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

Summary

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

Abstract

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.

Introduction

Antibodies play a central role in our defense against invading pathogens by directly neutralizing viruses^1,2 and bacteria^2,3, by activating the complement system^4,5 and the enhancement of phagocytosis⁶. Additionally, they are essential elements in cancer targeting and elimination of malignant cells⁷, and homeostasis maintenance^8,9.

Disorders of the immune system....

Protocol

1. Glycochips Production

1. Microarray preparation
   1. 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 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 × 14 columns) (Figure 1B).
      NOTE: Glycan-related information is provided in the Suppl....

Results

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.......

Discussion

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 tool^13,15,28. The proposed met.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Hierarchical Clustering Explorer application	University of Maryland, MD, USA	http://www.cs.umd.edu/hcil/hce/