One-step immunoassay for measuring protein concentrations in plasma, based on precipitate-enhanced ellipsometry.
Standard enzyme immunoassays (EIAs) require washing steps to remove excess enzyme-antibody complexes. Such washing is laborious, lengthens assay time, and increases assay scatter. Recently, so-called precipitate-enhanced immunoassays (PEIAs) were introduced. Instead of color formation due to enzymatic conversion of a chromogenic substrate, this technique measures the rate of precipitate formation due to conversion of a substrate with a precipitating product. Such precipitation can be measured in the presence of active enzyme-antibody complexes in the buffer and no washing is required. In the present study this technique was used in a one-step PEIA, without washing steps, for the measurement of plasma concentrations of fatty-acid-binding protein. Horseradish peroxidase was used as tagging enzyme and diaminobenzidine as precipitating substrate. Precipitate formation was measured by ellipsometry. Assay time of the one-step PEIA was much shorter than that for an existing standard EIA. Test results can be obtained within minutes, depending on the sensitivity required. Assay precision of the one-step PEIA was better than that of the standard EIA. In the one-step assay, loss of surface-bound conjugate due to washing is prevented, which could explain part of the improved sensitivity compared to that of the two-step PEIA. More importantly, the presence of substrate-converting enzyme-antibody complexes in the buffer caused a large enhancement of precipitation.
Cardiovascular Research Institute Maastricht, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands. h.speijer@carim.unimaas.nl
Han
H
Speijer
Ria H
RH
Laterveer-Vreeswijk
Jan F C
JF
Glatz
Willem
W
Nieuwenhuizen
Wim T
WT
Hermens
D015415:Biomarkers / Q000097:blood; D011232:Chemical Precipitation; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques; D011506:Proteins / Q000032:analysis*; D012825:Silicon / Q000737:chemistry; D013499:Surface Properties
D015415:Biomarkers; D011506:Proteins; D012825:Silicon