Name: profiling anti-neu5gc igg in human sera with a sialoglycan microarray assay
Uploaded: 2017-07-13T15:00:00
Description: Watch this Scientific Journal Video about Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay at JoVE.com

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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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) ...

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