Identification of Immunohistochemical Reagents for In Situ Protein Expression Analysis of Coronavirus-associated Changes in Human Tissues

Abstract

We studied the suitability of commercially available monoclonal antibodies (mAbs) for the immunohistochemical (IHC) detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) in standard archival specimens. Antibodies were screened on HEK293 cells transfected with viral nucleoprotein, S1 subunit and S2 subunit of spike protein and on untransfected cells, as well as a panel of normal tissue. Lung tissue with presence of SARS-CoV2 confirmed by in situ hybridization (ISH) was also used. A total of 7 mAbs were tested: (1) mAb 001 (Sino Biological, 40143-R001), (2) mAb 007 (Sino Biological, 40150-R007), (3) mAb 019 (Sino Biological, 40143-R019), (4) mAb 1A9 (GeneTex, GTX632604), (5) mAb ABM19C9 (Abeomics, 10-10007), (6) FIPV3-70 (Santa Cruz, SC-65653), and (7) mAb 6F10 (BioVision, A2060). Only 2 mAbs, clone 001 to the nucleoprotein and clone 1A9 to the S2 subunit spike protein displayed specific immunoreactivity. Both clones showed strong staining in the acute phase of COVID-19 pneumonia, mostly in areas of acute diffuse alveolar damage, but were not completely congruent. Viral protein was also found in kidney tubules, endothelia of multiple organs and a nasal swab of a patient with persistent SARS-CoV2 infection. The other tested reagents were either poorly reactive or demonstrated nonspecific staining in tissues and lesions not infected by SARS-CoV2. Our study demonstrates that rigid specificity testing is mandatory for the evaluation of mAbs to SARS-CoV2 and that clones 001 to nucleoprotein and 1A9 to S2 subunit spike protein are useful for the in situ detection of SARS-CoV2.

FIGURE 1

Pellets of HEK293 cells transfected with SARS-CoV2: S1 and S2 subunit of spike protein, nucleoprotein and untransfected cells. A–D, Hematoxylin eosin stain/H&E. E–H, mAb FIPV3-20 to nucleoprotein, no immunolabeling of any pellet. I–L, mAb 019, intense immunostaining of all cell pellets. M–P, mAb 1A9, exclusive immunoreactivity of HEK293 cells transfected with spike protein S2 subunit. Q–T, mAb 001, homogeneous staining of HEK293 cells expressing nucleoprotein. U–X. In situ hybridization with probe to S1 subunit positive in corresponding HEK293 cells.

FIGURE 2

H&E stain (A), in situ hybridization (B) and immunohistochemical staining (C–H) of SARS-CoV2 positive lung (A–E) and SARS-CoV2 negative tissues (F–H); ISH probe to S1 spike protein (B) demonstrating SARS-CoV2 viral RNA in lung tissue(B); SARS-CoV2-positive lung tissues negative for mAb FIPV3-20 (C) and mAb 007 (D); mAb 019, intense immunolabelling of hyaline membranes in lung (E), extensive nonspecific immunolabelling of skin (F), colon (G), and spleen (H).

FIGURE 3

Analysis of autopsy tissue from patients who died from COVID-19: H&E stain (A, D) immunoreactivity with mAb 1A9 to S2 subunit spike protein (B, E) and mAb 001 to nucleoprotein (C, F–I); COVID-19 pneumonia in acute phase diffuse alveolar damage with serial sections stained with H&E (A), mAb 1A9 (B), and mAb 001 (C); extensive immunostaining of hyaline membranes and alveolar macrophages for both spike protein and nucleoprotein. mAb 001 staining in endothelia of focal septal vessels and pneumocytes (inset); mucus filled bronchus (D) H&E stain with weakly positive S2 spike protein content (E) and strong signal for nucleoprotein (F); heart muscle with mAb 001 immunopositive endothelia; mAb 001/nucleoprotein-positive content in tubule of SARS-CoV2-positive autopsy kidney (H) and smear of nasal swab stained with mAb 001 positive for SARS-CoV2 (I).