In line with the urgent need for screening for type 1 diabetes, celiac disease, and coronavirus disease 2019, we developed a high throughput 6-Plex electrochemiluminescence assay to simultaneously detect all four islet autoantibodies, tissue transglutaminase autoantibodies, and antibodies to the receptor binding domain of severe acute respiratory syndrome coronavirus 2.
An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and celiac disease in the United States. With the coronavirus disease 2019 (COVID-19) pandemic, the epidemiology of COVID-19 in the general population and knowledge about the association between COVID-19 infection and T1D development are urgently needed. The currently standard screening method of the radio-binding assay (RBA) has met two great challenges: low efficiency with a single assay format and low disease specificity with a large proportion of low-affinity antibodies generated in screening. With the platform of the multiplex electrochemiluminescence (ECL) assay we established previously, a novel 6-Plex ECL assay was developed that combines, in a single well, all four islet autoantibodies (IAbs) to insulin, glutamic acid decarboxylase (GAD65), insulinoma antigen 2 (IA-2), and Zinc transporter 8 (ZnT8) for T1D, transglutaminase autoantibodies (TGA) for celiac disease, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antibodies for COVID-19. The assay was validated in blind using 880 samples from the ASK study, including 325 positive samples and 555 all antibody-negative samples, and compared with the standard RBAs and a single ECL assay. With the advantages of high efficiency, low cost, and low serum volume, this assay has been accepted as the primary screening tool for the ASK study.
Large epidemiologic studies worldwide demonstrate that the incidence of type 1 diabetes (T1D) has been rapidly increasing by 3%-5% annually, and the prevalence of T1D has doubled in the last 20 years, especially in young children1,2. Islet autoantibodies (IAbs) usually appear years before clinical symptoms and are currently the most reliable predictive and diagnostic biomarkers for T1D3. Screening for T1D autoantibodies can identify people at risk for progressing to clinical T1D, educate the public, largely reduce the life-threatening complication of ketoacidosis, and benefit clinical trials for prevention therapies. Worldwide prevention efforts for T1D are underway and multiple interventional trials are being carried out among subjects with IAbs positive to delay the progression to clinical T1D, and the Barbara Davis Center for Diabetes launched the first US clinical trial of screening in the general population in 2016 for T1D and celiac disease, Autoimmunity Screening for the Kids (ASK)4. Celiac disease (CD) is a chronic intestinal inflammatory disease related to immune and genetic factors. The prevalence of CD in children with T1D can reach up to 24.5%5. More than half of individuals with CD may not have typical symptoms at presentation6, so serological screening for celiac disease is quite necessary.
Since the pandemic of coronavirus disease 2019 (COVID-19) started, over 200 million cases have been reported globally, and children account for up to 16% of laboratory-confirmed cases7. Many studies have demonstrated the impact of existing T1D on the severity and fatality of COVID-19 infection8, while the impact of COVID-19 infection on T1D development is not clear. The investigation of the total rate of COVID-19 infection in the general population by the determination of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) antibodies and the study of the association between COVID-19 infection and triggering T1D autoimmunity or accelerating T1D progression are urgently needed and very important, while the ongoing ASK study is an excellent platform for this. Due to the single assay format and generation of a large proportion of low-affinity antibody positivity, the standard radio-binding assay (RBA) has met a bottleneck of low efficiency and low disease specificity9. In the present paper, we present a novel 6-Plexed electrochemiluminescence (ECL) assay built on our previous multiplex ECL assay platform using a multiple-Plex plate, combining six autoantibody assays in one single well, including all four major IAbs to insulin (IAA), glutamic acid decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and Zinc transport-8 (ZnT8A), autoantibodies to transglutaminase (TGA) for celiac disease, and SARS-CoV-2 receptor-binding domain (RBD) antibodies (COVID-19A). This is the first multiplex assay that recruited a complete panel of four major IAbs and further combined this with TGA and COVID-19A. It has been validated and formally accepted as the primary screening tool replacing the standard RBA in the ASK study.
The research protocol was approved by the Colorado Multiple Institutional Review Board.
1. Buffer Preparation
2. Antigen protein labeling with biotin and Ru Sulfo-NHS separately
NOTE: A higher concentration of antigen protein ≥0.5 mg/mL is recommended for higher labeling efficiency.
3. Define the best concentration and ratios for biotin- and Ru Sulfo-NHS-labeled antigens for the 6-Plex ECL assay (checkerboard assay)
NOTE: The checkerboard assay for each antigen is necessary before integration into the multiplexed assay.
4. Create linker-coupled antigen solution
5. Incubate serum samples with the labeled antigen
6. Prepare the 6-Plex plate
7. Transfer serum/antigen incubates into the 6-Plex plate
8. Wash the 6-Plex plate and add reading buffer
9. Read the plate and analyze the data
Table 1, Table 2, and Table 3 illustrate the representative results. The raw CPS obtained from the reading machine are illustrated in Table 1. In Table 2, raw CPS were arranged and sorted by the six linkers and corresponding antibodies. Table 3 shows the calculated index values with CPS as described in the assay protocol. All raw counting values should be checked to avoid wrong final index results caused by bad duplicates. In Table 1, examples of bad duplicates are given, which resulted in an error for the final index calculation in Table 3.
This assay was validated in a blind manner using 880 selected samples from the ASK study with 325 IAbs positive samples and 555 all Abs negative samples. The levels of autoantibodies from 6-Plex ECL assay for the 880 samples were compared point-by-point with the levels of corresponding single ECL assays (Figure 2) and with corresponding single standard RBA (Figure 3). The cutoff, sensitivity, and specificity for each antibody are listed in Table 4. The sensitivity and specificity of this ECL-COVID-19A assay have been identified as 100% and 99.9%, respectively10.
Figure 1: Illustration of the 6-Plex ECL assay. Serum autoantibodies make the connection of the Ru Sulfo-NHS-labeled antigen to the biotinylated antigen, which is coupled with a specific linker and forms a complex of antigen-antibody-antigen-linker. The complexes are captured through each specific linker onto the specific spots in the 6-Plex plate. For each antigen, the specific linker numbers were assigned: GAD-linker-1, Covid-19-linker-2, IA-2-linker-3, tTG-linker-8, ZnT8-linker-9, and Proinslulin-linker-10. This figure has been modified with permission from He et al.11. Please click here to view a larger version of this figure.
Figure 2: Comparison of six antibody levels in 880 samples from the ASK study between the single ECL assay and the 6-Plex ECL assay. The panels display comparisons of antibody levels for GADA, IAA, IA-2A, ZnT8A, TGA, and COVID-19A, respectively (index of COVID-19A was presented as (100 x calculated index value)). The dotted lines represent the assay cutoffs for each antibody assay. This figure has been modified with permission from He et al.11. Please click here to view a larger version of this figure.
Figure 3: Comparison of five antibody levels in 880 samples from the ASK study between the RBA and the 6-Plex ECL assay. The panels display comparisons of antibody levels for GADA, IAA, IA-2A, ZnT8A, and TGA, respectively. The dotted lines represent the assay cutoffs for each antibody assay. This figure has been modified with permission from He et al.11. Please click here to view a larger version of this figure.
Table 1: Raw CPS counts (left half of the plate). Raw CPS counts acquired from the left half of an assay plate. Each sample is performed in duplicate. Each CPS count within the same column (A1, A2, A3, etc.) represents the reading data from the 10 spots in the same well (corresponding linker numbers are marked). Examples of bad duplicates are highlighted in grey, as seen in row D-linker 3 column 5 and column 6. Please click here to download this Table.
Table 2: Arrangement of the Table 1 data, sorted with six linkers. CPS values from Table 1 were rearranged, calculating the mean values for duplicate readings of CPS and deleting unused values for linkers 4-7. The values of the internal standard high positive, low positive, and negative controls of each autoantibody assay are marked in dark bold. PC, positive control. NC, negative control. Please click here to download this Table.
Table 3: Results of index values. 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 defined as positive, marked in dark bold. The bad duplicate CPS values in Table 1, row D-linker 3-columns 5 and 6, led to a positive IA-2A index value of sample 11 (highlighted in grey), which was probably a false-positive result. Please click here to download this Table.
Table 4: Assay cutoff, sensitivity, and specificity for GADA, IA-2A, ZnT8A, IAA, and TGA among 880 ASK samples, including 325 IAbs positive samples and 555 all antibodies negative samples. The optimal cutoff index value of GADA, IA-2A, ZnT8A, IAA, and TGA assay was set to at least the 99th percentile of the 555 normal control samples. Please click here to download this Table.
The intervention for T1D has entered the pre-T1D era, with ongoing national and international large-scale screening programs for T1D and booming clinical trials being applied in stage 1 T1D to abrogate or slow the progression to clinical T1D12,13. Measurements of four IAbs using the current standard RBA with a single IAb assay format is laborious and inefficient for a mass screening program. With the urgent demand for a high-throughput multiplexed IAbs assay, multiplexed ECL assay, 3-Screen ICA ELISA, and antibody detection by agglutination-PCR (ADAP) have emerged as currently competitive technologies. In the Fr1da study of screening for T1D in a population of young children in Germany, 3-Screen ICA ELISA combining 3 IAbs (GADA, IA-2A, and ZnT8A) was used as the main test14. A major limitation of 3-Screen ICA ELISA is the lack of IAA, which is mostly the first IAb to appear with a high prevalence in young children with T1D. Furthermore, the assay is not able to distinguish which of the three IAbs is positive from a positive signal, and every positive sample needs to be repeated with three single assays for confirmation. ADAP15 combines GADA, IA-2A, and IAA and illustrated high assay sensitivity and specificity in the IASP workshop but lacks data regarding how predictive it is in population-based screening for T1D studies, which the IASP workshop is not able to determine. The multiplex ECL assay in the present study is built on the platform of a single ECL assay that has been validated against current standard RBA in multiple clinical trials like TrailNet9,16, DAISY17,18,19, and the ASK4,20,21 study. The present assay has been validated against both RBA and single ECL assay using 880 serum samples from participants of the ASK study. As shown in Figure 2 and Figure 3, antibody levels between 6-Plex and single ECL or between 6-Plex and RBA were mostly congruent with each other for the positivity of antibodies. The coincidence rates of IAbs tested by RBA and 6-Plex were 91.7%-97.8%, and the coincidence rates of IAbs tested by single ECL and 6-Plex were 95.3%-99.2%. Discordance between the ECL assay and RBA was mainly from those with single IAb. The levels of IAbs tested by 6-Plex and single ECL (r = 0.6431-0.9434, all p < 0.0001) and 6-Plex and RBA (r = 0.5775-0.8576, all p < 0.0001) were well correlated as well. TGA tested by 6-Plex assay were highly correlated with the results of both the RBA (99.4%) and single ECL assay (99.4%). Additionally, COVID-19A tested by 6-Plex reached 99.8% compliance with the results of the single ECL assay.
As a result, the 6-Plex ECL assay has formally been accepted as the primary screening method for the ongoing ASK study and replaced the standard RBA22. This assay demonstrated its excellent sensitivity and specificity with higher throughput, lower cost, and smaller volume of serum compared with standard RBA.
It has been documented that, in the T1D screening study, single IAb detected by RBA, which took up a large proportion of IAb positivities, were of low affinity, with low disease risk, and resulted in overall low predictive value19,21,23,24,25,26. Such low risk prediction caused huge extra costs of follow-up visits and resulted in difficulties for T1D preventive studies. The established ECL assay platform has been demonstrated in multiple clinical trials to discriminate high-affinity IAbs from low-affinity IAbs generated by RBA and significantly enhance the predictive values for all four IAbs, especially with single IAb positivity16,17,18,21. The multiplex ECL assay technology was built on the platform of the single ECL assay, with this distinct advantage of the detection of high-affinity Abs. In the present study, the 6-Plex ECL assay illustrated its similarity with the corresponding single ECL assays in sensitivity and specificity.
Some limitations and the technical concerns of the multiplex ECL assay using multiple Plex plates have already been covered in previous publications27,28. With six labeled antigens in one well, there may be some influences between different antigens, and the assay background could increase when adding each antibody assay for multiplexing in the same well. A large amount of assay optimization work is needed to adjust the assay conditions, especially for adjusting the final concentrations of each labeled antigen protein based on the checkerboard assay for each antigen, to maintain the sensitivity and specificity for each antibody assay. As mentioned in previous studies27,28, a small number of samples (<1%) result in false positivity on the multiple Plex plate without a clear underlying reason. All positive results should be repeated and confirmed by a single ECL assay as routine laboratory quality assurance; usually, the false positives will be removed. False-negative results caused by the "prozone effect" observed in the previous 7-Plex ECL assay27,28 were not observed in the present study. In the assay described here, we removed the step of the acid treatment of serum samples from the previous protocol; instead, we preheated the serum samples at 56 °C for 30 min (step 5.1.). On the second day of plate washing, we used a washing buffer containing 0.4 M NaCl (step 8.1.) instead of regular washing buffer to wash the plate more stringently. These modifications made the multiplex ECL assay simpler and lowerered background without losing the assay sensitivity.
In conclusion, this assay has outstanding performance for detecting four IAbs, TGA, and COVID-19A simultaneously. The multiplexing feature, as well as the high throughput, low cost, and small serum volume requirement, make large-scale screening for T1D and concomitant diseases much more feasible in the general population. Patients with T1D or any autoimmune diseases have a much higher risk for other autoimmune diseases. The multiplex ECL assay provides an excellent platform to screen for T1D and multiple autoimmune diseases simultaneously.
The authors have nothing to disclose.
The study was supported by JDRF grant 2-SRA-2019-695-S-B, 2-SRA-2020-965-S-B, 1-SRA-2017-564-M_N, NIH grant DK32083, and Diabetes Research Center (DRC) grant P30 DK116073.
5 mM NaCl | ThermoFisher | 1070832 | |
96-well Plate Shaker | VWR | 12620-926 | |
96-well round bottom plate | Fisher | 8408220 | |
Biotin | ThermoFisher | PI21329 | |
Blocker A | MSD | R93AA | |
Bottle-Top 500 ml-Filter Units | Fisher | 0974064A | |
Bovine Serum Albumin | Sigma | A-7906 | |
GAD65 protein | Diamyd Medical | 10-65702-01 | |
IA-2 protein (aa 605-979) | Creative BioMart | 283309 | |
MESO QuickPlex SQ120 | MSD | R31QQ-3 | Electrochemiluminescence analyzer |
PBS 10x | ThermoFisher | 70011044 | |
PBS 1x | ThermoFisher | 10010023 | |
Proinsulin protein | AmideBio | 20160118B3 | |
Read buffer B | MSD | Y0800019 | |
SARS-CoV-2 RBD protein | Creative Biomart | Spike-190V | |
Stop Solution | MSD | Y0090019 | |
Sulfo-TAG | MSD | R91AO-1 | |
tTG protein | Diarect AG | 15201 | |
Tween 20 | Sigma | P-1379 | |
U-Plex 6-Assay SECTOR plate | MSD | Z00U0142E | 6-plex plate |
U-PLEX Linker 1 | MSD | L0010022 | |
U-PLEX Linker 10 | MSD | L0100020 | |
U-PLEX Linker 2 | MSD | L0020015 | |
U-PLEX Linker 3 | MSD | L0030018 | |
U-PLEX Linker 8 | MSD | L0080018 | |
U-PLEX Linker 9 | MSD | L0090014 | |
ZeBa Column | ThermoFisher | 89892 | Spin columns |
ZnT8 protein | Research Laboratory | n/a |