Name: Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals (Video) | JoVE Journal
Uploaded: 2019-02-14T13:00:00.000+00:00
Duration: 8 min 49 s
Description: Learn printed glycan array technology for analyzing circulating anti-carbohydrate antibodies in small animal serum. This high-sensitivity method enables simultaneous measurement of hundreds of glycan targets using minimal sample volume for immunological research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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 controls (8 rows &#215; 14 columns) (Figure 1B). 
		NOTE: Glycan-related information is provided in the Supplementary Table 1. The glycan library used for printing microchips is the result of a long-term synthetic effort of the IBCh team; examples of synthesis are described in related publications29,30,31,32,33,34,35,36,37,38. The glycan library included blood group antigens and some of the most frequently occurring terminal oligosaccharides, as well as core motifs of mammalian N- and O-linked glycoproteins and glycolipids, tumor-associated carbohydrate antigens, and polysaccharides from pathogenic bacteria.</li>
		<li>Incubate the slides in moisture box (relative humidity ~70%) at room temperature (25 &#176;C) for 1 h.</li>
		<li>Blocking microarrays: incubate the slides for 1.5 h with blocking buffer at room temperature (100 mM boric acid, 25 mM ethanolamine, 0.2% (v/v) Tween-20 in ultrapure water).</li>
		<li>Wash the glycochip with ultrapure water and dry it by air.</li>
	</ol>
	</li>
	<li>Glycochip quality control
	<ol>
		<li>Analyze two microarrays from each batch using 1mg/mL solution of complex immunoglobulin preparation (CIP, containing IgG, IgM and IgA), 10 &#181;g/mL solution of biotinylated goat anti-human immunoglobulins as a secondary antibody (IgM + IgG + IgA), followed by 1 &#181;g/mL solution of the corresponding fluorescent streptavidin conjugate (via protocol described below, see step 2).</li>
		<li>Scan and analyze the glycochips (see step 3, analysis of glycan array).</li>
		<li>Use microarray batches with intra- and inter-chip correlation higher than 0.9.</li>
	</ol>
	</li>
</ol>
2. Glycan Array Technique
<ol>
	<li>Prepare the following aqueous solutions (in ultrapure water) and store them at room temperature (25 &#176;C):
	<ul>
		<li>Buffer-1: 1% (w/v) bovine serum albumin (BSA) in PBS, 1% (v/v) Tween-20 and 0.01% (w/v) NaN3</li>
		<li>Buffer-2: 1% (w/v) BSA in PBS, 0.1% (v/v) Tween-20 and 0.01% (w/v) NaN3.</li>
		<li>Buffer-3: 0.1% (v/v) Tween-20 in PBS.</li>
		<li>Buffer-4: 0.001% (v/v) Tween-20 in PBS.</li>
	</ul>
	</li>
	<li>Glycochip and sample preparation
	<ol>
		<li>Put the storage box with the slides on the table until they reach room temperature (25 &#176;C). 
		NOTE: Use powder-free latex gloves. The glycochip must be manipulated by the bottom part of the glass slide, where the barcode is located. The barcode will help you to identify the right side, avoiding the contact with the surface where the glycans are printed.</li>
		<li>Open the box, take the glycochip and place it in the incubation chamber (25 &#176;C), already conditioned with wet filter paper to keep humidity constant inside the chamber.</li>
		<li>Meanwhile, dilute the mice serum with Buffer-1 (1:20) in 1.5 mL tubes. Homogenize the serum solution (5 s) with a vortex mixer. 
		NOTE: The volume needed to totally cover a single glycochip surface is approximately 1 mL.</li>
		<li>After the homogenization, incubate the diluted serum at 37 &#176;C for 10 min in a water bath to avoid immunoglobulin aggregation. Centrifuge the tubes for 3 min at 10,000 x g and 25 &#176;C, collect the supernatant and discard any precipitated material.</li>
		<li>Place the glycochip carefully in the incubation chamber. Incubate it for 15 min at 25 &#176;C with 1 mL of Buffer-3 to eliminate any residual material on the surface of the glycochip.</li>
		<li>Hold the glycochip in a vertical position and rewash it with some drops of Buffer-3 using a plastic Pasteur pipette. Carefully remove the buffer from the glycochip surface using filter paper.</li>
	</ol>
	</li>
	<li>Reaction: antibodies binding
	<ol>
		<li>Place the glycochip in the incubation chamber. Spread the diluted serum sample over the glycochip surface using a micropipette. Incubate with orbital agitation (30 rpm) at 37 &#176;C for 1.5 h. Ensure that all dry area of the glycochip surface is covered by the diluted serum sample using the tip of the pipette.</li>
		<li>Remove any excess sample and immerse the glycochip for 5 min in Buffer-3 at 25 &#176;C. Then, move the glycochip to a container with Buffer-4 (5 min) and finally wash (5 min) the glycochip with ultrapure water. Centrifuge the glycochip for 1 min at 175 x g and 25 &#176;C to remove the liquid.</li>
	</ol>
	</li>
	<li>Detection: secondary antibody
	<ol>
		<li>Place the glycochip in the incubation chamber. Spread over the glycochip surface a solution (5 &#181;g/mL) of goat anti-mouse (IgG + IgM) conjugated to biotin in Buffer-2. Incubate with orbital agitation (30 rpm) at 37 &#176;C for 1 h.</li>
		<li>Remove the unbound fraction and repeat the washing steps.</li>
		<li>After the centrifugation, incubate the glycochip in darkness at 25 &#176;C for 45 min (30 rpm) with 2 &#181;g/mL of the corresponding fluorochrome-labeled streptavidin solution (in Buffer-2).</li>
		<li>In darkness, remove the unbound fraction and repeat the washing steps.</li>
		<li>Dry the glycochip by air. 
		Note: Glycochip should be scanned as soon as possible. But if it&#39;s impossible to do scan immediately after staining, glycochips can be stored in a cool and dry place in darkness.</li>
	</ol>
	</li>
</ol>
3. Analysis of Glycan Array
<ol>
	<li>Scan the array
	<ol>
		<li>Leave the glycochip on the table until it reaches room temperature in dark. At the same time, turn on the slide scanner and the laser (excitation wavelength of 633 nm).</li>
		<li>Holding the microarray, slide the glycochip into the slot until it touches the back.</li>
		<li>Scan the glycochip (&#34;run easy scan&#34;), and save the scan as a &#34;.TIFF&#34; file.</li>
	</ol>
	</li>
	<li>Array quantification
	<ol>
		<li>Quantify the array using a ScanArray Analysis System. Open previously scanned images, by clicking &#34;File&#34; in the &#34;Configure &#38; File group&#34; on the Main Window (Figure 1B-D)</li>
		<li>Load the corresponding array file template in GAL format (disposition of printed glycans on the glass slide) (Figure 1C).</li>
		<li>Adjust GAL template by carefully aligning the array (grids) with the spots in the image and initiate quantification (Figure 1D).</li>
		<li>Select the quantification parameters:
		<ul>
			<li>Quantification type: Run Easy Quant.</li>
			<li>Quantification method: Fixed Circle</li>
			<li>Auto find Spots: unclick all options</li>
			<li>Normalization Method: LOWESS (Locally Weighted Scatter Plot Smoothing).</li>
		</ul>
		</li>
		<li>Save the quantified data as &#34;.CSV&#34; file (Figure 1D). Transfer this data into a common spreadsheet file using Microsoft Excel or another appropriate application.</li>
		<li>Use the interquartile range (IQR) as the main statistical method: calculation of the median (Quartile 2) of all signals for each ligand and the interquartile deviation (75th and 25th percentiles, or upper and lower quartiles Q3 and Q1, respectively).</li>
		<li>Perform interactive exploration of data by using a Hierarchical Clustering Explorer application.</li>
		<li>Use clustering parameters: Average Linkage (UPGMA) and Euclidean distance as similarity distance measure. Perform hierarchical clustering by rows without normalization.</li>
	</ol>
	</li>
</ol>

<ol><li>Karlsson, G. B., Fouchier, R. A., Phogat, S., Burton, D. R., Sodroski, J., Wyatt, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+challenges+of+eliciting+neutralizing+antibodies+to+HIV-1+and+to+influenza+virus%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">The challenges of eliciting neutralizing antibodies to HIV-1 and to influenza virus.</a> Nat Rev Microbiol. 6 (2), 143-155 (2008).</li>
<li>Lu, L. L., Suscovich, T. J., Fortune, S. M., Alter, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beyond+binding%3A+antibody+effector+functions+in+infectious+diseases%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">Beyond binding: antibody effector functions in infectious diseases.</a> Nat Rev Immunol. 18 (1), 46-61 (2017).</li>
<li>Bebbington, C., Yarranton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibodies+for+the+treatment+of+bacterial+infections%3A+current+experience+and+future+prospects%5BTitle%5D)+AND+%22Curr+Opin+Biotech%22%5BJournal%5D)">Antibodies for the treatment of bacterial infections: current experience and future prospects.</a> Curr Opin Biotech. 19 (6), 613-619 (2008).</li>
<li>Murphy, K., Travers, P., Walport, M. The complement system and innate immunity. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMurphy%2C+K+%22Janeway%27s+Immunobiology%22">Janeway's Immunobiology</a>. , 7th, Garland Science. New York. 61-80 (2008).</li>
<li>Botto, M., Kirschfink, M., Macor, P., Pickering, M. C., Wurzner, R., Tedesco, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complement+in+human+diseases%3A+lessons+from+complement+deficiencies%5BTitle%5D)+AND+%22Mol+Immunol%22%5BJournal%5D)">Complement in human diseases: lessons from complement deficiencies.</a> Mol Immunol. 46 (14), 2774-2783 (2009).</li>
<li>Borrok, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancement+of+antibody-dependent+cell-mediated+cytotoxicity+by+endowing+IgG+with+Fc%CE%B1RI+%28CD89%29+binding%5BTitle%5D)+AND+%22MAbs%22%5BJournal%5D)">Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.</a> MAbs. 7 (4), 743-751 (2015).</li>
<li>Weiner, L. M., Murray, J. C., Shuptrine, C. W. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22424219">Antibody-based immunotherapy of cancer.</a> Cell. 148 (6), 1081-1084 (2012).</li>
<li>Ricklin, D., Hajishengallis, G., Yang, K., Lambris, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complement%3A+a+key+system+for+immune+surveillance+and+homeostasis%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Complement: a key system for immune surveillance and homeostasis.</a> Nat Immunol. 11 (9), 785-797 (2010).</li>
<li>Prechl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+generalized+quantitative+antibody+homeostasis+model%3A+antigen+saturation%2C+natural+antibodies+and+a+quantitative+antibody+network%5BTitle%5D)+AND+%22Clin+Transl+Immunology%22%5BJournal%5D)">A generalized quantitative antibody homeostasis model: antigen saturation, natural antibodies and a quantitative antibody network.</a> Clin Transl Immunology. 6 (2), e131(2017).</li>
<li>Vojdani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibodies+as+predictors+of+complex+autoimmune+diseases%5BTitle%5D)+AND+%22Int+J+Immunopath+Ph%22%5BJournal%5D)">Antibodies as predictors of complex autoimmune diseases.</a> Int J Immunopath Ph. 21 (2), 267-278 (2008).</li>
<li>Liu, W., Peng, B., Lu, Y., Xu, W., Qian, W., Zhang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoantibodies+to+tumor-associated+antigens+as+biomarkers+in+cancer+immunodiagnosis%5BTitle%5D)+AND+%22Autoimmun+Rev%22%5BJournal%5D)">Autoantibodies to tumor-associated antigens as biomarkers in cancer immunodiagnosis.</a> Autoimmun Rev. 10 (6), 331-335 (2011).</li>
<li>Suurmond, J., Diamond, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoantibodies+in+systemic+autoimmune+diseases%3A+specificity+and+pathogenicity%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Autoantibodies in systemic autoimmune diseases: specificity and pathogenicity.</a> J Clin Invest. 125 (6), 2194-2202 (2015).</li>
<li>Bovin, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repertoire+of+human+natural+anti-glycan+immunoglobulins.+Do+we+have+auto-antibodies%3F%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Repertoire of human natural anti-glycan immunoglobulins. Do we have auto-antibodies?</a> Biochim Biophys Acta. 1820 (9), 1373-1382 (2012).</li>
<li>de los Rios, M., Criscitiello, M. F., Smider, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+genetic+diversity+in+antibody+repertoires+from+diverse+species%5BTitle%5D)+AND+%22Curr+Opin+Struc+Biol%22%5BJournal%5D)">Structural and genetic diversity in antibody repertoires from diverse species.</a> Curr Opin Struc Biol. 33, 27-41 (2015).</li>
<li>Pochechueva, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+printed+glycan+array%2C+suspension+array+and+ELISA+in+the+detection+of+human+anti-glycan+antibodies%5BTitle%5D)+AND+%22Glycoconjugate+J%22%5BJournal%5D)">Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.</a> Glycoconjugate J. 28 (8-9), 507-517 (2011).</li>
<li>Shilova, N., Navakouski, M., Khasbiullina, N., Blixt, O., Bovin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Printed+glycan+array%3A+antibodies+as+probed+in+undiluted+serum+and+effects+of+dilution%5BTitle%5D)+AND+%22Glycoconjugate+J%22%5BJournal%5D)">Printed glycan array: antibodies as probed in undiluted serum and effects of dilution.</a> Glycoconjugate J. 29 (2-3), 87-91 (2012).</li>
<li>Manimala, J. C., Roach, T. A., Li, Z., Gildersleeve, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+carbohydrate+microarray+profiling+of+27+antibodies+demonstrates+widespread+specificity+problems%5BTitle%5D)+AND+%22Glycobiology%22%5BJournal%5D)">High-throughput carbohydrate microarray profiling of 27 antibodies demonstrates widespread specificity problems.</a> Glycobiology. 17 (8), 17C-23C (2007).</li>
<li>Jacob, F., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/21351089">Serum anti-glycan antibody detection of non-mucinous ovarian cancers by using a printed glycan array.</a> Int. J. Cancer. 130 (1), 138-146 (2012).</li>
<li>Lewallen, D. M., Siler, D., Iyer, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factors+affecting+protein-glycan+specificity%3A+effect+of+spacers+and+incubation+time%5BTitle%5D)+AND+%22ChemBioChem%22%5BJournal%5D)">Factors affecting protein-glycan specificity: effect of spacers and incubation time.</a> ChemBioChem. 10 (9), 1486-1489 (2009).</li>
<li>Oyelaran, O., Li, Q., Farnsworth, D., Gildersleeve, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microarrays+with+varying+carbohydrate+density+reveal+distinct+subpopulations+of+serum+antibodies%5BTitle%5D)+AND+%22J.+Proteome+Res%22%5BJournal%5D)">Microarrays with varying carbohydrate density reveal distinct subpopulations of serum antibodies.</a> J. Proteome Res. 8 (7), 3529-3538 (2009).</li>
<li>Pochechueva, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplex+suspension+array+for+human+anti-carbohydrate+antibody+profiling%5BTitle%5D)+AND+%22Analyst%22%5BJournal%5D)">Multiplex suspension array for human anti-carbohydrate antibody profiling.</a> Analyst. 136 (3), 560-569 (2011).</li>
<li>Chinarev, A. A., Galanina, O. E., Bovin, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biotinylated+multivalent+glycoconjugates+for+surface+coating%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Biotinylated multivalent glycoconjugates for surface coating.</a> Methods Mol Biol. 600, 67-78 (2010).</li>
<li>Huflejt, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-carbohydrate+antibodies+of+normal+sera%3A+findings%2C+surprises+and+challenges%5BTitle%5D)+AND+%22Mol+Immunol%22%5BJournal%5D)">Anti-carbohydrate antibodies of normal sera: findings, surprises and challenges.</a> Mol Immunol. 46 (15), 3037-3049 (2009).</li>
<li>Buchs, J. P., Nydegger, U. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+an+ABO-ELISA+for+the+quantitation+of+human+blood+group+anti-A+and+anti-B+IgM+and+IgG+antibodies%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Development of an ABO-ELISA for the quantitation of human blood group anti-A and anti-B IgM and IgG antibodies.</a> J Immunol Methods. 118 (1), 37-46 (1989).</li>
<li>de Jager, W., Rijkers, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Solid-phase+and+bead-based+cytokine+immunoassay%3A+a+comparison%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Solid-phase and bead-based cytokine immunoassay: a comparison.</a> Methods. 38 (4), 294-303 (2006).</li>
<li>Galanina, O. E., Mecklenburg, M., Nifantiev, N. E., Pazynina, G. V., Bovin, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GlycoChip%3A+multiarray+for+the+study+of+carbohydrate+binding+proteins%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">GlycoChip: multiarray for the study of carbohydrate binding proteins.</a> Lab Chip. 3 (4), 260-265 (2003).</li>
<li>Willats, W. G., Rasmussen, S. E., Kristensen, T., Mikkelsen, J. D., Knox, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sugar-coated+microarrays%3A+a+novel+slide+surface+for+the+high-throughput+analysis+of+glycans%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Sugar-coated microarrays: a novel slide surface for the high-throughput analysis of glycans.</a> Proteomics. 2 (12), 1666-1671 (2002).</li>
<li>Bello-Gil, D., Khasbiullina, N., Shilova, N., Bovin, N., Mañez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repertoire+of+BALB%2Fc+mice+natural+anti-Carbohydrate+antibodies%3A+mice+vs.+humans+difference%2C+and+otherness+of+individual+animals%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Repertoire of BALB/c mice natural anti-Carbohydrate antibodies: mice vs. humans difference, and otherness of individual animals.</a> Front Immunol. 8, 1449(2017).</li>
<li>Pazynina, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+glyco-O-sulfatome+for+profiling+of+human+natural+antibodies%5BTitle%5D)+AND+%22Carbohydr+Res%22%5BJournal%5D)">Synthetic glyco-O-sulfatome for profiling of human natural antibodies.</a> Carbohydr Res. 445, 23-31 (2017).</li>
<li>Ryzhov, I. M., Korchagina, E. Y., Popova, I. S., Tyrtysh, T. V., Paramonov, A. S., Bovin, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Block+synthesis+of+A+%28type+2%29+and+B+%28type+2%29+tetrasaccharides+related+to+the+human+ABO+blood+group+system%5BTitle%5D)+AND+%22Carbohydr+Res%22%5BJournal%5D)">Block synthesis of A (type 2) and B (type 2) tetrasaccharides related to the human ABO blood group system.</a> Carbohydr Res. 430, 59-71 (2016).</li>
<li>Ryzhov, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Function-spacer-lipid+constructs+of+Lewis+and+chimeric+Lewis%2FABH+glycans.+Synthesis+and+use+in+serological+studies%5BTitle%5D)+AND+%22Carbohyd+Res%22%5BJournal%5D)">Function-spacer-lipid constructs of Lewis and chimeric Lewis/ABH glycans. Synthesis and use in serological studies.</a> Carbohyd Res. 435, 83-96 (2016).</li>
<li>Pazynina, G. V., Tsygankova, S. V., Sablina, M. A., Paramonov, A. S., Tuzikov, A. B., Bovin, N. V. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S0959943616300992">Stereo- and regio-selective synthesis of spacer armed α2-6 sialooligosaccharides.</a> Mendeleev Commun. 26 (5), 380-382 (2016).</li>
<li>Pazynina, G. V., Tsygankova, S. V., Sablina, M. A., Paramonov, A. S., Formanovsky, A. A., Bovin, N. V. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S0959943616000547">Synthesis of blood group pentasaccharides ALey, BLey and related tri- and tetrasaccharides.</a> Mendeleev Commun. 26 (2), 103-105 (2016).</li>
<li>Severov, V. V., Pazynina, G. V., Ovchinnikova, T. V., Bovin, N. V. <a target="_blank" href="https://link.springer.com/article/10.1134/S1068162015020120">The synthesis of oligosaccharides containing internal and terminal Galβ1-3GlcNAcβ fragments.</a> Russian J. Bioorgan. Chem. 41 (2), 147-160 (2015).</li>
<li>Pazynina, G. V., Tsygankova, S. V., Bovin, N. V. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S0959943615001212">Synthesis of glycoprotein N-chain core fragment GlcNAcβ1-4(Fucα1-6)GlcNAc.</a> Mendeleev Commun. 25 (4), 250-251 (2015).</li>
<li>Solís, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+guide+into+glycosciences%3A+How+chemistry%2C+biochemistry+and+biology+cooperate+to+crack+the+sugar+code%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">A guide into glycosciences: How chemistry, biochemistry and biology cooperate to crack the sugar code.</a> Biochim Biophys Acta. 1850 (1), 186-235 (2015).</li>
<li>Pazynina, G. V., et al. <a target="_blank" href="https://www.researchgate.net/publication/273432327_Divergent_Strategy_for_the_Synthesis_of_a2-3-Linked_Sialo-oligosaccharide_Libraries_Using_a_Neu5TFA-a2-3-Gal_Building_Block">Divergent strategy for the synthesis of α2-3-Linked sialo-oligosaccharide libraries using a Neu5TFA-(α2-3)-Gal building block.</a> Synlett. 24 (02), 226-230 (2013).</li>
<li>Blixt, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Printed+covalent+glycan+array+for+ligand+profiling+of+diverse+glycan+binding+proteins%5BTitle%5D)+AND+%22P+Natl+Acad+Sci+USA%22%5BJournal%5D)">Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.</a> P Natl Acad Sci USA. 101 (49), 17033-17038 (2004).</li>
<li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+minimum+information+required+for+a+glycomics+experiment+%28MIRAGE%29+project%3A+improving+the+standards+for+reporting+glycan+microarray-based+data%5BTitle%5D)+AND+%22Glycobiology%22%5BJournal%5D)">The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data.</a> Glycobiology. 27 (4), 280-284 (2017).</li>
<li>Song, X., Heimburg-Molinaro, J., Cummings, R. D., Smith, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemistry+of+natural+glycan+microarrays%5BTitle%5D)+AND+%22Curr+Opin+Chem+Biol%22%5BJournal%5D)">Chemistry of natural glycan microarrays.</a> Curr Opin Chem Biol. 18, 70-77 (2014).</li>
<li>Hoy, Y. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Variation+in+taxonomic+composition+of+the+fecal+microbiota+in+an+inbred+mouse+strain+across+individuals+and+time%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Variation in taxonomic composition of the fecal microbiota in an inbred mouse strain across individuals and time.</a> PLoS One. 10 (11), e0142825(2015).</li>
<li>D'Argenio, V., Salvatore, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+the+gut+microbiome+in+the+healthy+adult+status%5BTitle%5D)+AND+%22Clin+Chim+Acta%22%5BJournal%5D)">The role of the gut microbiome in the healthy adult status.</a> Clin Chim Acta. 451 (Pt A), 97-102 (2015).</li>
<li>Khasbiullina, N. R., Bovin, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypotheses+of+the+origin+of+natural+antibodies%3A+a+glycobiologist%27s+opinion%5BTitle%5D)+AND+%22Biochemistry+%28Mosc%29%22%5BJournal%5D)">Hypotheses of the origin of natural antibodies: a glycobiologist's opinion.</a> Biochemistry (Mosc). 80 (7), 820-835 (2015).</li>
<li>Butler, J. E., Sun, J., Weber, P., Navarro, P., Francis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibody+repertoire+development+in+fetal+and+newborn+piglets%2C+III.+Colonization+of+the+gastrointestinal+tract+selectively+diversifies+the+preimmune+repertoire+in+mucosal+lymphoid+tissues%5BTitle%5D)+AND+%22Immunology%22%5BJournal%5D)">Antibody repertoire development in fetal and newborn piglets, III. Colonization of the gastrointestinal tract selectively diversifies the preimmune repertoire in mucosal lymphoid tissues.</a> Immunology. 100 (1), 119-130 (2000).</li>
<li>Bos, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serum+immunoglobulin+levels+and+naturally+occurring+antibodies+against+carbohydrate+antigens+in+germ-free+BALB%2Fc+mice+fed+chemically+defined+ultrafiltered+diet%5BTitle%5D)+AND+%22Eur+J+Immunol%22%5BJournal%5D)">Serum immunoglobulin levels and naturally occurring antibodies against carbohydrate antigens in germ-free BALB/c mice fed chemically defined ultrafiltered diet.</a> Eur J Immunol. 19 (12), 2335-2339 (1980).</li>
<li>van der Heijden, P. J., Bianchi, A. T., Heidt, P. J., Stok, W., Bokhout, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Background+%28spontaneous%29+immunoglobulin+production+in+the+murine+small+intestine+before+and+after+weaning%5BTitle%5D)+AND+%22J+Reprod+Immunol%22%5BJournal%5D)">Background (spontaneous) immunoglobulin production in the murine small intestine before and after weaning.</a> J Reprod Immunol. 15 (3), 217-227 (1989).</li>
<li>Krasnova, L., Wong, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+the+chemistry+and+biology+of+glycosylation+with+glycan+synthesis%5BTitle%5D)+AND+%22Annu+Rev+Biochem%22%5BJournal%5D)">Understanding the chemistry and biology of glycosylation with glycan synthesis.</a> Annu Rev Biochem. 85, 599-630 (2016).</li>
<li>Overkleeft, H. S., Seeberger, P. H., et al. Chemoenzymatic synthesis of glycans and glycoconjugates. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aOverkleeft%2C+HS+%22Essentials+of+Glycobiology+%5BInternet%5D%22">Essentials of Glycobiology [Internet]</a>. Varki, A., et al. , 3rd, Cold Spring Harbor Laboratory Press. Cold Spring Harbor (NY). Chapter 54 2015-2017 (2017).</li>
</ol>

Nailya Khasbiullina and Alexey Nokel are employees of Semiotik LLC, which is the supplier of the glycochips used in this study.

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

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...

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 ...

printed-glycan-array-sensitive-technique-for-analysis-repertoire

Research

JoVE Journal

Immunology and Infection

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.

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

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.

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

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.

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

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

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.

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

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Microarray polymer profiling (MAPP) is a high-throughput technique for compositional analysis of glycans in biological samples.

Microarray Polymer Profiling (MAPP) for High-Throughput Glycan Analysis

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses.  This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Learn printed glycan array technology for analyzing circulating anti-carbohydrate antibodies in small animal serum. This high-sensitivity method enables simultaneous measurement of hundreds of glycan targets using minimal sample volume for immunological research.

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals (Video) | JoVE Journal

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

Explore More Videos