狂犬病毒核蛋白抗原ELISA定量法的建立及初步验证

Development of an ELISA to quantitatively determine the nucleoprotein of rabies virus

目的建立检测狂犬病疫苗各工序产品中核蛋白抗原含量的双抗体夹心ELISA法。方法以兔抗狂犬病毒多抗为包被抗体,辣根过氧化物酶标记的抗狂犬病毒核蛋白单抗作为酶标记抗体,对狂犬病毒核蛋白抗原含量进行定量测定,并对该方法进行初步验证。结果此方法线性相关系数大于0.97;最佳线性范围为0.000625～0.01IU/ml,定量限度为0.000625IU/ml;准确性为102%～109%;变异系数(CV)为7.2%～9.4%;与小牛血清、牛血清清蛋白(BSA)、卵清蛋白(OVA)、流感疫苗纯化液、乙脑疫苗纯化液、甲肝疫苗纯化液等均无交叉反应。结论该方法特异性强,灵敏度高,准确性、重复性和稳定性好,可用于狂犬病疫苗各工序产品中核蛋白抗原的定量检测。

Objective To develop a double-antibody sandwich ELISA for determining the concentration of nucleoprotein （NP） of rabies virus in various products of rabies vaccine. Methods The purified rabies antibodies from a rabbit were used to coat microwell plates. Horseradish peroxidase-conjugated anti-NP monoclonal antibody was used to probe the NP bound to coated antibodies. The assay was used to quantitatively determine the concentration of NP of rabies virus. Results The results showed that the coefficient of linear correlation was higher than 0.97. The optimal linear range was 0. 000625 - 0.01 IU/ml and the detection limit was 0. 000625 IU/ml. The recovery rate was 102 - 109% and the coefficient of variation was only 7.2% - 9.4%. No cross reactions were observed with bovine serum, bovine serum albumin, ovalbumin, refined solution of influenza vaccine, encephalitis B vaccine, and hepatitis A vaccine. Conclusions The results indicated that the assay is specific, sensitive, accurate, reproducible, and stable, and could be suitable for quantitative determination of different rabies vaccine＇s processes products.