Human IgG3 ELISpotBASIC kit (ALP)

Human IgG3 ELISpotBASIC can enumerate B cells that secrete total IgG3 and antigen-specific IgG3. It contains a pair of matched monoclonal capture and detection antibodies, Streptavidin-ALP and polyclonal activator.

The kit can detect B cells that specifically secrete IgG3 by coating antigen. If biotinylated antigen detection is preferred, it should be coated with subclass-specific antibodies. For the analysis of memory B cells, we recommend using R848 and recombinant human IL-2 (both included) for pre-stimulation. IgG3 ELISpotBASIC can also be used for streptavidin-hrp.

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.

IgG3

human

Sandwich

Sandwich

cell suspension

Recognizes natural human IgG3

blood or lymphoid tissue

4 plates

1.Vial 1 (yellow top):anti-human IgG mAb MT145 (1200 ul);Concentration: 0.5 mg/ ml.
2.Vial 2 (blue top):anti-human IgG3 mAb MTG34, biotinylated (50 ul);Concentration: 0.5 mg/ml.
3.Vial 3 (white top):Streptavidin-Alkaline Phosphatase (50 ul).
5.Polyclonal activator: R848 (100 μl):Concentration: 1 mg/ml.
5.Lyophilised recombinant human IL-2 (1 ug).

200 µL

2h

Pre-coated

1.Dilute the coating antibodies (MT145) to 15 ug/ml in sterile PBS, pH 7.4.
2.Remove the ELISpot plate from the package and if using a PVDF plate, pre-wet the membrane by adding ethanol.
3.Wash plate 5 times with sterile water, 200 ul/well.
4. Add 100 ul/well of the antibody solution and incubate overnight at 48°C.
5.Remove excess antibody and wash plate 5 times with sterile PBS, 200 ul/well.
6. Add 200 ul/well of medium containing 10% of the same serum as used for the cell suspensions Incubate for at least 30 minutes at room temperature.
7.Remove the medium and add the stimuli followed by the cell suspension. Alternatively, cells and stimuli can be mixed before addition to the plate.
8.Put the plate in a 37°C humidified incubator with 5% CO2, and incubate for 18-48 hours.
9.Remove the cells by emptying the plate and wash 5 times with PBS, 200 ul/well.
10.Dilute the detection antibody(MTG34-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.
11.Wash plate as above.
12.Dilute the Streptavidin-ALP(1:1000) in PBS-0.5% FCS and add 100 ul/well. Incubate for 1 hour at room temperature.
13.Wash plate as above.
14.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.
15.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.
16.Leave the plate to dry. Inspect and count spots in an ELISpot reader or in a dissection microscope.
17. Store plate in the dark at room temperature.

human

Capture mAb (MT145), Biotinylated detection mAb (MTG34), Streptavidin-ALP, R848, Recombinant human IL-2

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. Human R848 and IL-2 shoud be stored frozen at -20°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.

IgG3

3502

P01860

Among its related pathways are Innate Immune System and Role of phospholipids in phagocytosis.

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.