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. 2022 Apr 8;209:114222. doi: 10.1016/j.bios.2022.114222

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Fig. 2 — Production, purification, and validation of antibodies. (A) A pipeline of antibody production: expression and purification of the recombinant protein – Wuhan-N (truncated form of N protein); immunization of the rabbit; purification of rabbit antibodies. Right panel: SDS PAGE gel showing purified Wuhan-N protein, Left panel: SDS PAGE gel showing BSA standard titration and purified Wuhan-N-ab antibodies (NC – Negative control, PP – Purified Wuhan-N, PA – Purified Wuhan-N-ab). (B) Titration of the Wuhan-N-ab antibodies in ELISA tests, ELISA plates were coated with Wuhan-N protein, bacterial cell lysate from BL21 E.coli served as a negative control. (C) Cross-reactivity of the Wuhan-N-ab antibody with different forms of nucleoprotein antigens produced in heterologous expression systems. Purified Wuhan-N protein was produced in a bacterial system, full-length N protein - full-N was produced in insect cells. Bacterial cell lysate from BL21 E.coli, Sf9 insect cell lysate, and wild-type baculovirus lysate served as a negative control (NC - BL21 E.coli cell lysate, SF9 - Sf9 cell lysate, WT - WT baculovirus lysate and fN - full-N) (D) Cross-reactivity of the Wuhan-N-ab antibody with other respiratory tract viruses. Influenza A Virus (IVA), Influenza B virus (IVB), Respiratory Syncytial Virus (RSV), and Epstein-Barr Virus (EBV) inactivated viruses were used to coat the ELISA plate. Norovirus (NoV) served as a non-respiratory tract virus control, Wuhan-N and full-N served as a positive control, swab from the healthy individual served as a negative control. (E) Detection of SARS-CoV-2 virus using immunofluorescence assays in infected human airway epithelial cells using the Wuhan-N-ab antibody. Red: SARS-CoV-2 virus, blue: nucleus. Images were obtained at 40x magnification.