K-ASSAY 

KAMIYA BIOMEDICAL COMPANY

KAMIYA BIOMEDICAL COMPANY

Rat Tumor Necrosis Factor
Alpha (TNFa) ELISA
For the quantitative determination of Rat TNFa inserum, plasma, tissue homogenates, cell culture supernates, andother biological fluids

Cat. No. KT-30800

For Research Use Only. Not for use in diagnostic procedures.

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Product Information
Rat Tumor Necrosis Factor Alpha (TNFa) ELISA
Cat. No. KT-30800

INTENDED USEThis ELISA kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of Rat TNFa
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.
Deionized 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 be
kept 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 above.

PRINCIPLE
The microtiter plate provided in this kit has been pre-coated with an antibody specific to TNFa. Calibratorsand samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal
antibody preparation specific for TNFa. 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 those wells that contain TNFa, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibita change in color. The enzyme-substrate reaction is terminated by the addition of a sulfuric acid solution
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and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The
concentration of TNFa in the samples is then determined by comparing the O.D. of the samples to thecalibration curve.

SAMPLE COLLECTION AND STORAGE
Serum
Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at
4°C before centrifugation for 20 minutes at approximately 1000 x g. Assay freshly prepared serum
immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw
cycles.

PlasmaCollect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x
g at 2°C - 8°C within 30 minutes of collection. Remove plasma and assay immediately or store samples inaliquots 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, tissues
were rinsed in ice-cold PBS (0.02 mol/L, pH 7.0-7.2) to remove excess blood thoroughly and weighed
before homogenization. Mince the tissues into small pieces and homogenize them in 5-10 mL of PBS
with a glass homogenizer on ice (Micro Tissue Grinders work too). The resulting suspension wassonicated with an ultrasonic cell disrupter or subjected to two freeze thaw cycles to further break the cell
membranes. After that, the homogenates were centrifugated for 5 minutes at 5000 x g. Remove thesupernate and assay immediately or aliquot and store at ≤-20°C.

Cell Culture Supernates and Other Biological Fluids
Centrifuge samples for 20 minutes at 1000 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 2-8°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 excessive 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 0.5 mL of Calibrator Diluent and allow it to sit for 10 minutes at room
temperature. Then mix uniformly but gently, avoid foaming. The concentration of the stock calibrator
solution is now at 1,000 pg/mL. Use the stock calibrator solution and the Calibrator Diluent to produce adilution series. Pipette 250 µL of Calibrator Diluent into each tube. Then perform a serial dilution,
beginning with the stock calibrator solution, to create the other calibrators (as shown below). Make sure touse a new pipette tip for each transfer and mix each tube thoroughly before the next transfer. The
calibrators will have concentrations of 1,000, 500, 250, 125, 62.5, 31.2, 15.6 pg/mL, and the last tube
filled with only Calibrator Diluent will serve as the blank at 0 pg/mL.

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Tube
pg/mL

1
1,000

KAMIYA BIOMEDICAL COMPANY

2
500

3
250

4
125

5
62.5

6
31.2

7
15.6

8
0

Assay Diluent A and B
Dilute 6 mL of Assay Diluent A or B Concentrate (2X) with 6 mL of deionized or distilled water to prepare
12 mL of Assay Diluent A or B. The prepared working dilution can not be frozen.

Detection Reagent A and B
Briefly spin or centrifuge the stock Detection Reagent A and Detection Reagent B before use. Dilute to
the 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 deionized 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 to
the vial.

Note:
1. Prepare the calibrators within 15 minutes of beginning the assay. Do not dissolve the reagents at
37°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 to
draw 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.
6. Distilled water is recommended to be used to prepare reagents or samples. Contaminated water orcontainers for reagent preparation will influence the detection result.

SAMPLE PREPARATION
1. Kamiya Biomedical Company is only responsible for the kit itself, but not for the samples consumed
during the assay. The user should calculate the possible amount of the samples used in the wholetest. Please reserve sufficient samples in advance.
2. Please predict the concentration before assaying. If values for these are not within the range of thecalibration curve, users must determine the optimal sample dilutions for their particular experiments.

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3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of
the kit is necessary.
4. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA
results due to the impacts of certain chemicals.
5. Owing to the possibility of mismatching between antigen from other resource and antibody used in
our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or
recombinant proteins from other manufacturers may not be recognized by our products.
6. Influenced by the factors including cell viability, cell number and also sampling time, samples fromcell culture supernatant may not be detected by the kit.
7. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradationand denaturalization may occur in those samples and finally lead to wrong results.
8. If the humidity level in the laboratory does not reach 60%, a humidifier is recommended to be used.

ASSAY PROCEDURE
Estimate the sample TNFa 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 sharply striking the plate on absorbent paper. Repeat
wash 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting.
Invert the plate 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 (Do not exceed 30 minutes). Protect from light. The solution will turn blue after the
addition of Substrate Solution.
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 no
bubbles on the surface of the liquid. Then, run the microplate reader and conduct measurements at
450 nm immediately.

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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 10
minutes. 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 good
performance. 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 on log-log graph paper with TNFa concentration on
the y-axis and absorbance on the x-axis. Draw the best fit line through the calibration points and it can be
determined by regression analysis. It is recommended to use some related software to do thiscalculation. If samples have been diluted, the concentration 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 TNFa is typically less than 6.1 pg/mL. The sensitivity of this assay,
or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be
differentiated from zero. It was determined by the mean O.D. value of 20 replicates of the zero calibrator
plus three standard deviations.

Specificity
This assay has high sensitivity and excellent specificity for detection of Rat TNFa. No significant crossreactivity or interference was observed.

ASSAY PROCEDURE SUMMARY
1.
2.
3.
4.
5.
6.

Prepare all reagents, samples and calibrators;
Add 100 µL calibrator or sample to each well. Incubate 2 hours at 37°C;
Add 100 µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
Aspirate and wash 3 times;
Add 100 µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
Aspirate and wash 5 times;
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7. Add 90 µL Substrate Solution. Incubate 15-25 minutes at 37°C;
8. Add 50 µL Stop Solution. Read at 450 nm immediately.

IMPORTANT NOTES
1. The final experimental results will be closely related to operation skills of the end users and theexperimental environments. Please make sure that sufficient samples are available.
2. Kits from different batches may be a little different in detection range, sensitivity and color developing
time. Please perform the experiment exactly according to the instruction attached in kit whileelectronic ones from our website (www.k-assay.com) is only for information.
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 covered tightly to prevent the evaporation and contamination of microorganism.
5. There may be some foggy substance in the wells when the plate is opened at the first time. It will nothave any effect on the final assay results. Do not remove microtiter plate from the storage bag until
needed.
6. Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting
for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10 nm
or less and an optical density range of 0-3 O.D. or greater at 450 ± 10 nm wavelength is acceptablefor use in absorbance measurement. Please read the instruction carefully and adjust the instrument
prior to the experiment.
7. Even the same operator might get different results in two separate experiments. In order to get better
reproducible results, the operation of every step in the assay should be controlled. Furthermore, apreliminary experiment before assay for each batch is recommended.
8. Each kit has been strictly passed Q.C. test. However, results from end users might be inconsistent
with our in-house data due to some unexpected transportation conditions or different lab equipments.
Intra-assay variance among kits from different batches might arise from above factors, too.
9. Kits from different manufacturers for the same item might produce different results, since we haven’t
compared our products with other manufacturers.
10. The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, andclothing protection when using 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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