anti-IL10 IBD Initiative
Neutralising autoantibodies against IL-10 were initially detected in 2 patients with very early-onset inflammatory bowel disease and subsequently in 3.5% of patients with severe inflammatory bowel disease.
Neutralizing autoantibodies against IL-10 define a rare but clinically very relevant subset of patients with inflammatory bowel disease. An association of the autoantibodoes with the HLA subtype DRB1*01:03 was identified. HLA DRB1*01:03 is the strongest HLA risk factor for ulcerative colitis and Crohn's disease.
We developed several assays to measure the presence of anti-IL10 autoantibodoes. Those measure the binding of autoantibodies to IL-10 as well as the ability of autoantibodies to block surface area on IL10 (competitive Elisa) as well as functional neutralisation of IL10 signalling in a IL-10-STAT3-reporter assay.
Griffin, et al. NEJM 2026
Gharahdaghi et al. NEJM 2026
Protocols *Gharahdaghi et al. NEJM 2026
IL10 ELISA Assay Protocol If you use the antibody test please cite
1. Plate Coating
o Coat the ELISA plate according to the manufacturer's protocol the night
before the experiment.
o Seal the plate securely (e.g., plate sealer) and store it in the refrigerator
(4°C). Ensure the plate is kept free from contamination during storage.
2. Reagent Preparation
o On the day of the experiment, equilibrate all buHers, assay diluent, and
reagents to room temperature (18-25°C) before starting the assay.
o Ensure all reagents have been prepared and are free of particulates.
3. Sample Preparation
o Defrost all serum sample aliquots and ensure a minimum volume of 15 µL
per sample is available for the assay.
o Mix serum samples gently by pipetting to ensure homogeneity. Avoid too
many repeated freeze-thaw cycles as this may degrade the sample
quality.
o
4. Preparation of Anti-IL-10 and Recombinant IL-10
o Anti-IL-10 Antibody (Clone JES3-19F1, 100 µg/mL): Defrost the aliquot.
o Recombinant Human IL-10 (400 ng/mL): Defrost the aliquot and label it
similarly for freeze-thaw tracking.
*Note: for all samples, IL10 and anti-IL10 aliquot, mark the vial with a dot for each
freeze-thaw cycle to track usage.
5. Preparation of Standards
o All standards and cut-oH standards must be prepared in triplicate.
o IL-10 Standard Preparation:
§ Prepare a 500 pg/mL stock: 7.5 µL of 400 ng/mL stock + 5992.5 µL
assay diluent.
§ From the 500 pg/mL stock, prepare subsequent dilutions:
§ 250 pg/mL for standard and as the reference for the all
other wells: Mix 3 mls of 500 pg/mL + 3 mls assay diluent
(depending upon the number of samples). (this reference
should be validated in every new set up)
§ Take 120 ul from 250 pg/ml stock to perform 1:1 serial
dilutions to achieve 125, 62.5, 31.2, and 15.6 pg/mL
standards.
§ For the cut-oH point standard, prepare 77 pg/mL by mixing
77 µL of 500 pg/mL stock + 423 µL assay diluent.
o Anti-IL-10 Standard Preparation:
o Prepare a 625 ng/mL stock: 1 µL of 100 µg/mL + 159 µL assay diluent.
o Perform 1:1 serial dilutions with assay diluent to create 312 ng/mL and
156 ng/mL stocks.§ Follow the table below for final concentrations in the plate:
|
Final Anti-IL-10 Conc. (ng/mL) |
Assay Diluent (µL) |
Recombinant IL-10 (µL) |
Anti-IL-10 Stock (µL) |
|
62.5 |
44 |
55 |
11 (from 625 ng/mL) |
|
31.25 |
44 |
55 |
11 (from 312 ng/mL) |
|
15.6 |
44 |
55 |
11 (from 156 ng/mL) |
6. Sample and Standard Preparation in V-Bottom Plate
o Use a 96-well V-bottom plate to prepare samples and standards for
transfer to the coated plate.
o For IL-10 standards, add 120 µL of each concentration (prepared in
triplicate) into the V-bottom plate.
o For serum samples:
- Add 55 µL of 250 pg/mL IL-10 to each well.
- Add 11 µL of each serum sample to its corresponding well.
- Top up each well with 44 µL assay diluent using a multi-channel pipette.
- Incubate the plate at room temperature for 1 hour for preincubation.
7. Plate Blocking for Non-Specific Binding
o Wash the coated plate using the company’s recommended washing
buHer.
o Add 100 µL of assay diluent to each well to block non-specific binding.
o Incubate the plate on a shaker for 1 hour at room temperature.
8. Sample and Standard Transfer
o After blocking, wash the coated plate as per the company’s instructions.
o Transfer the prepared standards and serum samples from the V-bottom
plate to the coated plate.
9. Follow Manufacturer's Protocol
o Follow the manufacturer’s instructions for the remaining steps, including
incubation times, washing steps, and reagent additions.