Use of excess solid-phase capacity in immunoassays: advantages for semicontinuous, near-real-time measurements and for analysis of matrix effects

Anal Chem. 2004 Feb 1;76(3):767-72. doi: 10.1021/ac0302096.


A flow-based immunoassay system using solid-phase particles with high binding capacity was used for semicontinuous, near-real-time, measurement of 17beta-estradiol (E2). The high binding capacity of the solid phase was exploited to enable (i) a quantitative determination of E2 concentration, based on rate of accumulation of fluorescently labeled anti-E2 antibody on the solid phase, and (ii) the use of a single solid phase for more than a dozen competitive binding measurements. The high binding capacity of the solid phase also permitted the immobilization of a second capture antigen. Biotin was immobilized as a second antigen and used to evaluate a biotin anti-biotin system as a control for matrix effects in the E2 immunoassay. In phosphate-buffered saline, E2 could be quantified (in the range of 10-1000 pM) by using either the summation or ratio of the signals from the labeled anti-E2 and anti-biotin antibody in the presence of biotin at a constant concentration. The same referencing system was applied to estimate the matrix effects in selected environmental samples. Matrix effects that inhibited the binding of the anti-E2 antibody to the solid phase led to false positive responses, but these matrix effects could be identified and partially corrected using the response from the anti-biotin antibody.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antibodies, Monoclonal / immunology
  • Antigen-Antibody Reactions
  • Biotin / immunology
  • Environmental Monitoring / methods
  • Estradiol / analysis*
  • Estradiol / immunology
  • Immunoassay / methods*
  • Spectrometry, Fluorescence
  • Water Pollutants, Chemical / analysis


  • Antibodies, Monoclonal
  • Water Pollutants, Chemical
  • Estradiol
  • Biotin