K-ASSAY 

KAMIYA BIOMEDICAL COMPANY

KAMIYA BIOMEDICAL COMPANY

Rat Interleukin 1 Beta (IL1B)
ELISA
For the quantitative determination of rat IL1B inserum, plasma, tissue homogenates, cell culture supernates, andother biological fluids

Cat. No. KT-18885

For Research Use Only. Not for use in diagnostic procedures.
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KAMIYA BIOMEDICAL COMPANY

Product Information
Rat Interleukin 1 Beta (IL1B) ELISA
Cat. No. KT-18885

INTENDED USEThis ELISA kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of rat IL1B
in serum, plasma, tissue homogenates, cell culture supernates, and other biological fluids. For researchuse only. Not for use in diagnostic procedures.

COMPONENTS
Reagents

Quantity

Pre-coated, ready to use 96-well plate1

Calibrator (lyophilized)

2

Calibrator Diluent

1 × 20 mL

Detection Reagent A

1 × 120 µL

Detection Reagent B

1 × 120 µL

Assay Diluent A (2X concentrate)

1 × 6 mL

Assay Diluent B (2X concentrate)

1 × 6 mLTMB Substrate

1 × 9 mL

Stop Solution

1 × 6 mL

Wash Buffer (30X concentrate)

1 × 20 mL

Plate sealer for 96 wells

4

MATERIALS REQUIRED BUT NOT SUPPLIED
1.
2.
3.
4.
5.
6.

Microplate reader with 450 ± 10 nm filter.
Precision single and multi-channel pipettes and disposable tips.
Eppendorf Tubes for diluting samples.
De-ionized or distilled water.
Absorbent paper for blotting the microtiter plate.
Container for Wash Solution.

STORAGEAll reagents should be stored according to their label. The Calibrators, Detection Reagent A, Detection
Reagent B and the 96-well plate should be stored at -20°C upon receipt. The unused strips should bekept in a sealed bag with the desiccant provided to minimize exposure to damp air. Opened test kits willremain stable until the expiration date, provided they are stored as prescribed above.

PRINCIPLE
The microtiter plate provided in this kit has been pre-coated with an antibody specific to IL1B. Calibratorsand samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal
antibody preparation specific for IL1B. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added
to each microplate well and incubated. Then the TMB substrate solution is added to each well. Only thosewells that contain IL1B, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change incolor. The enzyme-substrate reaction is terminated by the addition of a sulfuric acid solution and the color
change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of IL1B
in the samples is then determined by comparing the O.D. of the samples to the calibration curve.
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K-ASSAY KAMIYA BIOMEDICAL COMPANYSAMPLE COLLECTION AND STORAGE
Serum
Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at4°C before centrifugation for 20 minutes at approximately 1,000 x g. Assay freshly prepared serum
immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze/thawcycles.

PlasmaCollect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1,000 xg at 4°C within 30 minutes of collection. Remove plasma and assay immediately or store samples inaliquot at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

Tissue Homogenates
The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissue wasrinsed with ice-cold PBS (0.02 mol/L, pH7.0-7.2) to remove excess blood and weighed before
homogenization. Tissues were minced to small pieces and homogenized in 5~10 mL of PBS with a glass
homogenizer on ice (Micro Tissue Grinder works too). The resulting suspension was sonicated with anultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. Afterthat the homogenates were centrifugated for 5 minutes at 5,000 x g. Remove the supernate and assay
immediately or store in aliquot at ≤ -20°C.

Cell Culture Supernates and Other Biological Fluids
Centrifuge samples for 20 minutes at 1,000 x g. Remove particulates and assay immediately or aliquot
samples and store at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

Note:
1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C(≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and contamination.
2. Before performing the assay, slowly bring samples to room temperature.
3. Avoid hemolysis as hemolysis will impact the result.

REAGENT PREPARATION
Bring all kit components and samples to room temperature (18-25°C) before use.

Calibrators
Reconstitute the Calibrator with 1.0 mL of Calibrator Diluent, kept for 10 minutes at room temperature.
Shake gently, but avoid foaming. The concentration of the stock calibrator solution is 1,000 pg/mL.
Prepare 7 tubes containing 0.5 mL Calibrator Diluent and use the stock calibrator solution to produce a
double dilution series according to the picture shown below. Mix each tube thoroughly before the nexttransfer. Set up 7 points of diluted calibrator such as 1,000, 500, 250, 125, 62.5, 31.2, 15.6 pg/mL, andthe last EP tubes with Calibrator Diluent will serve as the blank at 0 pg/mL.

Tubepg/mL

11,000

2500

3250

4125
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Assay Diluent A and B
Dilute 6 mL of Assay Diluent A or B Concentrate (2X) with 6 mL of de-ionized or distilled water to prepare
12 mL of Assay Diluent A or B. The prepared working dilution cannot be frozen.

Detection Reagent A and B
Briefly spin or centrifuge the stock Detection Reagent A and Detection Reagent B before use. Dilute tothe working concentration with working Assay Diluent A or B, respectively (1:100).

Wash Solution
Dilute 20 mL of Wash Solution Concentrate (30X) with 580 mL of de-ionized or distilled water to prepare
600 mL of Wash Solution (1X).

TMB Substrate
Aspirate the needed dosage of the solution with sterilized tips and do not return the residual solution tothe vial.

Note:
1. Prepare the calibrators within 15 minutes of beginning the assay. Do not dissolve the reagents at37°C.
2. Do not perform your serial dilutions directly in the wells.
3. Carefully reconstitute Calibrators or working Detection Reagent A and B according to the instruction.
Avoid foaming and mix gently until the crystals have completely dissolved. To minimize imprecisioncaused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended todraw more than 10 µL for pipetting.
4. The reconstituted Calibrators, Detection Reagent A and Detection Reagent B can be used onlyonce.
5. If crystals have formed in the Wash Solution concentrate (30X), warm to room temperature and mix
gently until the crystals have completely dissolved.

ASSAY PROCEDURE
Estimate the sample IL1B concentration before assaying. If the estimated values are not within the rangeof the calibration curve, users must determine the optimal sample dilutions for their particularexperiments.
1. Determine wells to be used for diluted calibrators, blank and samples. Prepare 7 wells for calibrators,
1 well for blank. Add 100 µL each of dilutions of calibrators (see Reagent Preparation), blank andsamples into the appropriate wells. Cover with the Plate sealer. Incubate for 2 hours at 37°C.
2. Remove the liquid from each well, do not wash.
3. Add 100 µL of the Detection Reagent A working solution to each well. Incubate for 1 hour at 37°C
after covering with the Plate sealer.
4. Aspirate the solution and wash each well with 350 µL of 1X Wash Solution using a squirt bottle, multichannel pipette, manifold dispenser or auto-washer, and let sit for 1~2 minutes. Remove theremaining liquid from all wells completely by snapping the plate onto absorbent paper. Repeat wash 3times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert theplate and blot it against absorbent paper.
5. Add 100 µL of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37°C
after covering with the Plate sealer.
6. Repeat the aspiration/wash process five times as in step 4.
7. Add 90 µL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 25
minutes at 37°C. Protect from light. The solution will turn blue after the addition of Substrate Solution.
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8. Add 50 µL of Stop Solution to each well. The liquid will turn yellow after the addition of Stop solution.
Mix the liquid by gently tapping the side of the plate. If color change does not appear uniform, gently
tap the plate to ensure thorough mixing.
9. Remove any drops of solution or fingerprints on the bottom of the plate and confirm there are nobubbles on the surface of the liquid. Then, run the microplate reader and conduct measurements at
450 nm immediately.

Note:
1. Assay preparation: Keep appropriate numbers of strips for 1 experiment and remove extra strips
from microtiter plate. Unused strips should be resealed and stored at -20°C until the expiration date.
2. Sample or reagent additions: Use freshly prepared Calibrators. Carefully add samples to wells andmix gently to avoid foaming. Do not touch the well walls if possible. For each step in the procedure,
total dispensing time for addition of reagents or samples to the assay plate should not exceed 10minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplicationof all calibrators and samples, although not required, is recommended. To avoid cross-contamination,change pipette tips between additions of each calibrator level, between sample additions, andbetween reagent additions. Also, use separate reservoirs for each reagent.
3. Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps isnecessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Afterreagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay.
Incubation time and temperature must be observed.
4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential to goodperformance. After the last wash, remove any remaining Wash Solution by aspirating or decanting
and remove any drops of solution or fingerprints on the bottom of the plate. Insufficient washing will
result in poor precision and falsely elevated absorbance reading.
5. Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g.
observation once every 10 minutes), if the color is too dark, add Stop Solution in advance to avoid
excessively strong reaction which will result in inaccurate absorbance reading.
6. TMB Substrate is easily contaminated. Protect from light.

CALCULATION OF RESULTS
Average the duplicate readings for each calibrator, control, and sample and subtract the average zero
calibrator optical density. Create a calibration curve by reducing the data using computer software
capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a calibration
curve by plotting the mean absorbance for each calibrator on the x-axis against the concentration on they-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting
the log of the IL1B concentrations versus the log of the O.D. and the best fit line can be determined byregression analysis. This procedure will produce an adequate but less precise fit of the data. It is
recommended to use some related software to do this calculation. If samples have been diluted, theconcentration read from the calibration curve must be multiplied by the dilution factor.

PERFORMANCE
Detection Range
The detection range is: 15.6-1,000 pg/mL.
The calibration curve concentrations used for the ELISA’s were 1,000, 500, 250, 125, 62.5, 31.2, 15.6
pg/mL.

Sensitivity
The minimum detectable dose of rat IL1B is typically less than 4.2 pg/mL. The sensitivity of this assay, orLower Limit of Detection (LLD) was defined as the lowest protein concentration that could bedifferentiated from zero. It was determined by the mean O.D. value of 20 replicates of the zero calibrator
plus three standard deviations.
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K-ASSAY KAMIYA BIOMEDICAL COMPANYSpecificity
This assay has high sensitivity and excellent specificity for detection of rat IL1B. No significant crossreactivity or interference was observed.

IMPORTANT NOTES
1. The final experimental results will be closely related to the operative skills of the end users and theexperimental environments. Please make sure that sufficient samples are available.
2. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, andother factors present in biological samples. However, until all factors have been tested the possibilityof interference cannot be eliminated.
3. Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied bymanufacturer.
4. Protect all reagents from strong light during storage and incubation. All the bottle caps of reagentsshould be tightly closed to prevent the evaporation or contamination by microorganisms.
5. There may be some foggy substance in the plate wells when first opened. This will have no effect on
the final assay results. Do not remove microtiter plate from the storage bag until needed.
6. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 O.D. orgreater at 450 nm wavelength is acceptable for use in absorbance measurement.
7. The Stop Solution used with this kit is an acid. Wear eye, hand, face, and clothing protection whenusing this material.

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

KAMIYA BIOMEDICAL COMPANY
12779 Gateway Drive, Seattle, WA 98168
Tel: (206) 575-8068 Fax: (206) 575-8094
Email: LifeScience@k-assay.com
www.k-assay.com

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