This work was supported by NIH grant DK32083, JDRF Grant 2-SRA-2015-51-Q-R.

1. Buffer Preparation
<ol>
	<li>Labeling buffer (2x PBS, pH 7.9): In 400 mL of distilled deionized (DD) water, add 100 mL of 10x PBS, adjust pH to 7.9 with 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. 
	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. 
	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 PCR plate. Use half of a plate for the high positive sample and the other half for the negative sample, as shown in Figure 2A.</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 Figure 2A. 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 Figure 2B.</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 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. 
	Note: 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>
9. Read the Plate and Analyze Data
<ol>
	<li>Count the plate on the plate reader machine. Antibody values are shown as counts per second (CPS).</li>
	<li>Convey the antibody levels received from the plate reader machine as a relative index. Calculate the index from the following equation: 
	Index value = [CPS (sample) - CPS (negative control)] / [CPS (positive control) - CPS (negative control)].</li>
	<li>Identify positive and negative antibody results using the cut-off for positivity, defined based on the 99th percentile of 100 healthy control subjects (non-diabetic individuals without any known autoimmune diseases and who have no family history of diabetes).</li>
</ol>

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	<li>Insel, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+presymptomatic+type+1+diabetes:+a+scientific+statement+of+JDRF,+the+Endocrine+Society,+and+the+American+Diabetes+Association.">Staging presymptomatic type 1 diabetes: a scientific statement of JDRF, the Endocrine Society, and the American Diabetes Association.</a> Diabetes Care. 38 (10), 1964-1974 (2015).</li><li>Atkinson, M. A., Eisenbarth, G. S. . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+diabetes:+new+perspectives+on+disease+pathogenesis+and+treatment.">Type 1 diabetes: new perspectives on disease pathogenesis and treatment.</a> Lancet. 358 (9277), 221-229 (2001).</li><li>Atkinson, M. A., Eisenbarth, G. S., Michels, A. W. . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+diabetes.">Type 1 diabetes.</a> Lancet. 383 (9911), 69-82 (2014).</li><li>Steck, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TEDDY+Study+Group.+Predictors+of+progression+from+the+appearance+of+islet+autoantibodies+to+early+childhood+diabetes:+The+Environmental+Determinants+of+Diabetes+in+the+Young+(TEDDY).">TEDDY Study Group. Predictors of progression from the appearance of islet autoantibodies to early childhood diabetes: The Environmental Determinants of Diabetes in the Young (TEDDY).</a> Diabetes Care. 38 (5), 808-813 (2015).</li><li>Krischer, J. 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G. . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+history+of+type+1+diabetes.">Natural history of type 1 diabetes.</a> Diabetes. 54 (Suppl. 2), S25-S31 (2005).</li><li>Steck, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+of+islet+autoantibody+appearance+and+mean+levels+of+insulin,+but+not+GAD+or+IA-2+autoantibodies,+predict+age+of+diagnosis+of+type+1+diabetes:+diabetes+autoimmunity+study+in+the+young.">Age of islet autoantibody appearance and mean levels of insulin, but not GAD or IA-2 autoantibodies, predict age of diagnosis of type 1 diabetes: diabetes autoimmunity study in the young.</a> Diabetes Care. 34 (6), 1397-1399 (2011).</li><li>Achenbach, P., Koczwara, K., Knopff, A., Naserke, H., Ziegler, A. G., Bonifacio, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mature+high-affinity+immune+responses+to+(pro)insulin+anticipate+the+autoimmune+cascade+that+leads+to+type+1+diabetes.">Mature high-affinity immune responses to (pro)insulin anticipate the autoimmune cascade that leads to type 1 diabetes.</a> J Clin Invest. 114 (4), 589-597 (2004).</li><li>Yu, L., Dong, F., Miao, D., Fouts, A. R., Wenzlau, J. M., Steck, A. K. . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proinsulin/insulin+autoantibodies+measured+with+electrochemiluminescent+assay+are+the+earliest+indicator+of+prediabetic+islet+autoimmunity.">Proinsulin/insulin autoantibodies measured with electrochemiluminescent assay are the earliest indicator of prediabetic islet autoimmunity.</a> Diabetes Care. 36 (8), 2266-2270 (2013).</li><li>Yu, L., Miao, D., Scrimgeour, L., Johnson, K., Rewers, M., Eisenbarth, G. S. . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinguishing+persistent+insulin+autoantibodies+with+differential+risk:+nonradioactive+bivalent+proinsulin/insulin+autoantibody+assay.">Distinguishing persistent insulin autoantibodies with differential risk: nonradioactive bivalent proinsulin/insulin autoantibody assay.</a> Diabetes. 61 (1), 179-186 (2012).</li><li>Miao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAD65+autoantibodies+detected+by+electrochemiluminescence+assay+identify+high+risk+for+type+1+diabetes.">GAD65 autoantibodies detected by electrochemiluminescence assay identify high risk for type 1 diabetes.</a> Diabetes. 62 (12), 4174-4178 (2013).</li><li>Yu, L. . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+Autoantibody+Detection+by+Electrochemiluminescence+(ECL)+Assay.">Islet Autoantibody Detection by Electrochemiluminescence (ECL) Assay.</a> Methods Mol Biol. 1433, 85-91 (2016).</li><li>Myler, H. 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The authors have nothing to disclose.

Islet autoantibodies are currently the most reliable biomarkers for autoimmunity of type 1 diabetes. They mark the onset of islet specific autoimmunity and determine overt disease risks. The ECL assay, for islet autoantibodies, has been extensively validated in multiple national and international type 1 diabetes clinical trials. The assay has shown increased sensitivity and specificity as compared to the current &#39;gold&#39; standard RBAs. The ECL assay has shown its superior advantage for higher disease specificity by discriminating high-affinity and high-risk islet autoantibodies from low-risk, low-affinity signals generated by the RBA. This is especially noticed in subjects who are only single islet autoantibody positive and have never progressed to type 1 diabetes. Most of these low affinity autoantibodies were found to be lost during follow up testing done within months to years, behaving as a &#39;transient positive.&#39;&#160;As previously hypothesized, these low affinity &#39;single&#39; autoantibodies likely resulted from immunization with a cross-reactive molecule. While higher affinity, higher risk islet autoantibodies resulted from immunization with the islet antigens themselves. In addition, ECL-assays have demonstrated the ability to antedate the time of &#39;seroconversion&#39; from current standard radio-binding assays (RBA) by years in children with pre-diabetes, who were followed to clinical diabetes from birth. An ongoing international clinical trial, The Environmental Determinants of Diabetes in the Young (TEDDY), depends on accurate detection to pinpoint the timing and appearance of the first islet autoantibody &#39;seroconversion&#39; to mark the very beginning of islet autoimmunity and for identifying environmental triggers. A possible reason for increased sensitivity in the ECL assay is that the assay captures all immunoglobulin classes: IgG, IgM, IgA, or IgE, rather than the traditional RBA or ELISA which rely only on the detection of IgG.
In ECL assays, for some particular autoantibodies like IAA, the binding of autoantibodies to the antigen seems to be inhibited by some component present in normal human serum. To remove or release this inhibition, acid treatment of serum samples was necessary to do before the serum was incubated with antigen. As shown in [Figure 5], ECL-IAA assay, the binding activities of both the mouse insulin monoclonal antibody and patients&#39; serum autoantibodies with the antigen was greatly enhanced. The acidification of serum samples, used in antibody assays, is usually applied to disassociate pre-existing bound complexes. The mechanism behind how IAA signals are inhibited in human serum samples and released by acidification of serum is not known, but this method has been used in other ECL based assays14,15.
In a few cases, when labeling molecules, either Biotin or Sulfo-tag, the labeling positionsinside of the antigen protein molecules are at, or very close to key epitopes of antibody binding, which may interfere with the binding activity to antibodies. This will reduce assay sensitivity and can completely tear down the assay. For routine labeling procedure, maximization or saturation is desired for each antigen protein molecule to generate maximum activity or signal per labeled molecule by maximizing labeling capacity of every possible labeling position. Unsaturated labeling should be performed by reducing the molar ratio of labeling molecules (Biotin and Sulfo-tag) to the antigen protein if labeling on the antigen becomes a possible reason for interruption of antibody binding activity. Major epitopes can be better reserved and interruption of antibody binding activity can be released using the unsaturated labeling strategy in most cases.
Each assay should include an internal standard high positive and negative control for index calculations of unknown samples. A low positive control near the assay&#39;s upper limit of normal controls is important to include for the monitoring of assay sensitivity. The laboratory should keep enough aliquots of standard positive and negative controls for long-term use and all aliquots should be stored at -20 &#176;C. For assay quality assurance, assays must be run in duplicates for each sample and every positive result should be confirmed by repeating the sample in a separate assay. A third assay is necessary when the second confirmatory assay does not agree with the first assay and the results of two assays, which agree (e.g., +,+ or -,-), will be the final determination of a positive or negative result.
With the platform set for single ECL assays, a multiplexed assay can be expanded upon from these. It can simultaneously determine up to 10 different autoantibodies in one single well with a tiny amount of serum. Currently, four islet autoantibodies including IAA, GADA, IA-2A, and ZnT8A are equal in importance for the risk prediction of progression to T1D in both relatives of patients with T1D and the general population. The methods utilized for screening these 4 autoantibodies using current single autoantibody measurements are laborious and inefficient, especially for large scale population screening. Importantly, up to 40% of patients with T1D have an additional autoimmune condition16,17,18. Unfortunately, there is no easy and inexpensive tool to screen for these conditions. The multiplex ECL assay is not only capable of combining 4 current major islet autoantibody assays into one, but also is able to further combine more autoantibody assays from other relevant autoimmune diseases. This makes it possible to efficiently conduct high throughput screening for multiple autoimmune diseases simultaneously in large scale populations. In the multiplex ECL assay, as shown in [Figure 6], each antibody-antigen complex formed in the fluid-phase will be restrained to a specific linker source spot in the same well. The signal receiver on the plate reader machine is able to recognize the signals from 10 different sources of spots. However, the spots with an extremely high signal can generate a high assay background and cause interference to neighboring spots through cross-talk. For this reason, the upper limit signals for each autoantibody assay should be limited to fewer than 20,000 counts. In our experience, the autoantibodies with lower backgrounds should be placed relatively far away from those spots having higher counts when the spot map is designed. For long-term studies using multiplex ECL assays, it is recommended that the same linker be used for the same autoantibody assay to keep the assay consistent.

A recent staging classification system has been created to assist with the diagnosis of initial stages of T1D in patients. Exact detection of human islet autoantibodies plays an important role in identifying and staging presymptomatic type 1 diabetes, as the presence of islet autoantibodies indicates the presence of &#946;-cell autoimmunity. The rate at which diabetes affects patients from the initial occurrence of &#946;-cell autoimmunity to the symptomatic disease, associated with the number and type of islet autoantibodies, is variable2,3.
The age of autoantibody seroconversion, titer, and affinity of islet autoantibodies can affect the rate of the progression to symptomatic type 1 diabetes4,5,6,7,8,9,10. Recently, developed ECL assays have been extensively validated, have demonstrated increased sensitivity, and are more disease-specific10,11,12,13. These assays enhance the prediction and staging of diabetes risk through earlier detection of islet autoantibodies. They more precisely mark the initiation of islet autoimmunity and ignore the low-affinity and low risk signals not relevant to diabetes.
In an ECL assay, the autoantibodies in the serum, if present, bridge the Sulfo-tag-conjugated antigen to the biotinylated capture antigen in the fluid phase. After bridging, the Biotin linker is caught in the solid phase and detected through ECL by the Sulfo-tag on the streptavidin coated plate (Figure 1).
In this review, single antibody ECL assays with human islet autoantibodies are primarily utilized. Briefly, multiplexed antibody assays based on single ECL assays will be discussed. The multiplex assay can be used to identify multiple, up to 10, autoantibodies within one single well, using 15 &#181;L of serum. This simple high throughput assay can be used to screen, simultaneously, multiple autoantibodies for multiple relevant autoimmune diseases in the general population.

<table border="0" cellpadding="0" cellspacing="0" width="643">
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:292px;">Human recombinant proinsulin protein(PINS)</td>
			<td style="width:151px;">AmideBio</td>
			<td style="width:137px;">Human Proinsulin, Rec</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human recombinant GAD65 protein</td>
			<td style="width:151px;">Diamyd</td>
			<td style="width:137px;">rhGAD65</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human recombinant IA-2 protein</td>
			<td style="width:151px;">Creative BioMart</td>
			<td style="width:137px;">IA2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x Phosphate Buffered Saline (PBS), pH 7.4</td>
			<td style="width:151px;">Thermo Fisher Scientific</td>
			<td style="width:137px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10x PBS, pH 7.4</td>
			<td style="width:151px;">Thermo Fisher Scientific</td>
			<td style="width:137px;">70011-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:151px;">SIGMA-ALORICH</td>
			<td style="width:137px;">A7906-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well PCR plate&#160;</td>
			<td style="width:151px;">Fisher</td>
			<td>14230232</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin coated plate</td>
			<td style="width:151px;">MSD</td>
			<td style="width:137px;">L15SA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeba sizing spin column</td>
			<td style="width:151px;">Thermo Fisher Scientific</td>
			<td>89890</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Twen-20</td>
			<td style="width:151px;">Fisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCl</td>
			<td style="width:151px;">Fisher</td>
			<td>A144-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH</td>
			<td style="width:151px;">Fisher</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trizma Base</td>
			<td style="width:151px;">Fisher</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EZ-Link Biotin</td>
			<td style="width:151px;">Thermo Fisher Scientific</td>
			<td>PI21329</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sulfo-tag</td>
			<td style="width:151px;">MSD</td>
			<td>R91AO-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Blocker A</td>
			<td style="width:151px;">MSD</td>
			<td style="width:137px;">R93AA-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4x Read Buffer T with Surfactant</td>
			<td>MSD</td>
			<td>R92TC-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-Link NHS-PEG4-Biotin</td>
			<td>ThermoScience</td>
			<td>21329</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sulfo-TAG</td>
			<td>MSD</td>
			<td>R91AO-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well polymerase chain reaction (PCR) plate</td>
			<td style="width:151px;">Fisher brand</td>
			<td style="width:137px;">14230232</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-Well Streptavidin plate</td>
			<td style="width:151px;">MSD GOLD</td>
			<td style="width:137px;">L 15SA-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeba Spin Desalting Column</td>
			<td style="width:151px;">ThermoScience</td>
			<td style="width:137px;">PL208984</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well Plate Shaker</td>
			<td style="width:151px;">Perkin Elmer</td>
			<td style="width:137px;">1296-003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plate Reader</td>
			<td style="width:151px;">MSD</td>
			<td style="width:137px;">QuickPlex SQ120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Benchtop centrifuge with bucket rotary</td>
			<td style="width:151px;">Beckman</td>
			<td style="width:137px;">Allegra X-15R</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 2 displays the checker board. It is shown that 250 ng/mL of Biotin and 250 ng/mL of Sulfo-tag labeled antigen are the most rational concentrations used in the assay considering the signals from the high positive control sample, the negative control sample as the assay&#39;s background, and the ratio of positive to negative signals. With the optimized concentrations of these two labeled antigens, the assay was performed with the serum samples from 100 newly diagnosed patients with T1D and 100 healthy controls. The index value of 0.023 was defined as the assay cut-off for positivity. This represents 85% sensitivity in patients and 99% specificity in healthy controls using the receiver operating characteristic (ROC) curve shown in Figure 3A. The assay for the unknown samples was conducted with internal standard high positives, low positives, and negative control samples. The results of the CPS counts are shown in Table 2A. The mean CPS of high and low positive controls are highlighted in red, 17903 [(19940+15866)/2] and 839 [(857+820)/2], and the negative controls are highlighted in green, 168 [(170+165)/2]. The index for unknown samples is calculated by &#34;(CPSsample-168)/(17903-168).&#34;&#160;Table 2B shows the calculated index values for all samples. The index values that are greater than 0.023 are written in red, corresponding to the CPS values also written in red in Table 2A. These values will be defined as the positive results that are greater than the 99th percentile of the healthy control population. When an irrational antigen concentration is used, the assay will have a high background, as shown in Table 2C. Low levels of positive antibodies will be missed like the values A2, A5, D1, and H4 highlighted in gray in Table 2D.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57227/57227fig1.jpg" /> 
Figure 1: Illustration of a bivalent plate capture on a single ECL-IAA assay. The islet autoantibody in the serum bridges the Sulfo-tag conjugated antigen to the biotinylated capture antigen, which is captured in the solid phase on the streptavidin-coated plate. Detection of plate-captured Sulfo-tag conjugated antigen is accomplished through ECL. This figure has been modified from Yu, et al.11. <a href="/files/ftp_upload/57227/57227fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57227/57227fig2.jpg" /> 
Figure 2: The ROC curve for determining the cut-off of assay positivity. The 99th 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. <a href="/files/ftp_upload/57227/57227fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57227/57227fig3.jpg" /> 
Figure 3: Illustration of normal human serum blocking ECL-IAA signals. A: The signals from ECL-IAA assays with an insulin monoclonal antibody (MoAb) were drastically blocked by the addition of normal human serum. When comparing MoAb in PBS, the signal was partially restored when the human serum was treated with acid. B: Signals from an ECL-IAA assay with 4 patient sera were significantly enhanced with acid treatment. This figure has been modified from Yu, et al.11. <a href="/files/ftp_upload/57227/57227fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57227/57227fig4.jpg" /> 
Figure 4: Illustration of the Mutiplex ECL assay. Antibody-antigen complexes are formed in the fluid-phase with specific linkers. The specific antibody-antigen-linker complexes are restrained on each of the specific linker-spots in the same well. The plate reader machine is able to distinguish the signals from different sources of spots and gives CPS counts on 10 different channels, respectively. <a href="/files/ftp_upload/57227/57227fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/57227/57227tbl1.jpg" /> 
Table 1: Checker board assay determines the concentrations and ratios of Biotin and Sulfo-tag labeled antigens. A: Raw CPS counts for the checker board plate with high positive serum on half of the plate and negative serum on the other half. The concentration of biotinylated antigen was diluted horizontally in series and the concentration of Sulfo-tag labeled antigen was diluted vertically in series. B: The ratio values of the CPS counts from the high positive serum against each of their corresponding CPS counts from the negative serum. The yellow highlighted wells represent the best ratio of positive to negative in Panel B. This ratio corresponds to the 250 ng/mL Biotin and 250 ng/mL Sulfo-tag labeled antigen concentrations in Panel A with an acceptably low CPS count for negative serum. <a href="/files/ftp_upload/57227/57227tbl1large.jpg" target="_blank">Please click here to view a larger version of this table.</a>
<img alt="Table 2" src="/files/ftp_upload/57227/57227tbl2.jpg" /> 
Table 2: Analysis of assay results. A: Raw CPS counts from the assay plate with the standard high and low positive controls highlighted in red and the standard negative controls highlighted in green. Each sample was duplicated in the assay. B: Index values were calculated, as described in the assay protocol. Any index value that was greater than 0.023 was defined as a positive result, highlighted in red. C: Raw CPS counts from the assay plate with the same set of samples when an irrational antigen concentration was used. This resulted in a high background and some low positives normally detected, as shown in Panel B, were converted to negatives, highlighted in gray in Panel D. <a href="/files/ftp_upload/57227/57227tbl2large.jpg" target="_blank">Please click here to view a larger version of this table.</a>

Watch this Scientific Journal Video about Electrochemiluminescence Assays for Human Islet Autoantibodies at JoVE.com

Electrochemiluminescence Assays for Human Islet Autoantibodies

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 ...

electrochemiluminescence-assays-for-human-islet-autoantibodies

Research

JoVE Journal

Immunology and Infection

15.4K Views.  University of Colorado Denver. 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.

Video: Electrochemiluminescence Assays for Human Islet Autoantibodies

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...