This work was supported by NIH grant DK32083, JDRF Grants 2-SRA-2015-51-Q-R and 2-SRA-2018-533-S-B.

The research protocol was approved by the Colorado Multiple Institutional Review Board.
1. Buffer preparation
<ol>
	<li>Make the labeling buffer (2x PBS, pH 7.9). Using 400 mL of distilled deionized (DD) water, add 100 mL of 10x PBS. To adjust the pH of the solution, add NaOH until it reaches 7.9.</li>
	<li>Create 3 mM biotin by dissolving 1 mg of biotin into 588 &#181;L of labeling buffer previously created. Make 3 mM Ru Sulfo-NHS by dissolving 150 nmol of Ru Sulfo-NHS into 50 &#181;L of labeling buffer.</li>
	<li>Make the antigen buffer (1% BSA) by taking 500 mL of 1x PBS and adding 5 g of bovine serum albumin (BSA) to the solution. Prepare 0.5 M of acetic acid solution. Prepare 1 M Tris-HCl buffer using Trizma Base and adjusting the pH to 9.0.</li>
	<li>For the coating buffer (3% Blocker A), take 500 mL of 1x PBS and add 15 g of Blocker A. Prepare the washing buffer (0.05 % Tween 20, PBST) by mixing 5000 mL of 1x PBS with 2.5 mL of Tween 20. Create reading buffer (2x Read Buffer T with surfactant) by adding 500 mL of DD water and 500 mL of 4x Read Buffer T with Surfactant. 
	NOTE: To keep consistency between assays it is important that both the biotin and Ru Sulfo-NHS solutions are created just before the labeling procedure and are not created and stored for future use.</li>
</ol>
2. Labeling each antigen protein with biotin and Ru Sulfo-NHS separately
NOTE: To have a more effective labeling reaction, use an antigen protein concentration of &#8805;0.5 mg/mL.
<ol>
	<li>Calculate the molar number of each antigen protein and the molar numbers of the biotin and Ru Sulfo-NHS. Add a proper amount of biotin or Ru Sulfo-NHS to antigen protein for labeling reaction according to the molar ratio of antigen protein to biotin or Ru Sulfo-NHS.
	<ol>
		<li>For the antigen that has the smaller molecular weight (&#8804;10 kDa), such as proinsulin protein, use a molar ratio of 1:5 (antigen: biotin and Ru Sulfo-NHS). For the antigen that has the larger molecular weight (&#62;50 kDa), such as GAD protein, use a molar ratio of 1:20. For the antigen that has a middle molecular weight (10-50 kDa), use a molar ratio adjusted between 1:5-1:20.</li>
	</ol>
	</li>
	<li>For both the biotin and Ru Sulfo-NHS, divide each antigen weight by their corresponding molecular weights to get the antigen molar number for each one. Divide the molar number by the concentration to get the volume for biotin. Repeat this for Ru Sulfo-NHS.</li>
	<li>Mix the antigen protein with biotin using the proper molar ratio, determined in step 2.2. Then do the same for Ru Sulfo-NHS. 
	NOTE: For efficiency of labeling reaction, any reducing chemicals like Tris or glycine in the buffer system need to be exchanged to 2x PBS buffer, pH 7.9 by the sizing spin column. The labeling protocols for biotin and Ru Sulfo-NHS are identical.</li>
	<li>Cover the reaction tubes with aluminum foil and incubate them at room temperature (RT) for 1 h. The reason covering the reaction tubes with foil is because both biotin and Ru Sulfo-NHS reagents are light sensitive.</li>
	<li>Prime the 2 mL or 5 mL spin column while the reaction tubes are incubating (the size of spin column is determined by the volume uploaded onto the column). Fill the spin column with 2x PBS buffer and then centrifuge it at 1,000 x g for 2 min each time, for a total of three times.</li>
	<li>Stop the labeling reaction after the reaction tubes have finished incubating. To stop the reaction, purify the labeled antigen protein by passing it through the spin column once. Then centrifuge the column at 1,000 x g for 2 min.</li>
	<li>Calculate the total labeled antigen concentration by dividing the amount of antigen protein present by the final volume. Aliquot 50 &#181;L of the purified labeled antigen protein per tube and store the aliquots at -80 &#176;C for long term use. 
	NOTE: It is important to be aware that for every time the spin column passes the antigen protein through, there will be about a 90-95% retention rate.</li>
</ol>
3. Define the best concentration and ratios for the two labeled antigens for the assay (checkerboard assay)
NOTE: Since the ECL-IAA assay in this 7-Plex assay requires acid treatment of serum samples, the checkerboard assay for each antigen has to go through this step before incubating with the labeled antigen mixture.
<ol>
	<li>Apply the checkerboard assay for each antigen separately before running the multiplex assay. Steps 3.2-3.6 will use GAD65 as an example.</li>
	<li>Calculate dilution of labeled GAD65 protein. The recommended targeted concentration of the first mixture solution of biotinylated GAD65 is 2000 ng/mL and Ru Sulfo-NHS labeled GAD65 is 1000 ng/mL. If the concentration of both biotinylated and Ru Sulfo-NHS labeled GAD65 in stock solution are 1.0 &#181;g/&#181;L, the volume needed for biotinylated GAD65 in 560 &#181;L of working solution will be 1.12 &#181;L, and the volume needed for Ru Sulfo-NHS labeled GAD65 in 700 &#181;L of working solution will be 0.7 &#181;L.</li>
	<li>Mix 1.12 &#181;L of biotinylated GAD65 protein with 240 &#181;L of streptavidin-conjugated linker 1 in one tube and 160 &#181;L of 1% BSA. Incubate the mixture at room temperature for 30 min. Add 160 &#181;L of stop solution to the tube and incubate the mixture at room temperature for another 30 min.</li>
	<li>Make a serial dilution. Take 280 &#181;L of the mixture to a new tube and add 280 &#181;L of stop solution to make a 1:2 dilution. Prepare several new tubes. Repeat this step to run a horizontal serial dilution for biotin labeled GAD65 antigen (refer to the previous publication12).</li>
	<li>Mix 0.7 &#181;L of Ru Sulfo-NHS labeled GAD65 (1 &#181;g/&#181;L) protein with 700 &#181;L of stop solution. Then take 350 &#181;L of the mixture to a new tube and add 350 &#181;L of stop solution to make a 1:2 dilution. Prepare several new tubes, repeat this step to run a vertical serial dilution for Ru Sulfo-NHS labeled GAD65 antigen.</li>
	<li>Prepare two serum samples, one sample highly positive for GADA and one sample negative for GADA, each having a volume of 0.75 mL. Aliquot 15 &#181;L of positive serum into every well on the left half of the 96-well PCR plate. Aliquot 15 &#181;L of negative serum into every well on the right half of the 96-well PCR plate.</li>
	<li>Add 18 &#181;L of 0.5 M acetic acid into each well and mix. Incubate for 45 min at RT. Prepare a new 96-well PCR plate. Add 17.5 &#181;L of the biotin labeled antigen and 17.5 &#181;L of the Ru Sulfo-NHS labeled antigen into each well according to the serial dilutions (refer to the previous publication12).</li>
	<li>Continue the rest of the assay steps described in step 5.2 through 9.1.</li>
	<li>Determine the signal ratio from the high positive samples against the corresponding negative sample signals. Select the best concentration for the Biotin labeled antigen and the Ru Sulfo-NHS labeled antigen by identifying the point that has the highest or near to the highest ratio of positive to negative signal. In this ratio calculation, consider the low background signal obtained from the negative samples. 
	NOTE: The optimal concentrations of Ru Sulfo-NHS and biotin labeled antigen proteins from checkerboard assays are shown below: 30 ng/mL and 200 ng/mL for GAD65, 120 ng/mL and 120 ng/mL for proinsulin, 10 ng/mL and 42 ng/mL for IA-2, 80 ng/mL and 80 ng/mL for TG, 8 ng/mL and 16 ng/mL for TPO, 31 ng/mL and 31 ng/mL for ThG, and 12 ng/mL and 12 ng/mL for IFN&#945;.</li>
</ol>
4. Create the mixed linker-coupled antigen solution
<ol>
	<li>Select the optimal concentration for each antigen based on the checkerboard assay. Dilute the biotin and Ru Sulfo-NHS labeled antigen to the rational working concentration.</li>
	<li>Bind different linkers to each of the uniquely biotinylated antigen proteins. For one 96-well plate assay, mix 4 &#181;L of biotinylated GAD65, TPO, tTG, ThG, proinsulin, IFN-&#945; and IA-2 protein with 240 &#181;L of streptavidin-conjugated linker 1, 2, 3, 4, 8, 9 and 10 into separate tube (Figure 1). Then add 156 &#181;L of PBS/1% BSA per tube. Incubate the mixture at room temperature for 30 min.</li>
	<li>Add 160 &#181;L of stop solution into each tube and incubate them at room temperature for another 30 min. Take 400 &#181;L of linker-coupled antigen from each tube, and combine all 7 antigens together. Add 1.2 mL of stop solution and then add 4 &#181;L of Ru Sulfo-NHS labeled GAD65, TPO, tTG, ThG, proinsulin, IFN-&#945; and IA-2 antigen to the mixture. Now the antigen solution is ready to be used in the assay.</li>
</ol>
5. Incubate serum samples with the labeled antigen
NOTE: Since the ECL-IAA assay in this 7-Plex assay requires acid treatment of serum samples, each serum requires the acid treatment step before incubating with labeled antigen mixture for this 7-Plex assay.
<ol>
	<li>Aliquot 15 &#181;L of serum per well for a 96-well PCR plate. Add 18 &#181;L of 0.5 M acetic acid into each well and mix. Incubate for 45 min at RT. Prepare a new 96-well PCR plate and aliquot 35 &#181;L of antigen solution into each well.</li>
	<li>While the plate is still incubating, add 13 &#181;L of 1 M Tris pH 9.0 buffer to every well in the antigen plate. Add buffer to the side of each well to limit the mixing of Tris buffer and antigen. After incubation is complete, take 25 &#181;L of the acid treated serum, and immediately pipette it into every well on the antigen plate. Agitate the solution and cover the plate with PCR sealing foil to avoid light exposure.</li>
	<li>At RT, put the plate on a shaker, set at a low speed, for 1 h. Afterwards, store the plate at 4 &#176;C and let the plate incubate for 18-24 h.</li>
</ol>
6. Prepare the multiplex plate
<ol>
	<li>Take a multiplex plate from the 4 &#176;C refrigerator and allow the plate to come to RT. Once the multiplex plate is at RT, add 150 &#181;L of 3% Blocker A to each well. Cover the multiplex plate with sealing foil. Incubate the plate in a 4 &#176;C refrigerator overnight. 
	NOTE: Assay Day 1 includes Steps 4 through 6.</li>
</ol>
7. Transfer serum/antigen incubates into the multiplex plate
<ol>
	<li>The following day, place paper towels on the table and take the incubating multiplex plate from the refrigerator. Empty all of the buffer out of the plate. To do this, flip the plate upside down and pat it onto the prepared paper towels until there is no buffer present in any of the wells.</li>
	<li>Wash the multiplex plate by adding 150 &#181;L of PBST into every well. Discard the buffer, as mention in step 7.1., and repeat this step three times. Add 30 &#181;L of serum/antigen incubates into every well in the multiplex plate. Cover the plate with foil to limit its exposure to light. Place the plate on a plate shaker, set to a low speed, at RT for 1 h.</li>
</ol>
8. Wash the plate and add read buffer
<ol>
	<li>Remove the serum/antigen incubates from the multiplex plate by holding the plate upside down and flicking out the solution. Add 150 &#181;L of PBST into all of the wells and remove the buffer from the plate by again holding the plate upside down and flicking out the solution. Repeat this step three times. After the third wash is complete, add 150 &#181;L of Reading buffer into each well. 
	NOTE: Air bubbles interfere with the plate reader machine&#8217;s ability to accurately analyze the plate&#8217;s results and should be avoided at all costs.</li>
</ol>
9. Read the plate and analyze data
<ol>
	<li>Count the prepared plate on the plate reader machine, reading all of the values in counts per second (CPS).</li>
	<li>Calculate the relative index for the assay, using the antibody levels obtained from the plate reader machine, with the following equation: 
	Index value = [CPS (sample) - CPS (negative standard)] / [CPS (positive standard) - CPS (negative standard)].</li>
	<li>Determine which antibody results are negative or positive using the cut-offs that have been established. 
	NOTE: Assay Day 2 includes Steps 7 through 9.</li>
</ol>

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The authors have nothing to disclose.

In numerous national and international clinical trials for type 1 diabetes, the performance of the single ECL assay to detect islet autoantibodies and autoantibodies to transglutaminase (TGA) for celiac disease, has been substantiated8,9,10,11. Throughout these trails, this assay has increased the sensitivity and specificity for autoantigen detection when assessed against the existing &#8216;gold&#8217; standard RBA. The enhanced disease specificity can be viewed when discriminating high-affinity and high-risk islet autoantibodies from low-risk, low-affinity signals, between the single ECL assay and the RBA14,15,16,17. Building on the single ECL assay, we pose a new high throughput multiplexed ECL autoantibody assay, to enable us to screen for T1D as well as several applicable autoimmune diseases at the same time.
The multiplex ECL assay uses the multiplex plate, which can combine up to 10 autoantibody assays into one single well. For the present study, 7 autoantibody assays are combined together. The autoantibodies comprise of 3 IAbs (IAA, GADA, and IA-2A), 2 autoimmune thyroid disease autoantibodies (TPOA and ThGA), celiac disease autoantibodies (TGA), and APS-1 autoantibodies to interferon alpha (IFN&#945;A). The assay mechanism is, in general, based on a single ECL assay as previously published9,10,12,18 with some modifications. The main difference of a multiplex ECL assay from a single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained to a specific linker (Figure 1). The biotin labeled antigen is incubated with the Streptavidin conjugated, a linker used to form a specific antigen-linker complex. Antibody-antigen immune complex forms after incubation with patient serum and is captured onto a specific spot through the specific linker system on each well of the multiplex plate. Ru Sulfo-NHS labeled antigen captured by antibodies at the same time provides the signal with electrochemiluminescence. The plate reader machine can detect up to 10 different signals from the spot sources located in each well. To keep autoantibody assays consistent between plates and while conducting long-term studies, we suggest that the same linker is used.
Before setting up a multiplex ECL assay, single ECL assays for each autoantibody need to be optimized onto a multiplex plate, respectively, and validated against both RBA and single ECL assays on a regular ECL plate. To enhance the checkerboard assay, to the related autoantibody assay on the multiplex plate, a high positive patient and a negative patient sample were used. After the checkerboard assay was conducted, the most ideal concentrations for Ru Sulfo-NHS and biotin labeled antigen where calculated for each of the 7 autoantibody assays. The following shows these concentrations: 30 ng/mL and 200 ng/mL for GAD65, 120 ng/mL and 120 ng/mL for proinsulin, 10 ng/mL and 42 ng/mL for IA-2, 80 ng/mL and 80 ng/mL for TG, 8 ng/mL and 16 ng/mL for TPO, 31 ng/mL and 31 ng/mL for ThG, and 12 ng/mL and 12 ng/mL respectively for IFN&#945;13. The concentrations of some of the antigens from the checkerboard assay may need to be further adjusted according to the outcomes in the actual multiplex assay after combining all of the assays together.
As IAA is included in the present multiplex ECL assay, acid treatment of serum samples is mandatory before incubating the serum with antigen, as reported in the previous study12. Generally, when an extra autoantibody is added to a multiplex assay, the assay background is affected and an extremely high signal from one spot, in the well, may obstruct the results of nearby spots via crosstalk. Therefore, the maximum CPS needs to be limited to 20,000 counts for each autoantibody, or lower, for the highest positive samples. From our knowledge, in order to reduce the amount of crosstalk involved, autoantibodies that have a lower background should be separated, when designing the spot map, far from spots with a higher frequency of counts.
High positives and negative controls were used internally in every assay to calculate an accurate index for the unknown samples being tested. To accurately assess and monitor the assay&#8217;s sensitivity, low positive controls, set near the assay&#8217;s upper limit were used. These standard positive and negative controls were created in bulk and aliquot for long-term use and stored at -20 &#176;C or below, for consistency between assays. For quality assurance purposes, samples were run twice in every assay and every positive result was reran and confirmed by running the sample in a new ECL assay the next day. If there was any disagreement with the first and second confirmatory assay, a third assay was necessary. Of the three assays conducted, the results of the two assays that agree (e.g., +, + or -,-), determined the final result (positive or negative) of the sample.
In the last decade, many study groups are seeking a high throughput assay utilizing the multiplex method to combine multiple autoantibody assays together into one well to screen large populations. There are a few studies that are using different types of technologies to conduct multiplex autoantibody assays19,20,21,22, but there is no comparison for any of these assays in sensitivity and specificity against the current &#8216;gold&#8217; standard RBA, when studying T1D. These different types of platforms used are not validated through the international Islet Autoantibody Standardization Program (IASP) workshop or through testing large cohorts in clinical trials. In a recent general population-based screening in Germany, a high throughput combined assay, 3 Screen ICATM ELISA distributed by Kronus, is being used as a tool for first line screening to detect three IAbs, GADA, IA-2A, and ZnT8A, to achieve early diagnosis of childhood T1D23. The 3-Screen ELISA assay measures 3 autoantibodies either in 3 separated wells, consuming a large volume of serum, or in a single well with all 3 assays mixed. If one well in the 3-Screen ELISA assay is positive, one is unable to distinguish which of three autoantibodies are present. The biggest disadvantage of this assay is its inability to include IAA measurement. All IAA results performed by ELISA, as proven in IASP workshops, do not have an acceptable sensitivity and specificity24. IAA is usually the first IAb to appear and has a high prevalence among young children. IAb screening, with IAA, is necessary for children and it is not deemed acceptable to conduct this screening without IAA to asses T1D risk in the community. In addition, there are no published studies or data that show the Kronus IAb kit assay as being more T1D disease specific and able to discriminate high risk from low risk IAbs. The 7-Plex ECL assay, in the present study, was validated using a large cohort of newly diagnosed patients with T1D13. Compared with the current standard RBA and well-established single ECL assay, the 7-Plex assay is able to retain 100% positivity with the same assay specificity (Table 4). Currently, the 4-Plex ECL assay, parallel with the standard RBA, is being applied to an ongoing large clinical trial: Autoimmunity Screening for Kids (ASK) study. This trial screens children in the general population, of the Denver metropolitan area, for T1D and celiac disease. Compared to standard RBA used in the ASK study, the multiplex ECL assay is showing excellent sensitivity and a higher disease specificity, identical to our previous reports using the single ECL study25. Furthermore, our 4-Plex ECL assay demonstrated a pronounced reduction in labor, cost, and serum volume by 70%, compared to the corresponding 4 single assays for ECL and RBA. Using the multiplexed ECL assay, we can customize each well with different numbers, representing different autoantibodies (up to 10), to test for different autoimmune diseases that are specific to the needs of a particular clinical location.
There are some limitations observed, shown in the present study, for a multiplexed ECL assay using the multiplex plate. The final dilution of serum, incubated with antigen, cannot be adjusted to yield the most optimal conditions for every single autoantibody assay that is combined in a single well. Nine samples (9/1026), from T1D patients, were observed to have a false negative result for particular autoantibodies. 7 of the false negatives were for TPOA and 2 were for ThGA, in the 7-Plex ECL assay, but high positive results were exhibited in both the single ECL assay and the RBA (Figure 2 &#38; 3). After further dilution of all 9 samples, they became positive on the multiplex plate. This result is caused by, what we describe as the &#8216;prozone&#8217; phenomenon. This phenomenon causes the sample to show a false negative result because the high antibody titers are affecting the formation of antigen-antibody lattices. When setting up a multiplex assay, samples with very high titers, for each of the combined autoantibodies, are recommended to have pre-tests ran to identify the optional dilution of serum for antigen incubation. Alternatively, autoantibody assays with similar optimized conditions should be selected to form a combined assay from which the best assay sensitivity and specificity is achieved for each autoantibody. In the present study, 7 samples (7/1022), from healthy normal controls, resulted in false positives for multiple autoantibodies in the 7-Plex assay, but after running a single ECL assay and RBA (Figure 2 &#38; 3) these autoantibodies were found to be negative in both assays. The reasons behind these false positive results occurring on the multiplex plate, for these small subset of samples, are currently unknown. For the current application of the multiplex ECL assay, all positive samples are repeated with their corresponding single ECL assay to confirm positivity, which removes this false positive error from the multiplex ECL assay.

Type 1 diabetes (T1D) is a serious chronic disease that is most common at the childhood. Currently, around 1.4 million people have the T1D in the United States; strikingly, the incidence of T1D is steadily increasing at 3-5% each year worldwide and has doubled in the last two decades, especially in young children1,2. Islet autoantibodies (IAbs) in blood circulation are the most reliable biomarker at present. The IAbs may appear years before clinical T1D develops3. Currently, four major IAbs are widely used in T1D diagnosis and risk screening including autoantibodies to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A). These four IAbs are equally important in prediction of T1D development. The classification of T1D has been re-defined recently as the presence of &#8805;2 of any 4 IAbs with a normal glucose metabolism as the disease stage 14.
In the Diabetes Autoimmunity Study in the Young (DAISY), it was revealed that one in four children at risk for T1D were likely to progress to islet, celiac, thyroid or rheumatoid autoimmunity and more strikingly, approximately 40% of patients who have been diagnosed with T1D eventually develop an additional autoimmune condition5,6,7. Identification of autoantibodies are essential for prediction and diagnosis of these autoimmune diseases and should provide better clinical care for patients. There is no easy and inexpensive way at present to screen for these multiple autoimmune conditions. Current screening methods using standard radio-binding assay (RBA), with a single autoantibody measurement, are laborious and inefficient for a largescale screening.
Here, we will be describing the newly developed simple multiplexed ECL assay, which we have authenticated using a single ECL assay platform8,9,10,11. The multiplex ECL assay combines 7 autoantibody tests into a single well, using only 15 &#181;L of serum, and is capable of screening for T1D and multiple relevant autoimmune diseases simultaneously including celiac disease, autoimmune thyroid disease, and APS-1. A ZnT8A ECL assay had not been developed at the time and was not included in the multiplexed assay. The multiplex ECL assay provides an excellent tool for high throughput in general population screening for T1D and multiple autoimmune diseases.

<table><tbody><tr><td>4 &amp;deg;C refrigerator</td><td></td><td></td><td></td></tr><tr><td>&amp;ndash;80 &amp;deg;C and -20 &amp;deg;C freezers</td><td></td><td></td><td></td></tr><tr><td>96-well Plate Shaker</td><td>Wallac - Delfi</td><td></td><td></td></tr><tr><td>96-well round bottom plate</td><td>Fisher</td><td>8408220</td><td></td></tr><tr><td>Acetic acid solution</td><td>Fisher</td><td></td><td></td></tr><tr><td>Aluminum foil</td><td></td><td></td><td></td></tr><tr><td>Antigen proteins</td><td></td><td></td><td></td></tr><tr><td>Human GAD65 full length protein</td><td>Diamyd</td><td></td><td></td></tr><tr><td>Human ThG full length protein</td><td>BioMart</td><td></td><td></td></tr><tr><td>Human TPO full length protein</td><td>BioMart</td><td></td><td></td></tr><tr><td>IA-2 intracellular domain protein</td><td>BioMart</td><td></td><td></td></tr><tr><td>IFN-&amp;alpha; protein</td><td>Abcam</td><td></td><td></td></tr><tr><td>Proinsulin protein</td><td>AmideBio</td><td></td><td></td></tr><tr><td>tTG protein</td><td>DiaRect</td><td></td><td></td></tr><tr><td>Biotin</td><td>Sigma</td><td></td><td></td></tr><tr><td>Bottle-Top 500 mL , Filter Units</td><td>Fisher</td><td>0974064A or B</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A-7906</td><td></td></tr><tr><td>Distilled deionized (DD) water</td><td></td><td></td><td></td></tr><tr><td>HCl</td><td>Fisher</td><td></td><td></td></tr><tr><td>Ice maker</td><td></td><td></td><td></td></tr><tr><td>Ice trays</td><td></td><td></td><td></td></tr><tr><td>MSD Sector</td><td>Perkin-Elmer</td><td></td><td></td></tr><tr><td>Multi-channel pipette</td><td></td><td></td><td></td></tr><tr><td>NaOH</td><td></td><td></td><td></td></tr><tr><td>Paper tower</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>pH meter</td><td></td><td></td><td></td></tr><tr><td>Pipette-Aid</td><td></td><td></td><td></td></tr><tr><td>Pipettes/tips</td><td></td><td></td><td></td></tr><tr><td>Ru Sulfo-NHS</td><td>MSD (R91AN)</td><td></td><td></td></tr><tr><td>Trizma Base</td><td>Fisher</td><td>BP152-5</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P-1379</td><td></td></tr><tr><td>Uplex Development Kit</td><td>MSD</td><td></td><td></td></tr><tr><td>96-well UPlex plate</td><td>MSD</td><td></td><td></td></tr><tr><td>Blocker A</td><td>MSD</td><td>R93AA</td><td></td></tr><tr><td>Linker-Streptavidin</td><td>MSD</td><td></td><td></td></tr><tr><td>Read buffer</td><td>MSD</td><td>R92TC</td><td></td></tr><tr><td>Stop Solution</td><td>MSD</td><td></td><td></td></tr><tr><td>Vortex mixer</td><td></td><td></td><td></td></tr><tr><td>ZeBa Column</td><td>Pierce</td><td>89892</td><td></td></tr></tbody></table>

a high-throughput electrochemiluminescence 7-plex assay simultaneously screening for type 1 diabetes and multiple autoimmune diseases

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFN&#945;A) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

Watch this Scientific Journal Video about A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases at JoVE.com

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

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate ...

a-high-throughput-electrochemiluminescence-7-plex-assay

Research

JoVE Journal

Medicine

2.4K Views.  University of Colorado School of Medicine. We modelleren een eenvoudige multiplexed ECL-test die 7 autoantilichaamtests samen combineert. De test is in staat om tegelijkertijd te screenen op T1D en meerdere andere auto-immuunziekten, waaronder coeliakie, auto-immuun schildklierziekte en auto-immuun polyglandulair syndroom 1.

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

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Bioengineering

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

Immunology and Infection

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Biology

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

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

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.

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 ...

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

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