rev: 5/16/2008
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Sample Kit Protocol: (see actual insert with kit for batch specific information):
Pelikine (tm)
Human TNF-ALPHA ELISA Kit
CAT# RDI-M1920clb, new cat#: 55R-M1920clb 96 wells
PRICE: $621.50/kit
INTRODUCTION
Tumor necrosis factor a (TNF) is an extremely potent peptide cytokine which serves as an endogenous mediator of inflammatory, immune and host defence functions. Several substances originally described for their biological activities have been identified as TNFa; cachectin, macrophage cytotoxin (MCT), necrosin, cytotoxin (CTX), haemorrhagic factor, macrophage cytotoxic factor (MCF) and differentiation-inducing factor (DIF). TNFa is capable of acting independently and in conjunction with a variety of other factors to affect the phenotype and metabolism of cells in every tissue of the body. It is generally thought that TNFa is not produced constitutively by normal cells, but rather to be induced potently by invasive stimuli in the setting of both neoplastic and infectious disease. In this role macrophages and monocytes are thought to be the cells which contribute most to the local and systemic TNFa response to bacterial, viral and parasitic organisms and products. Bioassays for the quantification of TNFa, including the cytotoxic assay on murine fibroblasts have been used for several years. However, TNFa shares many of the biological effects of IL-1 and for this reason the two commonly interfere in bioassays. Although the cytotoxic assay mentioned above, is unaffected by IL-1, it remains time consuming and might be susceptible to interference by other substances. The Pelikine human TNFa ELISA kit has been developed for faster, more reproducible and specific quantification of human TNFa in serum, plasma and other body fluids, as well as in cell-culture supernatant.
II. PRINCIPLE OF THE TEST
The Pelikine human TNFa ELISA kit is a "sandwich-type" of enzyme immunoassay in which a monoclonal anti-human TNFa antibody is bound onto polystyrene microplate wells. Human TNFa present in a measured volume of sample or standard is captured by the antibody on the microplate plate, and non-bound material is removed by washing.Subsequently, a biotinylated polyclonal antibody to human TNFa is added.This antibody binds to the TNFa-antibody complex present in the microplate well. Excess biotinylated antibody is removed by washing, followed by addition of horseradish peroxidase (HRP) conjugated streptavidin, which binds onto the biotinylated side of the TNFa sandwich. After removal of non-bound HRP conjugate by washing, a substrate solution is added to the wells. A colored product is formed in proportion to the amount of TNFa present in the sample or standard. After the reaction has been terminated by the addition of a stop solution, absorbance is measured in a microplate plate reader. From the absorbance of samples and those of a standard curve, the concentration of TNFa can be determined by interpolation with the standard curve.
III. STORAGE AND STABILITY
The Pelikine human TNFa ELISA kit should be stored at 2-8'C. At arrival the components of the Pelikine human TNFa Elisa kit will be stable for at least 3 months when kept at 2-8'C. Prolonged stability up to the expiration date shown on the case label can be achieved by storing the streptavidine-HRP conjugate separately below -18'C and store the remainder of the Pelikine human TNFa ELISA kit at 2-8'C.
IV. CONTENTS OF THE KIT
The Pelikine human TNFa ELISA kit contains material sufficient for 96 tests, including standard curve samples. The reagents provided are:
Quantity Kit Component Volume
1 pc precoated microplateplate 12 x 8 strips + frame -
2 vials TNFa standard 10pg/ml lyophilized 200ul
1 vial biotinylated TNFa antibody 100-fold concentrated 200ul
1 vial streptavidin-HRP conjugate 10,000-fold concentrated 20ul
1 bottle wash buffer 20-fold concentrated 50ml
1 bottle HPE dilution buffer 5-fold concentrated 60ml
1 bottle TMB substrate solution ready-for-use 12.5ml
1 bottle stop solution(0.18M H(2)So(4)) ready-for-use 13.5ml
5 pcs plate seals -
V. Additional Materials Required
- Pipetting devices for accurate delivery of 1-10 ul, 50 ul, 100 ul, and 1 ml volumes.
- Distilled or de-ionized water.
- Polypropylene or polyethylene tubes for making sample dilutions, do no use polystyrene, polycarbonate or glass tubes.
- Beakers, flasks, cylinders necessary for preparation of reagents.
- Device for delivery of wash buffer (wash bottle/ automated plate washer).
- microplate plate reader, capable of measuring absorbance at 450 nm.
VI. PRECAUTIONS FOR USE
1) The Pelikine human TNFa ELISA kit is intended for research purposes only.
2) Only use the reagents and microplate plates supplied with the kit, do not mix reagents from different kit lots.
3) Handle all plasma and serum samples with care to prevent transmission of blood-borne infections.
4) Sodium azide inactivates HRP, do not use sodium azide-containing solutions, nor add sodium azide to the supplied materials
5) All reagents contain thiomersal (0.001 % w/v) and may be toxic upon ingestion, inhalation or skin contact. Avoid contact of skin, eyes or clothing with dilution, washing or substrate buffer.In case of contact, wash skin or eyes with water and consult a physician.
6) With the exception of the substrate blank wells, do not allow wells to stand uncovered or dry for extended periods between incubation steps.
7) The TNFa standard contains human serum which has been found to be non-reactive for Hepatitis B surface Antigen (HBsAg),Hepatitis C Virus (HCV) and human Immunodeficiency Virus (HIV). Nevertheless the standard should be handled as potentially hazardous and capable of transmitting diseases.
VII. Assay Procedure
- Bring All Reagents To Room Temperature (18-25'C)
For your convenience an easy-reference manual with check list and plate plan
are available on the last pages of this leaf let.
- Mix all reagents thoroughly without foaming before use.
-It is advised to test all samples and standard dilutions in duplicate.
1.microplate PLATE
The kit contains one frame with twelve pre-coated strips of eight microwells, vacuum sealed in a plastic bag. The CLB TNFa ELISA provides the flexibility to run two partial plates on separate occasions. Before opening the plastic bag determine the number of strips required to test the desired number of samples plus 18 wells needed for running standards in duplicate. Remove extra strips from holder and repack these in the plastic bag with the desiccant.
4. BUFFERS buffer preparation
Wash buffer concentrate Prepare a working-strength solution by adding 50 ml of the wash buffer concentrate (total content of the bottle) to 950 ml distilled water. The working-strength solution wash buffer can be stored for up to 2 months at 2-8'C.
HPE dilution buffer
Calculate the quantity of dilution buffer required (approximately 2 ml undiluted buffer per microwell strip) and prepare a working-strength solution by diluting the opalescent concentrated buffer5 times in distilled water before use. The working-strength dilution buffer can be stored for up to one week at 2-8'C.
3. TNFa STANDARD standard curve preparation
The kit contains two vials with 10ng/ml of a lyophilized natural human TNFa standard, calibrated against the WHO International Standard (TNFA 87/650; National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, U.K. 1 WHO Unit =25 pg TNFA).
Reconstitute one vial by adding 200 ul of distilled water. Allow 10 minutes at room temperature to dissolve and mix gently. The reconstituted standard can be stored for 2 hours at 2-8'C and for 1 week at <18'C. Avoid repeated freeze-thawing of the standard, although experimental data have shown that up to 3 freeze-thaw cycles have no effect on the TNFa levels of the standard.The second vial of lyophilized standard can be used in later assays.
Label 8 tubes, one tube for each dilution: 1000, 300, 111, 37, 12.4, 4.1,and 1.4 and 0pg/ml. Pipette 360 ul of HPE-dilution buffer into the first tube (1000pg/ml) and 300ul to the other tubes.
Pipette 40 ul of the TNFa standard (10ng/ml) into the first tube (1000 pg/ml) and mix well.
Transfer 150 ul of this dilution into the next tube (333 pg/ml) and continue to obtain serial dilutions.
The last tube (0 pg/ml) contains only HPE-dilution buffer.
It is recommended to prepare two separate series for each assay.
4. PREPARATION OF SAMPLES
Serum, EDTA-anti-coagulated plasma, and culture fluids are suitable for use in the assay (caution: separate plasma/serum and blood cells within 4 hours after collection, non-separated samples must be kept on temperatures from 2 to 8'C). Do not use grossly haemolyzed or lipemic specimens. If samples are to be run within 24 hours, they may be stored at 2-8'C; otherwise samples should be stored frozen (<-18'C, preferably <-70'C). Up to 3 freeze-thaw cycles have no effect on the TNFa levels of serum or plasma samples. Nonetheless, excessive free-thaw cycles should be avoided. Prior to the assay, frozen samples should be thawed as quickly as possible in tap water (18-25'C), do not use 37'C or 56'C water baths for this purpose.
Dilute the samples at least 1:2 in HPE-dilution buffer. If TNFa levels of >100 pg/ml are expected in the test samples, additional dilutions of samples (i.e. 1:10 1:50) should also be prepared.
5. First Washing Step.
Wash the required microwells in the plate-frame five times with washing buffer. For manual washing, completely fill the wells (>300ul) with washing buffer and discard, repeat this procedure four times. Finally the wells should be completely empty: Subsequent reagent should be added immediately, do not let the wells stand dry for extended period of time.
6. Incubation of Standards and Samples
Add 100 ul of the diluted standards and samples into the appropriate wells, leaving the substrate blank wells empty (see recom-ended plate plan on page 11).
Cover plate with adhesive seal, gently agitate the
microplate plate by tapping the edge for a few seconds to mix contents of each
well. Incubate for 1 hour at room temperature (18-25'C).
Just before washing prepare next incubation reagent as described in point 8 .
7. Second Washing Step
Aspirate supernatant from the wells and wash the plate as described in point 5.
8. Incubation with anti-TNFa Biotin Conjugate
The kit contains one yellow-capped vial with concentrated anti-TNFa biotin conjugate.
Calculate the required amount of conjugate (10 ul per strip) and dilute 1:100
in HPE-dilution buffer.
Add 100 ul diluted conjugate to the wells, leaving the substrate blank wells
empty.
Cover plate with adhesive seal, gently agitate the
plate by tapping the edge for a few seconds to mix the contents of the wells.
Incubate for 1 hour at room temperature (18-25'C).
Just before washing prepare next incubation reagents as described in point 10.
9. Third Washing Step
Aspirate the supernatant from the wells and wash the plate as described in point 5.
10. INCUBATION WITH STREPTAVIDIN-POLY-HRP
The kit contains one brown-capped vial of concentrated streptavidin-poly-HRP, which must be stored at -18'C to -32'C to maintain maximal stability. The contents of the vial will not be frozen at this temperature.
Dilute the concentrated streptavidin-poly-HRP 1:10.000 in HPE-dilution buffer. For this dilution we advise to pipette 3 ul of concentrated streptavidin-poly-HRP in 30ml HPE-dilution buffer (regardless the amount of wells to be used).
Add 100 ul of diluted streptavidin-poly-HRP conjugate to the wells, leaving the substrate blank wells empty.
Cover the plate with adhesive seal, gently agitate the
plate by tapping the edge for a few seconds to mix contents of each well.
Incubate for 30 minutes at room temperature (18-25'C).
11. FOURTH WASHING STEP
Aspirate the supernatant from the wells and wash the plate as described in point 5.
The kit contains one brown-capped bottle with a ready-for-use TMB substrate solution containing a mixture of 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide.Protect from prolonged exposure to light. This solution must be on room temperature before use!
Add 100 ul of substrate solution to all wells, including the substrate blank
wells.
Cover the microplate, gently agitate by tapping the
edge for a few seconds to mix contents of each well. Do not cover the plate
with aluminium or adhesive foil.
Incubate for 30 minutes at room temperature (18-25') in the dark.
13. STOP ENZYMATIC REACTION
The kit contains one white-capped bottle with a ready-for-use stop solution of 0.18 M H(2) SO(4).
Add 100 ul of stop solution to all wells.
14. PLATE READ-OUT
Read within 30 minutes at 450 nm in an ELISA reader.
VIII. RESULTS
Substrate blank -Record the absorbance at 450 nm for the substrate blank wells and average the duplicate values.
Standard curve
-Record the absorbance at 450 nm for each well containing standard and average the duplicate values.
-Calculate the net average absorbances by subtracting the average of the substrate blank wells.
-Plot the net average absorbances (ordinate) versus the TNFa concentration in pg/ml (abscissa) on log-linear paper and draw the best fitting curve. An example of a standard curve is given on the next page.
SAMPLES
-Record the absorbance at 450 nm for each well, and average the duplicate values.
-Calculate the net average absorbances by subtracting the average of the substrate blank wells.
-Locate the net average absorbance value found for each sample on the ordinate and follow a horizontal line intersecting the standard curve. At the point of intersection, read the TNFa concentration (pg/ml) on the abscissa. Multiply the obtained TNFa concentration by the dilution factor of the sample and record this figure.
SAMPLE Standard Curve (do not use for calculating results) calculated mean absorbances at 450nm
|
|
Static Incubation |
Shaken Incubation |
|
substrate blank |
0 |
0 |
|
0 pg/ml |
0.014 |
0.028 |
|
1.4 pg/ml |
0.031 |
0.081 |
|
4.1 pg/ml |
0.065 |
0.159 |
|
12.4 pg/ml |
0.139 |
0.402 |
|
37 pg/ml |
0.415 |
1.069 |
|
111 pg/ml |
1.102 |
2.726 |
|
333 pg/ml |
2.196 |
>3.00 |
|
1000pg/ml |
2.962 |
>3.00 |
IX. FEATURES
Increased sensitivity
The assay sensitivity can be increased by a small adaptation of the incubation methodology. Just follow all the instructions as stated in the assay procedure (chapter VII), but incubate at room temperature (18-25'C) on a horizontal plate shaker at 700+ 100 rpm. All incubations, including the enzymatic colour development, have to be completed on the shaker, in the same time as stated in the static assay procedure. This will result in an increase in assay sensitivity, with little effects on the back-ground levels (see figure opposite page).
Sensitivity
MEAN calculated zero signal + 3 SD: 1-3 pg/ml (shake-static incubation)
2X (MEAN calculated zero signal) : 4-6 pg/ml (shake-static incubation)
EXPECTED VALUES
TNFa values in fresh serum and plasma samples of healthy indivi-uals are below 10 pg/ml.
SPECIFICITY
No cross reactivity was observed with the following recombinant human proteins: IL-1a, IL1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-11, Macrophage Colony Stimulating Factor (M-CSF), Granulocyte Colongy Stimulating Factor (G-CSF), Granulocyte/Macrophage Colony Stimulating Factor (GM-CSF), Leukaemia inhibitory Factor (LIF), RANTES, Stem Cell Factor/Mast Cell Factor (SCF/MCF) Transforming Growth FactorB-1 (TGFB-1), Tumor Necrosis FactorB (TNFB/Lymphotoxin), and Interferon g (IFNg).
X. REFERENCES
Aggerwal, B. B. Gutterman, J.U. (1992) Human cytokines. Blackwell Sci.Pub. ISBN 0-86542-183-8
Ammann, A.J. et al (1987) J. Clin.Immunol. 7:6
Beutler, B. et al (1985) Science 229: 869
Beutler, B. et al (1985) Mature 316: 552
Carswell, E.A. et al (1975) Proc.Natl.Acad.Sci. 72: 3666
Exely, A.R. et al (1990) Cytokine 2: 353
Kwiakowski,D.A. et al (1990) The Lancet 336: 1201
Pennica,D. et al (1984) Nature 312: 724
Silberstein, S.B. et al (1986) Proc.Natl.Acad.Sci. 83:1055
Sugarman,B.J. et al (1985) Science 230:943
Thomson,A.W. (1991) The cytokine handbook. Academic Press ISBN 0-12-689660-7
Tori,F.M. et al (1985) Science 229: 876
Tracey,K.J. et al (1986) Science 23: 470
Wong,G.H.W. et al (1986) Nature 323:819
MATERIAL SAFETY DATA SHEET
Hazardous ingredients
3,3',5,5',-Tetramethylbenzidine may be harmful by inhalation, ingestions, or skin absorption. May cause irritation. To our best knowledge the chemical, physical and toxicological properties have not been thoroughly investigated. CAS No. 54827-17-7
Thiomersal may be toxic upon ingestion, inhalation or skin contact. Avoid contact of skin, eyes or clothing with dilution, washing or substrate buffer. In case of contact, wash skin or eyes with water and consult a physician. CAS No. 54-64-8
Sulphuric Acid: CAS No. 7664-93-9
Physical data
No information is available on physical data for the chemical mixture as a whole.Health hazard.
Please refer to "Precautions for use", page 2 and 3 of this information leaflet.
Protection information
Please refer to "Precautions for use", page 2 and 3 of this information leaflet.
Disclaimer
The above information is believed to be accurate and represents the best information available to us. However, CLB neither warrants the accuracy of this information nor assumes any legal responsibility in connection with its dissemination. All materials and mixtures may present unknown hazards and should be used with caution. Users should make their own investigations to determine the suitability of this information for their particular purpose.
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phone (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
EMAIL:antibodies@fitzgerald-fii.com
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