C-reactive protein (CRP) is a circulating protein mainly secreted from the liver. This acute phase protein consists of five identical non-glycosylated subunits of 23 kDa, that give rise to a symmetrically arranged globular protein with molecular weight of approximately 120 kDa.1 It has long been recognized that CRP is closely related to immunology, inflammation and host defence; as a result it has been used as an inflammatory marker. However, the development of high-sensitivity CRP (hsCRP) ELISA had addressed its role in other clinical issues. There is accumulating evidence suggesting the important role that CRP plays in mediating cardiovascular diseases (CVD) and type 2 diabetes.2-4 Normally CRP is presenting only in a trace amount in circulation (<1 µg/ml)5,6 but can increase over 1,000-fold under acute inflammatory state. Individual with blood CRP levels <1 µg/ml, 1-3 µg/ml and >3 µg/ml is considered to have low, moderate and high risk, respectively, of CVD and myocardial infraction.7 Therefore, blood CRP level has become a promising measure of CVD risk.8,9

This assay is a quantitative sandwich ELISA using monoclonal antibodies against human CRP. The immunoplate is pre-coated with a monoclonal antibody specific for human CRP. Standards and samples are pipetted into the wells and any human CRP present is bound by the immobilized antibody. After washing away any unbound substances, a horseradish peroxidase (HRP)-linked monoclonal antibody specific for human CRP is added to the wells. After wash step to remove any unbound reagents, an HRP substrate solution is added and colour develops in proportion to the amount of human CRP bound initially. The assay is stopped and the optical density of the wells determined using a micro-plate reader. Since the increases in absorbance are directly proportional to the amount of captured human CRP, the unknown sample concentration can be interpolated from a reference curve included in each assay.

A. Typical representation of standard curve

The following standard curve is provided for demonstration only. A standard curve should be generated for each set of sample assay.  

CRP (ng/ml)	Absorbance (450 nm)	Blanked Absorbance
0	0.066	0
0.312	0.125	0.059
0.625	0.183	0.117
1.25	0.317	0.251
2.5	0.545	0.479
5	0.955	0.889
10	1.665	1.599
20	2.498	2.432

 

B. Sensitivity

The lowest level of CRP that can be detected by this assay is 0.312 ng/mL.

 

C. Specificity

The antibody pair used in this assay is specific to human CRP and does not cross-react with mouse and rat CRP, and other cytokine or hormone molecules.

D. Precision

Intra-assay C.V.: 4.3%

Inter-assay C.V.: 5.9%

 

E. Recovery

The recovery of the assay was determined by adding various amounts CRP to a sample. The measured concentration of the spiked sample in the assay was compared to the expected concentration. The average recovery was 98%. 

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