两种酶标抗体制备方法的比较及李斯特菌TASELISA试剂盒性能的优化
Comparison of two methods in preparation of enzyme conjugates and optimization of Listeria monocytogenes TAS-ELISA KIT
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摘要: 研制一种低成本、快速、准确的三抗体夹心酶联免疫（Three antibodies sandwich ELISA,TAS-ELISA）检测试剂盒。采用戊二醛法、改良过碘酸钠法分别制备碱性磷酸酶（Alkaline Phosphatase,AP）、辣根过氧化物酶（Horseradish Peroxidase,HRP）标记的山羊抗兔IgG,比较单核细胞增生性李斯特菌（Listeria monocytogenes,LM）TAS-ELISA试剂盒两种酶标抗体的检测效果,并考察了试剂盒的检测灵敏度、特异性、稳定性、保存周期等指标。结果表明,采用改良过碘酸钠法制备的HRP-IgG效果好,克分子比最高可达2.9,酶结合率达27%;试剂盒检测周期6h,检测成本为3.5元/孔,特异性实验、干扰实验、重复性实验、稳定性实验表明采用该方法制备的酶标抗体,特异性好、结果稳定可靠。自制TAS-ELISA试剂盒检测性能可达到商业化试剂盒水平,为该产品的产业化提供了一定的理论依据。Abstract: To develop a low-cost, rapid and accurate TAS-ELISA KIT.This study preparated AP-IgG and HRP-IgG by glutaraldehyde method and improved periodate method respectively and compared two enzyme conjugates of Listeria monocytogenes TAS-ELISA KIT, further investigated some indexes including the sensitivity, specificity, stability, shelf life.The results showed that HRP-IgG preparated with improved periodate method was more effective, the molar ratios was 2.9, the rate of enzyme-labeled was 27%.The detection time of KIT was 6h, the detection cost was 3.5/well.Specific experiment, interference experiment, repetitive experiment and stable experiment indicated that the HRP-IgG preparated with improved periodate method was specific and reliable.The performance of self-made TAS-ELISA KIT could reach commercial KIT, it also could provide theoretical basis for industrialization of the product.
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