Method Article

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

DOI:

10.3791/56094

July 13th, 2017

In This Article

Summary

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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

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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

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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 metastasis2,3. 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<....

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Protocol

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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....

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Results

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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.......

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Discussion

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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.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This work was supported in part by a Research Career Development Award from the Israel Cancer Research Fund, a grant from the Israeli National Nanotechnology Initiative and the Helmsley Charitable Trust for a Focal Technology Area on Nanomedicines for Personalized Theranostics (V.P-K), and National Institutes of Health grant R01GM076360 (to X.C.).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Primary-amine containing sialoglycansGlycohub, Inc., Davis, CA, USA (http://www.glycohub.com/services)Contact info@glycohubusa.com for compound requestsPrinted glycans
Monosodium phosphate monohydrateSigmaS9638Printing buffer component
Disodium phosphate heptahydrateSigmaS9390Printing buffer component
Phosphate buffered salineHy-LabsBP-507/500DPrinting buffer/ incubation/washing buffer
Tris-baseSigmaT1503Slide blocking reagent
GlycerolSigmaG-7893Printing buffer component
EthanolamineThermo-Fisher Scientific0700/08Slide blocking reagent
Ovalbumin (Grade V)SigmaA5503Slide Blocking protein
Tween-20SigmaP7949Slide washing detergent
Alexa 555-HydrazideThermo-Fisher ScientificA20501MPMarker on array
ChromPure Human IgG, whole moleculeJackson Immunoresearch009-000-003Printing component
Biotinylated- SNAVector LaboratoriesB-1305Plant Lectin – binding Sia-alpha2–6-linked
Biotinylated-MALIIVector LaboratoriesB-1265Plant Lectin – binding Sia-alpha2–3-linked
Chicken-anti Neu5Gc IgYBioLegend146903Primary detection
Cy3-StreptavidinJackson Immunoresearch016-160-0848Biotin binding
Cy3-anti Human IgGJackson Immunoresearch109-165-088Secondary detection against human IgG
Cy3-anti Chicken IgYJackson Immunoresearch703-165-155Secondary detection against chicken IgY
Human sera samplesIsraeli Blood BankPrimary detection
Compressed Nitrogen (Grade 5)General dusting/drying tool
Epoxy-coated slidesCorning40044Slides
Epoxy-coated slidesPolyAn2D 104-00-221Slides. 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 plateGenetix2070Printing plate
VWR lab markerVWR52877-310Slide labeling
Staining TubeArrayItMSTSlide developing tool
Staining bathVWR25608-904Slide developing tool
Slides glass holdersVWR631-9321Slide developing tool
GenePix ScannerMolecular devices4000BSlide scanner
LM-60 NanoPrinterArrayItLM-60Array printer
PinsArrayIt946MP3Printing pins
ProPlate ModuleGrace Bio-LabsP37004Slide developing module
Distilled waterBio-Lab2321020500Required for arrayer and humidifier
Electronic Multi Pippete, 8 Channel , volume range 2-125 μLThermo-Fisher Scientific (Matrix)MA-2131 Impact2 Equalizer 384Multi pippete for sample dispansing into 384-well plate

References

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  1. Padler-Karavani, V. Aiming at the sweet side of cancer: Aberrant glycosylation as possible target for personalized-medicine. Cancer Lett. 352 (1), 102-112 (2014).
  2. Amon, R., Reuven, E. M., Leviatan Ben-Arye,, Padler-Karavani, S., V, Glycans in immune recognition and resp....

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Tags

Glycan PrintingAntibody ProfilingHuman Sera AnalysisFluorescent DetectionEpoxy Coated SlidesNano Printer TechnologyPBST WashingSecondary Antibody

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