Using microparticle labeling and counting for attomole-level detection in heterogeneous immunoassay

Kazunori Okano*, Satoshi Takahashi, Kenji Yasuda, Daizo Tokinaga, Kazumichi Imai, Masataka Koga

*この研究の対応する著者

研究成果: Article査読

29 被引用数 (Scopus)

抄録

A new heterogeneous "sandwich" immunoassay utilizing microparticles as labels to realize high sensitivity is described. In this method, antibody fixed on the microparticles reacts with antigen previously trapped on a microplate surface, which makes the antigen molecules visible and countable with an inverted optical microscope. The method is highly sensitive because the reacted single microparticle, therefore single antigen molecule, can be detected. The sensitivity depends both on the reaction efficiency of the immunoreaction and on nonspecific adsorption of the microparticles on the microplate surface. Therefore, the protocol for preparing microparticle having antibody on the surface and a microplate having capture antibody was investigated to realize high sensitivity. Carboxylated microparticles of 0.76 μm in diameter were conjugated with affinity-purified antibody using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. It was determined that 1 g microparticles had 880 μg antibody (approximately 1100 antibody molecules per 1 microparticle). The immunoreaction efficiency reached 18% at 1 × 10-13 mol/liter antigen concentration. The lower detection limit was 3.1 × 10-14 mol/liter (1.6 amol) using human α-fetoprotien as a model antigen.

本文言語English
ページ(範囲)120-125
ページ数6
ジャーナルAnalytical Biochemistry
202
1
DOI
出版ステータスPublished - 1992 4
外部発表はい

ASJC Scopus subject areas

  • 生物理学
  • 生化学
  • 分子生物学
  • 細胞生物学

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