Name: printed glycan array: a sensitive technique for the analysis of the repertoire of circulating anti-carbohydrate antibodies in small animals
Uploaded: 2019-02-14T13:00:00
Description: Watch this Scientific Journal Video about Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals at JoVE.com

This work was supported by &#34;Fondo de Investigaciones Sanitarias&#34; (FIS) grant PI13/01098 from Carlos III Health Institute, Spanish Ministry of Health. DB-G was benefited from a post-doctoral research position funded by the European Union Seventh Framework Programme (FP7/2007-2013) under the Grant Agreement 603049 (TRANSLINK). Work of NK, NS, and NB was supported by grant #14-50-00131 of Russian Science Foundation. DB-G wants to express his gratitude to Marta Broto, J. Pablo Salvador and Ana Sanchis for excellent technical assistance, and Alexander Rakitko for assistance in statistical analysis.&#160;With the support of the &#34;Pla de Doctorats Industrials de la Secretaria d&#39;Universitats i Recerca del Departament d&#39;Empresa i Coneixement de la Generalitat de Catalunya (grant number 2018 DI 021). We thank CERCA Programme / Generalitat de Catalunya for institutional support.

1. Glycochips Production
<ol>
	<li>Microarray preparation
	<ol>
		<li>Print the glycans (50 mM) and polysaccharides (10 &#181;g/mL) in 300 mM phosphate buffered saline (PBS, pH 8.5) at 6 replicates onto N-hydroxysuccinimide-derivatized glass slides, using non-contact robotic arrayer (drop volume ~900 pL). Each slide contains 4 different blocks of sub-arrays (Figure 1A, in colors) repeated 6 times. Every single sub-array is formed by 112 different glycan spots, including co...

Glycan microarrays have become indispensable tools for studying protein-glycan interactions40. The present work describes a protocol based on PGA technology to study the repertoire of circulating of anti-carbohydrate antibodies in BALB/c mice. Since PGA offers the possibility to screen large numbers of biologically unknown glycans, it is an exceptionally convenient discovery tool13,15,28. The proposed met...

Antibodies play a central role in our defense against invading pathogens by directly neutralizing viruses1,2 and bacteria2,3, by activating the complement system4,5 and the enhancement of phagocytosis6. Additionally, they are essential elements in cancer targeting and elimination of malignant cells7, and homeostasis maintenance8,9.
Disorders of the immune system can result in autoimmune and inflammatory diseases10 and cancer11. All these pathological conditions ideally demand a prompt diagnosis for an efficient treatment. In the case of autoimmune disorders, the serological presence of autoantibodies in most of the cases is a predictor for diagnostic of autoimmunity10,12. These antibodies react with the cell surface and extracellular autoantigens and, they are often present for many years before the presentation of autoimmune disease10,12. Immune deficiencies and cancer are also diagnosed with blood tests that either measure the level of immune elements such as antibodies, or their functional activity11.
The identification of the repertoire of circulating antibodies and their serological levels are paramount to set a prognosis and evaluate the progression of all of the mentioned pathological conditions. We have previously demonstrated the potential of PGA technique for the analysis of circulating antibodies in different animal species13-16, minimizing the use of large volumes of serological samples, avoiding the problem associated with antibodies cross-reactivity17 and allowing high-throughput profiling of an extensive repertoire of antibodies15.
Glycan-based immunoassays are mainly conditioned, among other factors, by the origin and production of carbohydrates, which determine the affinity and binding of ligands15,18,19,20,21. Glycan-based immunoassays can be developed in suspension (microspheres)15,21,22 or in flat-activated surfaces15,21,22,23,24. The last include ELISA (the most conventional of these methods) and PGA. There is not much data comparing these methodologies in the same experimental setting15,25,26,27. We have previously compared the efficacy and selectivity of these immunoassays to profile anti-glycan antibodies in individual human plasma samples15. For some antibodies such as those targeting anti-A/B blood group, all the immunoassays could detect them with statistical significance and they positively correlated with each other15,18,21. Meanwhile, anti-P1 antibodies were primarily detected by PGA with the highest discriminative power, and there was no correlation in the determinations made by the different glycan-based immunoassays15,18,21. These differences between methods were mainly related to the antibody/antigen ratio and glycan orientation15. ELISA and suspension arrays are more susceptible to unspecific binding than PGA because there is an excess of antigen over antibodies in these methods15. Additionally, the orientation of glycans in the PGA is more restricted than in ELISA and suspension arrays15. ELISA is convenient when the study includes a limited panel of glycans. Along with suspension arrays, ELISA offers broader flexibility regarding assay reconfiguration. PGA is exceptionally convenient for discovery approaches15,18,21,28. Despite these clear advantages and disadvantages, the three mentioned immunoassays could be used to study different aspects of glycan-antibody interactions. The final goal of the study is the one will guide the selection of the more suitable methodology.
The present work aims to extend the use of PGA technology for the analysis of the repertoire of circulating anti-glycan antibodies in small animals. As a representative result, we present here a detailed protocol to assess the repertoire of natural anti-carbohydrate antibodies in adult BALB/c mice by PGA.

<table border="0" cellpadding="0" cellspacing="0" width="1138">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Antibodies</td>
			<td style="width:392px;"></td>
			<td style="width:335px;"></td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">biotinylated goat anti-human Igs</td>
			<td style="width:392px;">Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:335px;">Ref. #: 31782</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">biotinylated goat anti-mouse IgM + IgG</td>
			<td style="width:392px;">Thermo Fisher Scientific</td>
			<td style="width:335px;">Ref. #: 31807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Equipment</td>
			<td style="width:392px;"></td>
			<td style="width:335px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Robotic Arrayer sciFLEXARRAYER S5&#160;</td>
			<td style="width:392px;">Scienion AG, Berlin, Germany</td>
			<td style="width:335px;">http://www.scienion.com/products/sciflexarrayer/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Stain Tray (slide incubation chamber)</td>
			<td style="width:392px;">Simport, Beloeil, QC, Canada</td>
			<td style="width:335px;">Ref. #: M920-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Centrifuge</td>
			<td style="width:392px;">Eppendorf, Hamburg, Germany&#160;</td>
			<td style="width:335px;">Ref. #: 5810 R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Pipettes</td>
			<td style="width:392px;">Gilson, Middleton, WI, USA</td>
			<td style="width:335px;">http://www.gilson.com/en/Pipette/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Slide Scanner&#160;</td>
			<td style="width:392px;">PerkinElmer, Waltham, MA, USA</td>
			<td style="width:335px;">ScanArray GX Plus&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Shaking incubator</td>
			<td style="width:392px;">Cole-Parmer, Staffordshire, UK</td>
			<td style="width:335px;">Ref. #: SI50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Biological samples</td>
			<td style="width:392px;"></td>
			<td style="width:335px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">BALB/c mice sera</td>
			<td style="width:392px;">This paper</td>
			<td style="width:335px;">N/ A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Complex Immunoglobulin Preparation (CIP)</td>
			<td style="width:392px;">Immuno-Gem, Moscow, Russia</td>
			<td style="width:335px;">http://www.biomedservice.ru/price/goods/1/17531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:1059px;">Chemicals, Reagents and Glycans&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Glycan library</td>
			<td style="width:392px;">Institute of Bioorganic Chemistry (IBCh), Moscow, Russia</td>
			<td style="width:335px;">N/ A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Bovine serum albumin (BSA)</td>
			<td style="width:392px;">Sigma-Aldrich, St. Louis, MO,&#160;</td>
			<td style="width:335px;">Ref. #: A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Ethanolamine</td>
			<td style="width:392px;">Sigma-Aldrich</td>
			<td style="width:335px;">Ref. #: 411000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Tween-20</td>
			<td style="width:392px;">Merck Chemicals &#38; Life Science S.A., Madrid, Spain</td>
			<td style="width:335px;">Ref. #: 655204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Phospahte buffered saline (PBS)</td>
			<td style="width:392px;">VWR International Eurolab S.L, Barcelona, Spain</td>
			<td style="width:335px;">Ref. #: E404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Sodium azide</td>
			<td style="width:392px;">Sigma-Aldrich</td>
			<td style="width:335px;">Ref. #: S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Streptavidin Alexa Fluor 555 conjugate&#160;</td>
			<td style="width:392px;">Thermo Fisher Scientific</td>
			<td style="width:335px;">Ref. #: S21381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Streptavidin Cy5 conjugate</td>
			<td style="width:392px;">GE Healthcare, Little Chalfont, Buckinghamshire, UK</td>
			<td style="width:335px;">Ref. #: PA45001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:1059px;">Materials</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">N-hydroxysuccinimide-derivatized glass slides H&#160;</td>
			<td style="width:392px;">Schott-Nexterion, Jena, Germany</td>
			<td style="width:335px;">Ref. #: 1070936</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Whatman filter paper&#160;</td>
			<td style="width:392px;">Sigma-Aldrich</td>
			<td style="width:335px;">Ref. #: WHA10347509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">1.5 mL tubes</td>
			<td style="width:392px;">Eppendorf&#160;</td>
			<td style="width:335px;">Ref. #: 0030120086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:1059px;">Software and algorithms</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:332px;">ScanArray Express Microarray Analysis System</td>
			<td rowspan="2" style="width:392px;">PerkinElmer</td>
			<td style="width:335px;">http://www.per</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">kinelmer.com/microarray</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Hierarchical Clustering Explorer application</td>
			<td style="width:392px;">University of Maryland, MD, USA</td>
			<td style="width:335px;">http://www.cs.umd.edu/hcil/hce/</td>
			<td></td>
		</tr>
	</tbody>
</table>

printed glycan array: a sensitive technique for the analysis of the repertoire of circulating anti-carbohydrate antibodies in small animals

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the individual immune condition determines the success in combating internal and external potential threat signals, but also the existence of a particular pattern of circulating anti-glycan antibodies (and their serological level variation) could be a significant marker of the onset and progression of certain pathological conditions. Here, we describe a Printed Glycan Array (PGA)-based methodology that offers the opportunity to measure hundreds of glycan targets with very high sensitivity; using a minimal amount of sample, which is a common restriction present when small animals (rats, mice, hamster, etc.) are used as models to address aspects of human diseases. As a representative example of this approach, we show the results obtained from the analysis of the repertoire of natural anti-glycan antibodies in BALB/c mice. We demonstrate that each BALB/c mouse involved in the study, despite being genetically identical and maintained under the same conditions, develops a particular pattern of natural anti-carbohydrate antibodies. This work claims to expand the use of PGA technology to investigate repertoire (specificities) and the levels of circulating anti-carbohydrates antibodies, both in health and during any pathological condition.

Here, we present a summary of representative results obtained from the quantification of the repertoire of natural anti-glycan antibodies in a population of 20 BALB/c mice. The glycochips used in this study contained 419 different glycan structures. Most glycans were synthesized as -CH2CH2CH2NH2 spacer-armed O-glycosides, in several cases as -CH2CH2NH2 or -NHCOCH2NH2 glycosides. All...

Watch this Scientific Journal Video about Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals at JoVE.com

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals

This work shows the potential of printed glycan array (PGA) technology for the analysis of circulating anti-carbohydrate antibodies in small animals.

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the ...

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