Development of ELISA on microplate for serum C-reactive protein and establishment of age-dependent normal reference range

Abstract

Background: C-reactive protein (CRP), a marker of systemic inflammation, has been proposed to predict outcome in patients with unstable angina; and elevated levels of CRP were found to be associated with an increased risk of coronary events. Methods: Two enzyme-linked immunosorbent assays (ELISA) of different sensitivities were developed on microplate for CRP. Both ELISA established used Dako polyclonal anti-CRP antibody for coating and Dako horse radish peroxidase (HRP)-conjugated polyclonal anti-CRP antibody for detection. Results: The sensitivity of the high and regular sensitivity ELISA was 0.16 and 0.6 mg/l, respectively. Our assays demonstrated an excellent correlation with commercial CRP assays performed on a Behring Nephelometer Analyzer II (BNII) at both regular and ultrasensitive levels, with both correlation coefficients above 0.98 and slopes of approximately 1. Using our microplate assays, we established normal reference value for serum CRP. Based on ANOVA statistical test, we found that the mean±S.D. was 1.3±1.27 mg/l ( n=202) for normal individuals of 50–80 years and 0.43±0.42 mg/l ( n=148) for the group of 20–50 years. Conclusions: The normal serum CRP mean concentrations for two age groups were distinctively different ( p value<0.001). Our study suggests two different normal cutoffs of serum CRP to be employed for individuals in different age groups.