rev:   March 16, 2005

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                 PeliSPOT (tm)  Human ELISPOT Reagent Set

-manufactured by CLB Reagents


Cytokine Elispot Kits

cat#RDI-M2536clb   Human Granzyme B  $750.00

cat#RDI-M2533clb   Human IFN-gamma   $500.00

cat#RDI-M2514clb   Human IL-4               $500.00

Cytokine Pelispot Antibody pairs   NEW  $375.00/set  

cat#RDI-M9433clb  Human IFN-gamma

cat#RDI-M9414clb  Human IL-4

cat#RDI-M9410clb  Human Il-10

cat#RDI-M9413clb   Human IL-13

cat#RDI-M9515clb   MOUSE IL-5


 

ELISPOT protocol for the use of CLB PeliPairs cytokine (ELISA) reagent sets

-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

Kit

$1250.00/set

coat* biotin-ab* number of

plates per kit

IL-1beta 1:20 1:50 4
IL-4 1:50 1:75 20
IL-6 1:75 1:50 15
IL-8 1:40 1:50 8
IL-10 1:50 1:50 20
IL-13 1:20 1:50 4
TNF alpha 1:100 1:100 40

* 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.

Stimuli

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.


-please inquire for other matched pair antibodies for human cytokines

-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

USA

phone (978) 371-6446 or (800) 370-2222

fax      (978) 371-2266

EMAIL:antibodies@fitzgerald-fii.com

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