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. Quantum-dot-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 sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

Figure 1:. Schematic of protein microarray.

Each slide contains multiple pads each with a single array, which consists of hundreds of antigens printed onto spots arranged in a grid, with each spot containing one antigen adsorbed onto the 3-dimensional topography of the nitrocellulose surface to which antibodies from serum are bound.

Figure 2:. 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.

Figure 3:. Procedure for attaching probing chamber to microarray slide.

From A to F, the probing chamber is placed on top of slide in correct orientation, attached to the slide using horizontal clips on the sides, and placed in the probing tray.

Figure 4:. Representative results of influenza antigen microarray.

Heat maps represent antigen-specific antibody responses, with each row representing a single antigen arranged by molecule, subtype, and strain, and each column representing a probing run of a single specimen, arranged by antibody isotype and run (A, white = 0, black = 20000, red = 40000 fluorescence intensity). The antigen subtypes including all hemagglutinin subtypes from 1 to 18 and all neuraminidase subtypes from 1 to 10 are arranged vertically and labeled on the left. A comparison of the fluorescence intensity between two runs demonstrates good assay reproducibility by linear regression for IgA (B) and IgG(C).

Figure 5:. Breadth of serum antibodies measured on influenza antigen microarray.

Serum IgA (A) and IgG (B) are grouped by HA and NA molecular forms and subtypes to demonstrate high specificity of HA head group antibodies for clinical subtypes and high cross-reactivity of whole HA and trimerized whole HA antibodies with inclusion of stalk region.