C-Reactive Protein (hsCRP) EIA Kit From ALPCO Diagnostics

The CRP EIA (ELISA) Kit is a highly sensitive sandwich assay designed to determine the amount of C-Reactive Protein in serum, plasma, feces and urine. C-Reactive Protein (CRP) is produced by hepatocytes and is present during times of acute inflammation. CRP in healthy individuals is usually <5 µg/ml. As an acute phase protein, CRP is released within 24 hours after induction of an inflammatory process (fever, infection, myocardial infarction, etc.). Thus, CRP is a confirmed biomarker of inflammation.

The kit components include a holder, removable precoated microtiter strips (wells coated with polyclonal antibodies directed against CRP), ELISA wash buffer, prediluted rabbit-anti-CRP peroxidase-labeled antibody, 6 ready-to-use calibrators (standards), a control, dilution buffer, TMB substrate, and ELISA stop solution. The chance of false-negatives is considerably lowered due to the use of a combination of two specific antibodies, thereby presenting a reliable more diagnostic assay.

The total assay time, not including sample dilution, pipetting, and wash times, is approximately 2 hours and 15 minutes. We used this kit specifically for CRP measurement in human serum. According to the manufacturers’ instructions, serum and plasma samples must be diluted 1:100 or 1:500 prior to performing the assay. We found 1:100 an ideal dilution for our serum samples. Overall, the protocol is straightforward and permits multiple samples to be processed at one time. Use caution with the microtiter strips, which can break easily. Sample size is only 10 µl per well. Standards range from 1.9 – 150 ng/ml, with a sensitivity of 0.124 ng/ml. Initially, the precoated microtiter strips were washed 5 times with ELISA wash buffer. Samples, standards, and controls were pipetted in duplicate (which proved to be sufficient), and the plate was then incubated at room temperature for 1 hour on a horizontal shaker. Wells were aspirated and washed 5 times in ELISA wash buffer. Next, the pre-diluted peroxidase-labeled CRP antibody was added to the wells, followed again by a 1 hour incubation as before. After decanting plate contents, wells were washed again 5 times with ELISA wash buffer. TMB substrate solution was added to each well and the plate was incubated in the dark at room temperature for 10 minutes. Color developed quite well within this time frame. Stop solution was then added into each well and mixed briefly. The extinction of the samples was measured at 450 nm using an absorbance plate reader.

We analyzed over 200 human serum samples, and found our results were well within range. The kit was easy to use, the instructions were clear and detailed, and the short time needed to perform the assay itself was a plus. We received a 5% volume discount, which helped offset the cost of the assay. This kit is priced slightly higher than some other CRP ELISA kits on the market, but because of its citation in reputable journals, we chose it and were pleased that we did.