Procalcitonin(PCT) ELISA Kit

Catalog No. CSB-E09502h

(96 tests)

l This immunoassay kit allows for the in vitro quantitative determination of

PCT concentrations in serum, plasma and other biological fluids.

l Expiration date six months from the date of manufacture

l 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

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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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MATERIALS PROVIDED

Reagent Quantity

Assay plate 1

Standard 5 x 0.5 ml

HRP-conjugate 1 x 6 ml

Substrate A 1 x 7 ml

Substrate B 1 x 7 ml

Stop Solution 1 x 7 ml

Standard S1 S2 S3 S4 S5

Concentration

(pg/ml) 125 375 875 2000 4000

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.

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OTHER SUPPLIES REQUIRED

— Microplate reader capable of measuring absorbance at 450

nm, with the correction wavelength set at 540 nm or 570 nm.

— Pipettes and pipette tips.

— Deionized or distilled water.

— Squirt bottle, manifold dispenser, or automated microplate

washer.

SAMPLE COLLECTION AND STORAGE

l 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.

l 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.

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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.

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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.

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LIMITATIONS OF THE PROCEDURE

— The kit should not be used beyond the expiration date on the

kit label.

— Do not mix or substitute reagents with those from other lots or

sources.

— If samples generate values higher than the highest standard,

dilute the samples and repeat the assay.

— Any variation in operator, pipetting technique, washing

technique, incubation time or temperature, and kit age can

cause variation in binding.

— 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

— When mixing or reconstituting protein solutions, always avoid

foaming.

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— 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.

— 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.

— To ensure accurate results, proper adhesion of plate sealers

during incubation steps is necessary.

— 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.

— 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.