Viral Antigen Microarray Assay to Detect Antibody Isotypes in Serum Against Viral Antigen

Abstract

Source: Khan, S., et al., Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes. J. Vis. Exp. (2019)

In this video, we demonstrate the process for detecting antibody isotypes in multiple human serum samples using a microarray slide printed with antigenic subtypes of influenza virus strains.

Protocol

1. Produce influenza antigen microarrays Print antigens onto microarray slides Reconstitute each lyophilized antigen to a concentration of 0.1 mg/mL in phosphate-buffered saline (PBS) with 0.001% Tween-20 (T-PBS). Transfer 10 μL of each reconstituted antigen to individual wells of an untreated 384-well flat-bottom plate. Print antigens using a microarray printer with low-volume microarray spotting pins that aspirate antigen into the sample channel and deposit v…

Representative Results

Figure 1: Schematic of influenza antigen microarray printing and probing protocol. From left to right, microarray is printed using onto nitrocellulose-coated slides, which are used to probe sera for IgG and IgA antibodies using quantum-dot-conjugated secondary antibodies, with slides imaged using a portable imager, and results analyzed to generate a heat map. <p class="jove_c…

Materials

16-pad nitrocellulose-coated glass slides	Grace Bio Labs	305016
1x GVS FAST blocking buffer	Fischer Scientific	10485356
ArrayCam portable imager	Grace Bio Labs	400S	Other imaging devices can be used to visualize slides if capable of achieving the resolution of the microarray spots and the excitation and emission wavelengths of the quantum dots.
Biotin-conjugated goat anti-mouse-IgG antibody	Thermo Fischer	31800
HiBase 384-well plate	Greiner Bio-One	T-3037-11
Microarray pins	ArrayIt	GMP2	Each different microarray printer may require its own custom microarray pins.
Mouse monoclonal poly-His antibody	Sigma-Aldrich	H1029
OmniGrid 100 microarray printer	GeneMachines		The version of the microarray printer used in this work is no longer commercially available, but the updated similar equipment is the OmniGrid Accent microarray printer from Digilab (Hopkinton, MA), and the same protocol can be carried out with most commercially available microarray printers.
ProPlate slide chambers	Grace Bio Labs	246890
ProPlate slide clips	Grace Bio Labs	204838
ProPlate slide frames	Grace Bio Labs	246879
Quantum dot 585 nm conjugated goat anti-human-IgA antibody	Grace Bio Labs	110620
Quantum dot 585 nm streptavidin conjugate	Thermo Fischer	Q10111MP
Quantum dot 800 nm conjugated goat anti-human-IgG antibody	Grace Bio Labs	110610