Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

Abstract

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens. This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantumdot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera. Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in serosurveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies. Video Link The video component of this article can be found at https://www.jove.com/video/59973/ Protein microarrays, potentially consisting of up to thousands of antigens printed onto nitrocellulose-coated slides as shown in Figure 1, can fill this need 12. These microarrays can be produced and probed in a high throughput manner while consuming small quantities of clinical specimen

Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

“…These antigens were provided by Sino Biological Inc. (Wayne, PA) as either catalog products, or service products. The antigens were printed onto microarrays, probed with human sera, and analyzed as previously described ( Figure 1) 6,9,10 . Briefly, lyophilized antigens were reconstituted to a concentration of 0.1 mg/mL in phosphate-buffered saline (PBS) with 0.001% Tween-20 (T-PBS) and then printed onto nitrocellulose-coated slides from Grace Bio Labs (GBL, Bend, OR) using an OmniGrid 100 microarray printer (GeneMachines).…”

“…The protein microarray methodology has been widely used to simultaneously perform binding assays against hundreds of antigens printed onto a nitrocellulose-coated slide for detection of multiple antibody isotypes 5 . This methodology was recently demonstrated for simultaneous measurement of IgG and IgA antibodies against over 250 antigens from diverse strains and subtypes of influenza 6 . This methodology has previously been applied to detect antibodies against the S1 domains of SARS and MERS coronaviruses 7 .…”

Analysis of SARS-CoV-2 Antibodies in COVID-19 Convalescent Blood using a Coronavirus Antigen Microarray

“…These antigens were provided by Sino Inc. (Wayne, PA) as either catalog products or custome synthesis service products. The antigens were printed onto microarrays, probed with human sera, and analyzed as previously described 9,13,14 . Briefly, lyophilized antigens were reconstituted to a concentration of 0.1 mg/mL in phosphate-buffered saline (PBS) with 0.001% Tween-20 (T-PBS) and then printed onto nitrocellulose-coated slides from Grace Bio Labs (GBL, Bend, OR) using an OmniGrid 100 microarray printer (GeneMachines).…”

“…However, the optimal set of antigens to detect strain-specific coronavirus antibodies remains unknown. Protein microarray technology can be used to detect antibodies of multiple isotypes against hundreds of antigens in a high throughput manner 8,9 so is well suited to serologic surveillance studies. This technology, which has previously been applied to other emerging coronaviruses 10 , is based on detection of binding antibodies, which are wellcorrelated with neutralizing antibodies 11 but do not require viral culture in biosafety level 3 facilities.…”

A Modular Microarray Imaging System for Highly Specific COVID-19 Antibody Testing

“…The slides were imaged using both the TinyArray imager and the commercial ArrayCam system (Grace Bio-Labs, Bend, OR). As the size and distances of the spots were known, the background-subtracted median spot fluorescence intensity was measured and quantified using Mapix (Innopsys) as previously described [22][23][24] . Mean fluorescence intensity (MFI) of the four replicate spots for each antigen was utilized for antigen heat map generation and data analysis.…”