Viral Antigen Microarray Assay to Detect Antibody Isotypes in Serum Against Viral Antigen
Published: June 29, 2023
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…
Divulgations
The authors have nothing to disclose.
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 |
Tags
Citer Cet Article
Viral Antigen Microarray Assay to Detect Antibody Isotypes in Serum Against Viral Antigen. J. Vis. Exp. (Pending Publication), e21455, doi: (2023).