primary goat anti human ace2 antibody  (R&D Systems)

Journal: bioRxiv

Article Title: SARS-CoV-2 hijacks neutralizing dimeric IgA for enhanced nasal infection and injury

doi: 10.1101/2021.10.05.463282

Figure Lengend Snippet: (A) RBD-specific binding activities of sera derived from 3 (P1-P3) convalescent and 1 (P4) acute COVID-19 patients as measured by ELISA. (B) Spike-specific binding activities of sera derived from four COVID-19 patients as measured by ELISA. (C) Neutralization activities of sera derived from four COVID-19 patients as measured by pseudotyped SARS-CoV-2 inhibition in 293T-ACE2 cells. (D) Antibody gene repertoire analysis of reactive B cells derived from each patient. The number of cloned antibody genes from each patient is shown in the center of each pie chart for both the heavy (H) and light (L) chains. The colors represent specific variable gene family. Each fragment of the same color stands for one specific sub-family. (E) . The percentage of somatic hypermutation (SHM) compared to germline sequences and the CDR3 amino acid lengths of cloned antibody H and L gene sequences were analyzed for each subject. (F) RBD (left) and spike (right) specific binding activities of five HuNAbs, including A6, B4, B7, B8 and C5, were measured by ELISA. (G) Neutralization activities of 5 HuNAbs against pseudotyped (left) and authentic (right) SARS-CoV-2 were determined in HEK 293T-ACE2 and Vero-E6 cells, respectively. HIV-1 specific HuNAb VRC01 served as a negative control. Each assay was performed in duplicates and the mean of replicates is shown with the standard error of mean (SEM).s (H) The competition of four HuNAbs, including B4, B7, B8 and C5, with human soluble ACE2 for binding to SARS-CoV-2 RBD was measured by SPR. The curves show binding of ACE2 to SARS-CoV-2 RBD with (red) or without (black) pre-incubation with each HuNAb.

Article Snippet: For identification of ACE2 expression, the goat anti-ACE2 primary antibody (R&D) and Alexa Fluor 568 donkey anti-goat IgG (H+L) secondary antibodies (Invitrogen) according to the manufacturer’s instructions.

Techniques: Binding Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Inhibition, Clone Assay, Negative Control, Incubation

Journal: bioRxiv

Article Title: SARS-CoV-2 hijacks neutralizing dimeric IgA for enhanced nasal infection and injury

doi: 10.1101/2021.10.05.463282

Figure Lengend Snippet: (A) The effects of B8-dIgA2 on SARS-CoV-2 infection in the MucilAir™ model, consisting of primary human nasal epithelial cells but no DCs. B8-mIgA2 or B8-dIgA2 were pre-incubated at doses of 10, 100, and 1000 ng/ml, respectively, in the apical compartment with or without mucus for 1 hour, before adding 10 4 PFU of SARS-CoV-2 (BetaCoV/France/IDF00372/2020) for 4 hours. The viral RNA loads were measured by RT-PCR in both the apical and basal compartments and are shown in log-transformed units. (B) Representative confocal images (400×) of olfactory epithelium in NT showed the expression of CD209 (DC-SIGN) in green and ACE2 in magenta by immunohistochemical staining of experimental hamsters treated with B8-dIgA2 without (left) or with (middle and right) SARS-CoV-2 infection. Color-coding indicates specific antibodies used for double staining. Infected CD209 + cells are visualized in yellow as indicated by arrows (right). (C) The CD209 or CD299 overexpressed-HEK 293T cells were pre-treated for 6 hours with 10 ng/ml of B8-dIgA1 or B8-dIgA2 or control dIgA1 or control dIgA2 or PBS, respectively, prior to SARS-CoV-2 infection (MOI: 0.05). Two days after infection, SARS-CoV-2 NP expression (green) was quantified by the mean fluorescence intensity (MFI) after anti-NP IF staining. Statistics were generated using student- t tests. * p <0.05; ** p <0.01; *** p <0.001. (D) The effects of B8 antibodies on cell-cell fusion. 293T cells co-transfected with SARS-CoV-2 spike and GFP were pre-treated with 100× the IC 90 dose of B8-IgG1, B8-mIgA1, B8-mIgA2, B8-dIgA1, B8-dIgA2 or and IgG isotypic control for 1 hour, respectively. Vero-E6 cells transfected with TMPRSS2 were then added to the treated 293T-spike-GFP cells and co-cultured for 48 hours. Cell-cell fusion was imaged under a fluorescence confocal microscope at the 50× magnification.

Article Snippet: For identification of ACE2 expression, the goat anti-ACE2 primary antibody (R&D) and Alexa Fluor 568 donkey anti-goat IgG (H+L) secondary antibodies (Invitrogen) according to the manufacturer’s instructions.

Techniques: Infection, Incubation, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Expressing, Immunohistochemical staining, Staining, Double Staining, Fluorescence, Generated, Transfection, Cell Culture, Microscopy

Journal: bioRxiv

Article Title: SARS-CoV-2 hijacks neutralizing dimeric IgA for enhanced nasal infection and injury

doi: 10.1101/2021.10.05.463282

Figure Lengend Snippet: (A) Side and top views of the Spike-B8 3u cryo-EM map showing 3 up RBDs each bound with a B8 Fab. Protomer 1, 2, and 3 are shown in slate blue, dark sea green, and India red, respectively. Heavy chain and light chain of the B8-Fab are in blue and gold, respectively. This color scheme was used throughout panels (A)-(E) . (B) Side and top views of the Spike-B8 3u cryo atomic model. (C) Side and top views of the Spike-B8 2u1d cryo-EM map showing two up RBDs up (RBD-1 and RBD-2) and one RBD down (RBD-3), each bound to a B8-Fab. (D) Side and top views of the Spike-B8 2u1d cryo atomic model. (E) Structural comparation of RBDs between Spike-B8 3u (different colors) and Spike-B8 2u1d (gray). (F) ACE2 (chocolate color, PDB: 6M0J) may clash with the heavy chain (blue) and light chain (gold) of the B8-Fab. ACE2 and the Fab share overlapping epitopes on the RBM (dotted black circle), and the framework of the B8-VL appears to clash with ACE2 (dotted black frame). The RBD core and RBM are shown in light sky blue and green, respectively. (G) Atomic model of an RBD-B8 complex portion in cartoon mode, shown with the same color scheme as in (F) . (H) The residues involved in interactions between B8 and the RBM. The heavy and light chain of the B8-Fab are in blue and gold, respectively. The RBM is shown in green.

Article Snippet: For identification of ACE2 expression, the goat anti-ACE2 primary antibody (R&D) and Alexa Fluor 568 donkey anti-goat IgG (H+L) secondary antibodies (Invitrogen) according to the manufacturer’s instructions.

Techniques: Cryo-EM Sample Prep