Ultrarapid, ultrasensitive one-step kinetic immunoassay for C-reactive protein (CRP) in whole blood samples: measurement of the entire CRP concentration range with a single sample dilution.
BACKGROUND
Recently, measurement of very low concentrations of C-reactive protein (CRP) has gained popularity as a potential new means for predicting the risk of future cardiac complications. In this study, we demonstrate the feasibility of a kinetic, one-step microparticle assay for quantitative determination of extremely low and high CRP concentrations in the limited timeframe typical for point-of-care testing.


METHODS
A noncompetitive, kinetic CRP immunoassay was developed that uses individual, porous microparticles as the solid phase. The microparticles were covalently coated with a monoclonal capture antibody, and the monoclonal detection antibody was labeled with europium. The one-step binding reaction was stopped by washing after 2 min of incubation, and the fluorescence signal of individual particles was measured.


RESULTS
The analytical detection limit (mean of zero calibrator + 3 SD) was 0.00016 mg/L CRP. Clinical samples were diluted 400-fold before assay to cover the CRP concentration range of 0.064-1200 mg/L. The assay correlated well with the Dade Behring N High Sensitivity CRP assay (for 0-10 mg/L, r = 0.969, S(y/x) = 0.68, n = 54; for 0-350 mg/L, r = 0.969, S(y/x) = 11.7, n = 100). The within- and between-run CVs based on calculated concentrations were, respectively, 9-16% and 14% at 0.11 mg/L, 4.5-12% and 8.2% at 4.2 mg/L, and 3.5-6.3% and 4.4% at 105 mg/L, with a CV <15% at 0.2 mg/L and above.


CONCLUSIONS
Use of the kinetic microparticle approach combined with time-resolved fluorometry allows ultrasensitive quantification of CRP in whole blood in 2 min with a linear assay range spanning more than four orders of magnitude.
Tarkkinen
Piia
P
Department of Biotechnology, University of Turku, Tykistökatu 6A, 6th Floor, FIN-20520 Turku, Finland. piia.tarkkinen@utu.fi
Palenius
Tom
T
Lövgren
Timo
T
D000911:Antibodies, Monoclonal; D002097:C-Reactive Protein / Q000032:analysis*; D004852:Epoxy Compounds; D006801:Humans; D007118:Immunoassay; D007700:Kinetics; D010316:Particle Size; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D004852:Epoxy Compounds; D002097:C-Reactive Protein