Immunohistochemical Detection of SARS-CoV-2 Antigens by Single and Multiple Immunohistochemistry

Lonardi, Silvia; Bugatti, Mattia; Valzelli, Arianna; Facchetti, Fabio

doi:10.1007/978-1-0716-2111-0_17

Cited by 3 publications

(2 citation statements)

References 36 publications

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“…Using IHC and immunofluorescent-labeled secondary antibody, it seems that CU-P1-1 does not work well for IHC. However, mAbs CU-P2-20 and CU-28-24 show viral infection in infected lungs and brains of mice exposed to the virus, and this activity seems to be concentrated in focal regions, consistent with observations made by others [ 26 , 27 , 28 ]. While some studies use antibodies generated against nucleocapsid proteins [ 29 , 30 ], many others use antibodies against the RBD [ 31 ].…”

Section: Discussionsupporting

confidence: 90%

Generation and Characterization of a Multi-Functional Panel of Monoclonal Antibodies for SARS-CoV-2 Research and Treatment

Patterson,

Dubansky,

Dubansky

et al. 2023

Viruses

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The Coronavirus disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is an ongoing threat to global public health. To this end, intense efforts are underway to develop reagents to aid in diagnostics, enhance preventative measures, and provide therapeutics for managing COVID-19. The recent emergence of SARS-CoV-2 Omicron variants with enhanced transmissibility, altered antigenicity, and significant escape of existing monoclonal antibodies and vaccines underlines the importance of the continued development of such agents. The SARS-CoV-2 spike protein and its receptor binding domain (RBD) are critical to viral attachment and host cell entry and are primary targets for antibodies elicited from both vaccination and natural infection. In this study, mice were immunized with two synthetic peptides (Pep 1 and Pep 2) within the RBD of the original Wuhan SARS-CoV-2, as well as the whole RBD as a recombinant protein (rRBD). Hybridomas were generated, and a panel of three monoclonal antibodies, mAb CU-P1-1 against Pep 1, mAb CU-P2-20 against Pep 2, and mAb CU-28-24 against rRBD, was generated and further characterized. These mAbs were shown by ELISA to be specific for each immunogen/antigen. Monoclonal antibody CU-P1-1 has limited applicability other than in ELISA approaches and basic immunoblotting. Monoclonal antibody CU-P2-20 is shown to be favorable for ELISA, immunoblotting, and immunohistochemistry (IHC), however, not live virus neutralization. In contrast, mAb CU-28-24 is most effective at live virus neutralization as well as ELISA and IHC. Moreover, mAb CU-28-24 is active against rRBD proteins from Omicron variants BA.2 and BA.4.5 as determined by ELISA, suggesting this mAb may neutralize live virus of these variants. Each of the immunoglobulin genes has been sequenced using Next Generation Sequencing, which allows the expression of respective recombinant proteins, thereby eliminating the need for long-term hybridoma maintenance. The synthetic peptides and hybridomas/mAbs and quantitative antigen-binding data are under the intellectual property management of the Clemson University Research Foundation, and the three CDRs have been submitted as an invention disclosure for further patenting and commercialization.

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Section: Discussionsupporting

confidence: 90%

Generation and Characterization of a Multi-Functional Panel of Monoclonal Antibodies for SARS-CoV-2 Research and Treatment

Patterson,

Dubansky,

Dubansky

et al. 2023

Viruses

View full text Add to dashboard Cite

show abstract

“…In situ, hybridization techniques also could offer high sensitivity for virus detection and can be used easily alongside IHC as it can be performed on paraffinized tissue but has limited use for detection in other organs, as demonstrated by Massoth et al [47]. Immunohistochemistry (IHC) is particularly effective in retrospective studies using paraffin-embedded tissue (FFPE) thanks to its unique combination of long-term tissue preservation, established compatibility, and flexibility in marker selection [48]. Paraffin-embedded tissues, stored for decades at room temperature maintain good morphological and antigenic integrity, enabling researchers to analyze past cases, compare data across time points, and understand disease progression, treatment efficacy, and long-term outcomes.…”

Section: Immunohistochemistry As a Methods Of Virus Detectionmentioning

confidence: 99%

Immunohistochemical and Morphometric Analysis of Lung Tissue in Fatal COVID-19

Gheban-Roșca,

Gheban,

Pop

et al. 2024

Diagnostics

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The primary targets of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the lungs are type I pneumocytes, macrophages, and endothelial cells. We aimed to identify lung cells targeted by SARS-CoV-2 using viral nucleocapsid protein staining and morphometric features on patients with fatal COVID-19. We conducted a retrospective analysis of fifty-one autopsy cases of individuals who tested positive for SARS-CoV-2. Demographic and clinical information were collected from forensic reports, and lung tissue was examined for microscopic lesions and the presence of specific cell types. Half of the evaluated cohort were older than 71 years, and the majority were male (74.5%). In total, 24 patients presented diffuse alveolar damage (DAD), and 50.9% had comorbidities (56.9% obesity, 33.3% hypertension, 15.7% diabetes mellitus). Immunohistochemical analysis showed a similar pattern of infected macrophages, infected type I pneumocytes, and endothelial cells, regardless of the presence of DAD (p > 0.5). The immunohistochemical reactivity score (IRS) was predominantly moderate but without significant differences between patients with and without DAD (p = 0.633 IRS for type I pneumocytes, p = 0.773 IRS for macrophage, and p = 0.737 for IRS endothelium). The nucleus/cytoplasm ratio shows lower values in patients with DAD (median: 0.29 vs. 0.35), but the difference only reaches a tendency for statistical significance (p = 0.083). Our study confirms the presence of infected macrophages, type I pneumocytes, and endothelial cells with a similar pattern in patients with and without diffuse alveolar damage.

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Generation and characterization of a multi-functional panel of monoclonal antibodies for SARS-CoV-2 research and treatment

Patterson,

Dubansky,

Dubansky

et al. 2023

Preprint

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The Coronavirus disease 2019 (COVID19) pandemic caused by Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is an ongoing threat to global public health. To this end, intense efforts are underway to develop reagents to aid in diagnostics, enhance preventative measures, and provide therapeutics for managing COVID-19. The recent emergence of SARS-CoV-2 Omicron variants with enhanced transmissibility, altered antigenicity, and significant escape of existing monoclonal antibodies and vaccines underlines the importance of the continued development of such agents. The SARS-CoV-2 spike protein and its receptor binding domain (RBD) are critical to viral attachment and host cell entry and are primary targets for antibodies elicited from both vaccination and natural infection. In this study, mice were immunized with two synthetic peptides (Pep 1 and Pep 2) within the RBD of the original Wuhan SARS-CoV-2, as well as the whole RBD as a recombinant protein (rRBD). Hybridomas were generated and a panel of three monoclonal antibodies, mAb CU-P1-1 against Pep 1, mAb CU-P2-20 against Pep 2, and mAb CU-28-24 against rRBD, were generated and further characterized. These mAbs were shown by ELISA to be specific for each immunogen/antigen. Monoclonal antibody CU-P1-1 has limited applicability other than in ELISA approaches and basic immunoblotting. Monoclonal antibody CU-P2-20 is shown to be favorable for ELISA, immunoblotting, and immunohistochemistry (IHC), however, not live virus neutralization. In contrast, mAb CU-28-24 is most effective at live virus neutralization as well as ELISA and IHC. Moreover, mAb CU-28-24 was active against rRBD proteins from Omicron variants B.2 and B.4/B5 as determined by ELISA, suggesting this mAb may neutralize live virus of these variants. Each of the immunoglobulin genes has been sequenced using Next Generation Sequencing, which allows the expression of respective recombinant proteins, thereby eliminating the need for long-term hybridoma maintenance. The synthetic peptide sequences, hybridomas, and related mAbs are now protected by Intellectual Property agreements with the Clemson University Research Foundation and are Patent Pending based on their unique amino acids within the complementary determining regions (CDRs).

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Immunohistochemical Detection of SARS-CoV-2 Antigens by Single and Multiple Immunohistochemistry

Cited by 3 publications

References 36 publications

Generation and Characterization of a Multi-Functional Panel of Monoclonal Antibodies for SARS-CoV-2 Research and Treatment

Generation and Characterization of a Multi-Functional Panel of Monoclonal Antibodies for SARS-CoV-2 Research and Treatment

Immunohistochemical and Morphometric Analysis of Lung Tissue in Fatal COVID-19

Generation and characterization of a multi-functional panel of monoclonal antibodies for SARS-CoV-2 research and treatment

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