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J Vis Exp. 2019 Jul 26;(149). doi: 10.3791/59973.
Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes.
Khan S1, Jain A2, Taghavian O2, Nakajima R2, Jasinskas A2, Supnet M2, Felgner J2, Davies J2, de Assis RR2, Jan S2, Obiero J2, Strahsburger E2, Pone EJ2, Liang L2, Davies DH2, Felgner PL3.
Author information
1 Division of Infectious Diseases, Department of Medicine, University of California Irvine Health. 2 Vaccine Research and Development Center, Department of Physiology, University of California Irvine. 3 Vaccine Research and Development Center, Department of Physiology, University of California Irvine; pfelgner@uci.edu.
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.
PMID: 31403629 DOI: 10.3791/59973
Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes.
Khan S1, Jain A2, Taghavian O2, Nakajima R2, Jasinskas A2, Supnet M2, Felgner J2, Davies J2, de Assis RR2, Jan S2, Obiero J2, Strahsburger E2, Pone EJ2, Liang L2, Davies DH2, Felgner PL3.
Author information
1 Division of Infectious Diseases, Department of Medicine, University of California Irvine Health. 2 Vaccine Research and Development Center, Department of Physiology, University of California Irvine. 3 Vaccine Research and Development Center, Department of Physiology, University of California Irvine; pfelgner@uci.edu.
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.
PMID: 31403629 DOI: 10.3791/59973