mouse rabv g antibody  (Bio-Rad)

Journal: Emerging Microbes & Infections

Article Title: A nucleoside-modified mRNA vaccine forming rabies virus-like particle elicits strong cellular and humoral immune responses against rabies virus infection in mice

doi: 10.1080/22221751.2024.2389115

Figure Lengend Snippet: Design and Characterization of RABV mRNA vaccines. (A) Schematic of five mRNA candidates for rabies. RABV G contains a mRNA encoding the full length of wild type RABV G. RABV tG-MTQ contains a mRNA encoding soluble truncated trimeric form of RABV G. RABV preG contains a mRNA encoding stabilized prefusion form of RABV G. RABV VLP contains two mRNAs encoding preG and M proteins that can self-assemble into VLPs. RABV VLP/N contains three mRNAs encoding preG, M, and N proteins. (B) Western blotting of RABV M protein. Expression of RABV M proteins were probed using mouse anti-RABV M polyclonal antibodies. (C) Immunofluorescence assays of the expression of RABV G, preG, and N proteins. 293 T cells were transfected with RABV G, preG, and N mRNA for 48 h, respectively, and detected by anti-rabies virus glycoprotein antibody and FITC-rabies virus nucleoprotein antibody. Scar bar: 50 μm. (D and E) Expression of the trimeric form of RABV G protein (tG-MTQ) was analyzed by SDS-PAGE, Native-PAGE, and western blottings, which showed that the monomeric and trimeric forms of tG-MTQ were approximately 57 and 150 kD, respectively. (F) Expression of the trimeric form of RABV G protein (tG-MTQ) was analyzed using a sandwich ELISA which were probed with mAbs 1112 and D1-25. (G) Western blotting assays of RABV VLPs. Expression of RABV preG, M or N proteins was probed using mouse anti-RABV G mAb, mouse anti-RABV M polyclonal antibodies and mouse anti-RABV N mAb. (H) Electron microscopy of VLPs. RABV VLP and VLP/N were concentrated by ultracentrifugation and prepared to negative staining for electron microscopy. Scar bar: 200 nm. (I) Immunoelectron microscopy of VLPs. RABV VLP and VLP/N were stained with mouse anti-RABV G mAb and then incubated with gold-labeled goat anti-mouse IgG antibody. Scar bar: 200 nm. (J) Stability of mRNA-LNPs stored at 4°C. Particle size and Zeta potential of six mRNA-LNPs detected in 28 d.

Article Snippet: The VLPs collected in the supernatants of 293 T cells that co-transfected with preG, M mRNAs (RABV VLP), or co-transfected with preG, M, N mRNAs (RABV VLP/N) were analyzed by Western blotting with mouse anti-RABV G mAb (3R7-4F1, HyTest, Finland) and mouse anti-RABV M polyclonal antibodies.

Techniques: Vaccines, Western Blot, Expressing, Immunofluorescence, Transfection, Virus, SDS Page, Clear Native PAGE, Sandwich ELISA, Electron Microscopy, Negative Staining, Immuno-Electron Microscopy, Staining, Incubation, Labeling, Zeta Potential Analyzer

Journal: Emerging Microbes & Infections

Article Title: A nucleoside-modified mRNA vaccine forming rabies virus-like particle elicits strong cellular and humoral immune responses against rabies virus infection in mice

doi: 10.1080/22221751.2024.2389115

Figure Lengend Snippet: Humoral immune responses after RABV mRNA vaccination. (A) Schematic of the immunization, rabies virus challenge, and sampling strategy. Six- to eight-week-old female BALB/c mice were immunized intramuscularly twice at day 0 and 14 with RABV G, tG-MTQ, preG, VLP, and VLP/N mRNA vaccine, respectively, followed by virus challenge and tissue sample collection. (B to E) RABV-specific IgG and IgG antibody isotype (IgG1, IgG2a, and IgG2b) titres were quantified by ELISA, respectively, from serum samples collected on day 28 ( n = 6). (F) The ratio of IgG2a/IgG1 and IgG2b/IgG1 were determined from the absorbance values of 100-fold diluted serum at day 28 measured at 450 nm. (G and H) Neutralizing antibody (Nab) titres of serum collected at day 14 and 28 were analyzed by Rapid Fluorescent Focus Inhibition Test (RFFIT) assay ( n = 6). The dashed line at 0.5 IU/mL represents a protective titre for RABV Nab titre. (I) Spearman correlation of (B) RABV-specific IgG titres and (H) Nab titres. (J) Kinetic of Nab in mice immunized with RABV mRNA vaccines or inactivated vaccines after a single vaccination ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, no significant difference. Data represent mean ± SEM.

Article Snippet: The VLPs collected in the supernatants of 293 T cells that co-transfected with preG, M mRNAs (RABV VLP), or co-transfected with preG, M, N mRNAs (RABV VLP/N) were analyzed by Western blotting with mouse anti-RABV G mAb (3R7-4F1, HyTest, Finland) and mouse anti-RABV M polyclonal antibodies.

Techniques: Virus, Sampling, Enzyme-linked Immunosorbent Assay, Inhibition, Vaccines

Journal: Virologica Sinica

Article Title: An inactivated recombinant rabies virus chimerically expressed RBD induces humoral and cellular immunity against SARS-CoV-2 and RABV

doi: 10.1016/j.virs.2022.12.006

Figure Lengend Snippet: Construction and identification of the recombinant viruses. A The construction scheme of the recombinant viruses SRV-nCoV-S1 and SRV-nCoV-RBD based on the reverse genetic operating system of RABV SRV9. B NA cells were infected with SRV-nCoV-S1, SRV-nCoV-RBD or wt-RABV SRV9. The cells were permeabilized or not at 48 h post-infection and then immunostained with antibodies against RABV G protein (red fluorescence) and SARS-CoV-2 RBD protein (green fluorescence). The nuclei were stained with DAPI (blue fluorescence). Scale bar: 10 μm. C The sucrose density gradient centrifugation-purified virions of recombinant viruses and wt-RABV SRV9 were analyzed by SDS-PAGE. The RABV proteins (RNA-dependent RNA polymerase (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M)) and the S1 and RBD protein of SARS-CoV-2 were indicated on the corresponding bands. D Sucrose-purified virions were used to detect the expression of the exogenous proteins SARS-CoV-2 S1 and RBD. E Electron microscopy (EM) and dual-label immunogold EM detection of recombinant viruses. Gold particles (10 nm, red arrow) were used to label RABV G, and gold particles (18 nm, green arrow) were used to label the SARS-CoV-2 RBD protein. Scale bars represent 50 nm.

Article Snippet: The cells were blocked with 1% BSA for 30 min at 20∼25 °C) and incubated with a rabbit anti-SARS-CoV-2 RBD polyclonal antibody (PAb) (40592-T62, Sino Biological, 1:200) and mouse anti-RABV-G monoclonal antibody (mAb) (MAB8727, Abcam, 1:200) at 37 °C for 1 h. An FITC-conjugated anti-rabbit antibody (ab6717, Abcam, 1:300) and TRITC-conjugated anti-mouse antibody (T5393, Sigma, 1:500) were added and then incubated at 37 °C for 1 h. After staining with 4,6-diamidino-2-phenylindole (DAPI), the fluorescence was observed by confocal laser microscopy.

Techniques: Recombinant, Infection, Fluorescence, Staining, Gradient Centrifugation, Purification, SDS Page, Expressing, Electron Microscopy