Calprotectin ELISA 试剂盒

(1) Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.
(2) ELISA Wash Concentrate must be diluted to working solution prior to use. Please see REAGENTS section for details.
(3) Reconstitute all assay standard level 1 to level 7 and controls by adding 0.5 mL of deminerialized water to each vial. Allow the standards and controls to sit undisturbed for 5 minutes, and then mix well by inversions or gentle vortexing. One must make sure that all solid is dissolved completely prior to use. These reconstituted standards and controls may be stored at 2 ? 8 C for up to 3 days or at ?10 C or below for long-term storage. Do not exceed 3 freeze-thaw cycles.
(4) Test Configuration
(5) Place a sufficient number of calprotectin-coated microwell strips in a holder to run human calprotectin standards, controls and unknown samples in duplicate.
(6) Prepare Tracer Antibody working solution by 1:21 fold dilution of the Calprotectin Tracer Antibody by adding the tracer antibody into the Tracer Antibody Diluent . Following is a table that outlines the relationship of strips used and antibody mixture prepared.

If the Epitope Diagnostics Fecal Sample Collection Tube is used, there is no sample preparation required.

(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 diluted 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.

It is recommended to use a point-to-point or 4-parameter standard curve fitting.
1. Calculate the average absorbance for each pair of duplicate test results.
2. Subtract the average absorbance of the level 1 standard (0 ng/mL) from the average absorbance of all other readings to obtain corrected absorbance.
3. The standard curve is generated by the corrected absorbance of all standard levels on the ordinate against the standard concentration on the abscissa using point-to-point or log-log paper. Appropriate computer assisted data reduction programs may also be used for the calculation of results. The fecal human calprotectin concentrations for the controls and the patient samples are read directly from the standard curve using their respective corrected absorbance. The use of the two absorbance wavelength at A 620 nm and A450/620 nm allows for two ways to calculate sample results. It is recommended to get sample results by using the primary standard curve at A 450/620 nm for samples with value below standard level
5. For samples with calprotectin value above standard level 5, it is recommended to use the secondary standard curve at A 620 nm.