Bioworld Technology,Inc
Human IL-1β ELISA Kit (96T)
Cat No. BE0001
The Human IL-1β ELISA Kit is an enzyme-linked immunosorbent assay for the quantitative detection of Human
IL-1β concentrations in cell culture supernatants, serum, plasma, and tissue. This package insert must be r ead
in its entirety before using this product.
This kit is for research use only. Do not use in diagnostic procedures.
INTRODUCTION
Interleukin 1 (IL-1) includes two distinct proteins, IL-1α and IL-1β, that play central roles in acute and chronic
inflammation, both locally and systemically. Human IL-1βis synthesized as a 269 amino acid (aa), 31 kDa
precursor protein (prepro-IL-1β) that is cleaved by IL-1β-converting enzyme (ICE) to the 153 aa, 17 kDa
mature IL-1β plus a prosegment. Some combination of the mature form, the prosegment and prepro-IL-1βis
released from the cell. IL-1β is produced primarily by monocytes and macrophages but also by astrocytes,
oligodendroglia, adrenal cortical cells, NK cells, endothelial cells, keratinocytes, megakaryocytes, platelets,
neurons, neutrophils, osteoblasts, Schwann cells, trophoblasts, T cells, and fibroblasts. The most extensively
studied function of IL-1 is initiation of inflammation. Bacterial endotoxin or a variety of non-microbial
inflammatory substances induce production of IL-1, which is released into the local environment. IL-1 induces
capillary endothelial cells to secrete chemokines (e.g. MCP-1) and to increase expression of cell adhesion
molecules (e.g. E-selectin, ICAM-1 and VCAM-1).
PRINCIPLES OF THE TEST
This assay employs the Sandwich immunoassay technique. An anti-h IL-1βmonoclonal coating antibody is
adsorbed onto microwells. IL-1βpresent in the sample or standard binds to antibodies adsor bed to the
microwells. Following incubation unbound sample or standard are removed during a wash step. a Biotinylated
anti-h IL-1βantibody is added and binds to IL-1βcaptured by the first antibody. Following incubation unbound
Biotinylated anti-h IL-1βantibody is removed during a wash step. A Streptavidin-HRP is added and binds to
Biotinylated anti-h IL-1βantibody. Following incubation unbound Streptavidin-HRP is removed during a wash
step. A coloured product is formed in proportion to the amount of IL-1βpresent in the sample. The reaction is
terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from
seven IL-1βstandard dilutions and IL-1βsample concentration deter-mined.
Step 1
Step 2
Step 3
coloured
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REAGENTS PROVIDED
Name
96 Tests
Store
1．Aluminium pouches with a Microwell Plate
8×12
4℃
2．Standard
2 Vial
4℃
3．Biotinylated antibody
2 Vial
4℃
4．Streptavidin-HRP
2 Vial
4℃(Avoid light.)
5．Assay Buffer (25ml/Vial)
6．Wash Buffer Concentrate 20x (30ml/Vial)
2 Vial
1 Vial
4℃
4℃
7．TMB Substrate Solution
8．Stop Solution (12ml/Vial)
12ml /1 Vial
1 Vial
4℃(Avoid light.)
4℃
9．Plate Covers- Adhesive strips
3 strips
10．Instruction
1
MATERIALS REQUIRED BUT NOT PROVIDED
1. Microwell strip reader capable of reading at 450 nm (If wavelength correction is available, set to 570 nm or
630 nm.)
2. 0.5-10ul, 2-20ul, 20-200ul, 200-1000ul adjustable micropipettes with disposable tips.
3. Deionized or distilled water.
4. 37℃ Incubator.
5. Statistical calculator with program to perform linear regression analysis.
SAMPLE COLLECTION AND STORAGE
1. The test tubes of collect sample must be clean.
2. Collect plasma using EDTA as an anticoagulant. Samples containing a visible precipitate must be clarified
prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens.
3. Samples must be stored frozen at -20℃ to avoid loss of bioactive. If samples are to be run within 24 hours,
they may be stored at 2℃ to 8℃. Avoid repeated freeze-thaw cycles. Prior to assay, frozen sera or plasma
should be brought to room temperature slowly and gently mixed by hand.
4. Serum and plusma samples require dilution. Dilution of 1:2 with Assay Buffer is recommend.
PRECAUTIONS FOR USE
1. Store kit reagents between 2℃ to 8℃. Immediately after use reagents should be returned to cold storage
(2℃ to 8℃).
2. Expiry of the kit and reagents is stated on labels.
3. Use clean, dedicated reagent trays for dispensing the conjugate and substrate reagents.
4. Store reconstituted standard at 2℃ - 8℃ for up to 60 days or aliquot and store at -70℃ for up to 6 months.
5. Do not mix or substitute reagents with those from other lots or other sources.（Except wash buffer and stop
solution）
6. Thorough mixing is very important .Gently tap the plate, if available on a rotator set at 100 rpm to ensure
thorough mixing.
7. It is recommended that all samples and standards be assayed in duplicate.
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PREPARATION OF REAGENTS
1. Bring all reagents and sample to room temperature before use.
2. Pour entire contents (30 ml) of the Wash Buffer Concentrate into a clean 1,000 ml graduated cylinder. Bring
final volume to 600 ml with glass-distilled or deionized water(1:20).
3. Reconstitute the standard with 1.0 ml of Assay buffer. This reconstitution produces a stock solution of 1000
pg/ml. Mix the standard to ensure complete reconstitution and allow the standard to sit for a minimum of 15
minutes with gentle agitation prior to making dilutions. A seven point standard curve using 2-fold serial
dilutions in assay buffer, and a high standard of 500 pg/ml is recommended. The assay buffer serves as the
zero standard (0 pg/mL).
Preparation of standard dilutions：
4. Preparation of Biotinylated antibody
The Biotinylated antibody must be diluted 1:100 with Assay Buffer just prior to use in a clean plastic test
tube.
5. Preparation of Streptavidin-HRP
The Streptavidin-HRP must be diluted 1:100 with Assay Buffer just prior to use in a clean plastic test tube.
Wash plate:
Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by
filling each well with Wash Buffer (350ul) using a squirt bottle, 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.
ASSAY PROCEDURE
1. Bring all reagents and samples to room temperature before use. Remove excess microplate strips from the
plate frame, return them to the foil pouch containing the desiccant pack, reseal.
2．Add 100µl of Assay Buffer to the blank wells.
3．Add 100ul of Standard or Sample per well. Cover with the adhesive strip provided. Incubate for 90 minutes
at 37℃.
4．Prepare Biotinylated antibody.
5．Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling
each well with Wash Buffer (350ul) using a squirt bottle, manifold dispenser or autowasher. Complete
removal of liquid at each step is essential to good performance. After the last wash, remove any remaining
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Phone: 6123263284(USA) 0086-025-68037686(CHN)
Bioworld Technology,Inc
Wash Buffer by aspirating or decanting. Invert the plate and blo t it against clean paper towels.
6．Add 100µl of diluted Biotinylated antibody to all wells( Except the blank well). Cover with the adhesive
strip provided. Incubate for 60 minutes at 37℃.
7．Prepare HRP-Conjugate.
8．Repeat the aspiration/wash as in step 5.
9．Add 100µl of diluted HRP-Conjugate to all wells( Except the blank wells). Cover with the adhesive strip
provided. Incubate for 30 minutes at 37℃.
10．Repeat the aspiration/wash as in step 5.
11．Add 100ul of TMB Substrate Solution to each well(Including the blank wells). Incubate for 15
minutes at 37℃. Protect from light.
12．Add 100ul of Stop Solution to each well(Including the blank wells). Determine the optical density of each
well within 30 minutes, using a microplate reader set to 450 nm. If wavelength correction is available, set
to 570 nm or 630 nm.
CALCULATION OF RESULTS
1．Create a standard curve by plotting the mean absorbance for each standard concentration on the ordinate
against the standard concentration on the abscissa. Draw a best fit curve through the
points of the graph.
2．To determine the concentration of 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 sample concentration.
3．If samples have been diluted, the concentration read from the standard curve must be multiplied by the
dilution factor.
Representative standard curve
Standard
500
250
125
62.5
31.25
15.625
7.8
0
OD
2.132
1.523
0.964
0.553
0.348
0.201
0.129
0.054
A standard curve should be generated for each set of samples assayed.
Sensitivity, Specificity and Precision:
1． Sensitivity: The minimum detectable dose of Human IL-1βis 2pg/ml.
2． Specificity: There was no detectable cross reactivity with any of the tested proteins following IL-2,3,4,5,6,
8,10,12,TNF,VEGF,TGF and IFN.
3． Precision: The overall variation coefficient of precision between assays and precision within an assay are
less than 10%.
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Email: [email protected]
Phone: 6123263284(USA) 0086-025-68037686(CHN)