Human IL-6 ELISpotPLUS kit (ALP)

This kit is ideal for users who want a convenient and sensitive assay.

This experiment aims to count the cells that secrete human IL-6. The kit includes monoclonal antibodies, biotinylated detection antibodies, Streptavidin-ALP and ELISpot plates pre-coated with BCIP/NBT-plus substrates. The pre-coated plate reduces the measurement time and minimizes variability. The human IL-6 ELISpotPLUS kit also contains streptavidin-hrp and TMB substrates.

ELISpot

Washing of plates can be done using a multi-channel micropipette. In washing steps not requiring sterile conditions(C1-C5), a regular ELISA plate washer can also be used, provided that the washing head isadapted to the ELISpot plates. Avoid getting liquid on the underside of the membrane as this may causeleakage due to capillary drainage. If using ELISpot PVDF (ELIIP) plates, always remove the plate from the plate traybefore manually emptying the plate.

IL6

human, non-human primates

Sandwich

Sandwich

cell suspension

Recognizes natural human IL6

2 plates

1.2 pre-coated plates, mAb 13A5.
2.Vial 1 (blue top):Biotinylated detection mAb 39C3 (40 μl);Concentration 1 mg/ml.
3.Vial 2 (white top):Streptavidin-HRP (40 μl).
4.TMB substrate (25 ml):The detection antibody is supplied in sterile fltered (0.2um) PBS with 0.02% sodium azide. Streptavidin-HRP is supplied in PBS with 0.002% Kathon CG. Vials have been overflled to ensure recovery of the specified amount.

200 µL

2h

Pre-coated

1.Remove the plate from the sealed package and wash 4 times with sterile PBS(200 ul/well).
2.Condition the plate with medium (200 ul/well) containing 10% of the same serum as used for the cell suspensions. Incubate for at least 30 minutes at room temperature.
3.Remove the medium and add the stimuli followed by the cell suspension. Alternatively, cells and stimuli can be mixed before addition to the plate.
4.Put the plate in a 37°C humidified incubator with 5% CO, and incubate for 12-48 hours. Do not move the plate during this time and take measures to avoid evaporation.
5.Remove the cells by emptying the plate and wash 5 times with PBS, 200 ul/well.
6.Dilute the detection antibody(39C3-biotin) to 0.05 ug/ml in PBS containing 0.5% fetal calf serum(PBS-O.5%FCS). Add 100 ul/well and incubate for 2 hours at room temperature.
7.Wash plate as above.
8.Dilute the Streptavidin-ALP(1:1000) in PBS-0.5% FCS and add 100 ul/well. Incubate for 1 hour at room temperature.
9.Wash plate as above.
10.Filter the ready-to-use substrate solution(BCIP/NBT-plus) through a 0.45 um filter and add 100 ul/well. Develop until distinct spots emerge.
11.Stop color development by washing extensively in tap water. If desirable, remove the underdrain (the soft plastic under the plate) and rinse the underside of the membrane.
12.Leave the plate to dry. Inspect and count spots in an ELISpot reader or in a dissection microscope.13. Store plate in the dark at room temperature.

human, non-human primates

Biotinylated detection mAb (39C3), Streptavidin-ALP, Substrate (BCIP/NBT-plus), Pre-coated MSIP white plates (mAb 13A5)

We recommend the use of PVDF-based membrane plates. Maximal antibody binding capacity of these platesis obtained by a brief treatment with ethanol.

PBS for washing and dilution should be filtered (0.3 um) for optimal results. Although possible to use, we do not recommend the inclusion of Tween or other detergents in the washing and incubation buffers.

4°C-8°C

Plates should be kept at room temperature.

The expiry date indicates how long unopened products, stored according to instructions, are recommended for use.

The serum should be selected to support cell culture and give low background staining. We recom-mend the use of fetal calf serum. Alternatively serum-free medium evaluated for cell culture can beused.

For Research Use Only. Not for use in diagnostic procedures.

B-cell stimulatory factor 2; CDF; HSF; BSF-2; Interferon beta-2; IL-6; IFNB2; CTL differentiation factor; Interleukin-6; HGF; Hybridoma growth factor; BSF2; IFN-beta-2

BSF-2, BSF2, CDF, HGF, HSF, IFN-beta-2, IFNB2, IL-6

This gene encodes a cytokine that functions in inflammation and the maturation of B cells. In addition, the encoded protein has been shown to be an endogenous pyrogen capable of inducing fever in people with autoimmune diseases or infections. The protein is primarily produced at sites of acute and chronic inflammation, where it is secreted into the serum and induces a transcriptional inflammatory response through interleukin 6 receptor, alpha. The functioning of this gene is implicated in a wide variety of inflammation-associated disease states, including suspectibility to diabetes mellitus and systemic juvenile rheumatoid arthritis.

3569

P05231

Among its related pathways are Tuberculosis and ErbB signaling pathway.

1.Antibody coating:Cytokine-specific monoclonal capture antibodies are immobilized on an ethanol-treated PVDF membrane plate.
2.Cell incubation:Cells are added to the wells in the presence or absence of activating stimuli, and then incubated to allow for cytokine secretion.
3.Cytokine capture:Secreted cytokines bind to the capture antibodies on the membrane immediately surrounding the activated cells.
4.Detection antibodies:Following removal of the cells and washing of the plate wells, biotinylated cytokine-specific detection antibodies are added to the wells.
5.Streptavidin-enzyme conjugate:To enable the formation of spots on the membrane, a streptavidin-enzyme conjugate is added to the wells.
6.Addition of substrate:Colorimetric substrate is added to the wells and will form an insoluble precipitate when catalyzed by the enzyme; a visible representation of cytokine release by a single activated cell.
7.Analysis Spots are counted in an automated ELISpot reader or under a dissection microscope, and the frequency of secreting cells is calculated.