MODIFICATION, OPTIMIZATION AND SIMPLIFICATION OF THE SPOT ELISA TECHNIQUE FOR THE ENUMERATION OF CELLS SECRETING ANTIHAPTEN ANTIBODIES
MODIFICATION, OPTIMIZATION AND SIMPLIFICATION OF THE SPOT ELISA TECHNIQUE FOR THE ENUMERATION OF CELLS SECRETING ANTIHAPTEN ANTIBODIES
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DOI：
10.1016/0022-1759(90)90438-2
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发表时间：
1990-05-25
影响因子：
2.2
通讯作者：
DINTZIS, HM
中科院分区：
文献类型：
--
作者：
GREENE, G;HODOUS, J;DINTZIS, HM
我们在这里描述了一个修改的斑点ELISA检测和计数的抗体分泌细胞。该修饰提高了灵敏度、方便性和简单性，同时优化了时间、温度和试剂的参数。通常用于标准ELISA测定的实验室试剂和96孔微量滴定板适用于斑点ELISA测定。特别强调的是放置在检测抗半抗原IgM或IgG生产的脾细胞在刺激的范围内的剂量的抗原。此外，描述了两个程序，其中斑点ELISA测定被改变以产生可测量的光密度：（1）溶解斑点EILSA测定中获得的蓝色沉淀，并测量所得蓝色的光密度，和（2）用标准ELISA底物代替斑点ELISA底物以产生有色溶液而不是沉淀。这两种方法都显示与斑点数量相关，因此可以补充或替代斑点计数（由抗体分泌细胞产生），从而加速和机械化测定。
We describe here a modification of the spot ELISA assay for the detection and enumeration of antibody-secreting cells. The modification increases sensitivity, convenience and simplicity while optimizing parameters of time, temperature and reagents. Laboratory reagents and 96 well microtiter plates commonly used for the standard ELISA assay are adapted for use in the spot ELISA assay. Special emphasis is placed on the detection of anti-hapten IgM or IgG production by splenocytes in response to stimulation by a range of doses of antigen. In addition, two procedures are described whereby the spot ELISA assay is altered to yield a measurable optical density: (1) the blue precipitate obtained in the spot EILSA assay is dissolved and the optical density of the resultant blue color is measured, and (2) a standard ELISA substrate is substituted for the spot ELISA substrate to yield a colored solution rather than a precipitate. Both of these are shown to correlate with spot numbers and can therefore supplement or substitute for the enumeration of spots (produced by antibody-secreting cells), thus speeding and mechanizing the assay.