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

Summary

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.

Abstract

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5'monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

Introduction

Sias are acidic sugars covering glycan chains on cell-surface glycoproteins and glycolipids in vertebrates. Sia expression is modified in cancer cells¹ and correlates with progression and/or metastasis^2,3. Two of the major Sia forms in mammals are Neu5Ac and its hydroxylated form, Neu5Gc². Humans cannot synthesize Neu5Gc due to a specific inactivation of the gene encoding the CMAH enzyme . This non-human Sia metabolically incorporates into human cells as "self," originating from dietary Neu5Gc-rich foods (e.g., red meat)⁴<....

Protocol

Human sera samples were obtained from the Israeli Blood Bank and were used in accordance with the Helsinki declaration and Tel Aviv University Institutional Review Board.

1. Array Fabrication Planning and Layout

1. Determine the slide layout.
   NOTE: Each slide contains 16 sub-arrays divided into 16 identical blocks numbered B1 to B16 (Figure 1B, Supplementary Figure 1A).
2. Determine the pin layout. Use four pins, each printing four sub-arrays (blocks) per slide:
   Pin 1 prints Blocks 1, 2, 9, and 10;
   Pin 2 prints Blocks 3, 4, 11, and 12;
   Pin 3 prints Bl....

Results

Array Printing, Development, and Analysis:

Printing a sialoglycan microarray with multiple glycan samples and human IgG STD curves in 16 different blocks requires thorough calibration to ensure that all samples are printed as uniformly as possible in all 16 blocks per slide and to all slides in the same print run. Therefore, multiple calibration experiments are required before the specific printing parameters are determined, includ.......

Discussion

A successful glycan microarray fabrication requires careful planning and includes several important steps in the protocol. These include: (1) planning the block and plate layouts that define all subsequent parameters (e.g., distances, spacing, amount of samples, and printing); (2) cleaning the pins and ensuring pin integrity, which is critical for controlling spot homogeneity; (3) maintaining high humidity during printing, critical to avoiding sample evaporation during long print runs, which could compromise spo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Primary-amine containing sialoglycans	Glycohub, Inc., Davis, CA, USA (http://www.glycohub.com/services)	Contact info@glycohubusa.com for compound requests	Printed glycans
Monosodium phosphate monohydrate	Sigma	S9638	Printing buffer component
Disodium phosphate heptahydrate	Sigma	S9390	Printing buffer component
Phosphate buffered saline	Hy-Labs	BP-507/500D	Printing buffer/ incubation/washing buffer
Tris-base	Sigma	T1503	Slide blocking reagent
Glycerol	Sigma	G-7893	Printing buffer component
Ethanolamine	Thermo-Fisher Scientific	0700/08	Slide blocking reagent
Ovalbumin (Grade V)	Sigma	A5503	Slide Blocking protein
Tween-20	Sigma	P7949	Slide washing detergent
Alexa 555-Hydrazide	Thermo-Fisher Scientific	A20501MP	Marker on array
ChromPure Human IgG, whole molecule	Jackson Immunoresearch	009-000-003	Printing component
Biotinylated- SNA	Vector Laboratories	B-1305	Plant Lectin – binding Sia-alpha2–6-linked
Biotinylated-MALII	Vector Laboratories	B-1265	Plant Lectin – binding Sia-alpha2–3-linked
Chicken-anti Neu5Gc IgY	BioLegend	146903	Primary detection
Cy3-Streptavidin	Jackson Immunoresearch	016-160-0848	Biotin binding
Cy3-anti Human IgG	Jackson Immunoresearch	109-165-088	Secondary detection against human IgG
Cy3-anti Chicken IgY	Jackson Immunoresearch	703-165-155	Secondary detection against chicken IgY
Human sera samples	Israeli Blood Bank		Primary detection
Compressed Nitrogen (Grade 5)			General dusting/drying tool
Epoxy-coated slides	Corning	40044	Slides
Epoxy-coated slides	PolyAn	2D 104-00-221	Slides. In this type of slides the surface is more hydrophobic (compared to Coring slides) therefore the glycans Print Buffer would need to be supplemented with 0.005% Tween-20 to obtain 100 µm size spots.
384-well microtiter plate	Genetix	2070	Printing plate
VWR lab marker	VWR	52877-310	Slide labeling
Staining Tube	ArrayIt	MST	Slide developing tool
Staining bath	VWR	25608-904	Slide developing tool
Slides glass holders	VWR	631-9321	Slide developing tool
GenePix Scanner	Molecular devices	4000B	Slide scanner
LM-60 NanoPrinter	ArrayIt	LM-60	Array printer
Pins	ArrayIt	946MP3	Printing pins
ProPlate Module	Grace Bio-Labs	P37004	Slide developing module
Distilled water	Bio-Lab	2321020500	Required for arrayer and humidifier
Electronic Multi Pippete, 8 Channel , volume range 2-125 μL	Thermo-Fisher Scientific (Matrix)	MA-2131 Impact2 Equalizer 384	Multi pippete for sample dispansing into 384-well plate