Tu162 - Clinical Quantification of Cardiovascular Disease Marker C-reactive Protein Using Radioimmunoassay and ELISA Immunoassay

Abstract Text: C-reactive protein (CRP) is an acute-phase protein synthesized in the liver, an inflammatory marker. CRP levels in the blood reflect acute or chronic inflammation; recently, CRP has become a potent cardiovascular marker. However, detection methods are not sensitive enough. Hence, sensitive radioimmunoassay and direct ELISA for CRP have been developed. We optimized two sets of standards for CRP radioimmunoassay: (1) a high standard range (0-6400 ng/mL), which can measure up to 1,60,000 ng/mL. (2) a highly sensitive assay (0-200 ng/mL), which can measure the CRP level in human serum as low as 3 ng/mL. CRP was radiolabelled using the chloramine-T method. A magnetizable cellulose particle-based separation system was adopted, which is user-friendly with minimum pipetting steps and an incubation time of 1 h at room temperature. The anti-CRP antibody was conjugated with HRP using the sodium periodate oxidation method for direct ELISA. Optimized assay protocol: Add 100 µL standards (0 to 100 ng/mL) to each well and incubate for 2 hrs at RT, then the plates were washed. The blocking was carried out for 30 minutes, followed by washing. Then, add 100 μL of anti-CRP-pAb-HRP to each well and incubate for 1 hr. Afterwards, 50 µL TMB was added to wells, 0.2M H2SO4 was added to stop the reaction, and the absorbance was measured at 450 nm to plot the standard graph. Optimized assays were analyzed and validated for assay sensitivity, precision, dilution linearity, recovery, and cross-reactivity. The assay covers the clinical range of human CRP analysis for clinical tests.