Assay Genie ELISpot is a highly specific immunoassay for the analysis of IL-6 production and secretion from T-cells at a single cell level in conditions closely comparable to the in-vivo environment with minimal cell manipulation. This technique is designed to determine the frequency of IL-6 producing cells under a given stimulation and the comparison of such frequency against a specific treatment or pathological state. Utilising sandwich immuno-enzyme technology, Assay Genie ELISpot assays can detect both secreted IL-6 (qualitative analysis) and single cells that produce IL-6 (quantitative analysis). Cell secreted IL-6 is captured by coated antibodies avoiding diffusion in supernatant, protease degradation or binding on soluble membrane receptors. After cell removal, the captured IL-6 is revealed by tracer antibodies and appropriate conjugates.
A capture antibody highly specific for IL-6 is coated to the wells of a PVDF bottomed 96 well microtitre plate either during kit manufacture or in the laboratory. The plate is then blocked to minimise any non-antibody dependent unspecific binding and washed. Cell suspension and stimulant are added and the plate incubated allowing the specific antibodies to bind any IL-6 produced. Cells are then removed by washing prior to the addition of Biotinylated detection antibodies which bind to the previously captured IL-6. Enzyme conjugated streptavidin is then added binding to the detection antibodies. Following incubation and washing, substrate is then applied to the wells resulting in coloured spots which can be quantified using appropriate analysis software or manually using a microscope.
Step
Procedure
1.
For PVDF membrane activation, add 25 µl of 35% ethanol to every well.
2.
Incubate plate at room temperature (RT) for 30 seconds.
3.
Empty the wells by flicking the plate over a sink & gently tapping on absorbent paper. Thoroughly wash the plate 3x with 100 µl of PBS 1X per well.
4.
Add 100 µl of diluted capture antibody to every well.
5.
Cover the plate and incubate at 4°C overnight.
6.
Empty the wells as previous and wash the plate once with 100 µl of PBS 1X per well.
7.
Add 100 µl of blocking buffer to every well.
8.
Cover the plate and incubate at RT for 2 hours.
9.
Empty the wells as previous and thoroughly wash once with 100 µl of PBS 1X per well.
10.
Add 100 µl of sample, positive and negative controls cell suspension to appropriate wells providing the required concentration of cells and stimulant.
11.
Cover the plate and incubate at 37°C in a CO2 incubator for an appropriate length of time (15-20 hours). Note: do not agitate or move the plate during this incubation.
12.
Empty the wells and remove excess solution then add 100 µl of Wash Buffer to every well.
13.
Incubate the plate at 4°C for 10 min.
14.
Empty the wells as previous and wash the plate 3x with 100 µl of Wash Buffer.
15.
Add 100 µl of diluted detection antibody to every well.
16.
Cover the plate and incubate at RT for 1 hour 30 min.
17.
Empty the wells as previous and wash the plate 3x with 100 µl of Wash Buffer.
18.
Add 100 µl of diluted Streptavidin-conjugate to every well.
19.
Cover the plate and incubate at RT following the supplier's instructions.
20.
Empty the wells and wash the plate 3x with 100 µl of Wash Buffer.
21.
Peel of the plate bottom and wash both sides of the membrane 3x under running distilled water, once washing complete remove any excess solution by repeated tapping on absorbent paper.
22.
Add 100 µl of ready-to-use substrate buffer to every well.
23.
Following the supplier's instructions, incubate the plate for 5-15 min monitoring spot formation visually throughout the incubation period to assess sufficient colour development.
24.
Empty the wells and rinse both sides of the membrane 3x under running distilled water. Completely remove any excess solution by gentle repeated tapping on absorbent paper Read Spots: allow the wells to dry and then read results. The frequency of the resulting coloured spots.
