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Sample Kit Protocol: (see actual insert with kit for batch specific information):
Pelikine Compact (tm) Human IFN-Gamma
ELISA Kit
CAT# RDI-M1933clb, new cat#: 55R-M1933clb 288 tests PRICE: $525.00
INTRODUCTION
At this moment fifteen interferon alpha (IFNa), one interferon Beta (IFNB) and one interferon Gamma (IFNG) have been reported. IFN-G is produced during an immune response by CD8+, NK,(gamma- ) 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. IFNG has documented antiviral and antiprotozoal activities, although IFNa and IFNB seem to have more potent antiviral activities than IFNG. 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 compact(tm) 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 compact 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 compact human IFNG ELISA kit should be stored at 18'C to -32'C. The performance of the kit is guaranteed until the expiration date shown on the case label.
IV. CONTENTS OF THE KIT
The Pelikine compact human IFNG ELISA kit contains material sufficient for 288 tests, including standard curve samples. The reagents provided are:
Quantity Kit Component Volume Cap color
1 vial coating antibody 100-fold concentrated 375ul red
1 vial blocking reagent 50-fold concentrated 2ml transparent
2 vial IFNG standard lyophilized 7200pg/ml 1ml grey
1 vial biotinylated IFNG antibody 100-fold concent 375ul yellow
1 vial streptavidin-HRP conjugate 10,000-fold concen 20ul brown
1 bottle dilution buffer 5-fold concentrated 60ml
3 pcs microtiter plates + lid
10 pcs plate seals
V. PRECAUTIONS FOR USE
1) The compact 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 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) The IFNG 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.
7) Centrifuge all vials before use (1 minute 3000 x g).
8) With the exception of the substrate blank wells, do not allow wells to stand uncovered or dry for extended periods between incubation steps.
VI. ADDITIONAL BUFFERS & SOLUTIONS REQUIRED (or see the Pelikine (tm)Tool Set )
Coating buffer: 0.1M Carbonate/bicarbonate buffer pH 9.6
Solution A: 1.24 g Na2CO3.H2O in 100 ml distilled water
Solution B: 1.68 g NaHCO3 in 200 ml distilled water
Take 70ml of solution A, and add solution B until the pH is 9.6 (approximately 175 ml of solution B required).
The prepared buffer can be stored up to one week at 2-8'C.
PBS stock solution [20 x]: 0.2 M Phosphate Buffered Saline (PBS)
Dissolve 32 g Na2HPO4.2H20
6 g NaH2PO4.2H20
164 g NaC1
in 900 ml distilled water
(intensive stirring and some heating will speed dissolution). Bring the temperature of the solution back to room temperature (18-25'C) and check pH; if necessary adjust pH to 6.8 - 6.9 with concentrated HCl or NaOH, and add distilled water to a volume of 1 liter (when diluted 20 times the obtained buffer will have a pH of 7.2 -7.4). Add 20 mg thiomersal as preservative. Do not use sodium azide (NaN3) since this preservative reduces the quality of the enzymatic label. The prepared buffer can be stored up to three months at 2-8'C.
Note: in the concentrated buffer salt crystals may appear when stored at 2-8'C. Before preparing the working-strength buffer, first warm the concentrated buffer BRIEFLY to 37'C to dissolve the precipitate.
Washing buffer: PBS with 0.005 % TWEEN 20
Make 1 liter of working-strength PBS by diluting the PBS stock solution (see above) 20-fold with distilled water.
Add 50 ul TWEEN 20.
The prepared buffer can be stored up to one month at 2-8'C.
Substrate buffer: 0.11 M acetate buffer pH 5.5
Dissolve 15.0 g sodium-acetate (CH3COONa.3H20) in 800 ml distilled water.
Adjust pH to 5.5 with glacial acetic acid, add distilled water to a volume of 1 liter.
Do not add any preservative (e.g. merthiolate,sodium azide) since this may affect the quality of the enzymatic color development.
The prepared buffer can be stored up to two weeks at 2-8'C.
3,5,3',5'-tetramethylbenzidine (TMB) stock solution: 6mg/ml TMB in DMSO
Dissolve 30 mg 3,5,3',5'-tetramethylbenzidine (TMB) in 5 ml dimethylsulfoxide (DMSO).
The prepared stock solution can be stored up to 1 month at room temperature (18-25'C) and protected against light.
Hydrogen peroxide stock solution: 3% H202 solution in distilled water.
The prepared stock solution can be stored up to one month at 2-8'C.
Substrate solution
For each plate mix the following reagents:
12 ml Substrate buffer
200 ul TMB stock solution
12 ul H202 stock solution
The substrate solution should be prepared just before use and has to be at room temperature (18-25'C) for optimal reproducible results.
Stop solution: 1.8 M H2S04 solution in distilled water.
VII. ADDITIONAL INFORMATION
Additional materials required
-Pipetting devices for accurate delivery of 1-10ul, 50 ul, 100 ul and 1 ml volumes.
-Beakers, flasks, cylinders necessary for preparation of reagents.
-Device for delivery of washing buffer(wash bottle/automated plate washer).
-Microtiter plate reader.
Sensitivity
MEAN calculated zero signal + 3 SD : 1-2pg/ml (shaken-static incubation)
2 x (MEAN calculated zero signal): 4-8 pg/ml(shaken-static incuba tion).
Note: the sensitivity is dependent of the type and quality of enzymatic substrate.
Expected values
IFN-G values in fresh serum,plasma and urine samples of healthy individuals are below 10 pg/ml
Specificity
No cross reactivity was observed with the following recombinant human proteins: IL-1a, 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 InhibitoryFactor LIF), RANTES, Stem Cell Factor/Mast Cell Factor (SCF/MCF), Transforming Growth Factor B-1 (TGFB-1),Tumor Necrosis Factor a (TNFa) and Tumor Necrosis Factor B (TNFa/Lymphotoxin).
REFERENCES
1.Adolf,G.R.(1985) Oncology 42:33
2.Aggerwal,B.B. Gutterman,J.U.(1992) Human cytokines.Blackwell Sci.Pub. ISBN 0-86542-183-8
3.Balkwill,F. (1989) The Lancet (1): 1060
4.Billiau,A. (1988) Immunology Today 9: 37
5.Billiau,A. et al (1990) Biochem.Pharmacol. 40: 1433
6.Bruserud,O. et al (1993) Eur.J.Hematol. 51: 73
7.Celada,A. et al (1989) Eur.J.Immunol. 19: 1103
8.Doldi,K. et al (1985) J. Interferon Res. 5: 55
9.Farrar,M.A. et al (1993) Ann.Rev. Immunol. 11: 571
10.Gray,P.W. et al (1892) Nature 295: 503
11.Grossberg,S.E. et al (1989) Experientia 45: 508
12.Ijzermans,J.M. et al (1989) Immunobiol. 179: 456
13.Kwiatkowski,D.A. et al (1990) The Lancet 336: 1201
14.Locksley,R.M. et al (1991) Immunoparasitology Today: A58
15.
16.O'Garra,A. (1989) The Lancet (1): 1003
17.Paliard,X. (1988) et al J.Immunol. 141: 849
18.Rieter, Z. (1993) J.Interferon Res. 13: 247
19.Samuel, C.E. (1991) Virology 183:1
20.Thomson,A.W. (1991) The cytokine handbook.Academic Press ISBN 0-12-689660-7
VIII. ASSAY PROCEDURE
1. BRING ALL REAGENTS TO ROOM TEMPERATURE (18-25'C), with the exception of the streptavidin-HRP conjugate which has to be kept at -18'C to -32'C to ensure stability. Centrifuge all vials before use (1 minute 3000 x g). For your convenience an easy-reference manual with check list and plate plan are available on the last pages of this leaflet
2. 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 (approximately15 ml
undiluted buffer per microtiter plate) 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.
3. MICROTITER PLATES Coating antibody
Coating The kit contains three microtiter plates for 96 tests each,including the standard curve samples.
Prepare coating buffer as described on page 3 of the information leaflet.
Per microtiter plate, add 120 ul of coating antibody (red-capped vial) to 12 ml coating buffer.
Add 100 ul to all wells, cover microtiter plate(s) with lid and incubate overnight at room temperature (18-25'C).
Washing procedure
Prepare working-strength PBS (1:20 dilution of stock PBS as described on page 3 of the information leaflet).Aspirate supernatants from wells and completely fill the wells (>300 ul) with working-strength PBS and aspirate. Repeat this four times, after the final aspiration the wells should be dry.
Blocking procedure
The kit contains one transparent-capped vial with 2 ml blocking reagent.
Prepare blocking buffer by adding 500 ul blocking reagent to 25 ml working-strength PBS (1:20 dilution of stock PBS as described on page 3 of the information leaflet). Add 200 ul blocking buffer to all wells, cover microtiter plate(s) 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).
4. IFN-G 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. 1 WHO Unit= 53pg IFNG).The kit contains two lyophilized grey-capped vials with 7200 pg/ml natural IFNG.
Reconstitute one lyophilized standard by adding 1 ml 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. Pipette 300 ul of working-strength dilution buffer into all tubes.Transfer 100ul of the IFNG standard (7200 pg/ml) into the first tube labelled 1800pg/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 Q 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).
5. SAMPLES
Serum,heparin or 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).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 bathes 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.
6. FIRST WASH STEP
Prepare washing buffer as described on page 3 of this leaflet.
Wash the required microtiter plates 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.
7. FIRST INCUBATION STEP Standards 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). Cover plate(s) with adhesive seal, gently agitate the micro titer 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).
8. SECOND WASH STEP
Aspirate supernatant from wells and wash the microtiter plate (s) as described in point 6 above.
9. SECOND INCUBATION STEP biotinylated-IFNG antibody The kit contains one
yellow-capped vial with concentrated IFNG antibody -biotin conjugate. Per
microtiter plate, add 120 ul biotinylated IFNG antibody to 12 ml
working-strength dilution buffer just before use.
Leaving the substrate blank wells empty, add 100 ul of diluted biotinylated IFNG antibody to all wells.
Cover plate(s) with adhesive seal, gently agitate the micro titer 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).
10.THIRD WASH STEP
Aspirate supernatant from wells and wash the microtiter plate(s) as described in point 6 above.
11.THIRD INCUBATION STEP Streptavidin-HRP conjugate The kit contains one brown
vial of concentrated streptavidin- HRP conjugate, 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.
Per microtiter plate, add 3 ul streptavidine-HRP conjugate to 30 ml of working-strength dilution buffer just before use. Do not prepare in advance of assay.
Leaving the substrate blank wells empty, add 100 ul of streptavidin-HRP conjugate to all wells.
Cover the microtiter plate(s) 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).
12. FOURTH WASH STEP
Aspirate supernatant from wells and wash the microtiter plate (s) as described in point 6 above.
13. FOURTH INCUBATION STEP Enzymatic colour development
Approximately 10 minutes before use, prepare the substrate solution as described on page 4 of this leaflet.
The substrate solution should be at room temperature (18-25' C) for optimal reproducible results
Add 100 ul of substrate solution to all wells, including the substrate blank wells.
Cover microtiter plate(s) with lid, gently agitate the microti ter 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.
Note: the speed of enzymatic colour development is influenced by many factors including temperature and quality of the used TMB.
14. STOP ENZYMATIC REACTION
Add 100 ul of stop solution to all wells. After stopping the colour is stable for maximally 30 minutes.
15. PLATE READ-OUT
Read at 450 nm in an ELISA reader.
IX. RESULTS
Substrate blank
- Record the absorbance at 450 nm for the substrate blank wells 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 IL-6 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 standard well, and a verage 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 (pg/ml) from the horizontal axis. Multiply the obtained IFNG concentration with the dilution factor of the sample and record this figure.
X. 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 VIII), 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, without effects on the background levels (see figure next page).
SAMPLE DATA (DO NOT USE FOR CALCULATING ACTUAL RESULTS)
calculated mean absorbance at 450nm
|
|
static incubation |
shaken incubation |
|
substrate blank |
0 |
0 |
|
0 pg/ml |
0.046 |
0.056 |
|
2.5 pg/ml |
0.073 |
0.094 |
|
7.4 pg/ml |
0.130 |
0.177 |
|
22.2 pg/ml |
0.301 |
0.439 |
|
66.7 pg/ml |
0.731 |
1.129 |
|
200 pg/ml |
1.652 |
2.348 |
|
600 pg/ml |
2.470 |
2.918 |
|
1800 pg/ml |
2.727 |
>3.000 |
Do not use these data to construct a standard curve for sample value calculations.
For In Vitro Research Use Only-Not for use in Diagnostics
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RDI Division of Fitzgerald Industries Intl
phone (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
EMAIL:antibodies@fitzgerald-fii.com
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