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

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

30 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)120-125
Number of pages6
JournalAnalytical Biochemistry
Issue number1
Publication statusPublished - 1992 Apr
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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