DBC High Sensitivity C-Reactive Protein (hs-CRP) Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows a typical two-step capture or ‘sandwich’ type assay. The assay makes use of two highly specifi c monoclonal antibodies: A monoclonal antibody specifi c for CRP is immobilized onto the microplate and another monoclonal antibody specific for a different region of CRP is conjugated to horse radish peroxidase (HRP). CRP from the sample and standards are allowed to bind to the plate, washed, and subsequently incubated with the HRP conjugate. After a second washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured on a microtiter plate reader. The intensity of the colour formed by the enzymatic reaction is directly proportional to the concentration of CRP in the sample. 
A set of standards is used to plot a standard curve from which the amount of CRP in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
C-reactive protein (CRP) is a pentameric acute phase reactant that is synthesized by the liver. Its production is controlled primarily by interleukin-6. The serum CRP concentration may increase by up to 1000-fold with infection, trauma, surgery, and other acute infl ammatory events. Chronic infl ammatory disorders such as auto-immune diseases and malignancy can produce persistent high levels of serum CRP. 
Traditionally, CRP has been used clinically for the diagnosis and monitoring of auto-immune and infectous disorders. Recent studies have shown that chronic infl ammation is an important component in the development and progression of atherosclerosis. As a result, increased serum CRP concentration are positively associated with the risk of future coronary events.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.1 mL of serum is required per duplicate determination. Collect 4–5 mL of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

CALCULATIONS 
1. Calculate the mean optical density of each calibrator duplicate. 
2. Calculate the mean optical density of each unknown duplicate. 
3. Subtract the mean absorbance value of the “0” calibrator from the mean absorbance values of the calibrators, controls and serum samples. 
4. Draw a calibrator curve on log-log paper with the mean optical densities on the Y-axis and the calibrator concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curve is recommended. 
5. Read the values of the unknowns directly off the calibrator curve. 
6. If a sample reads more than 10,000 ng/mL, then dilute it with calibrator A at a dilution of no more than 1:10 from the original 1:20 diluted serum (or 1:200 from neat serum). The result obtained should be multiplied by the dilution factor.