Determination of antibodies binding specificities by printed glycan array technology

doi:10.1038/protex.2017.060

Method Article

Determination of antibodies binding specificities by printed glycan array technology

https://doi.org/10.1038/protex.2017.060

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posted 23 Jun, 2017

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The present protocol is aimed to determine the natural anti-carbohydrate antibody repertoire (binding specificities) of alphaGal-KO mice. Glycochips (microchips printed with 577 different carbohydrate structures) were printed by Semiotik LLC (Moscow, Russia) using a collection of different amine-functionalized glycans and bacterial polysaccharides covalently coupled to N-hydroxysuccinimide-derivatized glass slides (slide H, Schott-Nexterion, Mainz, Germany). 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 with fully characterized structures (Zelinsky Institute of Organic chemistry, Moscow, Russia).

Biological techniques

Computational biology and bioinformatics

printed glycan array

glycochips

anti-carbohydrate antibodies

The Printed Glycan Array (PGA) technology has a high sensitivity and offers the possibility to analyze hundred of different glycan antigens to explore circulating natural anti-carbohydrate antibodies in different species (Pochechueva et al., 2011; Bovin et al., 2012; Shilova et al., 2012; de los Rios et al., 2015). The present work is aimed to describe the natural anti-carbohydrate antibody repertoire (binding specificities) of alphaGal-KO mice.

1- Glycochips (Semiotik LLC, Moscow, Russia).

2-alphaGal-KO mouse serum.

3- Phosphate saline buffer (PBS, Sigma-Aldrich, St. Louis, MO, USA).

4-Tween-20 (Sigma-Aldrich, St. Louis, MO, USA).

5- Whatman® filter paper (Sigma-Aldrich, St. Louis, MO, USA).

6- bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) 7- Goat anti-mouse IgG+IgM (H+L) conjugated to biotin (Thermo Fisher Scientific, Waltham, MA, USA)

8-Streptavidin labeled with Cy5 dye (GE Healthcare, Little Chalfont, Buckinghamshire, UK).

1- Slide incubation chamber

2- Thermostat (37 °C)

3- Orbital shaker (30 rpm, 37 °C)

3-Centrifuge with rotor for microplates and slide holder.

4- ScanArray GX Plus scanner (PerkinElmer, Waltham, MA, USA) with a laser (excitation wavelength of 633 nm) or similar.

5- Pipettes

6- High-speed centrifuge for vials

Glycochips (microchips printed with 577 different carbohydrate structures, Semiotik LLC Moscow, Russia) were incubated in an incubation chamber for 15 min at 25 °C with PBS plus 0.1% (v/v) Tween-20 (buffer 3) (Sigma-Aldrich, St. Louis, MO, USA), and the buffer was then carefully removed from the microchip surface using Whatman® filter paper (Sigma-Aldrich, St. Louis, MO, USA). αGal-KO mouse serum was diluted (1:15) in PBS plus 1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) and 1% (v/v) Tween-20 (buffer 1). Diluted serum was spread over the slide surface and incubated with agitation (30 rpm) at 37 °C for 1.5 h. After a round of washing steps with buffer 3, buffer 4 (PBS with 0.001% v/v Tween-20), and distilled water (Milli-Q grade), the glycochips were drained by mild centrifugation (1 min, 175g, Eppendorf, Hamburg, Germany). The glycochips were then incubated for 1 h at 37 °C (30 rpm) with goat anti-mouse IgG+IgM (H+L) conjugated to biotin (Thermo Fisher Scientific, Waltham, MA, USA) and diluted 200-fold in PBS plus 1% BSA and 0.1% Tween-20 (buffer 2). The unbound fraction was removed by repeating the same round of washing steps. Glass slides were incubated in darkness at 25 °C for 45 min (30 rpm) with streptavidin labeled with Cy5 dye (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and diluted 1:500 in buffer 2. After another round of washing, the glycochips were dried by airflow in darkness. Finally, the glycochips were scanned using a ScanArray GX Plus scanner (PerkinElmer, Waltham, MA, USA) with a laser (excitation wavelength of 633 nm). All data analysis was performed with the ScanArray® Express Microarray Analysis System (PerkinElmer, Waltham, MA, USA). Interactive exploration of multidimensional data (heat mapping and clustering analysis) was performed with the Hierarchical Clustering Explorer (HCE) application developed by the University of Maryland, Maryland, USA (http://www.cs.umd.edu/hcil/hce/).

5 hours

Bovin, N., Obukhova, P., Shilova, N., Rapoport, E., Popova, I., Navakouski, M., et al. (2012). Repertoire of human natural anti-glycan immunoglobulins. Do we have auto-antibodies? Biochim Biophys Acta. 1820(9): 1373-82. doi: 10.1016/j.bbagen.2012.02.005
Pochechueva, T., Jacob, F., Goldstein, D.R., Huflejt, M.E., Chinarev, A., Caduff, R., et al. (2011). Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies. Glycoconj J. 28(8-9): 507-17. doi: 10.1007/s10719-011-9349-y.
Shilova, N., Navakouski, M., Khasbiullina, N., Blixt, O., Bovin, N. (2012). Printed glycan array: antibodies as probed in undiluted serum and effects of dilution. Glycoconj J. 29(2-3): 87-91. doi: 10.1007/s10719-011-9368-8.

Authors declare no conflicting financial interests

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Determination of antibodies binding specificities by printed glycan array technology

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