Thermo Fisher Scientific Human IL-8 Chemiluminescent ELISA User guide

INSTRUCTIONS

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Number Description

84774 Human Interleukin-8 (IL-8) Chemiluminescent ELISA

Kit Contents:

Anti-Human IL-8 Pre-coated 96-well Strip Plate, 1 each

Lyophilized Recombinant Standard, 2 vials

Sample Diluent, 25mL, contains 0.1% sodium azide

Biotinylated Antibody Reagent, 7mL, contains 0.1% sodium azide

Streptavidin-HRP Reagent, 7mL

SuperSignal® Stable Peroxide Solution, 14mL

SuperSignal Luminol/Enhancer Solution, 14mL

Wash Buffer, (30X), 50mL

Plate Sealers, 3 each

Note: For research use only − not for use in diagnostic procedures.

Storage: For maximum stability, store in a non-defrosting -20ºC freezer and refer to the expiration

date for frozen storage on the label. Alternatively, store at 2-8ºC and refer to the expiration date for

refrigerated storage. Once thawed, store at 4ºC until the expiration date for refrigerated storage. Kit is

shipped on dry ice.

Table of Contents

Introduction ................................................................................................................................................................................. 1

Procedure Summary ..................................................................................................................................................................... 2

Additional Materials Required ..................................................................................................................................................... 2

Precautions ................................................................................................................................................................................... 2

Sample Preparation ...................................................................................................................................................................... 3

Reagent Preparation ..................................................................................................................................................................... 3

Assay Procedure .......................................................................................................................................................................... 4

Performance Characteristics ........................................................................................................................................................ 5

Data Templates ............................................................................................................................................................................ 6

Introduction

The Thermo Scientific Human IL-8 Chemiluminescent ELISA is a sandwich enzyme-linked immunosorbent assay (ELISA)

for measuring human IL-8 in serum; EDTA and heparin plasma; and culture supernatants. Each well of the provided

microplate has been pre-coated with a capture antibody specific to human IL-8. After the sample containing human IL-8 is

bound and washed, the biotinylated-detecting antibody is added and binds to a second site on the IL-8 protein. Excess

detecting antibody is then removed and streptavidin-horseradish peroxidase is added. The enzyme reacts with Thermo

Scientific SuperSignal ELISA Femto Chemiluminescent Substrate to produce signal that is detected with a plate-based

luminometer. The amount of signal produced is proportional to the amount of human IL-8 in the original standard or sample.

1212.3

84774

Human IL-8 Chemiluminescent ELISA

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Procedure Summary

1. Add 50µL of Standards

and samples to each well in

duplicate.

2. Cover plate and incubate

at room temperature

(20-25°C) for 1 hour.

3. Wash plate THREE

times.

4. Add 50µL of Biotinylated

Antibody Reagent to each

well.

5. Cover and incubate plate at

room temperature for

30 minutes.

6. Wash plate THREE

times.

7. Add 50µL of

Streptavidin-HRP Reagent

to each well.

8. Cover and incubate plate

at room temperature for

30 minutes.

9. Wash plate THREE times.

10. Prepare the

SuperSignal™ Working

Solution.

11. Add 50 µl of the

working solution to each

well. Evaluate plate within

1-15 minutes.

12. Measure the signal using

a plate-based luminometer.

Calculate results.

Additional Materials Required

• Luminometer

• Ultrapure water

• Precision pipettes with disposable plastic tips to deliver 5-1000µL

• Plastic pipettes to deliver 5-15mL

• Disposable reagent reservoirs, if a multi-channel pipette will be used

• A glass or plastic 2L container for preparation of Wash Buffer

• A squirt wash bottle or automated plate washer

• Polypropylene or polyethylene tubes for preparation of standards − do not use polystyrene, polycarbonate or glass tubes

• 15mL plastic tube for biotinylated detecting antibody and substrate preparation

Precautions

• Review all instructions carefully and verify all components against the Kit Contents list (page 1) before beginning.

• Equilibrate all specimens and reagents to room temperature (20-25ºC) before use in the assay.

• Do not mix reagents from different kit lots. Do not use kit beyond expiration date.

• Use new disposable pipette tips for each transfer to avoid cross-contamination.

• Use a new disposable reservoir and plate sealer for each step in the assay.

• Avoid exposure of reagents to excessive heat or light during storage and incubation.

• Individual components of this assay kit contain preservatives and antibiotics. Wear gloves while performing the assay to

avoid contact with samples and reagents.

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Sample Preparation

• Dilute Serum and plasma samples 1:5 (1 part sample added to 4 parts diluent) before testing in the Human IL-8

Chemiluminescent ELISA.

• When testing serum or plasma or if the concentration of cytokine in a sample is expected to exceed the highest point the

standard curve, prepare dilutions of the test sample. For example, prepare a five-fold dilution by adding 50µL of test

sample to 200µL of Sample Diluent.

Note: Serum and plasma samples must be diluted at least five-fold before testing in this assay.

• Store samples to be assayed within 4 hours at 2-8ºC. For overnight and long-term storage, aliquot samples and store at

-70ºC. Avoid multiple freeze-thaw cycles. Do not use a water bath to thaw samples.

• Equilibrate samples to room temperature before performing the assay. Mix samples by gently inverting the tubes. Do not

use a heated water bath to thaw samples.

• If using samples that are clotted, grossly hemolyzed or microbially contaminated or if there is any question about the

integrity of a sample, make a note on the template and interpret results with caution.

• When testing culture supernatants and using a medium other than RPMI with 5-20% FCS, validate the medium to

determine how it affects the assay. For best results, use a culture medium that contains a carrier protein such as FCS to

stabilize the cytokine. Lack of a carrier protein in the media or addition of other compounds may compromise assay

results.

Reagent Preparation

Wash Buffer

1. Label a clean glass or plastic bottle “Wash Buffer.” Add the entire contents of the 30X Wash Buffer bottle and dilute

with ultrapure water to a final volume of 1.5L.

2. Thoroughly mix the solution. Store Wash Buffer at room temperature.

Standards

3. Reconstitute one vial of lyophilized standard in the Sample Diluent provided to the volume stated on the standard vial

label. The standard will take approximately 1 minute to dissolve. Mix by gently inverting the vial. Use standard within

1 hour of reconstitution. The solution contains 5000pg/mL and is used as the highest concentration standard.

4. Label five tubes, one for each additional standard curve point (i.e., 500pg/mL, 50pg/mL, 5pg/mL, 0.5pg/mL and

0pg/mL).

5. Pipette 450 µL of Sample Diluent into each of the labeled tubes.

Note: For best results, when testing culture supernatants generated with media containing special additives validate the

media to confirm that it will not interfere with the assay. To perform this validation, prepare two standard curves, one

using your culture medium and the other using Sample Diluent. If the mean values for each point on the two curves are

within 10% of each other, the medium will not interfere with the assay. The culture samples may be evaluated using a

standard curve prepared with the Sample Diluent provided.

6. Pipette 50µL of the reconstituted standard) into the first tube (i.e., 500pg/mL) and mix. Pipette 50µL of this dilution into

the next tube (i.e., 50pg/mL) and mix. Perform two additional serial dilutions. Do not add cytokine to the final tube (i.e.,

0pg/mL).

Standard Dilutions using 50µL

500pg/mL

50pg/mL

5pg/mL

0.5pg/mL

0pg/mL

5000pg/mL

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Assay Procedure

1. Use the Data Template provided to record placement of standards and test samples. Perform five standard points and a

zero standard in duplicate with each series of unknown samples.

2. Add 50µL of standard or sample to each well. Cover plate with an adhesive plate sealer. Incubate for 1 hour at room

temperature (20-25ºC).

Note: Dilute serum and plasma samples 1:5 (1 part sample added to 4 parts diluent) before testing in this assay.

3. Thoroughly wash wells THREE times with prepared Wash Buffer and pat dry.

Note: See the Wash Procedure section at the end of the protocol.

4. Add 50µL of Biotinylated Antibody Reagent to each well. Cover plate with adhesive plate sealer. Incubate plate for

30 minutes at room temperature.

5. Thoroughly wash the plate THREE times with prepared Wash Buffer and pat dry.

6. Add 50µL of Streptavidin-HRP Reagent to each well. Cover plate with adhesive plate sealer. Incubate for 30 minutes at

room temperature.

7. Thoroughly wash the plate THREE times with prepared Wash Buffer and pat dry.

8. Prepare SuperSignal Working Solution by adding 4 ml of the Stable Peroxide to 4mL of the Luminol Enhancer and mix

gently. Add 50µL of the Working Solution to each well. Evaluate the plate within 1-15 minutes.

9. Measure the luminescence on a plate-based luminometer using a read time of 1 second per well.

Wash Procedure

Note: Automated plate washers may produce sub-optimal results. For best results, perform a manual wash the first time the

assay is performed. Automated plate washing may require validation to determine the number of washes necessary to achieve

optimal results; typically, 3-5 washes are sufficient.

1. Gently squeeze the long sides of the plate frame before washing to ensure all strips securely remain in the frame.

2. Empty plate contents. Use a squirt bottle to vigorously fill each well completely with Wash Buffer, then empty plate

contents. Repeat procedure two additional times for a total of THREE washes. Blot plate onto paper towels or other

absorbent material.

Note: For automated washing, aspirate all wells and wash by overfilling wells with Wash Buffer. Blot plate onto paper

towels or other absorbent material.

Calculation of Results

A standard curve is generated by plotting the average relative light

units (RLU) obtained for each of the standards on the vertical Y-axis

vs. the corresponding cytokine concentrations on the horizontal X-axis.

Determine the cytokine amount in each sample by interpolating from

the RLU value to cytokine concentration using the standard curve. For

best results, use a four-parameter curve fit. Multiply calculated pg/ml

values for unknowns by the dilution factor(s) to calculate protein

concentrations of the original samples.

Example Standard Curve

Relative Light Units

1,000

10,000

100,000

1,000,000

10,000,000

0 1 10 100 1,000 10,000

Human IL-8 (pg/mL)

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Performance Characteristics

Instrumentation: The Human IL-8 Chemiluminescent ELISA was validated using the Orion Microplate Luminometer from

Berthold using a read time of 1 second per well.

Standard Curve Range: 0.5-5000pg/mL

Sensitivity: 0.5pg/mL

Note: Sensitivity calculations were based on analysis of serial dilutions

of the low standard.

Intra-assay Precision: To determine intra-assay precision, 20 replicates

of samples containing high, medium and low levels of recombinant

human IL-8 were performed on a single plate. The calculated standard

deviation and %CV (coefficient of variation) are indicated in Table 1.

Inter-assay Precision: To evaluate inter-assay precision, two different

operators tested samples. Forty replicate sample values were used to

calculate inter-assay precision data. The calculated standard deviation

and %CV (coefficient of variation) are indicated in Table 2.

Calibration: The Human IL-8 standard was calibrated to the NIBSC

international reference standard lot 89/520.

One (1) pg = 0.8 NIBSC Units

Recovery: Human serum and plasma samples and diluent controls were spiked with recombinant human IL-8. Expected

values were calculated by adding endogenous cytokine levels from non-spiked samples to those of spiked diluent controls.

Percent recovery was calculated by dividing observed by expected values (Table 3).

Table 3. Recovery of samples spiked with recombinant human IL-8.

Sample Type

Mean Recovery

Median Recovery

Recovery Range

Serum (n =5)

96%

98%

86-101%

EDTA (n =5)

104%

92%

92-121%

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