以兔抗体偶联玻璃棒为固相的大分子抗原酶联夹心免疫测定法。

Enzyme-linked sandwich immunoassay of macromolecular antigens using the rabbit antibody-coupled glass rod as a solid phase.

作者信息

Hamaguchi Y, Kato K, Fukui H, Shirakawa I, Okawa S

出版信息

Eur J Biochem. 1976 Dec 11;71(2):459-67. doi: 10.1111/j.1432-1033.1976.tb11134.x.

DOI:10.1111/j.1432-1033.1976.tb11134.x

Abstract

A highly sensitive sandwich immunoassay of macromolecular antigens using the rabbit antibody Fab' - beta-D-galactosidase complex and the rabbit antibody immunoglobulin-G-coupled glass rod as a solid phase is described. The Fab' fragments of rabbit antibody IgG are conjugated with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide. Rabbit antibody IgG is coupled to the aminoalkylsilyl glass rods (3 mm in diameter and 5 mm in length) using glutaraldehyde. A wide range of the concentrations of rabbit IgG fraction (20-2000 mug/ml) is effective for coupling, and the amount of rabbit immunoglobulin G coupled can be controlled. The smallest amounts of ornithine delta-aminotransferase from rat liver, human immunoglobulin G and 2,4-dinitrophenyl human immunoglobulin G that can be determined are 0.03, 0.3 and 0.04 fmol, respectively. The sensitivity of the assay for these antigens is affected mainly by the non-specific binding of the complexes to the solid phase and the ability of antigen molecules, adsorbed on the solid phase, to bind specifically the complexes. The assay with the rabbit antibody immunoglobulin-G-coupled glass rods is simpler and more sensitive than that with the rabbit antibody immunoglobulin-G-coupled Sepharose 4B.

摘要

描述了一种使用兔抗体Fab'-β-D-半乳糖苷酶复合物和兔抗体免疫球蛋白G偶联玻璃棒作为固相的大分子抗原高灵敏度夹心免疫测定法。兔抗体IgG的Fab'片段使用N，N'-邻苯二甲酰亚胺与大肠杆菌的β-D-半乳糖苷酶缀合。兔抗体IgG使用戊二醛偶联到氨基烷基硅烷玻璃棒（直径3mm，长度5mm）上。宽范围的兔IgG级分浓度（20-2000μg/ml）对偶联有效，并且可以控制偶联的兔免疫球蛋白G的量。大鼠肝脏鸟氨酸δ-氨基转移酶、人免疫球蛋白G和2,4-二硝基苯基人免疫球蛋白G的最小可测定量分别为0.03、0.3和0.04fmol。这些抗原测定的灵敏度主要受复合物与固相的非特异性结合以及吸附在固相上的抗原分子特异性结合复合物的能力影响。使用兔抗体免疫球蛋白G偶联玻璃棒的测定比使用兔抗体免疫球蛋白G偶联琼脂糖4B的测定更简单、更灵敏。