Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array
Published: April 30, 2024
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…
開示
The authors have nothing to disclose.
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 |
