Enzyme Immunoassay for the Quantitative Determination of Calprotectin Concentration in Human Stool
Introduction
Inflammatory Bowel Diseases (IBDs) are among the most common digestive tract disorders, affecting millions worldwide. Crohn’s disease and ulcerative colitis are the two primary types of IBDs, often leading to complications and significant healthcare costs (Feagan et al., 2019). Additionally, IBD is associated with an increased risk of colorectal malignancy (Ungaro et al., 2017).
Irritable Bowel Syndrome (IBS), another gastrointestinal disorder, presents with chronic abdominal pain, bloating, and altered bowel habits without a clear organic cause (Chey et al., 2015). Since IBS and IBD share overlapping symptoms, their differentiation is crucial for determining appropriate therapeutic strategies. The gold standard for diagnosing IBD includes endoscopic and radiological assessments, which are invasive procedures. Therefore, identifying non-invasive biomarkers such as calprotectin has gained substantial clinical significance (Schreiner et al., 2019).
Calprotectin, a 36-kDa protein, is primarily produced by granulocytes, monocytes, and squamous epithelial cells. It plays a vital role in innate immunity by exerting antimicrobial effects against bacteria and fungi (Manolakis et al., 2011). In IBD patients, intestinal inflammation leads to leukocytosis in the mucosa, causing granulocytes to release cytoplasmic contents, including calprotectin, into the intestinal lumen. Given that over 60% of granulocyte cytosolic proteins consist of calprotectin, it serves as a reliable marker for detecting intestinal inflammation (Røseth et al., 2004).
Clinical Significance
Calprotectin testing has emerged as a valuable non-invasive diagnostic tool for distinguishing IBD from IBS. Elevated fecal calprotectin levels indicate active inflammation, guiding clinicians in determining the necessity for further invasive investigations (Schreiner et al., 2019). Moreover, calprotectin levels help monitor disease activity and treatment response in IBD patients (Sipponen & Kolho, 2015).
Test Principle
The Calprotectin ELISA Kit utilizes a sandwich enzyme-linked immunosorbent assay (ELISA) for quantitative detection. This method employs a monoclonal anti-calprotectin antibody immobilized on microtiter wells, while a horseradish peroxidase (HRP)-conjugated monoclonal anti-calprotectin antibody is used as the enzyme conjugate. Stool samples are extracted using a specially formulated buffer. Both samples and standards are incubated with solid-phase antibodies. After washing, the HRP-conjugated antibody binds to the captured calprotectin. Following an additional wash, the chromogenic substrate is added, resulting in color development proportional to the calprotectin concentration. The reaction is terminated using a stop solution, and absorbance is measured at 450 nm using an ELISA reader. The calprotectin concentration in stool is determined by comparing sample absorbance with a standard calibration curve.
References
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