rev: 5/16/2008
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Sample Kit Protocol: (see actual insert with kit for batch specific information):
Pelikine (tm)
Human Interferon Gamma ELISA Kit
CAT# RDI-M1921clb, new cat#: 55R-M1921clb 96 wells
PRICE: $621.50/kit
INTRODUCTION
At this moment fifteen interferon a (IFNa), one interferon B (IFNB) and one
interferon G (IFNG) have been reported. IFNG is produced during an immune
response by CD8+, NK,(gamma-delta) and TH1 T helper
cells. It differs structurally and functionally from IFNa and IFNB; binds to
distinct receptors and has pronounced immuno-regulatory effects, including
activation of macrophages to enhance phagocytosis and tumor killing capability,
activation and growth enhancement of cytolytic T-cells and NK-cells, and
induction to class II MHC antigen and FcG receptor on macrophages and many
other cell types. IFNG also regulates humeral immune responses: it induces
immunoglobulin secretion by activated B-cells stimulated with IL-2 and
potentiates IL-4 induced proliferation of human
B-cells. Several substances originally described for their biological
activities have been identified as IFNG; macrophage activating factor (MAF),
T-cell replacing factor (TRF), Type II interferon and immune interferon.
Bioassays for the quantification of IFNG, based on cytopatic reductive effects
of IFNG on cultured cells have been used for several years. In this assay IFNG
reduces the killing of a target cell line such as L929 (murine), HEp2C or A549
(human) cells by for, example,encephalomyocarditis
virus.
An alternative assay system involves measurement of induction of HLA-DR
antigens on tumour cells, which can be detected in a cell ELISA. These assays
although sensitive, are time consuming and might be susceptible to interference
by other substances.
The Pelikine human IFNG ELISA kit has been developed for faster, more reproducible and specific quantification of human IFNG in serum, plasma and other body fluids, as well as in cell-culture supernatant.
II. PRINCIPLE OF THE TEST
The Pelikine human IFNG ELISA kit is a "sandwich-type" of enzyme immunoassay in which a monoclonal anti-human IFNG antibody is bound onto polystyrene microtiter wells. Human IFNG present in a measured volume of sample or standard is captured by the antibody on the microtiter plate, and non-bound material is removed by washing.Subsequently, a biotinylated polyclonal antibody to human IFNG is added.This antibody binds to the IFNG-antibody complex present in the microtiter 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 IFNG 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 IFNG present in the sample or standard. After the reaction has been terminated by the addition of a stop solution, absorbance is measured in a microtiter plate reader. From the absorbance of samples and those of a standard curve, the concentration of IFNG can be determined by interpolation with the standard curve.
III. STORAGE AND STABILITY
The Pelikine human IFNG ELISA kit should be stored at 2-8'C. At arrival the components of the Pelikine human IFNg 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 IFNg standard, the biotinylated IFNg antibody conjugate, and the streptavidine-HRP conjugate separately below -18'C and store the remainder of the Pelikine human IFNg ELISA kit at 2-8'C.
IV. CONTENTS OF THE KIT
The Pelikine human IFNG ELISA kit contains material sufficient for 96 tests, including standard curve samples. The reagents provided are:
Quantity Kit Component Volume
1 pc precoated microtiterplate 12 x 8 strips + frame -
2 vials IFNg standard 7200pg/ml lyophilized 500ul
1 vial biotinylated IFNg 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).
- Microtiter plate reader, capable of measuring absorbance at 450 nm.
VI. PRECAUTIONS FOR USE
1) The Pelikine human IFNG ELISA kit is intended for research purposes only.
2) Only use the reagents and microtiter 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 sodiium 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) The IFNG standard contains human serum which has been found to be non-reactive for Hepatitis B surface Antigen (HBsAg),Hepa titis C Virus (HCV) and human Immunodeficiency Virus (HIV).
Nevertheless the standard should be handled as potentially hazardoous and capable of transmitting diseases.
7) With the exception of the substrate blank wells, do not allow wells to stand uncovered or dry for extended periods between incubation steps.
VII. Assay Procedure
1. 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.
2. Mix all reagents thoroughly without foaming before use.
3. MICROTITER PLATE
The kit contains one frame with twelve pre-coated strips of eight microwells, vacuum sealed in a plastic bag. The CLB IFNgELISA 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 16 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
The kit contains one bottle with 5-fold concentrated dilution buffer. For optimal assay results, dilute samples and standard in working-strength 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 buffer 5 times in distilled water before use. The working-strength dilution buffer can be stored for up to one week at 2-8'C.
5. IFNg STANDARD standard curve preparation
A natural human IFNg standard has been calibrated against the WHO reference preparation (IFNg 88/606; National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, U.K. 1WHO Unit = 53pg IFNg).
The kit contains two lyophilized vials with 7200 pg/ml natural IFNg. Reconstitute one lyophilized standard by adding 500 ul of distilled water to the vial. Incubate for 10 minutes at room temperature and mix gently. After reconstitution the standard must be kept cold (2-8'C) and stored frozen after use (<-18'C, preferably <-70'C).
Label 7 tubes, one tube for each dilution: 1800, 600, 200, 66.7, 22.2, 7.4, and 2.5 pg/ml. Pipette 300 ul of working-strength dilution buffer into all tubes.
Transfer 100 ul of the IFNg standard (7200 pg/ml) into the first tube labelled 1800 pg/ml, mix well and transfer 150 ul of this dilution into the second tube labelled 600 pg/ml.Repeat the serial dilutions six more times by adding 150 ul of the previous tube of diluted standard to the 300 ul of dilution buffer.
The standard curve will contain 1800, 600, 200, 66.7, 22.2, 7.4, 2.5 and 0 pg/ml (dilution buffer).
It is recommended to prepare two separate series for each assay.
Avoid repeated freeze-thawing of the standard, although experimental data have shown that up to 3 freeze-thaw cycles have no effect on the IFNg levels of the standard. Thaw the reconstituted standard in tap water (18-25'C), do not use 37'C or 56'C water baths for this purpose. The second vial of standard can be used in case of prolonged storage of the reconstituted standard (> 2 months).
6. SAMPLES sample preparation
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 form 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 store frozen (<-18'C, preferably <-70'C). Avoid freezing and thawing samples more than once. 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.
It is recommended to dilute the test samples at least 1:2 in working-strength
dilution buffer. If high levels of IFNg (> 500pg/ml) are expected in the
test samples, additional dilutions of sample i.e. 1:10 and 1:50 should also be
prepared.
7. FIRST WASH STEP
Prepare washing buffer as described on page 3 of this leaflet.
Wash the required microtiter plate five times with washing buffer in a plate washer. In case of manual washing, completely fill the wells (>300ul) with washing buffer and aspirate, repeat this four times. After the final aspiration the wells should be dry.
8. FIRST INCUBATION STEP standard and samples
Leaving the substrate blank wells empty, transfer 100 ul of the prepared standards and samples in duplicate into the appropriate wells (see recommended plate plan on the last pages of this leaflet).
Cover plate with adhesive seal, gently agitate the microtiter plate by tapping the edge of the plate for a few seconds to mix contents of each well and incubate for 1 hour at room temperature (18-25'C).
9. SECOND WASH STEP
Aspirate supernatant from wells and wash the microtiter plate as described in point 7 above.
10. SECOND INCUBATION STEP biotinylted-IFNg antibody
The kit contains one yellow-capped vial with concentrated IFNg antibody-biotin conjugate. Calculate the quantity of IFNg antibody-biotin conjugate required (10 ul conjugate antibody per microwell strip) and prepare a working-strength solution by diluting the conjugate 1:100 in working-strength dilution buffer just before use.
Leaving the substrate blank wells empty, add 100ul of diluted biotinylated IFNg antibody to all wells.
Cover plate with adhesive seal, gently agitate the microtiter plate by tapping
the edge of the plate for a few seconds to mix contents of each well and
incubate for 1 hour at room temperature (18-25'C).
11. THIRD WASH STEP
Aspirate supernatant form wells and wash the microtiter plate as described in point 7 above.
12. THIRD INCUBATION STEP Streptavidin-HRP conjugate
The kit contains one brown capped vial of concentrated streptavidin-HRP conjugate, which must be stored at -18'C to -32'C to maintains maximal stability. The contents of the vial will not be frozen at this temperature.
Add 3ul streptavidine-HRP conjugate to 30 ml of working-strength dilution buffer just before use. Do not prepare in advance to assay.
Leaving the substrate blank wells empty, add 100 ul of streptavidin-HRP conjugate to all wells.
Cover the microtiter plate with adhesive seal, gently agitate the microtiter plate by tapping the edge of the frame for a few seconds to mix contents of each well and incubate for 30 minutes at room temperature (18-25'C).
13. FOURTH WASH STEP
Aspirate supernatant from wells and wash the microtiter plate as described in point 7 above.
14. FOURTH INCUBATION STEP enzymatic colour development
The kit contains one brown capped bottle with a ready for use solution of 3,3',5,5'-tetramenthylbenzidine (TMB) and Hydrogen peroxide. Take care not to contaminate the TMB substrate reagent; if the solution is blue prior to use the reagent cannot be used any more. Protect from prolonged exposure to light.
Add 100 ul of substrate solution to all wells, including the substrate blank
wells.
Cover microtiter plate, gently agitate the microtiter plate by tapping the edge
of the frame for a few seconds to mix contents of each well and incubate for 30
minutes at room temperature (18-25'C) in the dark.
Do not cover the plate with aluminium foil.
15. 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.
After stopping the color is stable for maximally 30
minutes.
16. PLATE READ-OUT
Read at 450 nm in an ELISA reader.
Note: It is important that Stop Solution has been added to the wells before reading at 450 nm. The addition of Stop Solution causes an increase in absorbance of the chromogen and a shift in absorption spectrum (the blue color should turn to yellow).
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 substracting the average of the substrate blank wells.
Plot the net average absorbances (ordinate) versus the IFNg 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 of 450 nm for each standard 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 vertical axis and follow a horizontal line intersecting the standard curve. At the point of intersection, read the IFNg concentration with the dilution factor of the sample and record this figure.
SAMPLE Standard Curve (do not use for calculating results) calculated mean absorbances at 450
|
|
Static Incubation |
Shaken Incubation |
|
substrate blank |
0 |
0 |
|
0 pg/ml |
0.042 |
0.057 |
|
2.5 pg/ml |
0.069 |
0.122 |
|
7.4 pg/ml |
0.136 |
0.275 |
|
22.2 pg/ml |
0.295 |
0.676 |
|
66.7 pg/ml |
0.744 |
1.718 |
|
200 pg/ml |
1.432 |
2.986 |
|
600 pg/ml |
2.285 |
>3.00 |
|
1800pg/ml |
2.965 |
>3.00 |
IX. ADDITIONAL INFORMATION
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 t 700 +100rpm. All incubations, including the ennzymatic color 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)
2 x (MEAN calculated zero signal): 4-6 pg/ml(shake-static incubation)
Expected values
IFNg values in fresh serum and plasma samples of healthy individuals are below 10 pg/ml.
Specificity
No cross reactivity was observed with the following recombinant human proteins: IL1a, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, Macrophage Colony Stimulating Factor (M-CSF), Granulocyte Colony 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 Factor B-1 (TGFB-1), Tumour Necrosis Factor a (TNFa) and Tumor Necrosis Factor B TNFB/Lymphotoxin).
X. REFERENCES
Adolf, G.R. (1985) Oncology 42: 33
Balkwill, F. (1989) The Lancet (I): 1060
Billiau,A. (1988) Immunology Today 9: 37
Billiau,A. et al (1990) Biochem.Pharmacol. 40:1433
Bruserud,O. et al (1993) Eur. J. Hematol. 51: 73
Celada, A. et al (1989) Eur.J.Immunol. 19: 1103
Doldi,K. et al (1985) J.Interferon Res. 5: 55
Farrar,M.A. et al (1993) Ann. Rev..Immunol. 11: 571
Gray,P.W. et al (1892) Nature 295:503
Grossberg, S.E. et al (1989) Experientia 45: 508
Ijzermans, J.M. et al (1989) Immunobiol. 179: 456
Kwiatkowski,D.A. et al (1990) The Lancet 336:1201
Locksley,R.M. et al (1991) Immunoparasitology Today: A58
O'Garra,A. (1989) The Lancet (1): 1003
Paliard,X. (1988) et al J.Immunol. 141: 849
Reiter,Z. (1993) J.Interferon Res. 13: 247
Samuel, C.E. (1991) Virology 183:1
Thomson,A.W. (1991) The cytokine handbook.Academic Press ISBN 0-12-689660-7
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 propertieshave not been thoroughly investigated. CAS No. 54827-17-1.
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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EMAIL:antibodies@fitzgerald-fii.com
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