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Human Procalcitonin (PCT) ELISA Kit
Catalog No. CSB-E09502h
(96 tests)
This immunoassay kit allows for the in vitro quantitative determination of human PCT concentrations in serum, plasma and other biological fluids.
Expiration date six months from the date of manufacture
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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INTRODUCTION
Procalcitonin (PCT) is a precursor of the hormone calcitonin, which is involved with calcium homeostasis, and is produced by the C-cells of the thyroid gland. It is there that procalcitonin is cleaved into calcitonin, katacalcin and a protein residue.
Measurement of procalcitonin can be used as a marker of severe sepsis and generally grades well with the degree of sepsis, although levels of procalcitonin in the blood are very low. PCT has the greatest sensitivity (85%) and specificity (91%) for differentiating patients with SIRS from those with sepsis, when compared with IL-2, IL-6, IL-8, CRP and TNF-alpha. Evidence is emerging that procalcitonin levels can reduce unnecessary antibiotic prescribing to people with lower respiratory tract infections. Currently though, procalcitonin assay is not used outside the context of clinical trials.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an antibody specific to PCT. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP) -conjugated antibody preparation specific for PCT and incubated. Then substrate solutions are added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ±2 nm. The concentration of PCT in the samples is then determined by comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
125 pg/ml-4000 pg/ml. The standard curve concentrations used for the ELISA’s were 4000 pg/ml, 2000 pg/ml, 875 pg/ml, 375 pg/ml, 125pg/ml.
SPECIFICITY
This assay recognizes human PCT. No significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human PCT is typically less than 62.5 pg/ml.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.
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STORAGE
1. Unopened test kits should be stored at 2-8 C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date.
2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.
3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.
OTHER SUPPLIES REQUIRED
1 Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.
2 Pipettes and pipette tips.
3 Deionized or distilled water.
4 Squirt bottle, manifold dispenser, or automated microplate washer.
SAMPLE COLLECTION AND STORAGE
Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20℃. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.
Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20℃. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. All the reagents should be added directly to the liquid level in the well. The pipette should avoid contacting the inner wall of the well.
1 Set a Blank well without any solution. Add 50μl of Standard or Sample per well.
2 Add 50μl of HRP-Conjugate to each well (Not to Blank!). Incubate for 1 hour at 37°C.
3 Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with ddH2O (200μl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
4 Add 50μl of Substrate A and 50μl Substrate B to each well. Incubate for 15 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark.
5 Add 50μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
6 Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the PCT concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
LIMITATIONS OF THE PROCEDURE
1 The kit should not be used beyond the expiration date on the kit label.
2 Do not mix or substitute reagents with those from other lots or sources.
3 If samples generate values higher than the highest standard, dilute the samples and repeat the assay.
4 Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.
5 This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Quantikine Immunoassay, the possibility of interference cannot be excluded.
TECHNICAL HINTS
1 When mixing or reconstituting protein solutions, always avoid foaming.
2 To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
3 When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision.
4 To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
5 Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue.
6 Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.
