Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Abstract

Source: Olivera-Ardid, S. et al., Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals. J. Vis. Exp. (2019)

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.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Glycochips Production Microarray preparation Print the glycans (50 mM) and polysaccharides (10 µg/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 block…

Representative Results

Figure 1: Schematic representation (not at scale) of the glycan array configuration, printing, and analysis. (A) Printed microchips are developed with a library of 419 different glycan structures, followed by the detection with an appropriate fluorescently labeled secondary antibody. Each slide contains 4 different blocks of sub-arrays (in colors), repeated 6 times. Every si…

Materials

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