A.C. was supported by a postdoctoral fellowship from the Heart and Stroke Foundation of Canada and the Training Program in Regenerative Medicine (Canadian Institutes of Health Research). F.Y.Y.H. was supported by the Training Program in Regenerative Medicine. This work was funded by a grant from Astellas Pharma Canada. We also would like to thank Dr. Mark Menenghini (University of Toronto) for use of his Axon microarray scanner.

1. Diluting Antigens and Generating Antigen Microarrays
<ol>
	<li>Dilute antigens in PBS to a final concentration of 0.2 mg/ml. Print up to 162 unique antigens in duplicate with a microarrayer configuration with 9 pins. Include IgG and IgM antigens in the antigen library as positive controls. Include PBS only as a negative control.</li>
	<li>Add 20 &#181;l of each antigen to the 384 well source plate. Add antigens to the source plate in groups that mirror the setup of the printhead (e.g., arrange antigens in groups of 9 when 9 pins are used for printing).</li>
	<li>Cover source plate with foil and freeze at -80 &#176;C until ready to print arrays.</li>
	<li>Clean solid microarrayer pins by incubating them in a sonicating bath with deionized water for 3 x 1 min. Place pins in rack to dry and then arrange pins in microarray printhead. For 9 pins, use a 3 x 3 configuration.</li>
	<li>Program microarrayer for print run by setting number pins on printhead, number of slides to print, number of pads on each slide, and number of replicate spots for each antigen. Typically, use 9 pins, 70 slides, 2 pads/slide, and 2 replicate spots for each antigen. Program microarrayer to sonicate pins in water between different groups of antigens.</li>
	<li>Thaw source plate and then centrifuge plate for 1 min at 100 x g. Place source plate in designated spot in microarrayer. Remove the section of foil that is covering the first group of antigens to be printed.</li>
	<li>Arrange unprinted slides on arrayer surface and run print program. Print slides at RT with humidity set on the arrayer at 55 - 60% with the build in humidifier of the array machine.</li>
	<li>After each group of antigens is printed on all the slides, pause the microarrayer. Cover the antigens that were just printed with foil (to prevent evaporation) and uncover the next group of antigens to be printed. Continue print program.</li>
	<li>After all antigens have been printed, cover source plate with new piece of foil and freeze at -80 &#176;C. Place printed slides in slide box and vacuum seal. Slides may be used the next day or up to one month later.</li>
</ol>
2. Probing Antigen Microarrays with Diluted Sera
<ol>
	<li>Place slides into frames using incubation chambers. Add 700 &#181;l of blocking buffer (2.5% [vol/vol] Fetal calf serum (FCS), 0.1% [vol/vol] Tween 20 in PBS) to each array surface.</li>
	<li>Place adhesive film over frame and place in a sealed container with a piece of wet tissue. Incubate O/N at 4 &#176;C on a rocker.</li>
	<li>Dilute serum samples 1:100 in blocking buffer. Aspirate blocking solution from arrays and add 500 &#181;l of diluted sample to each array surface.</li>
	<li>Cover with adhesive film and incubate for 1 hr at 4&#176; C with rocking.</li>
	<li>Dilute secondary antibodies in blocking buffer. For human studies, dilute a Cy3 labeled goat anti-human IgG antibody to 0.33 &#181;g/ml and a Cy5 labeled goat anti-human IgM antibody to 0.25 &#181;g/ml. For mouse studies, dilute a Cy3 labeled goat anti-mouse IgG antibody to 0.38 &#181;g/ml and a Cy5 labeled goat anti-mouse IgM antibody to 0.7 &#181;g/ml.</li>
	<li>Aspirate samples from arrays and rinse array surfaces 4 times with rinse buffer (0.1% Tween 20 in PBS). Pour buffer onto to slides and flick off quickly.</li>
	<li>Add 700 &#181;l of blocking buffer to wash each array surface and incubate 10 min at RT with rocking. Repeat wash step two more times.</li>
	<li>Add 500 &#181;l of diluted secondary antibody to each slide surface and cover with adhesive film. Incubate 45 min at 4&#176; C with rocking.</li>
	<li>Aspirate secondary antibody mixture from slides and rinse 4 times as above. Wash slides 3 times with 700 &#181;l of blocking/dilution buffer as above.</li>
	<li>Remove slides from frames and place in metal slide rack that is immersed in PBS. Incubate 20 min at RT with orbital shaking. Place in new container of PBS and incubate another 20 min with shaking.</li>
	<li>Place slide rack in container of deionized water 15 sec. Place rack in a new container of water and incubate another 15 sec.</li>
	<li>To dry slides, place slide rack on an ELISA plate adapter in centrifuge and spin at 220 x g for 5 min at RT.</li>
	<li>Place slides in light-tight box until ready for scanning.</li>
</ol>
3. Scanning Antigen Microarrays and Exporting Data
<ol>
	<li>Scan slides using a microarray scanner that can detect Cy3 and Cy5 fluorescent signals. In order to adjust the photomultiplier tube (PMT) levels of the scanner, pre-scan a slide that was only probed with secondary antibodies. 
	Note: This slide should have the full antigen library printed on it including positive (IgG and IgM) as well as negative (PBS) controls. The pre-scan is a low resolution scan that allows the user quickly to find the optimal PMT settings.</li>
	<li>Set the PMT values so that the human IgG features (on the Cy3 channel) and the human IgM features (on the Cy5 channel) have a similar median fluorescence intensity minus background (MFI-B) (typically 40,000). The PMT levels are set by pressing the &#34;hardware&#34; button. Once set, keep these PMT settings constant.</li>
	<li>Scan the experimental slides on the two channels by pressing the &#34;scan&#34; button. Save the slide images (both channels) after each scan.</li>
	<li>Load the slide to be analyzed into the microarray software by using the &#34;file&#34; button.</li>
	<li>Load the gene array list (GAL) file by using the &#34;file&#34; button. The GAL file has the layout of the array with the identities of the array features.</li>
	<li>Place the array template over the scanned image so that the arrays features match the template as closely as possible.</li>
	<li>Align features in all blocks with the template by pressing the &#34;align&#34; button. The program will generally find the outline of the circular features but some manual adjustments may be necessary. These adjustments can be made by entering the feature mode. The software will then calculate a MFI-B for each of the antigens.</li>
	<li>Use the &#34;file&#34; button to export these results as a text file.</li>
</ol>
4. Analyzing Array Data with Significance Analysis of Microarrays (SAM)
<ol>
	<li>Load individual text files into excel and identify the columns that have MFI-B for the Cy3 and Cy5 channels.</li>
	<li>Calculate the average for the duplicate features.</li>
	<li>For any negative MFI-B, replace the negative number with 10. Transform data by dividing raw MFI-B by 100 and then by calculating log base 2.</li>
	<li>Analyze log transformed data with Significance Analysis of Microarrays (SAM) as described previously7.</li>
	<li>For a study using two groups (e.g., healthy controls vs. patients), label one group &#34;1&#34; and the group &#34;2.&#34; Use a two-class, unpaired analysis in SAM to identify antigens reactivities that are significantly different between the two groups (q value &#60; 0.05).</li>
	<li>Use clustering and heat-map generating software to make images for presentation8.</li>
</ol>

<ol><li>Naparstek, Y., Plotz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+autoantibodies+in+autoimmune+disease%5BTitle%5D)+AND+%22Annu+Rev+Immunol%22%5BJournal%5D)">The role of autoantibodies in autoimmune disease.</a> Annu Rev Immunol. 11, 79-104 (1993).</li>
<li>Damoiseaux, J., Andrade, L. E., Fritzler, M. J., Shoenfeld, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoantibodies+2015%3A+From+diagnostic+biomarkers+toward+prediction%2C+prognosis+and+prevention%5BTitle%5D)+AND+%22Autoimmun+Rev%22%5BJournal%5D)">Autoantibodies 2015: From diagnostic biomarkers toward prediction, prognosis and prevention.</a> Autoimmun Rev. 14, 555-563 (2015).</li>
<li>Robinson, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoantigen+microarrays+for+multiplex+characterization+of+autoantibody+responses%5BTitle%5D)+AND+%22Nature.+medicine%22%5BJournal%5D)">Autoantigen microarrays for multiplex characterization of autoantibody responses.</a> Nature. medicine. 8, 295-301 (2002).</li>
<li>Quintana, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigen+microarrays+identify+unique+serum+autoantibody+signatures+in+clinical+and+pathologic+subtypes+of+multiple+sclerosis%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Antigen microarrays identify unique serum autoantibody signatures in clinical and pathologic subtypes of multiple sclerosis.</a> Proc. Natl. Acad. Sci. USA. 105, 18889-18894 (2008).</li>
<li>Hueber, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigen+microarray+profiling+of+autoantibodies+in+rheumatoid+arthritis%5BTitle%5D)+AND+%22Arthritis+Rheum%22%5BJournal%5D)">Antigen microarray profiling of autoantibodies in rheumatoid arthritis.</a> Arthritis Rheum. 52, 2645-2655 (2005).</li>
<li>Price, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+microarray+analysis+reveals+BAFF-binding+autoantibodies+in+systemic+lupus+erythematosus%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus.</a> J. Clin. Invest. 123, 5135-5145 (2013).</li>
<li>Tusher, V. G., Tibshirani, R., Chu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Significance+analysis+of+microarrays+applied+to+the+ionizing+radiation+response%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Significance analysis of microarrays applied to the ionizing radiation response.</a> Proc. Natl. Acad. Sci. USA. 98, 5116-5121 (2001).</li>
<li>Eisen, M. B., Spellman, P. T., Brown, P. O., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cluster+analysis+and+display+of+genome-wide+expression+patterns%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Cluster analysis and display of genome-wide expression patterns.</a> Proc. Natl. Acad. Sci. USA. 95, 14863-14868 (1998).</li>
<li>Chruscinski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+Antigen+Microarrays+to+Screen+for+Autoantibodies+in+Heart+Failure+and+Heart+Transplantation%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation.</a> PloS one. 11, e0151224(2016).</li>
<li>Price, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+influenza+vaccine+immunogenicity+using+influenza+antigen+microarrays%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Characterization of influenza vaccine immunogenicity using influenza antigen microarrays.</a> PloS one. 8, e64555(2013).</li>
<li>Singh, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reactivity+profiles+of+broadly+neutralizing+anti-HIV-1+antibodies+are+distinct+from+those+of+pathogenic+autoantibodies%5BTitle%5D)+AND+%22Aids%22%5BJournal%5D)">Reactivity profiles of broadly neutralizing anti-HIV-1 antibodies are distinct from those of pathogenic autoantibodies.</a> Aids. 25, 1247-1257 (2011).</li>
<li>Porcheray, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+humoral+rejection+of+human+kidney+allografts+associates+with+broad+autoantibody+responses%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Chronic humoral rejection of human kidney allografts associates with broad autoantibody responses.</a> Transplantation. 89, 1239-1246 (2010).</li>
<li>Haddon, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+epitopes+of+U1-70K+autoantibodies+at+single-amino+acid+resolution%5BTitle%5D)+AND+%22Autoimmunity%22%5BJournal%5D)">Mapping epitopes of U1-70K autoantibodies at single-amino acid resolution.</a> Autoimmunity. 48, 513-523 (2015).</li>
<li>Schroeder, H. W. Jr, Cavacini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+and+function+of+immunoglobulins%5BTitle%5D)+AND+%22J+Allergy+Clin+Immunol%22%5BJournal%5D)">Structure and function of immunoglobulins.</a> J Allergy Clin Immunol. 125, S41-S52 (2010).</li>
</ol>

The authors have nothing to disclose.

The protocol described here allows for the quantification of autoantibodies using the antigen microarray technique. Antigen microarrays offer several advantages over conventional ELISA in screening for autoantibodies. First of all, a variety of antigens including nucleic acids, proteins, peptides, and cell lysates can be arrayed onto the nitrocellulose-coated slides, thus allowing for multiplexed screening of autoantibodies. In addition, only micrograms of antigen are necessary to generate the arrays since nanoliters of ...

Autoantibodies are present in many disease states and can often have direct pathogenic activity1. Identification of autoantibodies is important for diagnosis of certain diseases, for prognosis of disease outcome, and for the classification of patients who may benefit from specific therapies2. Autoantibodies are typically identified in patient serum using the ELISA technique; however, screening for multiple antigens with this technique is laborious and consumes a large quantity of patient sample. New technologies are therefore needed to profile autoantibodies on a larger scale.
The antigen microarray technique is a proteomic technology that allows autoantibodies to be profiled in a multiplex fashion3. In the first step of this process, an antigen library is arrayed onto a slide surface using a robotic microarrayer. The slides are probed with diluted serum and then fluorescent-labeled secondary antibodies are added. Antibody reactivities are visualized by scanning the slides with a microarray scanner and quantified by fluorescent intensities. Antigen microarrays offer multiple advantages over the ELISA technique in screening for autoantibodies: 1) they require only microliters of serum to profile autoantibodies to multiple antigens simultaneously, 2) they use antigen sparingly, as only nanoliters of antigen are spotted onto the arrays, 3) they have enhanced sensitivity3 compared to ELISA and 4) they allow for the simultaneous yet, separate detection of more than one antibody isotype. Antigen microarrays have been used to profile autoantibodies in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus4-6. In all three of these diseases, new insight into disease pathogenesis was obtained from profiling autoantibodies on a large scale with the arrays. 
Here we describe a protocol to generate antigen microarrays using nitrocellulose-coated slides. A variety of antigens including proteins, peptides, and cell lysates can be arrayed onto the slides using this technique. The arrays can be easily customized by including antigens of interest in the source plate that holds the antigen library. In addition, we show how a pair of secondary antibodies can be used to separate IgG and IgM reactivities on the same slide surface. We have now optimized this technique to measure autoantibodies in both humans and mice.

<table><tbody><tr><td>Ribosomal P0</td><td>Diarect</td><td>14100</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>human IgG</td><td>Jackson Immuno</td><td>009-000-003</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>human IgM</td><td>Jackson Immuno</td><td>009-000-012</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>mouse IgG</td><td>Sigma-Aldrich</td><td>I5381</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>mouse IgM</td><td>Biolegend</td><td>401601</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>double-stranded DNA</td><td>Sigma-Aldrich</td><td>D1626</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>single-stranded DNA</td><td>Sigma-Aldrich</td><td>D8899</td><td>dilute to 0.2 mg/ml in PBS</td></tr><tr><td>microarrayer</td><td>Virtek</td><td>VersArray Chipwriter Pro</td><td>many types of arrayers are suitable</td></tr><tr><td>solid printing pins</td><td>Arrayit Corporation</td><td>SSP015</td><td></td></tr><tr><td>software for robotic microarrayer</td><td>Virtek</td><td>Chipwriter Pro&amp;nbsp;</td><td></td></tr><tr><td>FAST slides (2 Pad)</td><td>GVS Northa America</td><td>10485317</td><td></td></tr><tr><td>FAST frame</td><td>GVS Northa America</td><td>10486001</td><td></td></tr><tr><td>FAST incubation chambers (2 Pad)</td><td>GVS Northa America</td><td>10486242</td><td></td></tr><tr><td>384 well plates</td><td>Whatman</td><td>7701-5101</td><td></td></tr><tr><td>plate sealers</td><td>VWR</td><td>60941-062</td><td></td></tr><tr><td>foil plate covers</td><td>VWR</td><td>60941-124</td><td></td></tr><tr><td>Tween-20</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Fetal calf serum</td><td>Invitrogen</td><td>12483020</td><td></td></tr><tr><td>Cy3 goat anti-human IgG</td><td>Jackson Immuno</td><td>109-165-096</td><td>use working stock in 50% glyercol</td></tr><tr><td>Cy5 goat anti-human IgM</td><td>Jackson Immuno</td><td>109-175-129</td><td>use working stock in 50% glyercol</td></tr><tr><td>Cy3 goat anti-mouse IgG</td><td>Jackson Immuno</td><td>115-165-071</td><td>use working stock in 50% glyercol</td></tr><tr><td>Cy5 goat anti-mouse IgM</td><td>Jackson Immuno</td><td>115-175-075</td><td>use working stock in 50% glyercol</td></tr><tr><td>Microarray Scanner</td><td>Molecular Devices</td><td>Axon 4200A</td><td></td></tr><tr><td>Microarray software</td><td>Molecular Devices</td><td>Genepix 6.1</td><td></td></tr><tr><td>Clustering software</td><td>eisenlab.org</td><td>Cluster 3.0</td><td></td></tr><tr><td>Heatmap software</td><td>eisenlab.org</td><td>Treeview 1.60</td><td></td></tr><tr><td>Microarray statistical software</td><td>Stanford University</td><td>SAM 4.0 (Significance Analysis of Microarrays)</td><td></td></tr></tbody></table>

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.

Antigens are arranged in a 384 well plate and printed onto slides by a robotic microarrayer as shown in Figure 1. Figure 2 shows slides placed in a frame with incubation chambers and a scanned slide after processing. Figure 3 shows positive and negative control slides. The negative slide is only probed with secondary antibodies, and the positive control slide is probed with serum from a patient with systemic lupus erythematosus. By using ...

Watch this Scientific Journal Video about Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies at JoVE.com

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

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Research

JoVE Journal

Immunology and Infection

12.8K Views.  University Health Network. 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. 

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

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 ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Biology

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

Multiplex Immunoassay for Borrelia-Specific IgG and IgM in Serum

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

Author Spotlight: Expanding the Scope of Multiplex Immunoassays for Lyme Borreliosis Diagnostics and Pathogen Research 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America.

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different ...

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.

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

Medicine

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Source: Olivera-Ardid, S. et al., <a href="https://app.jove.com/v/57662">Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates printed glycan array technology for analyzing circulating anti-glycan antibodies in mouse serum. The antibodies in the serum interact with specific glycans on the chip, which is confirmed by immunostaining followed by fluorescence scanning.

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

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JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

A Microfluidic Cartridge-Based Detection System for Allergen-Specific Immunoglobulin E

Source: Huang, Z., et al. <a href="https://app.jove.com/v/59100">Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE).</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates a protocol for detecting serum-specific immunoglobulin E using a microfluidic cartridge-based chemiluminescence system. Serum IgEs interact with distinct allergens immobilized in the reaction zone of the cartridge, which are subsequently detected with peroxidase-conjugated anti-IgE antibodies and a chemiluminescent substrate, allowing simultaneous detection of multiple allergen-specific IgEs.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Basic information of the study ...

A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip

Source: Blanco, Y. et al., <a href="https://app.jove.com/v/54994">Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method to detect the Cyanobacterial strains in an environmental sample using an antibody microarray chip. Samples carrying various bacteria interact with printed antibodies, and immunostaining with fluorophore-labeled anti-cyanobacterial-strain antibodies reveals the presence of specific cyanobacterial strains through fluorescence in distinct spots.