使用聚偏二氟乙烯膜作为固相支持物，通过改进的ELISPOT技术对大鼠体内产生天然抗体的B细胞进行定量分析。

Quantification of natural antibody producing B cells in rats by an improved ELISPOT technique using the polyvinylidene difluoride membrane as the solid support.

作者信息

Schielen P, van Rodijnen W, Tekstra J, Albers R, Seinen W

机构信息

Department of Immunotoxicology, Utrecht University, Netherlands.

出版信息

J Immunol Methods. 1995 Dec 15;188(1):33-41. doi: 10.1016/0022-1759(95)00199-9.

DOI:10.1016/0022-1759(95)00199-9

Abstract

We describe here a new type of solid support for the ELISPOT assay, the PVDF membrane. In parallel tests, spot yields on this membrane were superior to those obtained with the frequently used nitrocellulose (NC) membrane, coated with the same rat anti-IgM and anti-IgG antibodies, incubated with the same rat spleen cell suspensions, and developed with the same combination of AP-labeled conjugates and substrate. We therefore used the PVDF membrane, coated with anti-rat IgM and IgG antibodies, ssDNA or bromelain-treated mouse erythrocytes (BrMRBC) (exposing phosphatidylcholine (PC) as major autoantigen) to develop ELISPOT assays for the quantification of isotype-specific natural antibody secreting cells (ASC) in rats. We confirmed the isotype specificity of the binding of the anti-rat IgM and anti-rat IgG coating antibodies and conjugates with the secreted rat antibodies in this assay, and, by inhibition of spot formation with soluble antigen, their specificity for ssDNA and BrMRBC. An in-house 18-well culture device for the easy manufacture of PVDF-lined culture wells greatly facilitated coating, blocking, and washing procedures, as compared to the original method in 24 well culture plates. This simple, fast, specific and sensitive ELISPOT assay was used to make an inventory of the numbers of natural splenic ASC in Wistar and Fischer rats.

摘要

我们在此描述一种用于酶联免疫斑点分析（ELISPOT）的新型固相支持物——聚偏二氟乙烯（PVDF）膜。在平行试验中，在该膜上的斑点产出优于使用常用的硝酸纤维素（NC）膜时的产出，两种膜均包被相同的大鼠抗IgM和抗IgG抗体，与相同的大鼠脾细胞悬液孵育，并使用相同组合的碱性磷酸酶（AP）标记的结合物和底物进行显色。因此，我们使用包被有抗大鼠IgM和IgG抗体、单链DNA（ssDNA）或菠萝蛋白酶处理的小鼠红细胞（BrMRBC）（暴露磷脂酰胆碱（PC）作为主要自身抗原）的PVDF膜来开发ELISPOT分析方法，以定量大鼠中同种型特异性天然抗体分泌细胞（ASC）。我们在该分析中证实了抗大鼠IgM和抗大鼠IgG包被抗体以及结合物与分泌的大鼠抗体结合的同种型特异性，并且通过用可溶性抗原抑制斑点形成，证实了它们对ssDNA和BrMRBC的特异性。与在24孔培养板中的原始方法相比，一种便于制造PVDF内衬培养孔的内部18孔培养装置极大地促进了包被、封闭和洗涤程序。这种简单、快速、特异且灵敏的ELISPOT分析方法被用于清点Wistar大鼠和Fischer大鼠脾脏中天然ASC的数量。