Two-photon excitation fluorometric measurement of homogeneous microparticle immunoassay for C-reactive protein.
Recent developments in infrared laser technology have enabled the design of a compact instrumentation for two-photon excitation microparticle fluorometry (TPX). The microparticles can be used in immunoassays as the antibody-coated solid phase to capture an antigen and then detect it with a fluorescently labeled tracer antibody. Unlike most other methods, TPX technology allows low-volume, homogeneous immunoassays with real-time measurements of assay particles in the presence of a moderate excess of fluorescent tracer. In this study, the TPX assay system was used for the reagent characterization and the measurement of C-reactive protein (CRP) in diluted plasma samples, targeting the assay range useful in infectious disease diagnosis. The pentameric structure of the CRP permitted the optimization of an assay with the lowest detectable concentration of 1 microg/L (7.5 pM) by using a single monoclonal antibody both for capture and as the tracer. With a 1:200 predilution of samples, the measurement range of the assay was 1-150 mg/L, but an additional 1:10 dilution was required for higher concentrations. The TPX method showed a good correlation with the reference result obtained in a routine hospital laboratory, demonstrating the feasibility of the technology for immunodiagnostic applications.
Waris
Matti E
ME
Laboratory of Biophysics, Institute of Biomedicine, Turku University Central Hospital, University of Turku, P.O. Box 123, FIN-20521, 20520 Turku, Finland. matti.waris@utu.fi
Meltola
Niko J
NJ
Soini
Juhani T
JT
Soini
Erkki
E
Peltola
Olli J
OJ
Hänninen
Pekka E
PE
D000911:Antibodies, Monoclonal / Q000737:chemistry; D002097:C-Reactive Protein / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D002138:Calibration; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007118:Immunoassay; D007700:Kinetics; D010316:Particle Size; D017785:Photons; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D002097:C-Reactive Protein