In-House Immunofluorescence Assay for Detection of SARS-CoV-2 Antigens in Cells from Nasopharyngeal Swabs as a Diagnostic Method for COVID-19

Abstract

Immunofluorescence is a traditional diagnostic method for respiratory viruses, allowing rapid, simple and accurate diagnosis, with specific benefits of direct visualization of antigens-of-interest and quality assessment. This study aims to evaluate the potential of indirect immunofluorescence as an in-house diagnostic method for SARS-CoV-2 antigens from nasopharyngeal swabs (NPS). Three primary antibodies raised from mice were used for immunofluorescence staining, including monoclonal antibody against SARS-CoV nucleocapsid protein, and polyclonal antibodies against SARS-CoV-2 nucleocapsid protein and receptor-binding domain of SARS-CoV-2 spike protein. Smears of cells from NPS of 29 COVID-19 patients and 20 non-infected individuals, and cells from viral culture were stained by the three antibodies. Immunofluorescence microscopy was used to identify respiratory epithelial cells with positive signals. Polyclonal antibody against SARS-CoV-2 N protein had the highest sensitivity and specificity among the three antibodies tested, detecting 17 out of 29 RT-PCR-confirmed COVID-19 cases and demonstrating no cross-reactivity with other tested viruses except SARS-CoV. Detection of virus-infected cells targeting SARS-CoV-2 N protein allow identification of infected individuals, although accuracy is limited by sample quality and number of respiratory epithelial cells. The potential of immunofluorescence as a simple diagnostic method was demonstrated, which could be applied by incorporating antibodies targeting SARS-CoV-2 into multiplex immunofluorescence panels used clinically, such as for respiratory viruses, thus allowing additional routine testing for diagnosis and surveillance of SARS-CoV-2 even after the epidemic has ended with low prevalence of COVID-19.

Imaging Techniques: Essential Tools for the Study of SARS-CoV-2 Infection

“…The presence of the nucleocapsid (indirect immunofluorescence positivity) (but not RBD) correlates with the viral load (RT-PCR). On the other hand, no trend was seen for some samples, probably due to the quality of these samples (Lam et al, 2021).…”

“…Microscopy can be used as a tool for the development of rapid detection assays as numerous techniques are available in this field. Lam et al, 2021 demonstrated the potential of immunofluorescence as a simple diagnostic tool; however, due to sensitivity problems, other detection methods have to be developed (Arevalo- Rodriguez et al, 2020).…”

“…ISH has a more complex methodology compared to RT-PCR so it would not be recommended for diagnosis (Lam et al, 2021;Von Stillfried and Boor, 2021). Lam et al, 2021, propose immunofluorescence as a first step of diagnostic (with a routine and large-scale/automated machine), followed by more sensitive RT-PCR, for the samples identified as negative by antigen detection method.…”

Sensors Based on the Carbon Nanotube Field-Effect Transistors for Chemical and Biological Analyses

“…Therefore, a sensitive detection of these biomarkers can greatly improve the disease diagnosis and provide a more objective and quantifiable basis for clinical decision-making. However, most of the current detection methods, such as the enzyme-linked immunosorbent assay (ELISA) [ 4 ], the high-performance liquid chromatography (HPLC) [ 5 ] and the immunofluorescence assay (IFA) [ 6 ], have problems involving a lengthy consumption, complex steps, a low sensitivity, and radioactive pollution, which cannot be used to detect these trace substances. Moreover, some detection processes need secondary labeling, which has potential radiation hazards.…”

“…The advent of biochemical sensors provides a feasible solution for the rapid, sensitive and efficient detection and analysis of the disease-related microbiomarkers. Compared with the traditional detection methods, biochemical sensors are more and more widely used to detect various trace substances due to their advantages in offering a fast response, high sensitivity, strong specificity, a label-free detection and less sample demand [ 6 , 8 , 9 , 10 ].…”