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Benchmarks Benchmarks

Development of a high density hemagglutinin protein microarray to determine the breadth of influenza antibody responses Anthony L. Desbien1

, Neal Van Hoeven1 Casey1 , John D. Laurance1 Steven G. Reed1 , and Darrick Carter1,2

1 Infectious Disease Research Institute, Seattle, WA, and 2 Incorporated, Seattle, WA

Protein Advances

BioTechniques 54:345-348 (June 2013) doi 10.2144/ 000114041 Keywords: influenza hemagglutinin; antibody; vaccination; breadth; protein microarray Supplementary material for this article is available at

We have developed an influenza hemagglutinin protein microarray to assess humoral recognition of diverse influenza strains induced by vaccination and infection. Each array consists of controls and 127 hemagglutinin antigens from 60 viruses, spotted in replicates to generate a single array of 1296 spots. Six ar- rays are configured on a single slide, which in the following analysis was probed simultaneously with 2 isotype-specific fluorescent secondary antibodies yield- ing over 15,000 data points per slide. Here we report the use of this system to evaluate mouse, ferret, and human sera. Te array allows simultaneous exami- nation of the magnitude of antibody responses, the isotype of such antibodies, and the breadth of influenza strain recognition. We are advancing this technol- ogy as a platform for rapid, simple, high-throughput assessment of homologous and heterologous antibody responses to influenza disease and vaccination.

Considering the plasticity of the influenza virus, evaluating the ability of vaccines to induce broadly reactive humoral immune responses is critical (1–6). To facilitate this process, a protein microarray of influenza hemagglutinin antigens (HA) was developed by adsorbing picogram amounts of protein onto an activated glass surface via an epoxy attachment method (Arrayit Corporation, Sunnyvale,CA) (7,8). Te resulting covalent interaction yields randomly oriented proteins, promoting exposure of potential antigenic determinants configured in 100 µm size spots,

Method summary:

A protein microarray of influenza hemagglutinin antigens was generated by adsorbing picogram amounts of 127 hemagglutinin antigens from 60 viruses onto a single array. Sera to be evaluated are diluted and incubated with the microarray, followed by simultaneous probing with two isotype-specific fluorescent secondary antibodies. Arrays are then scanned using a conventional gene array scanner.

Vol. 54 | No. 6 | 2013 345

thus allowing for highly dense arrays. We developed an array composed of controls and 127 hemagglutinin antigens from 60 influenza viruses (Protein Sciences Meriden, CT; Sino Biologicals, Bejing, China), including repre- sentatives from the circulating H5 and H1 strains that are potential and known causes of pandemic flu (Figure 1A, Supplementary Table S1) (9). Each protein is spotted a total of 9 times in triplicate spots for each of 3 10-fold dilutions. Previously reported arrays were smaller, limiting their breadth of assessment and sample throughput (10,11).

, Steven J. Reed1 , Susan L. Baldwin1 , Allen C. , Malcolm S. Duthie1 , Execution of the microarray exper-

iment is performed in a manner much like a traditional ELISA, with the exception that fluorescently labeled secondary antibodies mediate detection rather than the HRP conjugates generally used for ELISA. For most samples, a common dilution ranging from 1:10 to 1:1000 is made in a simple buffer of PBS and 1% BSA for a total volume of 300 µL. Each experiment requires 0.3 to 30 µL of sera, which is incubated with the array for 30 min. Te protein microarray slide is composed of six partitioned arrays allowing simultaneous testing of analytes and minimizing intra-assay variability (Figure 1A). Arrays are washed, then exposed to secondary antibodies for 30 min and re-washed. Following development, arrays are scanned using a conventional array scanner and soſtware (GenePix 4000b, GenPix6.1, Molecular Devices, Sunnyvale, CA). Te experiment is performed in less than two hours. Adsorption of protein to a solid

substrate, as well as protein purification and production processes, could alter the structures of the hemagglutinin antigens on the array. To evaluate the conformational integrity of the antigens, arrays were probed with two broadly reactive, conformation- sensitive monoclonal antibodies, KB2 and 6F12, which react with the stalk region of hemagglutinin, spanning the HA2 and HA1 subunits (a kind giſt of Peter Palese and Florian Krammer) (12,13). KB2 reacts with H1 and H5 viruses, while 6F12 reacts with H1 viruses. A third antibody that reacts with the HA1 subunit of H5N1 viruses, which binds independently of conformation, was also used (7C2, MyBioSource, San Diego, CA). As shown, the stalk-specific antibodies KB2 and 6F12 reacted only with the HA0 (head and stalk) proteins on the array (Supplementary Figure S1A). Examination of the subtypes recognized by the antibodies demonstrated that the array recapitulated the reported specificities of KB2 and 6F12 (Supplementary Figure S1B). While this experiment does not validate each protein on the array or address the concern that some of the proteins may be misfolded, it does demonstrate that the processes used to create the array do not broadly alter the confor- mation of the antigens to such an extent that conformation-dependent neutralizing antibodies cannot bind. It remains to be determined if the breadth of array recog- nition correlates to protection.

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