Calprotectin level analysis in patients with inflammatory bowel disease.

Calprotectin is a protein produced by intestinal neutrophils and monocytes in a situation of intestinal inflammation. Thus, in some inflammatory bowel diseases, such as Crohn’s disease, said cells produce calprotectin, which is released into the lumen of the intestine and excreted together with the faeces.

The amount of calprotectin in feces has proven to be an effective biomarker both in the diagnosis and in the evolution of inflammatory bowel disease, as well as in monitoring the response to different treatments.

The sandwich ELISA method

The most widely used method to quantify calprotectin in feces is the sandwich ELISA method.

Briefly, this method employs antibodies that specifically bind to calprotectin, emitting a detectable signal that is directly proportional to the amount of calprotectin in the stool. However, for this quantification to be successful, it is necessary to previously isolate calprotectin from the rest of the molecules and debris present in the faeces, thus preventing them from interfering with detection.

Calprotectin Extraction

The first step to extract calprotectin is to take approximately 100mg of feces and dilute it 1:50 in extraction buffer, keeping in mind that 1g of feces corresponds to 1mL in terms of volume.

That is to say:

100mg feces + 4900μl extraction buffer = 5mL (5mL tubes) 

To ensure that the extraction buffer is adequately mixed with the stool sample, the 5mL tubes containing the mixture are homogenized for approximately 25 min. Next, the calprotectin is extracted, centrifuging the 5mL tubes for 20 min at 10,000 rpm, keeping the samples at a temperature between 2-8ºC.

Centrifugation is an exothermic process that produces heat due to friction with the air inside the centrifugation chamber of the different parts of the rotor. To carry out this centrifugation step, a refrigerated centrifuge is needed, such as our Digicen 21 and Bioprocen 22 R models that allow temperature regulation from -20°C (-4°F) to 40°C (104°F) in steps of 1°C.

After the centrifugation of the tubes, as a result, the molecules separate based on their density, giving rise to the formation of two phases, called supernatant (containing calprotectin) and pellet. Thus, after centrifugation, part of the supernatant is taken and diluted again 1:50 in dilution buffer.

For example:

20μl supernatant + 980μl dilution buffer = 5mL (in 5mL tubes).

After this process, 5mL of sample is obtained, with a 1:2500 dilution, containing the extracted calprotectin. Finally, 100μl of the sample diluted 1:2500 are transferred to a 96-well plate, and the immunoassay is started, following the manufacturer’s instructions in each case.

Centrifugation of the stool sample to extract calprotectin is a determining step for the correct functioning of the immunoassay (ELISA), and therefore for the correct detection of calprotectin, with the clinical relevance that this entails both in diagnosis and treatment of patients with inflammatory bowel diseases.