Human IgG/IgM Dual-Color B Cell ELISpot Kit (CAT#: ITS-0322-P155)

1 Kit

1.Human IgG/IgM Microplate.
2.Biotin-conjugated Detection Antibody.
3.HRP-conjugated Detection Antibody.
4.Streptavidin-conjugated to Alkaline Phosphatase.
5.Dilution Buffers.
6.Wash Buffer Concentrate.
7.BCIP/NBT Chromogen.
8.AEC Chromogen.
9.Human IgG/IgM Positive Controls.

1.Pipettes and pipette tips.
2.Deionized or distilled water.
3.Squirt bottle, manifold dispenser, or automated microplate washer.
4.500 mL graduated cylinder.
5.37 °C CO2 incubator.
6.Sterile culture media.
7.Dissection microscope or an automated ELISpot reader.

100 µL

3 hours 35 mins to 4 hours 50 mins*

Pre-coated

1.Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. To prepare Wash Buffer, add 50 mL of Wash Buffer Concentrate to 450 mL of deionized water and mix well.
2.Capture Antibody Mixture (Concentrate A + Concentrate B) - Tap or vortex each vial to release reagent collected in the cap. Transfer 100 µL of Capture Antibody Concentrate A and 100 µL of Capture Antibody Concentrate B into 12 mL of PBS and mix well.
3.Detection Antibody Mixture (Concentrate A + Concentrate B) - Tap or vortex each vial to release reagent collected in the cap. Transfer 100 μL of Detection Antibody Concentrate A and 200 μL of Detection Antibody Concentrate B into the vial labeled Dilution Buffer 1 and mix well.
4.Streptavidin-AP Concentrate A - Tap or vortex the vial to release reagent collected in the cap. Transfer 100 μL of Streptavidin-AP Concentrate A into the vial labeled Dilution Buffer 2 and mix well.
5.AEC Chromogen Solution - Transfer 250 μL of AEC Chromogen to the vial labeled AEC Chromogen Buffer and mix well.

1.1. For Detection of antigen-specific IgG/IgM producing B cells (Antigen-Down Assay Principle): Dilute the antigen (5-15 µg/mL) in 1X PBS and add 100 μL to each well. Incubate at 2-8 °C overnight. For Detection of total IgG/IgM producing B cells (Sandwich Assay Principle: Add 100 µL of the diluted Capture Antibody Mixture (A + B) into each well and incubate at 2-8 °C overnight.
2.Aspirate each well and wash, repeating the process once for a total of two washes.
3.Fill all wells in the microplate with 200 µL of Block Buffer and incubate for 2 hours at room temperature.
4.Aspirate each well. Fill all wells in the microplate with 200 µL of sterile culture media and incubate for 20 minutes at room temperature.
5.When cells are ready to be plated, aspirate the culture media from the wells. Immediately add 100 µL of the appropriate cells to each well.
6.Incubate cells in a humidified 37°C CO2 incubator. Optimal incubation time for each stimulus should be determined by the investigator.
7.Aspirate each well and wash as in step 2, using 1X Wash Buffer, repeating the process three times for a total of four washes.
8.Add 100 µL of diluted Detection Antibody Mixture (A + B) into each well and incubate at 2-8°C overnight.
9.Aspirate/wash as in step 7.
10.Add 100 µL of diluted Streptavidin-AP Concentrate A into each well and incubate for 2 hours at room temperature.
11.Aspirate/wash as in step 7.
12.Add 100 µL of BCIP/NBT Substrate into each well and incubate for 1 hour at room temperature. Protect from light.
13.Discard the BCIP/NBT Substrate solution from the microplate and rinse the microplate with deionized water. Invert the microplate and tap to remove excess water.
14.Add 100 µL of the prepared AEC Chromogen Solution into each well and incubate for 20 minutes at room temperature. Protect from light.
15.Decant the AEC Chromogen Solution 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).

Human

96-well PVDF-backed microplate

Some components of this kit contain sodium azide, which may react with lead and copper plumbing to form explosive metallic azides. Flush with large volumes of water during disposal.
AEC Chromogen may cause skin, eye, and respiratory irritation. Avoid breathing fumes.
BCIP/NBT is toxic if swallowed, in contact with skin, or if inhaled. It is a highly flammable liquid and vapor may cause serious irritation and damage to organs. Do not eat, drink, or smoke when using this product. Do not breathe fumes. Use only in a well-ventilated area. Keep away from heat, sparks, open flames, and hot surfaces. Keep the container tightly closed.

Some components in this kit contain a preservative which may cause an allergic skin reaction. Avoid breathing mist.
Wear protective gloves, clothing, eye, and face protection. Wash hands thoroughly after handling. Refer to the SDS on our website prior to use.

Store the unopened kit at 2-8 °C.

Do not use past kit expiration date. This kit is validated for single use only.

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

For Research Grade Use Only.

1.Coat PVDF-backed microplate with antigen provided by user.
2.Add the stimulated cells into the wells.
3.Incubate in a humidied 37°C CO2 incubator.
4.Wash away any cells and unbound substances.
5.Add a biotinylated polyclonal antibody specic for the first immunoglobulin and an HRP-conjugated polyclonal antibody specic for the second immunoglobulin to the wells.
6.Wash away any unbound biotinylated antibodies.
7.Add alkaline phosphatase conjugated streptavidin.
8.Wash away any unbound enzyme.
9.Add the BCIP/NBT Substrate.
10.Incubate until a blue-black precipitate forms.
11.Add the AEC Chromogen.
12.Identify and count the spots with an automated ELISpot reader or manually with a stereomicroscope.