Human IL-6 ELISPOT KIT

INTENDED USE

This ELISPOT kit is used to quantitatively determine the frequency of cells releasing human Interleukin-6 (IL-6). The Reagent Diluent recommended may be suitable for most cell culture supernate, serum, and plasma samples.

This kit contains sufficient materials to run ELISPOTs on half 96 well plate, provided the following conditions are met:

• The reagents are prepared as described in this package insert.
• The assay is run as described in the ASSAY PROCEDURE.
• The recommended microplates, buffers, diluents, substrates, and solutions are used.

MATERIALS PROVIDED & STORAGE CONDITIONS

 Store the unopened kit at 2-8 °C. Do not use past kit expiration date.

Note: This kit is validated for single use only. Results obtained using previously opened or reconstituted reagents may not be reliable.

 PART DESCRIPTION

6. Human IL-6 Microplate: Part #ES1001-1, 96-well PVDF-backed microplate coated with a monoclonal antibody specific for human IL-6.
7. Human IL-6 Detection Antibody Concentrate: Part #ES1001-2, 150 μL of a 120X concentrated solution of biotinylated polyclonal antibody specific for human IL-6 with preservatives.
8. Streptavidin-AP Concentrate A: Part #ES1001-3, 150 μL of a 120X concentrated solution of Streptavidin conjugated to Alkaline Phosphatase with preservatives.
9. Dilution Buffer 1: Part #ES1001-4, 12 mL of a buffer for diluting Human IL-6 Detection Antibody Concentrate with preservatives.
10. Dilution Buffer 2: Part #ES1001-5, 12 mL of a buffer for diluting Streptavidin-AP Concentrate A with preservatives.
11. Wash Buffer Concentrate: Pat #ES1001-6, 50 mL of a 10X concentrated solution of a buffered surfactant with preservative.
12. BCIP/NBT Substrate: Part #ES1001-7, 12 mL of a stabilized mixture of 5-Bromo-4-Chloro-3′ Indolylphosphate p-Toluidine Salt (BCIP) and Nitro Blue Tetrazolium Chloride (NBT).
13. Human IL-6 Positive Control: Part #ES1001-8, 2 ng of recombinant human IL-6 with preservatives; lyophilized.

REAGENT PREPARATION

1. Keep the Human IL-6 Detection Antibody Concentrate and Dilution Buffer 1 at 2-8 °C, bring all reagents to room temperature.
2. Wash Buffer: To prepare Wash Buffer, add 50 mL of Wash Buffer Concentrate to 450 mL of deionized water and mix well.
3. Human IL-6 Positive Control: Reconstitute the lyophilized Human IL-6 Positive Control with 250 μL of culture medium.
4. Detection Antibody: Transfer 100 μL of Human IL-6 Detection Antibody Concentrate into the vial labeled Dilution Buffer 1 and mix well. Prepare antibody mixture immediately before use.
5. Streptavidin-AP Concentrate A – Transfer 100 μL of Streptavidin-AP Concentrate A into the vial labeled Dilution Buffer 2 and mix well. Prepare the Streptavidin-AP immediately before use.

SAMPLE PREPARATION

The types of effector and responder cells used, method of cell separation, mode of stimulation, and length of incubation are to be determined by each end user.

ASSAY PROCEDURE

1. Add 200 μL of sterile culture media to each well and incubate for approximately 20 minutes at room temperature.
2. When cells are ready to be plated, aspirate the culture media from the wells. Immediately add 100 μL of the appropriate cells or controls to each well.
3. Incubate cells in a humidified 37 °C CO₂ Optimal incubation time for each stimulus should be determined by end user. Do not disturb the cells during the incubation period.
4. Aspirate each well and wash, repeating the process three times for a total of four washes using Wash buffer. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5. Add 100 μL of Detection Antibody mixture into each well, and incubate overnight at 2-8 °C. Alternatively, incubation with detection antibodies can be done for 2 hours at room temperature on a rocking platform.
6. Repeat the wash procedure described in step 4.
7. Add 100 μL of diluted Streptavidin-AP Concentrate A into each well, and incubate for 2 hours at room temperature.
8. Repeat the wash procedure described in step 4.
9. Add 100 μL of BCIP/NBT Substrate into each well, and incubate for 1 hour at room temperature. Protect from light.
10. Decant the BCIP/NBT Substrate from the microplate and rinse the microplate with deionized water. Invert the microplate and tap to remove excess water. Remove the flexible plastic underdrain from the bottom of the microplate, wipe the bottom of the plate thoroughly with paper towels and dry completely either at room temperature (60-90 minutes) or 37 °C (15-30 minutes).

CALCULATION OF RESULTS

The developed microplate can be analyzed by counting spots using either a dissection microscope or an ELISpot reader. Quantitation of results can be done, for example, by calculating the number of spot forming cells per number of cells added to the well.