Amersham Interleukin-1 Beta [(h)IL-1β] Human, Biotrak ELISA System
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Warning:For research use only. Not recommended
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 statement(s) for specific advice.
3. Contents of the assay systemThis pack contains the following assay components, sufficient material for 96 wells.
All reagents are stored refrigerated at 2–8°C. Refer to the expiry date on the kit box. (h)IL-1β microplate - 96 well polystyrene microplate (12 strips of 8 wells) coated with an antibody against (h)IL-1β. Biotinylated antibody reagent - antibody against (h)IL-1β conjugated to biotin, with preservative, 8 ml. (h)IL-1β standard - 2 vials of recombinant human IL-1β, lyophilized. Streptavidin-HRP concentrate - streptavidin conjugated to HRP, with preservative, 50 µl. Streptavidin-HRP dilution buffer - with preservative, 14 ml. Standard diluent - with preservative, 12 ml. Wash buffer concentrate - 30-fold concentrated solution, with preservative, 50 ml. Pre-mixed TMB substrate reagent - 12–13 ml. Stop solution - <1% sulfuric acid, 13–15 ml. Plate covers - 4 adhesive strips.
4. DescriptionThe Biotrak™ human interleukin-1 beta ELISA system from GE Healthcare provides a simple, specific, reliable and precise quantitative determination of (h)IL-1β in cell culture supernatants, plasma (sodium citrate and heparin), serum and urine.
The assay system is based on a solid phase ELISA, which utilizes an antibody for (h)IL-1β bound to the wells of a microplate together with a biotinylated antibody to (h)IL-1β and streptavidin conjugated to horseradish peroxidase. Although the Biotrak (h)IL-1β immunoassay contains recombinant (h)IL-1β and antibodies raised against recombinant (h)IL-1β it has been shown to quantitate accurately both natural (h)IL-1β and recombinant (h)IL-1β
(h)IL-1β can be measured in the approximate range 10–400 pg/ml (0.5–20 pg/well) in less than 5 hours using the protocol provided with the kit. Each pack contains sufficient material for 96 wells. If one standard curve is constructed, 42 unknowns can be measured in duplicate.
• Same day protocol • Pre-coated plate • Specific for (h)IL-1β
5. IntroductionIL-1β is one of a group of three related polypeptide hormones that also includes IL-1α and IL-1ra (receptor antagonist). The IL-1 molecules exert effects on a variety of cell types involved in the host response to injury and infection.
IL-1β is produced from a 269 amino acid precursor which is not biologically active. The mature form of IL-1β is a 153 amino acid protein of 17.5 kDa (determined by SDS-PAGE) which is produced from the precursor by IL-1β protease. Although many cell types synthesize the IL-1β precursor, expression of the IL-1β protease appears to be limited to monocytes and myeloid cell lines.
IL-1β can be induced by a variety of agents including: LPS, S. aureus, IL-1, leukotrienes, phorbol esters, TNF, C5a, GM-CSF, indomethacin and zymosan. Agents that inhibit IL-1β production include: dexamethasone, prednisolone, cAMP, PGE2, IL-4, IL-10, TGFβ, and
Two distinct IL-1 receptors (IL-1R) have been identified, termed type I (80 kDa) and type II (68 kDa). Both receptors bind IL-1α and IL-1β with high affinity (~10-10M). IL-1RI is expressed by T cells, endothelial cells and fibroblasts while IL-1RII is expressed by B cells and other monomyelocytic cell types. Receptor density ranges from 0–30 000 per cell with fibroblasts and keratinocytes displaying the highest levels. Both IL-1RI and IL-1RII are members of the Ig superfamily.
Although IL-1 has been demonstrated to play an important role in several in vivo phenomena, the detection of IL-1 in vivo using bioassays has been difficult. IL-1 is an endogenous pyrogen and induces fever in animal models. IL-1 is also involved in muscle proteolysis, bone resorption, wound healing, hematopoiesis and inflammatory diseases, including diabetes, periodontitis and rheumatoid arthritis.
6. Summary of the assayThis assay employs the quantitative ‘sandwich’ enzyme immunoassay technique. An antibody specific for (h)IL-1β has been coated on the microplate provided in the kit. Samples are pipetted into the wells along with biotinylated antibody reagent. If present, the (h)IL-1β is bound by the immobilized antibody and the biotinylated antibody. After washing away any unbound sample proteins and biotinylated antibody, a streptavidin-HRP conjugate is added to the wells. Any (h)IL-1β which was bound by both the immobilized and the biotinylated antibody during the first incubation will be bound by the streptavidin conjugate. Following a wash to remove unbound conjugate, a substrate solution is added to the wells and color develops in proportion to the amount of (h)IL-1β bound in the initial step.
In addition to the samples to be tested, a series of wells is prepared using known concentrations of the human IL-1β standard. A curve, plotting the optical density versus the concentration of the standard well, is prepared. By comparing the optical density of the samples to this standard curve, the concentration of the (h)IL-1β in the unknown samples is then determined.
7. Assay methodology Users are recommended to read this entire section before starting work.
7.1. Materials and equipment requiredThe following materials and equipment are required:
Pipettes or pipetting equipment with disposable tips (50 µl, 100 µl and 1.00 ml) Disposable polypropylene test tubes - do not use polystyrene, polycarbonate or glass Measuring cylinder, 2 L Distilled or deionized water A centrifuge for preparing streptavidin-HRP solution Plate reader capable of reading at 450 nm Optional equipment Assays may be performed with commercially available microplate washers to aid convenience and assay throughput.
7.2. Sample preparation Serum, plasma, urine and cell culture supernatants EDTA plasma interfere with plasma measurements of IL-1β in this ELISA. Use only plasma collected with sodium citrate or heparin. Serum, plasma, urine and culture supernatant samples that are to be assayed within 24 hours should be stored at 2–8°C. Specimens to be stored for longer periods of time should be frozen at -70°C to avoid loss of biologically active cytokine. Avoid freezing and thawing samples more than once. Test samples should be assayed in duplicate each time the ELISA is performed, 50 µl of sample per well is required in this way. Dilution of test samples If you suspect that the (h)IL-1β concentration of a sample exceeds the highest point of the standard curve, prepare one or more five fold dilutions of the test sample. Mix thoroughly between dilutions and before assaying.
7.3. Critical Parameters 1. First select the number of strips to be run and allow samples and
all reagents to reach room temperature prior to performing the assays. Do not use water baths to thaw samples or reagents. 2. Mix samples and all reagents thoroughly before use. 3. Avoid excessive foaming of reagents. Also avoid exposure of
reagents to excessive heat or light during storage and incubation. 4. Avoid handling the tops of the wells both before and after filling. 5. Standards and samples should be assayed in duplicate. 6. Run a separate standard curve for each assay. 7. The total dispensing time for each plate should not exceed 20 8. Use only coated wells from the same reagent batch for each
assay. Also do not mix reagents from different kit lots. 9. It is important that the wells are washed thoroughly and uniformly.
If using an automatic washer check operation of heads before starting. If washing by hand ensure that all wells are completely filled at each wash. 10. A small amount of precipitate may be present in some vials. It will
not affect assay performance and should be ignored.
8.1. Reagent preparation Wash buffer concentrate Any precipitate formed during storage will redissolve upon dilution. Dilute 30-fold to prepare 1500 ml of wash buffer. Store at 2–8°C until the expiry date of the kit. Do not use wash buffer if it becomes visibly contaminated on storage. Streptavidin-HRP solution Prepare the exact amount of streptavidin-HRP solution no more than 15 minutes prior to use.
The streptavidin-HRP concentrate may require spinning down to force the contents to the bottom of the vial. Add 30 µl of streptavidin-HRP concentrate per 12 ml of streptavidin-HRP dilution buffer in a plastic 15 ml tube and mix gently.
If running partial plates use 2.5 µl of streptavidin-HRP concentrate and 1 ml of streptavidin-HRP dilution buffer per strip being run.
8.2. Preparation of working standards a. Two vials of lyophilized standards are provided with this kit.
Reconstitute and use one vial per partial plate. b. Prepare standards shortly before use. Use within one hour of
reconstitution. Do not store reconstituted standards. c.Whenrunning culture supernatant samples, reconstitute
standard in distilled or deionized water. Reconstitution volume is stated on the standard vial label. The standard will take approximately 1 minute to dissolve. Mix by gently inverting the vial. Use your culture medium to prepare the dilutions of the standard curve, go to step e) below for further instructions. If running a partial plate, refer to step a) above. d.If running serum, plasma or urine samples, reconstitute standard
with distilled or deionized water. Reconstitution volume is stated on the standard vial label. The standard will take approximately 1 minute to dissolve. Mix by gently inverting the vial. Use the standard diluent provided to prepare the dilutions of the standard curve. If running a partial plate, refer to step a) above. e. Label 6 tubes, one for each standard curve point: 400 pg/ml,
160 pg/ml, 64 pg/ml, 25.6 pg/ml, 10.24 pg/ml and 0 pg/ml. Then prepare 1:2.5 serial dilutions for the standard curve as follows:
f. Pipette 240 µl of appropriate diluent (see steps c) and d) above) g. Pipette 160 µl of the reconstituted standard into the first tube, h. Pipette 160 µl of this dilution into the second tube labelled i. Repeat serial dilutions three more times. These concentrations,
400 pg/ml, 160 pg/ml, 64 pg/ml, 25.6 pg/ml, 10.24 pg/ml and 0 pg/ml are your standard curve. Running partial plates This ELISA provides the flexibility to run two partial plates on separate occasions. Decide the number of strips you wish to run, leaving the strips to be used in the frame. Remove the unnecessary strips and store them in the foil pouch with the desiccant provided at 2–8°C, making sure the foil pouch is sealed tightly.
When adding the TMB substrate reagent, pour out from the bottle only the amount needed to run the first half plate. Do not combine left over substrate with that reserved for the second half of the plate. Care must be taken to ensure that the remaining TMB substrate reagent is not contaminated. If the substrate reagent is bright blue prior to use, it has been contaminated. DO NOT USE.
8.3. Assay protocol 1. Prepare assay reagents and working standards as described in the 2. Set up the microplate with sufficient wells to enable the running of
all standards and samples as required (see figure 1). 3. Remove excess microplate strips from the frame and store in the 4. Add 50 µl of standard or sample per well, in duplicate. 5. Add 50 µl of the biotinylated antibody reagent to all wells being
used. Add 50 µl of appropriate sample diluent to any wells not being utilized. Cover with adhesive strip provided and incubate for 3 hours at room temperature (20–25°C).
6. Aspirate or decant each well and wash, repeating the process
twice for a total of three washes. Wash vigorously by filling each well with wash buffer using a washbottle, pipette or manifold dispenser. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towelling. 7. Add 100 µl of pre-diluted streptavidin-HRP conjugate. Cover
with a new adhesive strip and incubate for 30 minutes at room temperature (20–25°C). 8. Repeat the aspiration/wash step as in step 6. 9. Add 100 µl of TMB substrate solution into each well, incubate
for 30 minutes at room temperature (20–25°C). If the substrate reagent is bright blue prior to use, do not use. THE PLATE SHOULD BE DEVELOPED IN THE DARK. Do not cover the plate with aluminium foil or an adhesive strip. 10. Add 100 µl of stop solution to each well. 11. Determine the optical density of each well within 30 minutes,
using a spectrophotometer set to 450 nm. Figure 1. Recommended positioning of standard (0–400 pg/ml) and sample wells (S). Table 1. Assay protocol (all volumes are in microlitres) Standards Standard
Cover plate, incubate at room temperature for 3 hours.
Aspirate/decant and wash vigorously all wells three times
Cover plate, incubate at room temperature for 30 minutes.
Aspirate/decant and wash vigorously all wells three times
Incubate at room temperature for 30 minutes in the dark.
Determine optical density at 450 nm within 30 minutes. Summary of assay protocol
8.4. Calculation of resultsAverage the duplicate readings for each standard, control and sample and subtract the zero standard optical density.
Plot the optical density for the standards versus the concentration of the standards and draw the best curve. The data can be linearized by using a log/log plot and regression analysis can be applied to the log transformation.
Figure 2 shows such a plot of the data from table 2. The standard curve is provided for illustration only. A standard curve should be generated for each set of samples to be assayed. This allows for the measurement of 42 unknowns in duplicate. Figure 2. Standard curve Typical assay data The following data (table 2) were obtained for a standard curve using the protocol provided. Table 2. Typical assay data Tube Optical density Zero standard subtracted
9.1. SpecificityThis assay recognizes both natural and recombinant (h)IL-1β. It does not cross react with human IL-1α, IL-1ra, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, TNFα, IFNα, IFNγ or mouse IL-1β.
9.2. CalibrationThe standard in this ELISA is calibrated to the NIBSC reference lot 86/680. One (1)pg of Biotrak standard = 2 NIBSC pg.
9.3. Reproducibility Within-assay precision The within-assay coefficient of variation of the ELISA has been determined to be <10%. Between-assay precision The between-assay coefficient of variation of the ELISA has been determined to be <10%.
9.4. SensitivityThe minimum detectable dose of (h)IL-1β was determined to be <1 pg/ml (0.05 pg/wel ), by adding two standard deviations to the optical density value of zero and calculating the corresponding concentration from the standard curve.
9.5. ParallelismThe linearity of dilution was determined by serial y diluting six different positive samples. The dilutions were run in the ELISA and ‘found’ doses were plotted against ‘expected’ doses.
9.6. RecoveryRecovery in the ELISA has been determined by spiking low, medium and high levels of recombinant human IL-1β into normal human serum, plasma (sodium citrate), and urine samples, as well as a control buffer. The values below are typical recoveries:
Recovery from serum Recovery from plasma Recovery from urine
9.7. Expected valuesNormal average levels of (h)IL-1β in 9 serum samples is 0 pg/ml.
Normal average levels of (h)IL-1β in 9 plasma samples is 0 pg/ml.
Normal average levels of (h)IL-1β in 8 urine samples is 0 pg/ml.
10. References 1. DINARELLO, C.A. et al., Blood,77, p.1627, 1991. 2. CANNON, J.G. et al., J. Infect. Dis., 161, p.79, 1990. 3. GRASSI, J. et al., J Immunol. Methods,123, p.193, 1989. 4. LONNERMANN, G. et al., Lymphokine Res.,7, p.75, 1988. 5. ZUCALL, J.R. et al., Blood, 69, p.33, 1987. 6. DINARELLO, C.A. et al., J. Clin. Invest.,77, p.1743, 1987. 7. DINARELLO, C.A. et al., Year Immunol.,2, p.68, 1986. 8. KRONHEIM, S.R. et al., J. Exp. Med.,161, p.1490, 1985. Biotrak range of Easy ELISA systems Interferon-alpha [(h)IFNα]
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Cell Proliferation assay system and reagents Cell Proliferation Biotrak ELISA
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