Sensitive and Specific Immunohistochemistry Protocol for Nucleocapsid Protein from All Common SARS-CoV-2 Virus Strains in Formalin-Fixed, Paraffin Embedded Tissues

• Published in: Methods and protocols Vol. 4; no. 3; p. 47
• Main Authors: Sun, Yunguang, Ge, Linna, Udhane, Sameer S., Langenheim, John F., Rau, Mary J., Patton, Mollie D., Gallan, Alexander J., Felix, Juan C., Rui, Hallgeir
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 10-07-2021; MDPI
• Subjects: Amino acid sequence; Animal models; Antibodies; Antigens; Autopsy; Conserved sequence; Coronaviruses; COVID-19; Cytometry; Formaldehyde; Gene expression; Glycoproteins; Immunoglobulins; Immunohistochemistry; immunohistochemistry protocols; Infections; Lungs; Middle East respiratory syndrome; nucleocapsid protein; Nucleocapsids; Nucleotide sequence; Paraffin; Pneumonia; Polyclonal antibodies; Proteins; Protocol; Respiration; Respiratory diseases; SARS-CoV-2; Severe acute respiratory syndrome coronavirus 2; spike protein; Strains (organisms); Tropism; Viral infections; Viruses; United States > US
• ISSN: 2409-9279; 2409-9279
• DOI: 10.3390/mps4030047

Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specific detection of SARS-CoV-2 viral proteins in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism of this virus. We identified commercially available affinity-purified polyclonal antibodies raised against nucleocapsid and spike proteins of SARS-CoV-2 that provide sensitive and specific detection of the virus by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Two immunohistochemistry protocols are presented that are mutually validated by the matched detection patterns of the two distinct viral antigens in virus-infected cells within autopsy lung tissue of COVID-19 deceased patients. Levels of nucleocapsid protein in the lungs of COVID-19 decedents, as measured by quantitative histo-cytometry of immunohistochemistry images, showed an excellent log–linear relationship with levels of viral nucleocapsid RNA levels, as measured by qRT-PCR. Importantly, since the nucleocapsid protein sequence is conserved across all known viral strains, the nucleocapsid immunohistochemistry protocol is expected to recognize all common variants of SARS-CoV-2. Negative controls include autopsy lung tissues from patients who died from non-COVID-19 respiratory disease and control rabbit immunoglobulin. Sensitive detection of SARS-CoV-2 in human tissues will provide insights into viral tissue and cell distribution and load in patients with active infection, as well as provide insight into the clearance rate of virus in later COVID-19 disease stages. The protocols are also expected to be readily transferable to detect SARS-CoV-2 proteins in tissues of experimental animal models or animals suspected to serve as viral reservoirs.