A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

Electrochemiluminescence assay can simultaneously screen for Type-1 diabetes and other relevant autoimmune disease, including autoimmune cryo disease, celiac disease, and autoimmune polyglandular syndrome-1. The much complex ECR assay combines up to 10 multiple antibody assays in a single well, resulting in a high throughput, less libraries, and less expensive assay that requires less broad volume. Demonstrating the procedure will be Xiaofan Jia, a post from my laboratory.

To prepare a mixed linker-coupled antigen solution, first select the optimal concentration for each antigen, based on the checkerboard assay, and dilute the biotin and ruthenium labeled antigen to the rational working concentration. To bind different linkers to each of the uniquely biotinylated antigens, for one 96-well played assay, mixed 240 microliters of an appropriate STREPTAVIDIN conjugated linker with 4 microliters of the biotinylated proteins of interest. Then add 156 microliters of 1%BSA per tube and incubate the mixture at room temperature for 30 minutes.

At the end of the incubation, add 160 microliters of stop solution to each tube and incubate the tubes at room temperature for an additional 30 minutes. At the end of the incubation, remove 400 microliters of linker coupled antigen from each tube and pull all seven of the antigen suspensions into a single tube. Then, add 1.2 milliliters of stop solution and 4 microliters of Ru sulfo-NHS-labeled GAD65, TPO, tTg, ThG, Proinsulin interferon-alpha, and IA-2 antigen to the mixture.

Before incubating serum samples with the labeled antigen mixture, add 15 microliters of serum per well of a 96-well PCR plate, and mix 18 microliters of 0.5 molar acidic acid with each well of serum for a 45-minute incubation at room temperature. During the incubation, add 35 microliters of antigen and 13 microliters of one molar Tris buffer per well of a new 96-well PCR plate. At the end of the incubation, immediately transfer 25 microliters of the acid-treated serum into each well of the antigen plate.

Cover the plate with PCR ceiling foil, and placed the plate on a shaker at low speed at room temperature for one hour. At the end of the incubation, place the plate at 4 degrees Celsius for 18 to 24 hours. Take the plate out of the refrigerator, and allow it to come to room temperature.

Then add 150 microliters of 3%blocker A-buffer to each well. When all of the wells have been treated, cover the plate with ceiling foil and place the plate in a 4 degree Celsius refrigerator overnight. On the following morning, flip the plate upside down, and wash the samples three times with 150 microliters of PBS-T per well, per wash.

Pat the plate until no buffer remains in any of the wells. Add 30 microliters of serum antigen incubate to each well. When all of the incubates have been added, cover the plate with foil and place the plate on a shaker at low speed for one hour, at room temperature.

At the end of the incubation, remove the incubates by flicking and wash the wells three times with 150 microliters of PBS-T per well, as demonstrated. After the last wash, add 150 microliters of reading buffer to each well and read the plate on a plate reader, in counts per second. In this analysis of a large cohort of samples from 1026 newly diagnosed Type-1 diabetes patients, the levels of auto antibodies from a seven plus electro luminescence assay were compared with the levels from each corresponding established single electro luminescence assay and from each corresponding single standard radio binding assay and ELISA.

The assay specificities were identical at the 99th percentiles for all of the auto-antibody assays, except for thyroid auto antibodies, for which the 95th percentile was used. A small number of discordant samples for each of the seven auto antibodies were found around the borderline of the cutoffs for each assay. Here, the corresponding data for 1022 healthy controls for each assay can be observed.

The most important thing to remember is to find a different linker to each of the uniquely biotinylated antigens, and to mix each antigen with the properties to providing conjugated linker. This assay technique will allow researchers to plan screening for multiple autoimmune diseases simultaneously in large scale populations and allow clinics to easily screen multiple relevant diseases with Type-1 diabetes.