rev: 5/16/2008
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Sample Kit Protocol: (see actual insert with kit for batch specific information):
Pelikine Compact Human Soluble CD27 ELISA Kit
CAT# RDI-M1960clb, new cat#: 55R-M1960clb 288 tests PRICE:
$525.00
INTRODUCTION
CD27 is a lymphocyte specific member of a growing family of receptors for
membrane bound and soluble cytokines, the TNFr superfamily [1.2]. Next to the
type I and type II TNFr (CD120a and CD120b) this receptor family comprises the
Hodgkin's disease-related CD30 antigen CD40, FAS/APO-1 (CD95), 4-1BB and the
rat T-cell surface molecule OX40, all proteins that are predominantly expressed
on cells of the haemapoetic system [3.4]. The expression of CD27 is restricted
to cells of the lymphoid lineage and appears to be related to functional
differentiation programs of T-and B-cells [5-8]. With respect to the T-cell
compartment, thymocytes at the CD3(low) stage do not express CD27, but
expression can be induced after activation [9]. CD27 becomes constitutively
expressed on CD3(high) medullary thymocytes and this expressionis further
enhanced upon activation. Thymic emigrants express CD-45RA along with CD27 and
these naive T-cells have a very strongcapacity to upregulate CD27 after TCR/CD3
triggering [7,10]. In addition, activation induces the production of a soluble
32 kDa form of CD27, which is most likely generated by the proteolytic cleavage
of the putative ligand binding domain of the 55 kDa transmembrane molecule
[11,12]. This soluble receptor moleculesCD27) can not only be found in
supernatants of activated lymphocytes, but is also present in biological fluids
of healthy individuals. Levels of sCD27 have been found to increase in patients
suffering from a variety of immunopathological diseases which suggests that
measurement of sCD27 can serve as a marker for T-cell activation in vivo
[13-16].
This sCD27 ELISA [13] has been developed for fast, reproducible and specific
quantification of human sCD27 in serum and in plasma, as well as in
cell-culture supernatant.
II. PRINCIPLE OF THE TEST
The Pelikine compact human soluble CD27 ELISA kit is a"sandwich-type"of enzyme immunoassay in which a monoclonal CD27 antibody is coated to polystyrene microplate wells. Soluble CD27 molecules (sCD27), present in a measured volume of sample or standard are bound by the antibodies on the microplate plate, and non-bound material is removed by washing. Subsequently, a biotinylated second monoclonal CD27 antibody is added. This antibody binds to the sCD27-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 sCD27-antibody sandwich. After removal of non-bound streptavidine-HRP conjugate by washing, substrate solution is added to the wells. A colored product is formed in proportion to the amount of sCD27 present in the sample or standard. After the reaction has been terminated by the addition of a stop solution, absorbance is measured in a micro-titer plate reader. From the absorbance of samples and those of a standard curve, the concentration of sCD27 can be determined by interpolation with the standard curve.The compact sCD27 ELISA kit is intended for in vitro research use only.
III. STORAGE AND STABILITY
The Pelikine compact sCD27 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 sCD27 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 standard 400 U/ml 500ul black
1 vial biotinylated CD27-antibody 100-fold concentrated 375 ul yellow
1 vial streptavidin-HRP conjugate 1,000-fold concentrated 50 ul brown
1 bottle dilution buffer 10-fold concentrated 50 ml
3 pcs microplate plates + lid
10 pcs plate seals
V. PRECAUTIONS FOR USE
1) The compact sCD27 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) The sCD27standard contains human serum which have been found to be non-reactive for Hepatitis B surface Antigen (HBsAg), Hepatitis C and Human Immunodeficiency Virus. Nevertheless the standard should be handled as potentially hazardous and capable of transmitting disease.
7) Centrifuge all vials before use (1 minute 3000 x g).
VI. ADDITIONAL BUFFERS & SOLUTIONS REQUIRED (or obtain the Pelikine ToolSet)
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 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 2 liter of working-strength PBS by diluting the PBS stock solution (see above) 20-fold with distilled water.
Add 2 ml 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.
-Device for delivery of washing buffer(wash bottle/automated plate washer).
-microplate plate reader.
Sensitivity
MEAN calculated zero signal + 3 SD : 1.0 U/ml
2 x (MEAN zero signal) : 4.8 U/ml
Note: the sensitivity is dependent of the type and quality of enzymatic substrate.
Expected values
sCD27 values in fresh serum and plasma samples of healthy individuals are below 350 U/ml (Mean + 3x SD).
REFERENCES
1. Bazan J.F. (1993) Curr.Biol. 9:603
2. Hintzen R.Q. et al (1994) Immunol. Today 15: 307
3. Farrah T.,Smith
4. Mallet S., Barclay A.N. (1991) Immunol.Today 12: 220
5. De Jong R. et al (1992) Eur.J. Immunol. 22: 993
6. De Jong R. et al (1992) J. Immunol. 149: 2795
7. Hintzen R. Q. et al (1993) J.Immunol. 151: 2426
8. Maurer D. et al (1992) J.Immunol. 148: 3700
9. Martorell J. et al (1990) J.Immunol. 145: 1356
10.Sugita K. et al (1992) J. Immunol. 149: 3208
11.Hintzen R.Q. et al (1991) J. Immunol. 147: 29
12.Loenen W.A.M. et al (1992) Eur.J.Immunol. 22: 447
13.Hintzen R.Q. et al (1991) J.Neuroimmunol. 35: 211
14.Hol B.E. et al (1993) Am.Rev.Resp.Dis. 148: 643
15.Yazdanbakhsh M. et al (1993) Eur.J.Immunol. 23: 3312
16.Van Oers M.H.J. et al (1993) Blood 82: 3430
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 10 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 12 ml undiluted buffer per microplate plate) and prepare a working-strength solution by diluting the opalescent concentrated buffer 10 times in distilled water before use. The working-strength dilution buffer can be stored for up to one week at 2-8'C.
3. microplate PLATES Coating antibody
Coating The kit contains three microplate plates for 96 tests each,including the standard curve samples.
Prepare working-strength PBS (1:20 dilution of stock PBS (page 2 of information leaflet).
Per microplate plate, add 120 ul of coating antibody (red-capped vial) to 12 ml coating buffer.
Add 100 ul to all wells, cover microplate 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 (page 2 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
Add 200 ul working strength dilution buffer to all wells, cover microplate plates(s) with adhesive seal, gently agitate the microplate plate by tapping the edge of the plate for a few seconds to mix contents of each well and incubate for 30 minutes at room temperature (18-25'C).
4.sCD27 STANDARD Standard curve preparation
The kit contains one black-capped vial with 400 U/ml sCD27.
Label 7 tubes, one tube for each dilution:100, 50, 25, 12.5, 6.25, 3.12 and 1.56 U/ml. Pipette 450 ul of working-strength dilution buffer into the tube labelled 100 U/ml and 300 ul of working-strength dilution buffer into the other tubes. Transfer 150ul of the sCD27 standard into the first tube labelled 100 U/ml, mix well and transfer 300ul of this dilution into the second tube labelled 50 U/ml. Repeat the serial dilution six more times by adding 300 ul of the previous tube of diluted standard to the 300 ul of dilution buffer.
The standard curve will contain 100, 50, 25, 12.5, 6.25, 3.12, 1.56 and 0 U/ml (dilution buffer).
Avoid repeated freeze-thawing of the standard, although experimental data have shown that up to 3 freeze-thaw cycles have no effect on the sCD27 levels of the standard.
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). 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 sCD27 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 1:10 in working-strength dilution buffer. If high levels of sCD27(>800U/ml) are expected in the test samples, additional dilutions of sample i.e. 1:50 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 microplate 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
Transfer 100 ul of the prepared standards and samples in duplicate into the appropriate wells. Cover plate(s) with adhesive seal, gently agitate the microplate 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 microplate plate (s) as described in point 6 above.
9. SECOND INCUBATION STEP biotinylated-sCD27 antibody
The kit contains one yellow-capped vial with concentrated sCD27 antibody-biotin conjugate.
Per microplate plate, add 120 ul biotinylated sCD27 antibody to 12 ml working-strength dilution buffer just before use.
Add 100 ul of biotin conjugate 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 microplate 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 microplate plate, add 12ul streptavidine-HRP conjugate to 12 ml of working-strength dilution buffer just before use. Do not prepare in advance of assay.
Add 100 ul of streptavidin-HRP conjugate to all wells.
Cover the microplate plate(s) with adhesive seal, gently agitate the microplate 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 microplate 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.
Cover microplate plate(s) with lid, gently agitate the microplate 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. CALCULATIONS
-Record the absorbance at 450 nm for each well containing standard, and average the duplicate values.
-Plot the average absorbances (ordinate) versus the sCD27 concentration in U/ml (abscissa) on log-linear paper and draw the best fitting curve. An example of a standard curve is given below.
- Record the absorbance at 450 nm for each standard well, and average the duplicate values.
- Locate the 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 sCD27 concentration (U/ml) from the horizontal axis. Multiply the obtained sCD27 concentration with the dilution factor of the sample and record this figure.
For In Vitro Research Use Only-Not for use in Diagnostics
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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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