Use of excess solid-phase capacity in immunoassays: advantages for semicontinuous, near-real-time measurements and for analysis of matrix effects.
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.
Glass
Thomas R
TR
Department of Bioscience, Central Research Institute of the Electric Power Industry, 1646 Abiko, Abiko City, Chiba, Japan 270-1194.
Saiki
Hiroshi
H
Blake
Diane A
DA
Blake
Robert C
RC
Lackie
Steve J
SJ
Ohmura
Naoya
N
D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001710:Biotin / Q000276:immunology; D004784:Environmental Monitoring / Q000379:methods; D004958:Estradiol / Q000032:analysis* / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D013050:Spectrometry, Fluorescence; D014874:Water Pollutants, Chemical / Q000032:analysis
D000911:Antibodies, Monoclonal; D014874:Water Pollutants, Chemical; D004958:Estradiol; D001710:Biotin