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USER GUIDE
Human IL-13 Instant ELISA Kit
Enzyme-linked immunosorbent assay for quantitative detection of human IL-13
Catalog Number
BMS231INST (128 tests)
Pub. No.
MAN0016604
Rev.
A.0 (30)
WARNING!
Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.
Product description
The Human IL-13 Instant ELISA Kit is an enzyme-linked immunosorbent assay for the quantitative detection of human IL-13.
Summary
Interleukin-13 (IL-13) was first described as a protein product, designated P600, encoded by an RNA produced by activated mouse
Th2 cells. More recently, the cDNA cloning of the human homologue of P600, human IL-13, has been reported. Human IL-13 is a nonglycosylated protein of 132 amino acids with a molecular weight of 12 kDa. The IL-13 gene is located on chromosome 5q23-31, in the same region as the genes encoding IL-3, IL-4, IL-5, and GM-CSF. It is produced by activated CD4+ and CD8+ T-cells, but the expression seems to be more abundant in CD4+ T-cells. Although it is a Th2 cytokine in the mouse, it appears to be produced by Th0, Th1, and
Th2-like human T-cell clones.
IL-13 is a pleiotropic cytokine whose spectrum of action encompasses
B-cells, mononuclear phagocytes, and large granular lymphocytes.
IL-13 induces CD23 expression on B-cells, promotes B-cell proliferation in combination with anti-Ig or CD40 antibodies, and stimulates secretion IgE, and IgG4. IL-13 has also been shown to prolong survival of human monocytes and increases surface expression of MHC class II, CD23, and members of the integrin superfamily, like CD11b, CD11c, CD18, CD29 and CD49e. IL-13 inhibits the production of a series of cytokines like IL-1, IL-6, TNF-α, and IL-8 by activated human monocytes. IL-13 induces IFN-γ production by NK cells.
For literature update refer to our website.
Principles of the test
An anti-human IL-13 coating antibody is adsorbed onto microwells.
Human IL-13 present in the sample or standard binds to antibodies adsorbed to the microwells; a biotin-conjugated anti-human IL-13 antibody binds to human IL-13 captured by the first antibody.
Streptavidin-HRP binds to the biotin conjugated anti-human IL-13.
Fig. 1 First incubation
Following incubation unbound biotin conjugated anti-human IL-13 and Streptavidin-HRP is removed during a wash step, and substrate solution reactive with HRP is added to the wells.
Fig. 2 Second incubation
A colored product is formed in proportion to the amount of soluble human IL-13 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A standard curve is prepared from 7 human IL-13 standard dilutions and human
IL-13 sample concentration determined.
Fig. 3 Stop reaction
For Research Use Only. Not for use in diagnostic procedures.
Reagents provided
1 aluminum pouch with a Microwell Plate (12 strips with 8 wells each) coated with monoclonal Antibody to human IL-13, Biotin-Conjugate
(anti-human IL-13 monoclonal antibody), Streptavidin-HRP and
Sample Diluent, lyophilized.
2 aluminum pouches with a human IL-13 Standard curve (colored)
1 bottle (25 mL) Wash Buffer Concentrate 20x (phosphate-buffered saline with 1% Tween ™ 20)
1 vial (12 mL) Sample Diluent (Use when an external predilution of the samples is needed)
1 vial (15 mL) Substrate Solution (tetramethyl-benzidine)
1 vial (15 mL) Stop Solution (1M Phosphoric acid)
2 Adhesive Films
Storage instructions
Store ELISA plate and Standard curves or whole kit at -20°C. The plate and the standard can also be removed, stored at -20°C, remaining kit reagents can be stored between 2°C and 8°C. Expiry of the kit and reagents is stated on labels.
The expiry of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, the reagent is not contaminated by the first handling.
Sample collection
Serum, plasma (EDTA), and cell culture supernatant samples were tested with this assay. Other biological samples might be suitable for use in the assay. Remove serum or plasma from the clot or red cells as soon as possible after clotting and separation.
Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic samples.
Samples must be stored frozen at –20°C to avoid loss of bioactive human IL-13. If samples are to be run within 24 hours, they may be
stored at 2°C to 8°C (for sample stability refer to “Performance characteristics“ on page 4).
Avoid repeated freeze-thaw cycles. Prior to assay, frozen serum or plasma should be brought to room temperature slowly and mixed gently.
Materials required but not provided
• 5 mL and 10 mL graduated pipettes
• 5 µL to 1000 µL adjustable single channel micropipettes with disposable tips
• 50 µL to 300 µL adjustable multichannel micropipette with disposable tips
• Multichannel micropipette reservoir
• Beakers, flasks, cylinders necessary for preparation of reagents
• Device for delivery of wash solution (multichannel wash bottle or automatic wash system)
• Microwell strip reader capable of reading at 450 nm (620 nm as optional reference wave length)
• Glass-distilled or deionized water
• Statistical calculator with program to perform linear regression analysis
Precautions for use
• All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses, and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statements(s) for specific advice.
• Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures.
• Do not mix or substitute reagents with those from other lots or other sources.
• Do not use kit reagents beyond expiration date on label.
• Do not expose kit reagents to strong light during storage or incubation.
• Do not pipet by mouth.
• Do not eat or smoke in areas where kit reagents or samples are handled.
• Avoid contact of skin or mucous membranes with kit reagents or samples.
• Rubber or disposable latex gloves should be worn while handling kit reagents or samples.
• Avoid contact of substrate solution with oxidizing agents and metal.
• Avoid splashing or generation of aerosols.
• To avoid microbial contamination or cross-contamination of reagents or samples that may invalidate the test, use disposable pipette tips and/or pipettes.
• Use clean, dedicated reagent trays for dispensing substrate reagent.
• Glass-distilled water or deionized water must be used for reagent preparation.
• Substrate solution must be at room temperature prior to use.
• Decontaminate and dispose samples and all potentially contaminated materials as if they could contain infectious agents.
The preferred method of decontamination is autoclaving for a minimum of 1 hour at 121.5°C.
• Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1.0% sodium hypochlorite. Allow 30 minutes for effective decontamination. Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite.
Preparation of reagents and samples
1.
Buffer concentrate should be brought to room temperature and diluted before starting the test procedure.
2.
If crystals have formed in the buffer concentrate, warm it gently until crystals have completely dissolved.
Wash buffer (1x)
1.
Pour entire contents (25 mL) of the Wash Buffer Concentrate (20x) into a clean 500 mL graduated cylinder. Bring to final volume to
500 mL with glass-distilled or deionized water. Mix gently to avoid foaming.
2.
Transfer to a clean wash bottle and store at 2°C to 25°C. Please note that Wash Buffer (1x) is stable for 30 days.
Test protocol
Note:
·
Use plate immediately after removal from -20°C!
·
Do not wait until pellets have completely dissolved before applying samples - the binding reaction in the standard strips starts immediately after addition of water!
· Do not try to dissolve pellets by pipetting up and down in the wells
- some parts of the pellet could stick to the tip creating high variation of results
· Perform the washing step with at least 400 µL of washing buffer as stated in the manual or fill the wells completely - otherwise any pellet residues sticking to the rim of the well will not be removed and create high variation of results
·
Allow the washing buffer to sit in the wells for a few seconds before aspiration
·
Remove covers of the standard strips carefully in order that all the lyophilised pellets remain in the wells
2 Human IL-13 Instant ELISA Kit User Guide
1.
Determine the number of Microwell Strips required to test the desired number of samples plus Microwell Strips for blanks and standards (colored). Each sample, standard and blank should be assayed in duplicate. Remove extra Microwell Strips from holder and store in foil bag with the desiccant provided at -20°C sealed tightly. Place Microwell Strips containing the standard curve in
position A1/A2 to H1/H2 (see Table 1).
Table 1 Example of the arrangement of blanks, standards and samples in the microwell strips.
A
B
C
1
Standard 1
200.00 pg/mL
Standard 2
100.00 pg/mL
Standard 3
50.00 pg/mL
2
Standard 1
200.00 pg/mL
Standard 2
100.00 pg/mL
Standard 3
50.00 pg/mL
3
Sample 1
Sample 2
Sample 3
4
Sample 1
Sample 2
Sample 3
D
Standard 4
25.00 pg/mL
Standard 4
25.00 pg/mL
Sample 4 Sample 4
E
F
Standard 5
12.50 pg/mL
Standard 6
6.25 pg/mL
Standard 5
12.50 pg/mL
Standard 6
6.25 pg/mL
Sample 5
Sample 6
Sample 5
Sample 6
G
H
Standard 7
3.13 pg/mL
Blank
Standard 7
3.13 pg/mL
Blank
Sample 7
Sample 8
Sample 7
Sample 8
2.
Add distilled water to all standard and blank wells as indicated on the label of the standard strips (A1/A2 to H1/H2).
3.
Add 100 µL of distilled water to the sample wells.
4.
Add 50 µL of each Sample, in duplicate, to the designated wells and mix the contents.
5.
Cover with a adhesive film and incubate at room temperature
(18°C to 25°C) for 3 hours on a microplate shaker.
6.
Remove adhesive film and empty wells. Wash the microwell strips 3 times with approximately 400 µL Wash Buffer per well with thorough aspiration of microwell contents between washes.
Allow the Wash Buffer to sit in the wells for about 10 – 15 seconds before aspiration. Take care not to scratch the surface of the microwells.
After the last wash, tap microwell strips on absorbent pad or paper towel to remove excess Wash Buffer. Use the microwell strips immediately after washing or place upside down on a wet absorbent paper for no longer than 15 minutes. Do not allow wells to dry.
7.
Pipette 100 µL of TMB Substrate Solution to all wells, including the blank wells.
8.
Incubate the microwell strips at room temperature (18°C to 25°C) for 10 minutes. Avoid direct exposure to intense light.
The color development on the plate should be monitored and the substrate reaction stopped (see point 9. of this protocol) before positive wells are no longer properly recordable.
Determination of the ideal time period for color development has to be done individually for each assay.
It is recommended to add the stop solution when the highest standard has developed a dark blue color.
Alternatively the color development can be monitored by the
ELISA reader at 620 nm. The substrate reaction should be stopped as soon as Standard 1 has reached an OD of 0.9 – 0.95.
9.
Stop the enzyme reaction by quickly pipetting 100 µL of Stop
Solution into each well, including the blank wells. It is important that the Stop Solution is spread quickly and uniformly throughout the microwells to completely inactivate the enzyme.
Results must be read within 5 minutes after the Stop Solution is added.
10.
Read absorbance of each microwell on a spectro-photometer using 450 nm as the primary wave length (optionally 620 nm as the reference wave length; 610 nm to 650 nm is acceptable). Blank the plate reader according to the manufacturer's instructions by using the blank wells. Determine the absorbance of both the samples and the human IL-13 standards.
Note: In case of incubation without shaking the obtained O.D. values may be lower than indicated below. Nevertheless the results are still valid.
Calculation of results
• Calculate the average absorbance values for each set of duplicate standards and samples. Duplicates should be within 20% of the mean.
• Create a standard curve by plotting the mean absorbance for each standard concentration on the ordinate against the human IL-13 concentration on the abscissa. Draw a best fit curve through the points of the graph.
• To determine the concentration of circulating human IL-13 for each sample, first find the mean absorbance value on the ordinate and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding human IL-13 concentration.
• Samples have been diluted 1:2, thus the concentration read from the standard curve must be multiplied by the dilution factor (x 2).
Note: There is a common dilution factor for samples due to the conjugate which must then be included in the calculation. The samples contribute 100 µL to the final volume per well. These
100 µL are composed of 50 µL of sample diluent plus 50 µL of the sample. This is a 1:2 dilution.
The remaining 50 µL to give 150 µL are due to the addition of
50 µL conjugate to all wells.
50 µL sample diluent and 50 µL conjugate results in 100 µL reconstitution volume, addition of 50 µL sample (50 µL + 50 µL =
1:2 dilution)
• Calculation of samples with a concentration exceeding standard 1 may result in incorrect, low human IL-13 levels. Such samples require further external predilution according to expected human
IL-13 values with Sample Diluent in order to precisely quantitate the actual human IL-13 level.
• It is suggested that each testing facility establishes a control sample of known human IL-13 concentration and runs this additional control with each assay. If the values obtained are not within the expected range of the control, the assay results may be invalid.
Human IL-13 Instant ELISA Kit User Guide 3
•
A representative standard curve is shown in Figure 4. This curve
cannot be used to derive test results. Every laboratory must prepare a standard curve for each group of microwell strips assayed.
Fig. 4 Representative standard curve for human IL-13 Instant
ELISA. Human IL-13 was diluted in serial 2-fold steps in Sample
Diluent each symbol represents the mean of 3 parallel titrations.
Do not use this standard curve to derive test results. A standard curve must be run for each group of microwell strips assayed.
Note: If the sample is prediluted the value read from the standard curve must be multiplied by the dilution factor. For example, if the sample is prediluted 1:25 the value read from the standard curve must be multiplied by a factor of 25 to give the final result.
Table 2 Typical data using the human IL-13 Instant ELISA
Measuring wavelength: 450 nm
Reference wavelength: 620 nm
Standard human IL-13
Concentration
(pg/mL)
O.D. Mean C.V. (%)
1
2
3
4
5
6
7
Blank
200.00
100.00
50.00
25.00
12.5
6.25
3.13
0.00
O.D.
(450 nm)
1.891
1.846
1.234
1.137
0.670
0.625
0.356
0.359
0.171
0.162
0.147
0.132
0.069
0.068
0.018
0.018
1.869
1.186
0.648
0.357
0.167
0.140
0.069
0.018
2.3
3.6
4.2
4.7
5.0
5.1
6.5
0.0
Limitations
• Since exact conditions may vary from assay to assay, a standard curve must be established for every run.
• Bacterial or fungal contamination of either screen samples or reagents or cross-contamination between reagents may cause erroneous results.
• Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of all detergents before use.
• Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Empty wells completely before dispensing fresh wash solution, fill with Wash
Buffer as indicated for each wash cycle and do not allow wells to sit uncovered or dry for extended periods.
• The use of radioimmunotherapy has significantly increased the number of patients with human anti-mouse IgG antibodies
(HAMA). HAMA may interfere with assays utilizing murine monoclonal antibodies leading to both false positive and false negative results. Serum samples containing antibodies to murine immunoglobulins can still be analyzed in such assays when murine immunoglobulins (serum, ascitic fluid, or monoclonal antibodies of irrelevant specificity) are added to the Sample.
Performance characteristics
Sensitivity
The limit of detection of human IL-13 defined as the analyte concentration resulting in an absorbance significantly higher than that of the dilution medium (mean plus 2 standard deviations) was determined to be 0.99 pg/mL (mean of 6 independent assays).
Reproducibility
Intra-assay
Reproducibility within the assay was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 8 serum samples containing different concentrations of human IL-13. Two standard curves were run on each plate. The calculated overall intraassay coefficient of variation was 9.0%.
Inter-assay
Assay to assay reproducibility within one laboratory was evaluated in
3 independent experiments. Each assay was carried out with 6 replicates of 8 serum samples containing different concentrations of human IL-13. Two standard curves were run on each plate. The calculated overall coefficient of variation was 12.6%.
Spike recovery
The spike recovery was evaluated by spiking 4 levels of human IL-13 into serum. Recoveries were determined in 3 independent experiments with 6 replicates each. The unspiked serum was used as blank in these experiments. The overall mean recovery was 80.0%.
Dilution parallelism
Four serum samples with different levels of human IL-13 were analyzed at serial 2-fold dilutions with 4 replicates each. The overall mean recovery was 92%.
Sample stability
Freeze-Thaw stability
Aliquots of serum samples (unspiked or spiked) were stored at -20°C and thawed 5 times, and the human IL-13 levels determined. There was no significant loss of human IL-13 immunoreactivity detected by freezing and thawing.
Storage stability
Aliquots of serum samples (spiked or unspiked) were stored at –20°C,
2°C to 8°C, room temperature, and at 37°C, and the human IL-13 level determined after 24 hours. There was no significant loss of human
IL-13 immunoreactivity detected during storage under above conditions.
Specificity
The interference of circulating factors of the immune system was evaluated by spiking these proteins at physiologically relevant concentrations into serum. No cross-reactivity was detected.
4 Human IL-13 Instant ELISA Kit User Guide
Expected values
A panel of sera samples from randomly selected apparently healthy donors was tested for human IL-13. No detectable human IL-13 levels were found. Elevated human IL-13 levels depend on the type of immunological disorder.
Calibration
The immunoassay is calibrated with highly purified recombinant human IL-13 which has been evaluated against the international
Reference Standard NIBSC 94/622 and has been shown to be equivalent.
NIBSC 94/622 is quantitated in International Units (IU), 1IU corresponding to 1 ng human IL-13.
Reagent preparation summary
Wash buffer (1x)
Add Wash Buffer Concentrate 20 x (25 mL) to 475 mL distilled water
Test protocol summary
1.
Place standard strips in position A1/A2 to H1/H2.
2.
Add distilled water, in duplicate, to all standard and blank wells as indicated on the label of the standard strips.
3.
Add 100 µL distilled water to sample wells.
4.
Add 50 µL Sample to designated wells.
5.
Cover microwell strips and incubate 3 hours at room temperature
(18°C to 25°C) if available on a microplate shaker.
6.
Empty and wash microwell strips 3 times with 400 µL Wash
Buffer.
7.
Add 100 µL of TMB Substrate Solution to all wells including blank wells.
8.
Incubate the microwell strips for 10 minutes at room temperature
(18°C to 25°C).
9.
Add 100 µL Stop Solution to all wells including blank wells.
10.
Blank microwell reader and measure color intensity at 450 nm within 5 minutes.
Note: Samples have been diluted 1:2, thus the concentration read from the standard curve must be multiplied by the dilution factor (x 2).
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