rev: January 10, 2005
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PELIPAIR (tm) Human Cytokine Reagent Set
-manufactured by CLB Reagents
-see recommended Elispot protocols below
-The concept of the Pelipair reagent sets provides performance at the lowest price. the procedure recommended is the same as for our sucessful compact kits, after overnigth coating the assay is competed in 4 hours. The optimized and easy-to-follow protocol included in the sets warrants reliable assay results.
-When HEPE (High Performance ELISA, not included, order cat#RDI-M1940clb) dilution bufer is used linear dose response curves in different matrices are ensured. High sensitivities can be achieved by using POLY-HRP (a polymer of HRP-Streptavidin, order cat#RDI-PHRPX-SA).
-For even more convenience additional buffers ad TMB subsrates are available as the Pelikine (tm) toolset, cat#RDI-M1980clb $188.00 or $156.00 when ordered with kit, sufficient for 3 X 96 weels) (or TMB 1 liter cat#RDI-TMB-S-1L $312.0/1 Liter)
Each kit contains sufficient reagents for at least 1440 samples (including calibration curves)
5 vials coating antibody ( 5 X 375ul)
5 vials human cytokine standard
5 vials biotin-conjugated anti-human cytokine antibody ( 5 X 375ul)
*mean zero signal + 3 SD (when using POLY-HRP20-SA)
-see also new dedicated reagent sets for Elispot (PeliSpot)
-for BIO-Plex(tm) Luminex (tm) system see-
Jager et al, "Similtaneous Detection of 15 human Cytokines in a Single Sample of Stimulated Peripheral Blood Mononuclear Cells", Clinical and Diagnostic Laboratory Immunology, Jan 2003, p133-139
plates per kit
* Peilpair antibody dilutions (optimized dilution should be determined individually)
-see also Granzyme B Peli-Spot(tm kit) for Granzyme B
-see also IFN-Gamma Elispot kit
-Coat ( e.g. ethanol activated PVDF plates ) with catching antibody* to cytokine; 50 Ál/well in coating buffer overnight at room temperature (RT).
-Wash the plate 4x with PBS; 1x with IMDM or with RPMI 1640.
-Incubate with blocking medium; 100 ul/well 1 h at 37oC.
-Empty plate and fill with cells (titration) and stimuli.
-Incubate at 37 oC in CO2 incubator. Incubation time depends on stimulus and cytokine under study.
-Wash the plate 5x with washing buffer.
-Add biotinylated antibody* to cytokine 100 ul/well in HPE and incubate 1h at RT on a shaker.
-Wash 5x with washing buffer.
-Incubate for 5min at RT with 100 Ál streptavidin-poly HRP (1/2000 in PBS + 2% milk) on shaker.
-Wash 5 x with washing buffer.
-Stain for ~10 minutes at RT with 3-amino-9-ethylcarbazol.
-Wash with H2O and dry.
Coating buffer: 0.1 M NaHCO3, pH 9.5
PBS 10 mM sodium phosphate 0.14 M NaCl pH 7.2
Culture medium RPMI 1640 or IMDM with 5 % FCS, pen/strep,
50 µM 2-mercaptoethanol; human transferin 20 µg/ml.
Blocking medium RPMI or IMDM containing 10 % FCS, pen/strep and 50 M 2-mercaptoethanol
Washing buffer PBS + 0.02 % Tween-20 (PT).
HPE Elisa High Performance buffer produced by CLB
3-amino-9-ethylcarbazol stock solution 10 mg/ml in dimethylformamide; For use in this assay dilute 1:30 in 50 mM Na acetate pH 5.0 and filter through a 0.45 µM filter. Then add 0.015 % H2O2.
To measure T cell cytokines (IL-2, IL-4, IL-13, IFN, TNFá and GM-CSF) the following stimuli can be used:
- Ca-ionophore (1 µM ionomycine) + PMA 1ng/ml. Stimulation overnight or
- anti-CD3 1 µg/ml + anti-CD28 1 µg/ml. Stimulation is for 2,3 or 4 days.
To measure monokine production (IL-1, IL-6, IL-8, IL-10, IL-12 and TNFá) stimulation can be carried out with LPS 1ng/ml or with SAC (1:1000).
For a number of monokines the plates are stimulatory by themselves.
-see some combinations under the individual cytokine antibodies which can be accessed through the cytokine table
RDI Divison of Fitzgerald Industries Intl
34 Junction Square Drive
Concord MA 01742-3049
phone (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
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for recombinant cytokines and antibodies , click here
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