This work was supported in part by a Research Career Development Award from the Israel Cancer Research Fund, a grant from the Israeli National Nanotechnology Initiative and the Helmsley Charitable Trust for a Focal Technology Area on Nanomedicines for Personalized Theranostics (V.P-K), and National Institutes of Health grant R01GM076360 (to X.C.).

Human sera samples were obtained from the Israeli Blood Bank and were used in accordance with the Helsinki declaration and Tel Aviv University Institutional Review Board.
1. Array Fabrication Planning and Layout
<ol>
	<li>Determine the slide layout. 
	NOTE: Each slide contains 16 sub-arrays divided into 16 identical blocks numbered B1 to B16 (Figure 1B, Supplementary Figure 1A).</li>
	<li>Determine the pin layout. Use four pins, each printi...

<ol>
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B., 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=The+minimum+information+required+for+a+glycomics+experiment+(MIRAGE)+project:+sample+preparation+guidelines+for+reliable+reporting+of+glycomics+datasets.">The minimum information required for a glycomics experiment (MIRAGE) project: sample preparation guidelines for reliable reporting of glycomics datasets.</a> Glycobiology. 26 (9), 907-910 (2016).</li><li>Liu, Y., 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=The+minimum+information+required+for+a+glycomics+experiment+(MIRAGE)+project:+improving+the+standards+for+reporting+glycan+microarray-based+data.">The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data.</a> Glycobiology. , (2016).</li><li>Hara, S., Yamaguchi, M., Takemori, Y., Furuhata, K., Ogura, H., Nakamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+mono-O-acetylated+N-acetylneuraminic+acids+in+human+and+rat+sera+by+fluorometric+high-performance+liquid+chromatography.">Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography.</a> Anal Biochem. 179 (1), 162-166 (1989).</li><li>Padler-Karavani, V., 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=Cross-comparison+of+protein+recognition+of+sialic+acid+diversity+on+two+novel+sialoglycan+microarrays.">Cross-comparison of protein recognition of sialic acid diversity on two novel sialoglycan microarrays.</a> J Biol Chem. 287 (27), 22593-22608 (2012).</li><li>Padler-Karavani, V., Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+impact+of+the+non-human+sialic+acid+N-glycolylneuraminic+acid+on+transplant+rejection+risk.">Potential impact of the non-human sialic acid N-glycolylneuraminic acid on transplant rejection risk.</a> Xenotransplantation. 18 (1), 1-5 (2011).</li><li>Samraj, A. 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M., L&#228;ubli, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sialic+acids+in+cancer+biology+and+immunity.">Sialic acids in cancer biology and immunity.</a> Glycobiology. 26 (2), 111-128 (2016).</li><li>Alisson-Silva, F., Kawanishi, K., Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+risk+of+diseases+associated+with+red+meat+intake:+Analysis+of+current+theories+and+proposed+role+for+metabolic+incorporation+of+a+non-human+sialic+acid.">Human risk of diseases associated with red meat intake: Analysis of current theories and proposed role for metabolic incorporation of a non-human sialic acid.</a> Mol Aspects Med. 51, 16-30 (2016).</li><li>Pearce, O. 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A successful glycan microarray fabrication requires careful planning and includes several important steps in the protocol. These include: (1) planning the block and plate layouts that define all subsequent parameters (e.g., distances, spacing, amount of samples, and printing); (2) cleaning the pins and ensuring pin integrity, which is critical for controlling spot homogeneity; (3) maintaining high humidity during printing, critical to avoiding sample evaporation during long print runs, which could compromise spo...

Sias are acidic sugars covering glycan chains on cell-surface glycoproteins and glycolipids in vertebrates. Sia expression is modified in cancer cells1 and correlates with progression and/or metastasis2,3. Two of the major Sia forms in mammals are Neu5Ac and its hydroxylated form, Neu5Gc2. Humans cannot synthesize Neu5Gc due to a specific inactivation of the gene encoding the CMAH enzyme . This non-human Sia metabolically incorporates into human cells as &#34;self,&#34; originating from dietary Neu5Gc-rich foods (e.g., red meat)4,5. Neu5Gc is present at low levels on the cell surfaces of human epithelia and endothelia, but it especially accumulates in carcinomas. Neu5Gc is recognized as foreign by the human humoral immune system2,6. The antigenic complexity of Neu5Gc-glycans may arise at multiple levels, including Neu5Gc modification, linkage, underlying glycans and scaffolds, and their density, all reflected by the complexity of anti-Neu5Gc antibody response in humans6. Some of these antibodies serve as carcinoma biomarkers and potential immunotherapeutics7. The advent of the chemoenzymatic synthesis of different sialoglycans8 paved the way for the more in-depth analysis of such antibodies, facilitated by the use of glycan microarray technology9,10. Thus, with the facilitated preparation and manipulation of large libraries of natural and synthetic carbohydrates, glycan microarrays have become a powerful high-throughput technology for investigating the interactions of carbohydrates with a myriad of biomolecules10,11,12,13. In an array format, minimal amounts of materials are used, and this multivalent display of biologically relevant glycans allows for the investigation of thousands of binding interactions in a single experiment. Importantly, this technology can also be applied to biomarker discovery and to monitoring immune responses in various samples7,12.
Successful glycan microarray fabrication requires the consideration of three important aspects: the printer robot type, glycan conjugation chemistry, and detection optics. As to the printing instrument consideration, two techniques are available: contact and non-contact printers. In contact printing, 1-48 steel pins are dipped into a multi-well source plate containing glycan solution and are spotted on functionalized glass slides by directly contacting the glass slide surface. The solution amount delivered to the slide is a function of the lingering duration on the slide surface. Usually, the samples are first pre-spotted on a glass block (to reach homogenous spots) before they are printed on the slide surface. In non-contact printers (e.g., the piezo-electronic printer), the glycans are printed from a glass capillary using controlled electric signals. The electric signal can be finely calibrated to achieve more precise printing relative to contact printing. The size and morphology of the spots are also relatively more homogeneous. An additional advantage is the recycling of the sample back to the source plate after printing. Nevertheless, the major disadvantage of piezo-electronic printers is the printing tip limitation (4 or 8), resulting in a very long printing duration, which requires special attention to slide stability, temperature, humidity, and sample evaporation. The non-contact inkjet printer requires larger sample volumes14.
In contrast to the limited available options for printing methods, glycan conjugation chemistry is a more complex consideration, with many options to choose from. Selected immobilization chemistry must account for both the active groups on the glycans and the slide surface reactivity. The glycans to be immobilized onto a specific microarray surface, either synthetically synthesized or naturally isolated, all require an identical reactive group. In addition, the glycans need to be pure and homogenous. On the other hand, the immobilization surface and chemistry should provide reproducibility and reliable attachment density. Multiple immobilization methods have been developed with either covalent or non-covalent (physical absorption) attachment10,11,12,13. For highly detailed information on printed glycan microarray technology for the uninitiated investigator, refer to these excellent reviews 13,15. Importantly, the recent Minimum Information Required for a Glycomics Experiment (MIRAGE) initiative describes guidelines for sample preparation16 and for reporting data from glycan microarray analyses17 to improve the standards in this growing field.
Here, we describe a detailed protocol for the fabrication of sialoglycan microarrays using a specific contact nano-printer in a 16-well format. Each of the glycans have a primary amine that mediate their covalent link to epoxy-activated glass slides. We also describe the development and analysis of one slide using various human sera samples, antibodies, and Sia-binding plant lectins. Sialoglycan microarray assays involve several major steps that include array fabrication, processing, development, and analysis. Array fabrication requires planning the array layout, preparing the glycans and source plate, programming the nano-printer, and printing the slides. Subsequently, the slides are processed, developed, and analyzed (Figure 1).

<table border="0" cellpadding="0" cellspacing="0" width="1205">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:269px;">Primary-amine containing sialoglycans</td>
			<td style="width:235px;">Glycohub, Inc., Davis, CA, USA (http://www.glycohub.com/services)</td>
			<td style="width:227px;">Contact info@glycohubusa.com for compound requests</td>
			<td style="width:476px;">Printed glycans</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Monosodium phosphate monohydrate</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:227px;">S9638</td>
			<td style="width:476px;">Printing buffer component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Disodium phosphate heptahydrate</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:227px;">S9390</td>
			<td style="width:476px;">Printing buffer component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Phosphate buffered saline</td>
			<td style="width:235px;">Hy-Labs</td>
			<td style="width:227px;">BP-507/500D</td>
			<td style="width:476px;">Printing buffer/ incubation/washing buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Tris-base</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:227px;">T1503</td>
			<td style="width:476px;">Slide blocking reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Glycerol</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:227px;">G-7893</td>
			<td style="width:476px;">Printing buffer component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Ethanolamine</td>
			<td style="width:235px;">Thermo-Fisher Scientific</td>
			<td style="width:227px;">0700/08</td>
			<td style="width:476px;">Slide blocking reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Ovalbumin (Grade V)</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:227px;">A5503</td>
			<td style="width:476px;">Slide Blocking protein</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Tween-20</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:227px;">P7949</td>
			<td style="width:476px;">Slide washing detergent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Alexa 555-Hydrazide</td>
			<td style="width:235px;">Thermo-Fisher Scientific</td>
			<td style="width:227px;">A20501MP</td>
			<td style="width:476px;">Marker on array</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">ChromPure Human IgG, whole molecule</td>
			<td style="width:235px;">Jackson Immunoresearch</td>
			<td style="width:227px;">009-000-003</td>
			<td style="width:476px;">Printing component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Biotinylated- SNA</td>
			<td style="width:235px;">Vector Laboratories</td>
			<td style="width:227px;">B-1305</td>
			<td style="width:476px;">Plant Lectin &#8211; binding Sia-alpha2&#8211;6-linked</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Biotinylated-MALII</td>
			<td style="width:235px;">Vector Laboratories</td>
			<td style="width:227px;">B-1265</td>
			<td style="width:476px;">Plant Lectin &#8211; binding Sia-alpha2&#8211;3-linked</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Chicken-anti Neu5Gc IgY</td>
			<td style="width:235px;">BioLegend</td>
			<td style="width:227px;">146903</td>
			<td style="width:476px;">Primary detection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Cy3-Streptavidin</td>
			<td style="width:235px;">Jackson Immunoresearch</td>
			<td style="width:227px;">016-160-0848</td>
			<td style="width:476px;">Biotin binding</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Cy3-anti Human IgG</td>
			<td style="width:235px;">Jackson Immunoresearch</td>
			<td style="width:227px;">109-165-088</td>
			<td style="width:476px;">Secondary detection against human IgG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Cy3-anti Chicken IgY</td>
			<td style="width:235px;">Jackson Immunoresearch</td>
			<td style="width:227px;">703-165-155</td>
			<td style="width:476px;">Secondary detection against chicken IgY</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Human sera samples</td>
			<td style="width:235px;">Israeli Blood Bank</td>
			<td style="width:227px;"></td>
			<td style="width:476px;">Primary detection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Compressed Nitrogen (Grade 5)</td>
			<td style="width:235px;"></td>
			<td style="width:227px;"></td>
			<td style="width:476px;">General dusting/drying tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Epoxy-coated slides</td>
			<td style="width:235px;">Corning</td>
			<td style="width:227px;">40044</td>
			<td style="width:476px;">Slides</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:269px;">Epoxy-coated slides</td>
			<td style="width:235px;">PolyAn</td>
			<td style="width:227px;">2D 104-00-221</td>
			<td style="width:476px;">Slides. In this type of slides the surface is more hydrophobic (compared to Coring slides) therefore the glycans Print Buffer would need to be supplemented with 0.005% Tween-20 to obtain 100 &#181;m size spots.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">384-well microtiter plate</td>
			<td style="width:235px;">Genetix</td>
			<td style="width:227px;">2070</td>
			<td style="width:476px;">Printing plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">VWR lab marker</td>
			<td style="width:235px;">VWR</td>
			<td style="width:227px;">52877-310</td>
			<td style="width:476px;">Slide labeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Staining Tube</td>
			<td style="width:235px;">ArrayIt</td>
			<td style="width:227px;">MST</td>
			<td style="width:476px;">Slide developing tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Staining bath</td>
			<td style="width:235px;">VWR</td>
			<td style="width:227px;">25608-904</td>
			<td style="width:476px;">Slide developing tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Slides glass holders</td>
			<td style="width:235px;">VWR</td>
			<td style="width:227px;">631-9321</td>
			<td style="width:476px;">Slide developing tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">GenePix Scanner</td>
			<td style="width:235px;">Molecular devices</td>
			<td style="width:227px;">4000B</td>
			<td style="width:476px;">Slide scanner</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">LM-60 NanoPrinter</td>
			<td style="width:235px;">ArrayIt</td>
			<td style="width:227px;">LM-60</td>
			<td style="width:476px;">Array printer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Pins</td>
			<td style="width:235px;">ArrayIt</td>
			<td style="width:227px;">946MP3</td>
			<td style="width:476px;">Printing pins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">ProPlate Module</td>
			<td style="width:235px;">Grace Bio-Labs</td>
			<td style="width:227px;">P37004</td>
			<td style="width:476px;">Slide developing module</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Distilled water</td>
			<td style="width:235px;">Bio-Lab</td>
			<td style="width:227px;">2321020500</td>
			<td style="width:476px;">Required for arrayer and humidifier</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:269px;">Electronic Multi Pippete, 8 Channel , volume range 2-125 &#956;L</td>
			<td style="width:235px;">Thermo-Fisher Scientific (Matrix)</td>
			<td style="width:227px;">MA-2131 Impact2 Equalizer 384</td>
			<td style="width:476px;">Multi pippete for sample dispansing into 384-well plate</td>
		</tr>
	</tbody>
</table>

profiling anti-neu5gc igg in human sera with a sialoglycan microarray assay

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5&#39;monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

Array Printing, Development, and Analysis:
Printing a sialoglycan microarray with multiple glycan samples and human IgG STD curves in 16 different blocks requires thorough calibration to ensure that all samples are printed as uniformly as possible in all 16 blocks per slide and to all slides in the same print run. Therefore, multiple calibration experiments are required before the specific printing parameters are determined, includ...

Watch this Scientific Journal Video about Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay at JoVE.com

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases.

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

The overall goal of the sialoglycan microarray assay is to evaluate Anti-Neu5Gc IgG in human sera in a high-throughput manner against a diverse collection of sialoglycans. This method can help answer key questions in the glycoimmunology field, such as how Anti-Neu5Gc antibodies are involved with various human diseases including cancer, heart diseases, and xenotransplantation. The main advantage of this technique is that it allows high-throughput profiling of Anti-Neu5Gc antibodies against a myriad of sialoglycan antigens.Demonstrating the procedure will be Sharon Yehuda, a master student from my laboratory. Careful and detailed planning of array fabrication and layout is important to ensure success. This process is detailed in the text protocol and the supplementary information.Dilute each glycan to 100 micromolar in a total volume of 100 microliters of glycan printing buffer in microcentrifuge tubes. Next, prepare primary amine containing fluorescent dye to one milligram per millimeter with marker buffer. Then, dilute to one microgram per milliliter in a one milliliter total volume.Prepare human IgG standard curve dilutions as described in the text protocol. Place the 384 well plate on ice. Using an electronic multi-pipette, aliquot seven microliters of each glycan, marker, and stardard curve IgG according to the plate layout found in the text protocol.Cover the plate with parafilm and spin down for two minutes at 250 times g and 12 degrees Celsius. Printing must be carried out in dust-free area with a humidity of 60 to 70%Here, all steps are carried out in a clean room with appropriate gloves and clothing for protection. Turn on the nano printer machine and humidifier.After programming the printing machine as described in the text protocol, position the 384 well plate with aliquoted samples into its location on the arrayer deck and remove the parafilm cover. At this point, start printing. Number the slides with an alcohol solvent-resistant marking pen, and store them in a vacuum-sealed box in the dark.To process the arrays, first remove a slide from the vacuum-sealed box and place it in a chemical hood for five minutes, array facing up, to evaporate the excess liquid. Next, hydrate the slide by placing it it in a staining tube filled with deionized water. Cover the tube with foil and incubate for five minutes with slow and gentle shaking at room temperature.To block the reactive epoxy groups on the slide, dip the slide into the 50 milliliter staining tube filled with pre-warmed ethanolamine blocking solution. Cover the tube with foil and incubate for 30 minutes with slow and gentle shaking at room temperature. Discard the ethanolamine blocking solution into the appropriate biohazard trash receptacle.Then, place the slide in a new, clean staining tube filled with 50 degree celsius deionized water. Cover the tube with foil and incubate for 10 minutes with slow and gentle shaking at room temperature. Discard the water and transfer the slides to a glass staining holder.Place the apparatus into a swinging plate holder and centrifuge the slide dry. Next, place the slide onto a 16-well divider. After pre-wetting the wells, use a multi-pipette to aliquot 200 microliters of PBST into the slide wells.Then, place them in a covered, humid chamber and shake for five minutes. Remove the PBST by flicking and tapping gently on a clean paper towel. Then, aliquot 200 microliters of PBS-Ovalbumine blocking buffer into each well.Place the slides in a humid chamber and incubate for one hour at room temperature with gentle shaking. Next, prepare the primary detection, diluted in PBS-OVA blocking buffer as described in the text protocol. Flick PBS-OVA blocking buffer off the slide and add 200 microliters of primary antibody per well.Incubate the slides in a humid chamber for two hours. Then, flick the primary detection off the slide and wash three times with PBST. Incubate the slide in PBST for five minutes in a humid chamber before washing a final time with PBST.Then, wash once with PBS. Next, prepare the secondary antibody in PBS as described in the text protocol. Flick the PBS off the slide and add 200 microliters of secondary antibody.Flick the secondary antibody off the slide and wash four times with PBST as before. Carefully remove the frame, then place the slides into a 50 milliliter slide staining tube filled with PBS and shake for five minutes. Next, fill two slide staining baths with deionized water.Place the slide into the slide holder and dip the slide into the first bath. Quickly dip the slide ten times. Then, transfer the slide to the second bath and dip ten more times.After dipping, incubate the slide for ten minutes at room temperature with shaking. Following incubation, discard the water and transfer the slide to a glass staining holder. Place the glass staining holder in a swinging plate holder and centrifuge the slide dry.Open the scanner door and place the slide inside, array facing down. At this point, start the scanning. Save the scanned slides as a TIFF file.To prepare a slide for analysis, select Load Array List"and upload the appropriate GAL file that maps the arrays in each block on the slide. Slides were developed with 16 different primary detection moieties, one in each block. The plant Lectin SNA binds a2-6 linked sialic acid.Whereas the plant Lectin MALII binds a2-3 linked sialic acid. Polyclonal Anti-Neu5Gc IgY binds Neu5Gc-containing sialoglycans but does not bind Neu5Ac-containing sialoglycans. To profile Anti-Neu5Gc IgG, 12 sera from healthy human donors were analyzed on the printed sialoglycan microarray and developed using fluorescently labeled anti-human IgG in normalized to nanograms per microliter.All tested human sera contained various levels of Anti-Neu5Gc IgG with almost no recognition of the matched pairs of Neu5Ac-containing sialoglycans. Furthermore, Anti-Neu5Gc IgG recognition patterns are highly diverse between these 12 different sera, both in the level of intensity and diversity. After watching this video, you should have a good understanding of how to profile Anti-Neu5Gc antibodies in various human samples in a high-throughput manner, using a glycan microarray system.

profiling-anti-neu5gc-igg-human-sera-with-sialoglycan-microarray

Research

JoVE Journal

Behavior

Assessing Functional Performance in the Mdx Mouse Model

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions (e.g. forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories.

Assessing Functional Performance in the Mdx Mouse Model

The primary outcome measure in clinical trials for neuromuscular disorders is generally improved muscle function. Therefore, assessing the effect of potential therapeutic compounds on muscle performance pre clinically in mouse models is of great importance. We here describe several functional tests to address this.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential ...

Measurement of Fronto-limbic Activity Using an Emotional Oddball Task in Children with Familial High Risk for Schizophrenia

Adolescence is a critical developmental period where the early symptoms of schizophrenia frequently emerge. First-degree relatives of people with schizophrenia who are at familial high risk (FHR) may show similar cognitive and emotional changes. However, the neurological changes underlying the emergence of these symptoms remain unclear. This study sought to identify differences in frontal, striatal, and limbic regions in children and adolescents with FHR using functional magnetic resonance imaging. Groups of 21 children and adolescents at FHR and 21 healthy controls completed an emotional oddball task that relied on selective attention and the suppression of task-irrelevant emotional information. The standard oddball task was modified to include aversive and neutral distractors in order to examine potential group differences in both emotional and executive processing. This task was designed specifically to allow for children and adolescents to complete by keeping the difficulty and emotional image content age-appropriate. Furthermore, we demonstrate a technique for suitable fMRI registration for children and adolescent participants. This paradigm may also be applied in future studies to measure changes in neural activity in other populations with hypothesized developmental changes in executive and emotional processing.

This paper describes how to use the emotional oddball task and fMRI to measure brain activation in children and adolescents at familial high risk for schizophrenia (FHR).&#160;FMRI was used to measure differences in fronto-striato-limbic regions during an emotional oddball task. Children with FHR exhibited abnormal functional activation during adolescence.

Adolescence is a critical developmental period where the early symptoms of schizophrenia frequently emerge. First-degree relatives of people with ...

Evaluation of a Smartphone-based Human Activity Recognition System in a Daily Living Environment

An evaluation method that includes continuous activities in a daily-living environment was developed for Wearable Mobility Monitoring Systems (WMMS) that attempt to recognize user activities. Participants performed a pre-determined set of daily living actions within a continuous test circuit that included mobility activities (walking, standing, sitting, lying, ascending/descending stairs), daily living tasks (combing hair, brushing teeth, preparing food, eating, washing dishes), and subtle environment changes (opening doors, using an elevator, walking on inclines, traversing staircase landings, walking outdoors). 
To evaluate WMMS performance on this circuit, fifteen able-bodied participants completed the tasks while wearing a smartphone at their right front pelvis. The WMMS application used smartphone accelerometer and gyroscope signals to classify activity states. A gold standard comparison data set was created by video-recording each trial and manually logging activity onset times. Gold standard and WMMS data were analyzed offline. Three classification sets were calculated for each circuit: (i) mobility or immobility, ii) sit, stand, lie, or walking, and (iii) sit, stand, lie, walking, climbing stairs, or small standing movement. Sensitivities, specificities, and F-Scores for activity categorization and changes-of-state were calculated.
The mobile versus immobile classification set had a sensitivity of 86.30% &#177; 7.2% and specificity of 98.96% &#177; 0.6%, while the second prediction set had a sensitivity of 88.35% &#177; 7.80% and specificity of 98.51% &#177; 0.62%. For the third classification set, sensitivity was 84.92% &#177; 6.38% and specificity was 98.17 &#177; 0.62. F1 scores for the first, second and third classification sets were 86.17 &#177; 6.3, 80.19 &#177; 6.36, and 78.42 &#177; 5.96, respectively. This demonstrates that WMMS performance depends on the evaluation protocol in addition to the algorithms. The demonstrated protocol can be used and tailored for evaluating human activity recognition systems in rehabilitation medicine where mobility monitoring may be beneficial in clinical decision-making.

A standardized evaluation method was developed for Wearable Mobility Monitoring Systems (WMMS) that includes continuous activities in a realistic daily living environment. Testing with a series of daily living activities can decrease activity recognition sensitivity; therefore, realistic testing circuits are encouraged for valid evaluation of WMMS performance.

An evaluation method that includes continuous activities in a daily-living environment was developed for Wearable Mobility Monitoring Systems (WMMS) that ...

Substantiating Appropriate Motion Capture Techniques for the Assessment of Nordic Walking Gait and Posture in Older Adults

Nordic walking (NW) has become a safe and simple form of exercise in recent years, and in studying this gait pattern, various data collection techniques have been employed, each with positives and negatives. The aim was to determine the effect of NW on older adult gait and posture and to determine optimal use of different data collection systems in both short and long duration analysis. Gait and posture during NW and normal walking were assessed in 17 healthy older adults (age: 69 &#177; 7.3). Participants performed two trials of 6 Minute Walk Tests (6MWT) (1 with poles (WP) and 1 without poles (NP)) and 6 trials of a 5m walk (3 WP and 3 NP). Motion was recorded using two systems, a 6-sensor accelerometry system and an 8-camera 3-dimensional motion capture system, in order to quantify spatial-temporal, kinematic, and kinetic parameters.
With both systems, participants demonstrated increased stride length and double support and decreased gait speed and cadence WP compared to NP (p &#60;0.05). Also, with motion capture, larger single support time was found WP (p &#60;0.05). With 3-D capture, smaller hip power generation and moments of force were found at heel contact and pre-swing as well as smaller knee power absorption at heel contact, pre-swing, and terminal swing WP compared to NP, when assessed over one cycle (p &#60;0.05). Also, WP yielded smaller moments of force at heel contact and terminal swing along with larger moments at mid-stance of a gait cycle (p &#60;0.05). No changes were found for posture.
NW seems appropriate for promoting a normal gait pattern in older adults. Three-dimensional motion capture should primarily be used during short duration gait analysis (i.e. single gait cycle), while accelerometry systems should be primarily employed in instances requiring longer duration analysis such as during the 6MWT.

The aim was to substantiate optimal use of data collection techniques for Nordic walking gait and posture analysis. Three-dimensional motion capture should be used during short duration analysis (i.e. single gait cycle), while accelerometry should be employed for longer duration analysis (i.e. repeated cycles) like a 6 Minute Walk Test.

Nordic walking (NW) has become a safe and simple form of exercise in recent years, and in studying this gait pattern, various data collection techniques ...

Using the Visual World Paradigm to Study Sentence Comprehension in Mandarin-Speaking Children with Autism

Sentence comprehension relies on the ability to rapidly integrate different types of linguistic and non-linguistic information. However, there is currently a paucity of research exploring how preschool children with autism understand sentences using different types of cues. The mechanisms underlying sentence comprehension remains largely unclear. The present study presents a protocol to examine the sentence comprehension abilities of preschool children with autism. More specifically, a visual world paradigm of eye-tracking is used to explore the moment-to-moment sentence comprehension in the children. The paradigm has multiple advantages. First, it is sensitive to the time course of sentence comprehension and thus can provide rich information about how sentence comprehension unfolds over time. Second, it requires minimal task and communication demands, so it is ideal for testing children with autism. To further minimize the computational burden of children, the present study measures eye movements that arise as automatic responses to linguistic input rather than measuring eye movements that accompany conscious responses to spoken instructions.

We present a protocol to examine the use of morphological cues during real-time sentence comprehension by children with autism.

Sentence comprehension relies on the ability to rapidly integrate different types of linguistic and non-linguistic information. However, there is ...

Impact of High-intensity Interval Exercise and Moderate-Intensity Continuous Exercise on the Cardiac Troponin T Level at an Early Stage of Training

An elevation in cardiac troponin T (cTnT), as a highly specific biomarker of cardiomyocyte damage, after moderate-intensity continuous exercise (MCE) has been described. The exercise-induced cTnT response distorts the diagnostic role of the cTnT assay. Although high-intensity interval exercise (HIE) is growing in popularity and concerns remain about its safety, available data related to cTnT release after HIE is limited, which hampers the use of HIE as a health intervention. Here, we present three representative HIE protocols [traditional HIE (repeated 4 min cycling at 90% V&#775;O2max interspersed with 3 min rest, 200 kJ/session); sprint interval exercise (SIE, repeated 1 min cycling at 120% V&#775;O2max interspersed with 1.5 min rest, 200 kJ/session); and repeated sprint exercise (RSE, 40 x 6 s all-out sprints interspersed with 9 s rest)] and one representative MCE protocol (continuous cycling exercise at an intensity of 60% V&#775;O2max, 200 kJ/session). Forty-seven sedentary, overweight young women were randomly assigned to one of four groups (HIE, SIE, RSE, and MCE). Six bouts of respective exercise were performed by every single group, with each being 48 h apart. Meanwhile, for four groups, the duration of the entire testing period was identical, being 10 days. Before and after the first and final exercise bouts, an assessment was conducted of cTnT. The current study provides a frame of reference giving a clear picture of how a specific exercise session affects the circulating cTnT concentration at the early stage of training. The information may assist with clinical interpretations of post-exercise cTnT elevation and guide the prescription of exercise, especially for HIE.

Here, we present protocols of high-intensity interval and moderate-intensity continuous exercise to observe the response of circulating cardiac troponin T (cTnT) concentration to acute exercise over 10 days. The information may assist with clinical interpretations of post-exercise cTnT elevation and guide the prescription of exercise.

An elevation in cardiac troponin T (cTnT), as a highly specific biomarker of cardiomyocyte damage, after moderate-intensity continuous exercise (MCE) has ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and offspring, including the development of neuropsychiatric disorders. Risk factors linked to the emergence of affective dysregulation in the maternal-infant dyad have been extensively studied. Exposure to psychosocial stress during pregnancy has consistently emerged as one of the strongest predictors. Several rodent models have been created to explore this association; however, these models rely on the use of physical stressors or a limited number of psychosocial stressors presented in a repetitive fashion, which do not accurately capture the type, intensity, and frequency of stressors experienced by women. To overcome these limitations, a chronic psychosocial stress (CGS) paradigm was generated that employs various psychosocial insults of different intensity presented in an unpredictable fashion. The manuscript describes this novel CGS paradigm where pregnant female mice, from gestational day 6.5 to 17.5, are exposed to various stressors during the day and overnight. Day stressors, two per day separated by a 2 h break, range from exposure to foreign objects or predator odor to frequent changes in bedding, removal of bedding, and cage tilting. Overnight stressors include continuous light exposure, changing cage mates, or wetting bedding. We have previously shown that exposure to CGS results in the development of maternal neuroendocrine and behavioral abnormalities, including increased stress reactivity, the emergence of fragmented maternal care patterns, anhedonia, and anxiety-related behaviors, core features of women suffering from perinatal mood and anxiety disorders. This CGS model, therefore, becomes a unique tool that can be used to elucidate molecular defects underlying maternal affective dysregulation, as well as trans-placental mechanisms that impact fetal neurodevelopment and result in negative long-term behavioral consequences in the offspring.

The chronic psychosocial stress (CGS) paradigm employs clinically relevant stressors during pregnancy in mice to model psychiatric disorders of mothers and infants. Here, we provide a step-by-step procedure of applying the CGS paradigm and downstream assessments to validate this model.

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and ...

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental factors, and the toxicological and pharmacological effects of numerous substances. Few methods currently implemented are amenable to high throughput measurement. The Microplate Feeder Assay (MFA) was developed for quantifying the consumption of liquid food for individual flies using absorbance. In this assay, flies consume liquid food medium from select wells of a 1536-well microplate. By incorporating a dilute tracer dye into the liquid food medium and loading a known volume into each well, absorbance measurements of the well acquired before and after consumption reflect the resulting change in volume (i.e., volume consumed). To enable high throughput analysis with this method, a 3D-printed coupler was designed that allows flies to be sorted individually into 96-well microplates. This device precisely orients 96- and 1536-well microplates to give each fly access to up to 4 wells for consumption, thus enabling food preference quantification in addition to regular consumption. Furthermore, the device has barrier strips that toggle between open and closed positions to allow for controlled containment and release of a column of samples at a time. This method enables high throughput measurements of consumption of aqueous solutions by many flies simultaneously. It also has the potential to be adapted to other insects and to screen consumption of nutrients, toxins, or pharmaceuticals.

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

The microplate feeder assay offers an economical, high throughput method for quantifying liquid food consumption in Drosophila. A 3D-printed device connects a 96-well microplate in which flies are housed to a 1536-well microplate from which flies consume a feeding solution with a tracer dye. The solution volume decline is measured spectrophotometrically.

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental ...

The Motivation for Alcohol Reward: Predictors of Progressive-Ratio Intravenous Alcohol Self-Administration in Humans

The Progressive Ratio (PR) self-administration paradigm is a common pre-clinical method used to examine the motivation for a drug attributed to a craving, reward, or the relief of negative affect. The Computer-assisted Alcohol Infusion System (CAIS) enables intravenous alcohol self-administration behavior in humans. This system provides the investigator with control over the trajectory of each incremental breath alcohol concentration (BrAC) reward and the maximum BrAC allowed in a session. This paradigm allows participants to earn these alcohol rewards using a sequence of button presses specified by the investigator. The system employs a physiologically-based pharmacokinetic model-based algorithm to achieve the same incremental BrAC exposure in every participant. Participants (n = 11) took part in two identical sessions to examine test-retest reliability, and an additional group (n = 73) completed a single session. Sessions began with a 25 min priming phase: participants were instructed to press a button an increasing number of times per reward, accumulating four standardized incremental BrAC trajectories. The second phase comprised an ad-lib, PR paradigm lasting 125 min. Each reward required an increasing number of button presses. Measures of self-administration included: average and peak BrAC, total rewards earned, total grams of ethanol consumed per unit of total body water, the total number of button presses, and the average rate of button pressing. Self-administration measures were highly correlated both between and within sessions, demonstrating test-retest reliability and internal consistency. Recent drinking history was strongly associated with self-administration measures; heavier drinkers chose greater alcohol self-administration. These results indicate the reliability and sensitivity of this progressive-ratio intravenous alcohol self-administration method for assessing the motivational properties of alcohol, with the potential for improved testing of the efficacy of new medications thought to reduce consumption of alcohol. This method can be used to understand the genetic and environmental determinants of alcohol self-administration in humans.

This study aims to show that the Progressive-Ratio Computer-assisted Alcohol-Infusion System (CAIS) paradigm is a reliable and sensitive method that can be used to examine the motivating properties associated with alcohol self-administration in humans.

The Progressive Ratio (PR) self-administration paradigm is a common pre-clinical method used to examine the motivation for a drug attributed to a craving, ...