Human C-Reactive Protein/CRP Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human CRP Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human CRP in cell culture supernates, serum, and plasma. It contains NS0-expressed recombinant human CRP and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human CRP showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring human CRP.

Preparation and Storage

Background: C-Reactive Protein/CRP

C-Reactive Protein (CRP), also known as Pentraxin 1, is a non-glycosylated protein in the Pentraxin family that also includes Pentraxin 2/SAP and Pentraxin 3/TSG-14. CRP functions as a sensor and activator of the innate immune response (1). In humans, it is a major acute-phase protein; its circulating concentration is dramatically elevated at the onset of inflammation (2). In mice, however, serum CRP levels increase only slightly during inflammation, and the analogous acute phase role is filled by Pentraxin 2 (3). CRP assembles non-covalently into a 110-120 kDa cyclical pentamer (4). Mature human CRP shares 71% and 64% amino acid (aa) sequence identity with mouse and rat CRP, respectively (5). 

CRP binds and opsonizes apoptotic cells (6-8) as well as bacteria such as S. pneumoniae (9, 10). It subsequently enhances the phagocytosis of these opsonized cells (6, 8-10). CRP additionally binds several proteins in the complement cascade including C1q, C4BP, and Factor H (8, 11-13). It enhances activation of the classical complement pathway and the deposition of C3b (9). In later stages of the response, CRP inhibits complement-mediated cell lysis through its binding to C4BP and Factor H (8, 12). These interactions induce the upregulation of complement inhibitory proteins CD46, CD59, and CD55/DAF and inhibit assembly of the membrane attack complex (MAC) (8, 14). 

CRP binds to Fc gamma RI, Fc gamma RIIA, and Fc gamma RIIB on macrophages and dendritic cells (15-17), and Fc receptors are required for the phagocytosis of CRP-opsonized target cells (6, 10, 18). CRP binding to Fc gamma RI induces Src activation which subsequently triggers the inhibitory Fc gamma RIIb and dampens the inflammatory response (15, 19). CRP additionally promotes dendritic cell maturation and humoral immunity (10). In cardiovascular disease, CRP binds to oxidized LDL, exacerbates tissue damage in coronary artery infarction, and inhibits the repair of injured vascular endothelium (7, 19, 20).

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.