The use of gold reagents to quantitate antibodies eluted from nitrocellulose blots: application to electron microscopic immunocytochemistry.
The use of gold reagents to quantitate antibodies eluted from nitrocellulose blots: application to electron microscopic immunocytochemistry.
复制标题
使用金试剂定量从硝酸纤维素印迹中洗脱的抗体：在电子显微镜免疫细胞化学中的应用。
DOI：
10.1016/0003-2697(89)90472-7
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发表时间：
1989
影响因子：
2.9
通讯作者：
Polans,AS
中科院分区：
文献类型：
--
作者：
Burton,MD;Onstott,LT;Polans,AS
描述了一种测定，其中使用金试剂对从固定在硝化纤维纸上的抗原洗脱的纳克量的抗体进行定量。通过将兔免疫球蛋白 G (IgG) 应用到组装在点印迹矩阵装置中的硝酸纤维素片上来生成标准曲线。使用胶体金或免疫金对印迹进行染色，从而可以通过扫描密度测定法定量 IgG 浓度。获得线性且可重复的标准曲线。使用任一金试剂都可以定量低至 1 ng IgG/点。与胶体金相反，免疫金可以专门用于定量兔 IgG，无论是否存在牛血清白蛋白或从硝酸纤维素印迹共洗脱的抗原。通过分离针对青蛙视网膜杆外节的 RIM 蛋白产生的复合兔抗血清，证明了免疫金测定的适用性。抗 RIM 抗体经过亲和纯化，通过免疫金测定进行定量，随后用于使用嵌入亲水性塑料 LR-Gold 中的视网膜薄片进行免疫细胞化学研究。
An assay is described in which gold reagents were used to quantitate nanogram amounts of antibody that had been eluted from antigens immobilized on nitrocellulose paper. Standard curves were generated by the application of rabbit immunoglobulin G (IgG) to nitrocellulose sheets assembled in a dot blot matrix apparatus. Blots were stained using either colloidal gold or immunogold, enabling quantitation of IgG concentration by scanning densitometry. Linear and reproducible standard curves were obtained. As little as 1 ng IgG/dot could be quantified using either gold reagent. In contrast to colloidal gold, immunogold could be used specifically to quantitate rabbit IgG regardless of the presence of bovine serum albumin or antigen coeluted from the nitrocellulose blot. The applicability of the immunogold assay was demonstrated by fractionating a complex rabbit antiserum raised against the RIM protein of frog retinal rod outer segments. Anti-RIM antibody was affinity-purified, quantitated by the immunogold assay, and subsequently employed in immunocytochemical studies using thin sections of retina embedded in a hydrophilic plastic, LR-Gold.