detection of human islet autoantibodies using an electrochemiluminescence assay

Detection of Human Islet Autoantibodies Using an Electrochemiluminescence Assay

Mix the human islet autoantigen with biotin or sulfo-tag in 1:5 molar ratio in a tube, and cover the tube with aluminum foil since both the tags are light-sensitive. Then, incubate the tube for an hour at room temperature. In the meanwhile, when the incubation is going on, prime the spin column with twofold phosphate-buffered saline.
Then, centrifuge the column at 1,000 times g for two minutes each time. Next, centrifuge the autoantigen tag mixture in the spin column at 1,000 times g for 2 minutes. Then, aliquot and store that labeled antigen at negative 80 degrees Celsius.
Next, use the rational concentration of the biotin sulfo-tag-labeled antigen based on the checkerboard assay for the antigen buffer to prepare 3 milliliters of antigen solution per 96-well plate.
Then in each well, add 4 microliters of serum and adjust the final volume to 20 microliters with 1-fold phosphate-buffered saline. Then, add 20 microliters of labeled antigen solution in each well, and cover the plate with sealing foil to prevent light. Then, transfer the plate on a shaker at room temperature for 2 hours.
Once the shaker stops, leave the plate at 4 degrees Celsius in the refrigerator for 18 to 24 hours. Now, mix 15 microliters of serum with 18 microliters of 0.5 molar acetic acid for each sample, and incubate the same at room temperature for 45 minutes.
On the basis of the checkerboard assay, use the rational concentration of the biotin sulfo-tag-labeled antigen to prepare the antigen solution.
Then, aliquot 35 microliters of the antigen buffer in each well of a new PCR plate. Just before the chorus of incubation of the serum mixture is completed, add 3.8 microliters of 1 molar Tris buffer at pH nine along the side of each well on the antigen plate.
Once the incubation is over, quickly transfer 25 microliters of the acetic acid-treated serum in each well of the antigen plate, and agitate the solution. Then, cover the PCR plate with sealing foil to avoid light, and place on a shaker at room temperature for 2 hours. Once done, transfer the plate at 4 degrees Celsius in the refrigerator, and incubate for 18 to 24 hours.
Remove the streptavidin plate from the refrigerator at 4 degrees Celsius, and let the plate reach room temperature. After the streptavidin plate attains room temperature, add 150 microliters of 3% blocker A in each well, and cover the PCR plate with sealing foil, and incubate in the refrigerator overnight.
The next day, remove the streptavidin plate from the refrigerator and expel the buffer from the wells. Then, dry the plate by setting it upside down on some dry paper towels for the remaining buffer to soak. Then, add 150 microliters of 1-fold PBST buffer to wash the well for three consecutive washes.
Next, transfer 30 microliters of serum antigen incubate in each well of the streptavidin plate, and cover the plate with foil to avoid light. Then, transfer the plate on a shaker at room temperature for an hour. After one hour, discard the serum antigen incubate from the plate, and add 150 microliters of 1-fold PBST buffer to wash the well three times. Once the washing is over, add 150 microliters of reading buffer to each well to read on a plate reader.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Buffer Preparation
<ol>
	<li>Labeling buffer (2x phosphate-buffered saline, (PBS), pH 7.9): In 400 mL of distilled deionized (DD) water, add 100 mL of 10x PBS, adjust pH to 7.9 with sodium hydroxide (NaOH).</li>
	<li>3 mM of Biotin: Dissolve 1 mg of Biotin in 588 &#181;L of labeling buffer.</li>
	<li>3 mM of Sulfo-tag: Dissolve 150 nmol of Sulfo-tag in 50 &#181;L of labeling buffer.</li>
	<li>Antigen buffer (5% BSA): In 500 mL of 1x PBS, add 25 g of Bovine Serum Albumin (BSA).</li>
	<li>Prepare 0.5 M acetic acid solution.</li>
	<li>1.0 M of Tris-HCl buffer pH 9.0: Prepare 1 M Tris-HCl buffer, and adjust pH to 9.0 with HCl.</li>
	<li>Coating buffer (3% Blocker A): In 500 mL of 1x PBS, add 15 g of Blocker A.</li>
	<li>Washing buffer (0.05 % Tween 20, PBST): In 5000 mL of 1x PBS, add 2.5 mL of Tween 20.</li>
	<li>Reading buffer (2x Read Buffer T with surfactant): In 500 mL of DD water, add 500 mL of 4x Read Buffer T with Surfactant (Table of Materials).</li>
	<li>Store Biotin and Sulfo-tag in a -20 &#176;C freezer.&#160;&#160; 
	NOTE: Both Biotin and Sulfo-tag solutions should always be freshly prepared just before the labeling procedure.</li>
</ol>
2. Label the Human Islet Autoantigen with Biotin and Sulfo-tag
NOTE: A high concentration of antigen, &#8805;0.5 mg/mL, is recommended for a more efficient labeling reaction.
<ol>
	<li>Determine the molar ratio of human islet autoantigen for Biotin and Sulfo-tag.
	<ol>
		<li>Obtain the antigen molar number by dividing the antigen weight by the molecular weight.</li>
		<li>Use the molar ratio of 1:5 for the antigen with smaller molecular weight (&#8804;10 kd), and the molar ratio of 1:20 for the antigen with larger molecular weight (&#62;50 kd).</li>
		<li>Calculate the volume for Biotin or Sulfo-tag by dividing the molar number by its concentration.</li>
	</ol>
	</li>
	<li>Mix the human islet autoantigen with Biotin or Sulfo-tag with the molar ratio of 1:5. 
	NOTE: The protein in either tris or glycine buffer systems should be exchanged to 2x PBS buffer with pH 7.9 using the sizing spin column.</li>
	<li>Since Biotin and Sulfo-tag are light sensitive, cover the reaction tubes with aluminum foil. Incubate the reaction at room temperature (RT) for 1 h.</li>
	<li>During the incubation, prime the spin column by dispensing 2x PBS buffer into the column three times. Centrifuge the solution at 1000 x g for 2 min each time. 
	NOTE: Currently the labeling reaction, through bridging, is still occurring and needs to be stopped.</li>
	<li>Stop the labeling reaction by purifying the labeled human islet autoantigen. To purify, pass the autoantigen through the spin column and centrifuge the column at 1000 x g for 2 min.</li>
	<li>Determine the labeled antigen concentration (&#181;g/&#181;L) using the amount of antigen protein and the final volume.&#160; 
	NOTE: Roughly, there will be 90 - 95% retention of labeled antigen after every spin column pass.</li>
	<li>Aliquot the labeled antigen and store labeled antigen at -80 &#176;C.</li>
</ol>
3. Define the Best Concentrations and Ratios for the Two Labeled Antigens for the Assay (Checker Board Assay)
<ol>
	<li>Prepare two serum samples, one high positive and one negative, with a total volume of 200 &#181;L for each.</li>
	<li>Aliquot 4 &#181;L of serum and add 1x PBS until the final volume is 20 &#181;L per well for a 96-well polymerase chain reaction (PCR) plate. Use half of a plate for the high positive sample and the other half for the negative sample, as shown in&#160;Figure 1A.</li>
	<li>Add 10 &#181;L of Biotin and 10 &#181;L of Sulfo-tag labeled antigen and conduct serial dilutions for the two differently labeled antigens, as shown in&#160;Figure 1A. Run a horizontal serial dilution for one labeled antigen and a vertical serial dilution for the other labeled antigen.</li>
	<li>Continue the rest of the assay steps described in 5.3 to 9.1.</li>
	<li>Calculate the ratio of signals from the high positive sample against each of the corresponding signals from the negative sample shown in&#160;Figure 1B.</li>
	<li>Identify the best concentrations for Biotin and Sulfo-tag labeled antigens by selecting a point with the highest or near highest ratio of positive to negative. Consider the low background signal from the negative sample to identify the ratio. 
	NOTE: Assay Day 1 includes Step 4 to Step 6.</li>
</ol>
4. Prepare the Antigen Buffer Using the Correct Concentration of Biotin/Sulfo-tag Labeled Antigen
<ol>
	<li>Select the rational concentration of each antigen based on the checker board assay.</li>
	<li>Prepare 3 mL of antigen solution per plate, using the rational concentration of Biotin/Sulfo-tag labeled antigen for the antigen buffer.</li>
</ol>
5. Incubate Serum Samples with Labeled Antigen
NOTE: There are two protocols in this section, one without serum acid treatment and one with serum acid treatment. All islet autoantibody assays except insulin autoantibody (IAA) assay use regular protocol without serum acid treatment from steps 5.1 to 5.5, whereas IAA assay skips these steps and uses protocol with serum acid treatment from steps 5.6 to 5.9.
<ol>
	<li>Aliquot 4 &#181;L of serum and add 1x PBS until the final volume is 20 &#181;L per well for a 96-well PCR plate.</li>
	<li>Add 20 &#181;L of labeled antigen solution per well.</li>
	<li>Cover the PCR plate with sealing foil to avoid light.</li>
	<li>Put the plate on a shaker (low speed) at RT for 2 h.</li>
	<li>Put the plate in the 4 &#176;C refrigerator and incubate overnight (18 - 24 h). 
	NOTE: The following protocol of steps from 5.6 to 5.9 is only designed for IAA assay and other autoantibody assays skip these steps.</li>
	<li>Mix 15 &#181;L of serum with 18 &#181;L of 0.5 M acetic acid for each serum sample. Incubate this mixture at RT for 45 min.</li>
	<li>Prepare the antigen solution with the rational concentration of Biotin and Sulfo-tag labeled antigen, based on the checker board assay, using antigen buffer. In each well, aliquot 35 &#181;L of antigen buffer, containing Biotin and Sulfo-tag labeled antigen, into a new PCR plate.</li>
	<li>Before the 45 min incubation (step 5.6) step is finished, add 8.3 &#181;L of 1 M Tris pH 9.0 buffer to the side of each well on the antigen plate (step 5.6). It is important to limit the mixing between the Tris buffer and the antigen. Immediately transfer 25 &#181;L of the serum, which is treated with acid, in step 5.6, into each well and agitate the solution. Cover the PCR plate with sealing foil to avoid light.</li>
	<li>Put the plate on a shaker (low speed) at RT for 2 h. Put the plate in the 4 &#176;C refrigerator and allow the plate to incubate overnight (18 - 24 h).</li>
</ol>
6. Prepare the Streptavidin Plate
<ol>
	<li>Take a streptavidin plate from the 4 &#176;C refrigerator and allow the plate to come to RT.</li>
	<li>Once the streptavidin plate is at RT, add 150 &#181;L of 3% Blocker A to each well.</li>
	<li>Cover the PCR plate with sealing foil.</li>
	<li>Incubate the plate in the 4 &#176;C refrigerator overnight.&#160;&#160;&#160; 
	NOTE:&#160;Assay Day 2 includes Step 7 to Step 9.</li>
</ol>
7. Transfer Serum/Antigen Incubates to the Streptavidin Plate
<ol>
	<li>The next day, take out the streptavidin plate from the refrigerator and discard the buffer from the plate. Set some dry paper towels out on the table and tap the plate upside down until there is no more buffer inside of the wells.</li>
	<li>Fill the empty streptavidin plate with 150 &#181;L of PBST per well and discard the PBST from the plate for a total of three washes.</li>
	<li>Transfer 30 &#181;L of serum/antigen incubates per well into the streptavidin plate.</li>
	<li>Cover the plate with foil to avoid light. Shake the plate, at a low setting, at RT for 1 h.</li>
</ol>
8. Wash the Plate and Add Read Buffer
<ol>
	<li>Discard incubates from the plate. Add 150 &#181;L of PBST per well and discard the PBST from the plate for a total of three washes.</li>
	<li>After washing, add 150 &#181;L per well of Reading buffer. 
	NOTE: It is important to prevent air bubbles in the solution because this will affect how the plate is read on the plate reader machine.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Human recombinant proinsulin protein(PINS)</td>
			<td>AmideBio</td>
			<td>Human Proinsulin, Rec</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant GAD65 protein</td>
			<td>Diamyd</td>
			<td>rhGAD65</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant IA-2 protein</td>
			<td>Creative BioMart</td>
			<td>IA2</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffered Saline (PBS), pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS, pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>SIGMA-ALORICH</td>
			<td>A7906-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well PCR plate&#160;</td>
			<td>Fisher</td>
			<td>14230232</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin coated plate</td>
			<td>MSD</td>
			<td>L15SA</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba sizing spin column</td>
			<td>Thermo Fisher Scientific</td>
			<td>89890</td>
			<td></td>
		</tr>
		<tr>
			<td>Twen-20</td>
			<td>Fisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher</td>
			<td>A144-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Fisher</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-Link Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>PI21329</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-tag</td>
			<td>MSD</td>
			<td>R91AO-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocker A</td>
			<td>MSD</td>
			<td>R93AA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>4x Read Buffer T with Surfactant</td>
			<td>MSD</td>
			<td>R92TC-1</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-Link NHS-PEG4-Biotin</td>
			<td>ThermoScience</td>
			<td>21329</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-TAG</td>
			<td>MSD</td>
			<td>R91AO-1</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well polymerase chain reaction (PCR) plate</td>
			<td>Fisher brand</td>
			<td>14230232</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Streptavidin plate</td>
			<td>MSD GOLD</td>
			<td>L 15SA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba Spin Desalting Column</td>
			<td>ThermoScience</td>
			<td>PL208984</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Plate Shaker</td>
			<td>Perkin Elmer</td>
			<td>1296-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Reader</td>
			<td>MSD</td>
			<td>QuickPlex SQ120</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge with bucket rotary</td>
			<td>Beckman</td>
			<td>Allegra X-15R</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Gu, Y. et al., <a href="https://app.jove.com/v/57227">Electrochemiluminescence Assays for Human Islet Autoantibodies.</a>&#160;J. Vis. Exp. (2018)
This video showcases the electrochemiluminescence detection of islet autoantibodies in human serum. The antibodies in the serum bridge the ruthenium-conjugated antigen to the biotinylated capture antigen, forming complexes that are detected by electrochemiluminescence measurements.

<img alt="Figure 1" src="/files/ftp_upload/22098/22098_Figure1.jpg" /> 
Figure 1: The ROC curve for determining the cut-off of assay positivity.&#160;The 99th&#160;percentile of specificity corresponding to 85% sensitivity was selected and is represented as an index value of 0.023. This upper limit of the assay was taken from 100 healthy controls.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Buffer Preparation

	Labeling ...

To detect islet autoantibodies &#8212; a biomarker of type-1 diabetes &#8212; take human serum samples and add acetic acid to inhibit interfering components.
Incubate and then add an alkaline buffer to neutralize the acid.
Transfer the treated serum to a multi-well plate containing a mixture of human islet autoantigens, including biotinylated capture and ruthenium-conjugated antigens, each at an optimal concentration for the assay.
The islet autoantibody in the positive sample bridges the ruthenium-conjugated antigen to the biotinylated capture antigen, forming an antigen-antibody complex.
Transfer the mixture into a streptavidin-coated electrochemiluminescence plate pre-treated with a blocking agent and equipped with working and counter electrodes.
Incubate to allow biotinylated complex interactions with the bound streptavidin and then wash.
Add a buffer containing tripropylamine or TPA.
In an electrochemiluminescence instrument, the voltage application between the electrodes initiates the oxidation of ruthenium and TPA, generating TPA radicals.
 The TPA radicals excite ruthenium complexes, which emit photons while returning to their ground state. 
A high chemiluminescence in the positive sample indicates the presence of islet autoantibodies.

15 ビュー. 電気化学発光アッセイを用いたヒト膵島自己抗体の検出

ビデオ: 電気化学発光アッセイを用いたヒト膵島自己抗体の検出 - 実験

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.

MeCP2タンパク質変異体に対するエレクトロケミセンスベースアッセイ

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...

JoVE Journal

生化学

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.

1型糖尿病、セリアック病、およびCOVID-19のハイスループット多重化スクリーニング

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and ...

免疫・感染学

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.

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.

Detection of Human Islet Autoantibodies Using an Electrochemiluminescence Assay

記録

Detection of Human Islet Autoantibodies Using an Electrochemiluminescence Assay