Establishment Of A Sandwich Elisa For Quantitative Measurement Of Human High Sensitivity C-Reactive Protein And Its Primary Clinical Application

Background:C-reactive protein(CRP) is a phylogenetically highly conserved plasma protein,with homologs in vertebrates and many invertebrates,that participates in the systemic response to inflammation.Its plasma concentration increases during inflammatory states,a characteristic that has long been employed for clinical purposes.CRP is a pattern recognition, binding to specific molecular configuration that are typically exposed during cell death or found on the surfaces of pathogens.Its rapid increase in synthesis within hours after tissue injury or infection suggests that it contributes to host defense and that it is part of the innate immune response. Recently,an association between minor CRP elevation and future major cardiovascular events has been recognized.Therefor,it is important to establish a high sensitive method for measurement of CRP and it is also a practical work to explore a method with high sensibility.Objective:To establish a sandwich ELISA for quantitative measurement of hs-CRP, and to explore its clinical application.Methods:1.Anti-CRP mAbs prepared by our laboratory were purified by Protein A affinity chromatography and analyzed by SDS-PAGE and Western-blot to test their characteristics.All the mAbs were labeled with horseradish peroxidase by sodium oxidation method.2.Human CRP was isolated from malignant ascites fluid using Immobilized p-Aminophenyl Phosphoryl Choline Gel.The malignant ascites fluid was obtained from cancer patients and the investigation conforms to the principles outlined in the Declaration of Helsinki for use of human tissue or subjects.Purified human CRP was assayed by SDS-PAGE and western-blot analysis.3.Antibody pairs were performed using anti-CRP mAb as coating antibody and HRP labeled anti-CRP mAb as labeled antibody,which optimal concentrations were defined by titration.Standard curve was performed using purified CRP and was judged by sensitivity,reproducibility and recovery rate.4.According to the result of coronary angiography,plasma hs-CRP level was detected in 68 normal patients(without coronary artery stenosis),59 coronary atherosclerotic patients(the severity of coronary artery stenosis＜50%) and 67 coronary artery disease patients(the severity of coronary artery stenosis≥50%).5.Statistics:The data are presented as M±QR.The Kruskal-Wallis and Mann-Whitney tests were performed with the use of SPSS 16.0 statistical software.Statistical significance was accepted at P＜0.05.Results:1.Anti-CRP purified from mice ascites fluid using protein A was in the dipolymer form(55KD,25KD) with no detection of other protein by SDS-PAGE(purity up to 95%) and western-blot.2.CRP purified from malignant ascites fluid using Immobilized p-Aminophenyl Phosphoryl Choline Gel was in the monomeric form (24KD) with no detection of other proteins by SDS-PAGE(purity up to 95%) and western-blot.3.The optimal paired antibodies were anti-CRP mAb 1C10 and HRP labeled anti-CRP mAb 2C11 which optimal concentrations were 10ug/ml and 1:2000,respectively.The sensitivity of this assay was 8.3ng/ml.The coefficients of variation were 3.1%to 9.7%within assay and 3.6%to 13.6%between assays.The recovery rate was 90%to 109%.4.The results showed that the plasma hs-CRP level in coronary atherosclerotic patients was significantly higher than normol patients[(1.15±3.65mg/L) vs(0.74±1.75mg/L)](P＜0.05),the plasma hs-CRP level in coronary artery disease patients was also significantly higher than coronary atherosclerotic patients[(3.29±8.93mg/L) vs(1.15±3.65mg/L)](P＜0.05).Conclusion:A sandwich ELISA assay for detecting hs-CRP was obtained.