A High-Throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19

The novel 6-Plex ECL assay is the first multiplex assay validated and now applied in clinical trial for general population screening. It simultaneously combines six autoantibody assays suitable for screening type 1 diabetes, Celiac disease, and COVID-19 in a large population. Compared to the current standard autoantibody assay, this technique can more efficiently screen multiple autoantibodies for multiple autoimmune disease simultaneously.

The implication of this technique will make the large-scale screening more feasible and disease-specific, leading to early disease prediction for prevention and more precise diagnosis for therapy. To begin, add four microliters of biotinylated GAD65, SARS-CoV-2 RBD, IA-2, tTG, ZnT8, and proinsulin antigen protein into a tube containing 156 microliters of 1%BSA and mix with 240 microliters of corresponding streptavidin-conjugated linkers, 1, 2, 3, 8, 9, and 10. Then, incubate the mixture for 30 minutes at room temperature.

Aliquot 160 microliters of stop solution into each tube and incubate the mixture at room temperature for 30 minutes. Now, take 400 microliters of the mixture solution from each tube and combine them together. Add four microliters of ruthenium-labeled GAD65, SARS-CoV-2 RBD, IA-2, tTG, ZnT8, and proinsulin antigen to the mixture.

Then, add 1.6 milliliters of stop solution and 3.2 milliliters of PBS. Mix all the components thoroughly. And now, the antigen solution is ready for use in the assay.

For a 96-well PCR plate, aliquot seven microliters of serum per well and cover the plate with a sealing film. Next, preheat the sera at 56 degrees Celsius for 30 minutes on a PCR machine and briefly centrifuge the plate at 100 times G for one minute. Add 63 microliters of prepared antigen solution per well, mix with the sera, and cover the PCR plate with sealing foil to avoid light.

Shake the plate on a shaker at a speed of 450 rpm for two hours at room temperature. And then, incubate the plate at four degrees Celsius for 18 to 24 hours. Take a 6-Plex plate from the four degree Celsius refrigerator, allowing the plate to come to room temperature.

Then, add 150 microliters of 3%Blocker A to each well of the 6-Plex plate. Cover the plate with foil to avoid light. Put the plate back in a four degree Celsius refrigerator and incubate overnight.

At the end of the incubation, once the plate is removed from the refrigerator, remove all the buffer from the plate. Pat the plate on the paper towels to dry out, and wash it thrice with 150 microliters of washing buffer per well each time. Dry the plate on the paper towel and aliquot 30 microliters of serum antigen incubate from each well of the overnight incubating PCR plate into two wells of the 6-Plex plate.

Cover the plate with foil, then put the plate on a plate shaker and shake with a speed of 450 rpm at room temperature for one hour. Dump the solution present in the 6-Plex plate and wash the plate three times with 150 microliters of 0.4 M sodium chloride washing buffer per well. After the third wash is complete, dry the plate against the paper towel and add 150 microliters of reading buffer into each well.

The levels of six autoantibodies for GADA, IAA, IA-2A, ZnT8A, TGA, and COVID-19A from the 6-Plex ECL assay were well-correlated with their corresponding six single ECL assay, and the current standard radio-binding assay. Index values for each sample for all six antibodies were calculated against the corresponding positive and negative controls. An index value greater than the cutoff value was observed, which indicated a positive result.

Autoantibodies in serum breached the ruthenium-labeled antigen to the biotinylated antigen, forming a complex with a specific linker to build a multiplexing platform. This tactic makes the large-scale screening more feasible and more efficient in the general population for type one diabetes and concomitant diseases, leading to early prediction and prevention of the diseases.