Sensitive and rapid quantification of C-reactive protein using quantum dot-labeled microplate immunoassay

Abstract

Abstract Background High-sensitivity C-reactive protein (hs-CRP) assay is of great clinical importance in predicting risks associated with coronary heart disease. Existing hs-CRP assays either require complex operation or have low throughput and cannot be routinely implemented in rural settings due to limited laboratory resources. Methods We developed a novel hs-CRP assay capable of simultaneously quantifying over 90 clinical samples by using quantum dots-labeled immunoassay within a standard 96-well microplate. The specificity of the assay was enhanced by adopting two monoclonal antibodies (mAbs) that target distinct hs-CRP epitopes, serving as the coating antibody and the detection antibody, respectively. In the presence of hs-CRP antigen, the fluorescence intensity of the mAb-Ag-mAb sandwich complex captured on the microplate can be read out using a microplate reader. Results The proposed hs-CRP assay provides a wide analytical range of 0.001-100 mg/L with a detection limit of 0.06 (0.19) μg/L within 1.5 h. The accuracy of the proposed assay has been confirmed for low coefficient of variations (CVs), 2.27% (intra-assay) and 8.52% (inter-assay), together with recoveries of 96.7-104.2%. Bland-Altman plots of 104 clinical samples exhibited good consistency among the proposed assay, commercial high-sensitivity ELISA, and nephelometry, indicating the prospects of the newly developed hs-CRP assay as an alternative to existing hs-CRP assays. Conclusion The developed assay meets the needs of the rapid, sensitive and high-throughput determination of hs-CRP levels within a short time using minimal resources. In addition, the developed assay can also be used to detect and quantify other diagnostic biomarkers by immobilizing specific monoclonal antibodies.

Keywords

• Cardiovascular disease
• C-reactive protein
• Fluoroimmunoassay
• Quantification
• Quantum dots