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Sample Kit Protocol: (see actual insert with kit for batch specific information):
Pelikine Compact Human IL6 ELISA Kit
CAT# RDI-M1916clb, new cat#: 55R-M1916clb 288 tests PRICE:
$525.00
INTRODUCTION
Interleukin 6 (IL-6) is a mediator of the inflammatory response and is involved in the induction of acute phase proteins [1,2,3, 4] and the development of fever [5]. A marked correlation between IL-6 levels and inflammatory processes has been demonstrated in synovial fluid and serum of rheumatoid arthritis patients [6,7,8] and in serum of patients with burns [9,10]. It was demonstrated that in recipients of kidney transplants the IL-6 levels in serum and urine hallmark the onset of rejection episodes [11,12]. Elevated Il-6 levels were also observed in sera of patients with septic shock, multiple myeloma and alcoholic hepatitis, and a significant difference between IL-6 levels of survivors and non-survivors was found [13,14,15].Bioassays for the quantification of IL-6,based on the proliferation of B-cell hybridomas have been used for several years [16,17,18]. These assays, although sensitive, are time consuming and susceptible to interference by other substances. This IL-6 ELISA [19] has been developed for faster, more reproducible and specific quantification of human IL-6 (hulL-6) in plasma and other body fluids, as well as in cell-culture supernatant.
II. PRINCIPLE OF THE TEST
The compact huIL-6 ELISA kit is a "sandwich-type" of enzyme immumoassay in which a monoclonal anti-huIL-6 antibody is
bound onto polystyrene microtiter wells. Human IL-6,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
sheep antibody to huIL-6 is added. This antibody binds to the huIL-6-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 huIL-6 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 huIL-6 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 huIL-6 can be determined by interpolation with the standard curve.
III. STORAGE AND STABILITY
The compact IL-6 ELISA kit should be stored at -18'C to -32'C.The performance of the compact IL-6 ELISA kit is guaranteed until the expiration date shown on the case label.
IV. CONTENTS OF THE KIT
The compact IL-6 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
1 vial IL-6 standard 3500 pg/ml (lyophilized) 1ml grey
1 vial biotinylated IL-6 antibody 100-fold concen 375ul yellow
1 vial streptavidin-HRP conjugate 10,000-fold concen 20ul brown
1 bottle dilution buffer 5-fold concentrated 60ml
3 pcs microtiter plastes + lid
10 pcs plate seals
V. PRECAUTIONS FOR USE
1) The compact IL-6 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) Centrifuge all vials before use (1 minute 3000 x g).
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 temperature to room temperature (18-25'C) and check pH; if necessary adjust pH to 6.8 - 6.9 with concentrated HC1 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 dis tilled 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 dev elopment.
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 : 0.2 - 0.4 pg/ml(shaken-static incubation)
2 x (MEAN calculated zero signal): 0.5 - 1 pg/ml (shaken-static incuba tion).
Note: the sensitivity is dependent of the type and quality of enzymatic substrate.
Expected values
IL-6 values in fresh serum,plasma and urine samples of healthy individuals are below 20 pg/ml
REFERENCES
1. Gauldie,J. et al (1987) Proc.Natl.Acad.Sci.(USA) 84: 7251
2. Le,J. and Vilcek,J. (1989) Lab.Invest. 61: 588
3. Heinrich,P.C. et al (1990) Biochem.J. 265: 621
4. Kishimoto,T. (1989) Blood 74: 1
5. Helle,M. et al (1988) Eur.J.Immunol. 18: 957
6. Houssiau,F.A. et al (1988) Arthritis Rheum. 31: 784
7. Swaak,A.J.G. et al (1988) Scand.J.Rheumatol. 17: 469
8. Waage,A. et al (1989) Clin.Immunol.Pathol. 50: 394
9. Nijsten,M.W.N. et al (1988) Lancet 11: 921
10.Guo,Y. et al (1990) Clin.Immunol.Pathol. 54: 361
11. Van Oers, M.H.J. et al (1988) Clin.Exp.Immunol. 71: 314
12. Yoshimura,N. et al (1991) Transplantation 51: 172
13.Hack,C.E. et al (1989) Blood 74: 1704
14.Ludwig,H. et al (1991) Blood 77: 2794
15.Sheron,N. et al (1991) Clin.Exp.Immunol. 84: 449
16.Aarden,L.A. et al (1985) Lymphokines 10: 175
17.Van Snick,J. et al (1987) J.Exp.Med 165: 641
18.Helle,M. et al (1988) Eur. J.Immunol. 18: 1535
19.Helle,M. et al (1991) J.Immunol.Methods 138: 47
20.Gaines Das,R.E. et al (1993) J.Immunol.Methods 160: 147
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 (approximately 15 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,in cluding 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. IL-6 STANDARD Standard curve preparation
A recombinant huIL-6 standard has been calibrated against the WHO International Standard (IL-6 89/548: National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, U.K. 1 WHO Unit= 10 pg IL-6,see ref [20]). In former CLB IL-6 compact kits [Batch IL6-CK0001 to IL6-CK0004],1 pg IL-6 standard is comparable with 3.15 pg of the new WHO standard). The kit contains one grey-capped vial with 3500 pg/ml recombinant IL-6.
Reconstitute the recombinant IL-6 standard, by adding 1 ml of distilled or de-ionized water to the vial. Incubate for 30 min at room temperature (18-25') and mix gently.
Label 7 tubes, one tube for each dilution: 450, 150, 50, 16.7, 5.6 , 1.9 and 0.6 pg/ml.
Pipette 610ul of working-strength dilution buffer into the tube labelled 450 pg/ml and 400ul of working-strength dilution buffer into the other tubes. Transfer 90ul of the IL-6 standard (3500 pg/ml) into the first tube labelled 450 pg/ml, mix well and transfer 200 ul of this dilution into the second tube labelled 150pg/ml. Repeat the serial dilutions six more times by adding 200ul of the previous tube of diluted standard to the 400 ul of dilution buffer.
The standard curve will contain 450, 150, 50, 16.7, 5.6, 1.9, 0.6 and 0 pg/ml (dilution buffer).
It is recommended to prepare two separate series for each assay.
The reconstituted standard can be stored below -18'C for up to 3 months.
Avoid repeated freeze-thawing of the standard, although experimental data have shown that up to 3 freeze-thaw cycles have no effect on the IL-6 levels of the standard.
5. SAMPLES
Serum,heparin or EDTA-anti-coagulated plasma, and culture fluids are suitable for use in the assay.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). Up to 3 freeze-thaw cycles have no effect on the IL-6 levels of of serum or plasma samples. Nonetheless, excessive freeze-thaw cycles should be avoided. Prior to the assay, frozen samples should be thawed as quickly as possible in a 37'C waterbath and then brought to room temperature (18-25'C).
It is recommended to dilute the test samples at least 1:2 in working-strength dilution buffer. If high levels of IL-6 (>400pg/ml) are expected in the test samples, additional dilutions of sample i.e. 1:10 and 1:100 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-IL-6 antibody
The kit contains one yellow-capped vial with concentrated IL-6 antibody -biotin conjugate. Per microtiter plate, add 120 ul biotinylated IL-6 antibody to 12 ml working-strength dilution buffer just before use.
Leaving the substrate blank wells empty, add 100 ul of the bio tin conjugate to all wells.
Cover 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).
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 strepta vidin-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.
Add 100 ul of substrate solution to all wells, including the substrate blank wells.
Cover microtiter plate(s) with lid, 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.
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 aver age 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 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 IL-6 concentration (pg/ml) from the horizontal axis. Multiply the obtained IL-6 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 |
0.014 |
0.019 |
|
0.6 pg/ml |
0.046 |
0.066 |
|
1.9 pg/ml |
0.110 |
0.179 |
|
5.6 pg/ml |
0.302 |
0.474 |
|
16.7 pg/ml |
0.793 |
1.245 |
|
50.0 pg/ml |
1.738 |
2.667 |
|
150 pg/ml |
2.497 |
>3.00 |
|
450 pg/ml |
2.750 |
>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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