Antibody microarray immunoassay for screening and differential diagnosis of upper respiratory tract viral pathogens

Abstract

Upper respiratory tract infections are the world's most common infectious disease. The etiologic agents behind upper respiratory tract infections (URTIs) are, in fact, a diverse set of pathogens such as influenza, parainfluenza, adenovirus, rhinovirus, and others. More than 200 pathogens are known to be involved. Differential diagnosis of viral infections is sometimes complicated by their diversity or similarity of clinical presentation. This work is devoted to the development of a method which enables simultaneous detection of six common viral URTI pathogens: IAV; IBV; RSV; hAdV; hPIV2; and hPIV3. Antibody microarray technology is utilized to accomplish the analysis. In preparation for protein microchip creation, we produced, characterized, and selected approximately 50 monoclonal antibodies; for each of the aforementioned pathogens, an optimal monoclonal antibody pair was selected. A protein microchip was created, and its core working conditions were optimized. With a balance between convenience and maximal assay sensitivity in mind, a one-step analysis approach was developed for accomplishing the ELISA-like “sandwich” interaction on the manufactured microchip (antibody microarray). Reference viral strains were used to establish the lower limits of detection (LoD) for the assay. For IAV, the LoD was 0.25 ng/ml total viral protein. For other viruses, the LoD ranged from 1 to 2 ng/ml total protein. These sensitivity limits are slightly better than those of standard ELISA, but inferior to those of PCR. Overall, we believe that the developed microchip is a good alternative to existing methods, allowing relatively quick (overnight), inexpensive, simultaneous screening of several pathogens. The design of the antibody microarray is conducive to further development, and the panel of analyzed pathogens can be expanded to include approximately 50 members.

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).