rev:  January 6, 2005

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Pelikine Compact soluble human IL-6 receptor ELISA Kit


CAT# RDI-M1926lb 288 tests    PRICE: $500.00


-for in Vitro Research Use Only-Not For Use in Diagnostics

-sample protocol-see batch specific insret inside each kit-


INTRODUCTION

Interleukin 6 (IL-6) is a pleiotropic cytokine that acts on a wide variety of cells [1-3]. It has been shown that IL-6 acts on target cells through surface IL-6 receptors, identified as two different membrane proteins, i.e. an 80-kDa protein (gp80) that binds IL-6 with low affinity (IL-6r) and a 130-kDa protein (gp 130) that is required for high-affinity binding and for signal transduction [4,5]. Not only membrane-bound receptors, but also soluble forms of membrane-bound cytokine and growth-factor receptors, which have retained their ability to specifically bind their ligands, have been identified in biological fluids and biochemically characterized [6]. Soluble IL-6 receptor (sIL-6r) is a 50-55 kDa ligand binding protein, derived from the extracellular part of the gp80 receptor by shedding [7,8]. The clinical significance of sIL-6r levels has been reported is several studies, including HIV [9], multiple myeloma [10],interstitial pneumonia [11] and sarcoidosis [11].

The Pelikine compact (tm) soluble human IL-6 receptor ELISA kit has been developed for faster, more reproducible and specific quantification of soluble human IL-6 receptor in serum, plasma and other body fluids, as well as in cell-culture supernatant.


II. PRINCIPLE OF THE TEST

The Pelikine compact soluble human IL-6 receptor (sIL-6r) ELISA kit is a"sandwich-type"of enzyme immunoassay in which a monoclonal anti-sIL-6r antibody is bound onto polystyrene microtiter wells. Soluble human IL-6 receptor, present in a 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 second monoclonal antibody to sIL-6r is added. This antibody binds to the sIL-6r 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 sIL-6r sandwich. After removal of non-bound HRP conjugate by washing, a substrate solution is added to the wells. A coloured product is formed in proportion to the amount of sIL-6r 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 sIL-6r can be determined by interpolation with the standard curve.


III. STORAGE AND STABILITY

The Pelikine compact soluble human IL-6 receptor 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(tm) soluble human IL-6 receptor 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

1vial                       sIL-6r standard (lyophilized) 7500 pg/ml                1000ul

1 vial                      biotinylated sIL-6r antibody 100-X concentrated     375ul                yellow

1 vial                      streptavidin-HRP conjugate 10,000-X concentrated   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(tm) soluble human IL-6 receptor ELISA kit is in tended 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 soluble IL-6 receptor 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 purchase the Pelikine ToolSet)

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 in a well closed bottle.


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 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 :    3-5pg/ml   (shake-static incubation)

2 x(MEAN calculated zero signal): 4-6pg/ml(shake-static incuba tion).

Note: the sensitivity is dependent of the type and quality of   enzymatic substrate.


Expected values

sIL-6r values in fresh serum and plasma samples of healthy individuals are in the range of 15 - 120 ng/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, IL-13, 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),Tumor Necrosis Factor a, Tumor Necrosis Factor B (TNFB/Lymphotoxin), and Interferon g (IFNG)

1. Kishimoto,T. (1989) Blood 74:1

2. Aggerwal,B.B.Gutterman,J.U.(1992) Human cytokines.Blackwell

Sci.Pub. ISBN 0-86542-183-8

3. Thomson,A.W. (1991) The cytokine handbook. Academic Press ISBN 0-12-689660-7

4. Taga,T. et al Cell 58: 573

5. Kishimoto,T. (1992) Int.Arch.Allergy Immunol. 99: 172

6. Rose-John,S.Heinrich,P.C. (1994) Biochem.J. 300: 281

7. Novick,D. et al (1989) J.Exp.Med. 170: 1409

8. Mullberg,J. et al (1993) Eur.J.Immunol. 23: 473

9. Honda,M. et al (1992) J.Immunol. 148: 2175

10. Gaillard,J-P, et al (1993) Eur.J.Immunol. 23: 820

11. Yokoyama,A. et al (1995) Clin.Exp.Immunol. 100: 325


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.  sIL-6r STANDARD Standard curve preparation

The kit contains a lyophilized vial with 7500 pg/ml natural soluble human IL-6 receptor. Reconstitute the lyophilized standard by adding 1 ml of distilled water to the vial. In cubate 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:2500, 1000, 400,160, 64, 25.6, and 10.2 pg/ml.

Pipette 300ul of working-strength dilution buffer into all the labelled tubes. Transfer 150 ul of the sIL-6r standard (7500 pg/ml) into the first tube labelled 2500 pg/ml, mix well and transfer 200 ul of this dilution into the second tube labelled 1000 pg/ml. Repeat the serial dilutions six more times by adding 200 ul of the previous tube of diluted standard to the 300 ul of dilution buffer.

The standard curve will contain 2500, 1000, 400, 160, 64, 25.6, 10.2 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 sIL-6r levels of the standard. Thaw the reconstituted standard in tap water (18-25'C), do not use 37'C or 56'C waterbaths for this purpose.


5. 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). Up to 3 freeze-thaw cycles have no effect on the sIL-6r levels 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 1:200 in working-strength dilution buffer. If high levels of sIL-6r (<200ng/ml) are expected in the test samples, additional dilution of samples (i.e. 1:1000) 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 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).


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-sIL-6r antibody

The kit contains one yellow-capped vial with concentrated sIL- 6r antibody-biotin conjugate.

Per microtiter plate, add 120 ul biotinylated sIL-6r antibody to 12 ml working-strength dilution buffer just before use.

Leaving the substrate blank wells empty, add 100 ul diluted biotinylated sIL-6r 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 main- tain 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.

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 aluminum 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.

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 sIL-6r 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 sIL- 6r concentration (pg/ml) from the horizontal axis. Multiply the obtained sIL-6r concentration with the dilution factor of the sample and record this figure.


X. INCREASED SENSITIVITY

The assay signals can be increased by a small adaptation of theincubation 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 signals, especially in the upper range of the assay (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 0.011 0.009
10.2    pg/ml 0.031 0.031
25.6    pg/ml 0.067 0.074
64       pg/ml 0.158 0.190
160     pg/ml 0.375 0.502
400     pg/ml 0.874 1.244
1000   pg/ml 1.503 2.459
2500   pg/ml 2.163 >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


RDI Divison of researchd Industries Intl

San Jose, 95123 CA Snell ave 658

USA

or (800) 370-2222

EMAIL:margaret@cellular-research.com

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for recombinant cytokines and antibodies , click here

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