Calprotectin ELISA Assay Kit

Additional Information

Assay Background

Quantitative determination of fecal calprotectin is an indication of the severity of bowel inflammation. Also, higher levels of calprotectin in the stool are associated with an increased risk of relapse in patients with inflammatory bowel disease (IBD).1 Low stool calprotectin levels correlate well with a low risk for intestinal allograft rejection. This assay uses specific monoclonal antibodies to ensure only calprotectin is detected.

Assay Principle

Assay standards, controls and patient samples are added directly to wells of a microtiter plate that is coated with antibody to calprotectin. After a short incubation period, the plate is washed and horseradish peroxidase (HRP) conjugated human calprotectin specific monoclonal antibody is added to each well. After the second incubation period, a “sandwich” of solid-phase antibody – human calprotectin – HRP conjugated monoclonal antibody” is formed. The unbound monoclonal antibodies and buffer matrix are removed in the subsequent washing step. For the detection of this immunocomplex, the well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to the wall of each microtiter well is directly proportional to the amount of human calprotectin in the test sample. A standard curve is generated by plotting the absorbance versus the respective human calprotectin concentration for each standard on a point-to-point or 4-parameter curve fitting. The concentration of fecal human calprotectin in test samples is determined directly from this standard curve of the Calprotectin ELISA Assay.

Assay Procedure

1. Add 50 µL of Assay Buffer into the designated microwells. Gently tap the plate to coat the wells evenly.
2. Add 50 µl of Standards, Controls and extracted patient samples into the designated microwells
3. Seal the plate wells securely, cover with foil or other material to protect from light, and rotate on an ELISA plate shaker (small orbit radius) for 1 hr. ± 5 minutes at 400 to 450 rpm.
4. Just prior to the end of the incubation time, dilute the proper amount of Tracer Antibody for the assay.
5. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
6. Add 100 µL of above Tracer Antibody to each well.
7. Seal the plate wells securely, cover with foil or other material to protect from light, and rotate on an ELISA plate shaker (small orbit radius) for 45 minutes ± 5 minutes at 400 to 450 rpm.
8. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
9. Add 100 µL of ELISA HRP Substrate into each of the wells.
10. Cover the plate with aluminum foil to or other material to avoid exposure to light. Incubate plate static, at room temperature, for 12 minutes (Optional 8 – 15 minutes).
11. Remove the aluminum foil. Read the absorbance at 620 nm (optional wavelengths from 595 nm to 650 nm depending on available filters) immediately.
12. Immediately add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
13. Read the absorbance at 450 nm with reference filter at 620 nm or 650 nm.

Typical Standard Curve (LOW)

Typical Standard Curve (High)