Research paperAntibody microarray immunoassay for screening and differential diagnosis of upper respiratory tract viral pathogens
Introduction
Upper respiratory tract infections (URTIs) are a group of acute infectious diseases of the respiratory system (nose, sinuses, pharynx, or larynx), a main feature of which is airborne transmission. URTIs are widespread, and they are the most common infectious disease. With individuals often affected multiple times per year, the global burden is substantial. A systematic analysis of impacts from hundreds of diseases estimated that there were likely 17.2 billion URTIs in 2015 globally GBD 2015 Disease and Injury Incidence and Prevalence Collaborators, 2016). Most infections are viral in nature, although in other instances, the cause is bacterial Rhinitis Versus Sinusitis in Children (PDF), 2019). Diagnosis of URTI is mainly based on the patient's clinical manifestation; confirmation by laboratory detection (PCR) is occasionally and inconsistently used. However, clinical determination of an URTI's character (viral versus bacterial) is highly desirable in cases involving the diagnosis of patients with severe forms, in young children, and in the elderly.
This study presents a novel method, based on protein microarray technology, for laboratory determination of viral etiologies in URTIs. Protein microarrays operate on the same principles behind traditional ELISA methods. The microarray format, however, permits the simultaneous detection of hundreds of proteins using small sample and reagent volumes (Duarte and Blackburn, 2017). The basis and mechanism of this approach is that proteins (antibodies), capable of specific binding to partner antigens (virus), are applied in a matrix arrangement to a solid substrate and immobilized; signal is visualized after binding of analyte containing antigen (Chang, 1983). The main advantage of protein microarrays is potential simultaneous determination of numerous factors (up to 500) in one sample.
The aim of this work was the development and validation of a protein diagnostic microchip capable of simultaneous identification of six viral pathogens in human nasal samples. Using the microchip developed here, the following pathogens can be specifically differentiated: influenza A (IAV); influenza B (IBV); human respiratory syncytial virus (RSV); human adenovirus (hAdV); parainfluenza virus type 2 (hPIV2); and parainfluenza virus type 3 (hPIV3).
Section snippets
Viruses
The viral strains used were obtained from the virus and cell culture collection of the Smorodintsev Research Institute of Influenza (St. Petersburg, Russia). The following influenza A and B viral strains were used for analysis: A/California/07/09 (H1N1pdm09); A/Texas/50/12 (H3N2); A/Vietnam/1194/04 (H5N1) vaccine strain NIBRG-14; A/duck/Potsdam/1402–6/86 (H5N2); B/Brisbane/46/15; B/Massachusetts/2/12; B/Beijing/184/93; B/Phuket/3073/13. The following non-influenza strains were also used for
Selection of capture and detection antibodies
For creation of the protein microchip, 50 monoclonal antibodies (mAb), most of which were produced at the Smorodintsev Research Institute of Influenza (SRII, div. Russian Ministry of Health), were characterized (Table 1, Appendices Fig. A1 and Fig. A2) (Timoshicheva et al., 2019). The main criteria for antibody selection were: specificity (lack of cross-reactivity); affinity for the target antigen; and features conducive to application in a reliable multiplex system.
Antibodies were mainly
Conclusion
In summary, an antibody microarray diagnostic system (and associated protocols) for the detection and differentiation of six clinically important upper respiratory tract pathogen groups (IAV, IBV, RSV, hAdV, hPIV2, hPIV3) from human nasal samples was developed. The protein array presented here is based on the well-understood and widely-exploited antibody-antigen-antibody (sandwich format) interaction. Sample analysis protocols were optimized for the array, and a single-step method (wherein
Funding
This work was supported by the Russian Ministry of Science and Higher Education of the Russian Federation via agreement on the provision of grants from the federal budget in the form of subsidies No. 075-15-2019-1241 dated June 10, 2019 (previously No. 14.604.21.0180 dated September 26, 2017) for the project entitled “Development of SARI-TEST, a multiplex diagnostic suite for detection of severe acute respiratory infections and prediction of their severity”, project identification number
Author contributions
Individual contributions to the work are as follows: conception and study design (MP, SK, AV); data acquisition and analysis (KL, AL, AT); drafting of the manuscript (MP, NG, SK); translation and editing of the manuscript (ER); and critical revision of the manuscript (MP, SK, ER). All authors have approved the final manuscript.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors thank Evgeny Sorokin and Aram Shaldjan for their invaluable help in obtaining and purifying monoclonal antibodies. The authors are also grateful to Irina Baranovskaya and Daria Danilenko for providing needed viral strains.
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