Thermo Fisher Scientific Human IL-8 Chemiluminescent ELISA User guide

Type
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
(815) 968-0747
www.thermoscientific.com/pierce
3747 N. Meridian Road
(815) 968-7316 fax
2
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
SuperSignalWorking
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
(815) 968-0747
www.thermoscientific.com/pierce
3747 N. Meridian Road
(815) 968-7316 fax
3
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
(815) 968-0747
www.thermoscientific.com/pierce
3747 N. Meridian Road
(815) 968-7316 fax
4
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
(815) 968-0747
www.thermoscientific.com/pierce
3747 N. Meridian Road
(815) 968-7316 fax
5
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%
Products are warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in
the Product documentation, specifications and/or accompanying package inserts (“Documentation”). No claim of suitability for use in applications regulated
by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this
warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to
anyone other than Buyer. Any model or sample furnished to Buyer is merely illustrative of the general type and quality of goods and does not represent that
any Product will conform to such model or sample.
NO OTHER WARRANTIES, EXPRESS OR IMPLIED, ARE GRANTED, INCLUDING WITHOUT LIMITATION, IMPLIED WARRANTIES OF
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CONFORMING PRODUCTS DURING THE WARRANTY PERIOD IS LIMITED TO REPAIR, REPLACEMENT OF OR REFUND FOR THE NON-
CONFORMING PRODUCT(S) AT SELLER’S SOLE OPTION. THERE IS NO OBLIGATION TO REPAIR, REPLACE OR REFUND FOR PRODUCTS
AS THE RESULT OF (I) ACCIDENT, DISASTER OR EVENT OF FORCE MAJEURE, (II) MISUSE, FAULT OR NEGLIGENCE OF OR BY BUYER,
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Unless otherwise expressly stated on the Product or in the documentation accompanying the Product, the Product is intended for research only and is not to
be used for any other purpose, including without limitation, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses, or
any type of consumption by or application to humans or animals.
Current product instructions are available at www.thermoscientific.com/pierce. For a faxed copy, call 800-874-3723 or contact your local distributor.
© 2012 Thermo Fisher Scientific Inc. All rights reserved. Unless otherwise indicated, all trademarks are property of Thermo Fisher Scientific Inc. and its
subsidiaries. Printed in the USA.
Table 1. Intra-assay results using the Thermo
Scientific Human IL-8 ELISA Kit.
Spike Level
High
Medium
Low
Mean (pg/ml)
2,164
210
24.6
Standard Deviation
84.9
9.6
1.1
%CV
3.9%
4.6%
4.6%
Table 2. Inter-assay results using the Thermo
Scientific Human IL-8 ELISA Kit.
Spike Level
High
Medium
Low
Mean (pg/ml)
1,445
186
19.6
Standard Deviation
130
12.2
1.8
%CV
9.0%
6.5%
9.2%
Pierce Biotechnology
(815) 968-0747
www.thermoscientific.com/pierce
3747 N. Meridian Road
(815) 968-7316 fax
6
Data Templates
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Thermo Fisher Scientific Human IL-8 Chemiluminescent ELISA User guide

Type
User guide

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