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


CONCLUSIONS
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.
Department of Laboratory Medicine, Southwest Hospital, The Third Military Medical University, Chong Qing 400038, Peoples' Republic of China.
Yang
Y
Luo
Bo
B
Zhang
Ming
M
Chen
Tianlun
T
Jiang
Daiyang
D
Zhou
Junfu
J
Huang
Weiling
W
Fu
D002097:C-Reactive Protein / Q000378:metabolism*; D002138:Calibration; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005453:Fluorescence; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods*; D057230:Limit of Detection; D009391:Nephelometry and Turbidimetry; D045663:Quantum Dots; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D013997:Time Factors
D055767:Immobilized Proteins; D002097:C-Reactive Protein