量。结果与结论:建立了mNGF直接竞争酶联免疫检测方法;该方法的灵敏度约20 ng,变异系数为批内≤10%、批间≤15%,回收率为85%～115%;利用该方法检测了3批样品中mNGF的含量。
OBJECTIVE: To establish a mNGF direct competitive enzyme-linked immunosorbent assay using mouse neural growth factor (mNGF) and purified rabbit anti-mNGF IgG. Methods: Affinity purified rabbit anti-mNGF IgG was used as coating antibody, blocked, washed and then added with different concentrations of standard mNGF or sample solution and biotin-labeled mNGF diluted with diluent, incubated at 37 ℃ for 80 min or room temperature Incubated for 120 min, washed and then added horseradish peroxidase-labeled streptavidin, was added after washing color development, determination of D450nm value, the standard curve calculated by the sample mNGF content. RESULTS AND CONCLUSION: The method of mNGF direct competitive enzyme-linked immunosorbent assay (ELISA) was established. The sensitivity of this method was about 20 ng, the coefficient of variation was within 10% within batch, the difference between batches was less than or equal to 15% and the recovery was between 85% and 115% Three batches of samples were tested for mNGF content.