Journal: iScience

Article Title: SARS-CoV-2 antigen-carrying extracellular vesicles activate T cell responses in a human immunogenicity model

doi: 10.1016/j.isci.2023.108708

Figure Lengend Snippet: Characterization of 293F cell-derived EVs carrying SARS-CoV-2 spike protein (A) Schematic of EV engineering strategy using 293F cells including SARS-CoV-2 spike expression construct (see Figure S1 ) with spike protein depicted in blue. Schematic created with Biorender.com . (B) Histogram plot of particle size distribution comparing 293F EVs and Spike EVs as determined by NTA. The number of particles falling within each size bin was normalized to total particle counts and are presented as the proportion of total particles. Data represent the mean ± standard error of the mean (SEM). N = 3 independent experiments. Statistical comparisons were carried out by unpaired, two-tailed Student’s t test where p < 0.05 would be considered significant. (C) Mean and mode particle size of 293F EVs and Spike EVs as determined by NTA. Data represent the mean ± standard error of the mean (SEM). N = 3 independent experiments. Statistical comparisons were carried out by unpaired, two-tailed Student's t test where p < 0.05 would be considered significant. n.s. indicates not significant. (D) Multiplexed bead-based profiling assay of 37 surface antigens on 293F EVs and Spike EVs confirming the presence of expected EV surface markers. Data represent the median fluorescent intensity (MFI) of APC signal (reflecting CD63-APC, CD81-APC and CD9-APC counterstaining) following the subtraction of isotype controls. N = 1 independent experiment. (E) Representative western blots characterizing the presence of SARS-CoV-2 spike protein in Spike EVs and markers known to be present in EV preparations (CD63, CD9, Flotillin, TSG101, GAPDH) as well as the absence of cell contaminant markers (GM130, Calnexin). (F) Confirmation of SARS-CoV-2 spike protein presence in Spike EVs through competitive Spike-ACE2 binding assay. Spike EVs inhibit the binding of fluorescently labeled, recombinant SARS-CoV-2 spike protein competitively, in a dose-responsive manner, while 293F EVs demonstrate little inhibition. Data are mean ± SEM. N = 2 independent experiments and 2 technical replicates per sample per run. (G) Transmission electron micrographs of unstained Spike EVs confirming expected EV morphology. SARS-CoV-2 spike protein presence on 293F Spike EVs was also visually confirmed using immunogold labeling where 10 nm gold particles are seen associating with 293F Spike EVs.

Article Snippet: Mouse Anti-Flotillin-1, Clone 18 , BD Transduction Laboratories , Cat#610820; RRID: AB_398139.

Techniques: Derivative Assay, Expressing, Construct, Two Tailed Test, Western Blot, Binding Assay, Labeling, Recombinant, Inhibition, Transmission Assay

Journal: TH Open: Companion Journal to Thrombosis and Haemostasis

Article Title: Anti-Factor XII Autoantibodies in Patients with Recurrent Pregnancy Loss Recognize the Second Epidermal Growth Factor–Like Domain

doi: 10.1055/s-0039-1695709

Figure Lengend Snippet: A representative experiment showing binding of ASQ41-bound autoantibodies purified from patient plasma to recombinant mouse EGF, human EGF, and HB-EGF. ( A ) ELISA was performed using amino plates coated with 10 µM recombinant mouse EGF, human EGF, or HB-EGF. The eluate (1:10) from the ASQ41-affinity column that contained autoantibodies to ASQ41 that were purified from anti-ASQ41-positive patient plasma or normal control plasma was applied, followed by an alkaline-phosphatase-conjugated secondary antibody. The absorption at 405 nm was then measured. ( B ) SDS-PAGE was performed using a polyacrylamide gradient gel (5–20%). Recombinant human EGF, mouse EGF, or human HB-EGF (30 μM solution) under reducing conditions was applied to each lane. Subsequent transfer to a PVDF membrane was performed for 20 minutes at 0.1 amps. We used 1% BSA in TBS (pH 7.3) to block the membrane for 1 hour. Incubation was performed with the eluate (1:10) from the ASQ41-affinity column that contained autoantibodies to ASQ41 that were purified from anti-ASQ41-positive patient plasma or normal control plasma for 2 hours, followed by three washes in 0.05% Tween 20/TBS. The membranes were exposed to horseradish-peroxidase-conjugated polyclonal antibodies against human IgM for 1 h, followed by washing as described above. The immunoreactive bands were developed using 3,3′,5,5′-tetramethylbenzidine. BSA, bovine serum albumin; EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; HB, heparin binding; IgM, immunoglobulin M; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; TBS, Tris-buffered saline.

Article Snippet: Polyclonal antibodies against recombinant mouse EGF (antimouse EGF) were purchased from R&D Systems Inc. (Minnesota, United States), and recombinant mouse EGF was obtained from ProSpec-Tany TechnoGene Ltd. (Ness Ziona, Israel).

Techniques: Binding Assay, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Affinity Column, SDS Page, Blocking Assay, Incubation, Nucleic Acid Electrophoresis

Journal: TH Open: Companion Journal to Thrombosis and Haemostasis

Article Title: Anti-Factor XII Autoantibodies in Patients with Recurrent Pregnancy Loss Recognize the Second Epidermal Growth Factor–Like Domain

doi: 10.1055/s-0039-1695709

Figure Lengend Snippet: A representative experiment showing binding of polyclonal antibodies to EGF through FXII. FXII or activated FXII was subjected to SDS-PAGE under nonreducing (NR) or reducing (R) conditions. In reduced forms, activated FXII was divided into heavy chain and light chain. FXII or activated FXII (8 µL of 100 µg/mL solution) was applied to each lane. Subsequent transfer to PVDF membranes was performed for 20 minutes at 0.1 amps. Membranes were then blocked for 2 hours with 1% BSA in TBS, pH 7.3. After incubation with polyclonal antibodies to recombinant human EGF (antihuman EGF; 0.2 µg/mL) or mouse EGF (antimouse EGF; 0.2 µg/mL) for 2 hours, we performed three washes in 0.05% Tween 20/TBS. The membranes were then exposed to horseradish peroxidase-conjugated polyclonal antibodies to goat IgG for 1 hour, followed by washing as described above. The immunoreactive bands were developed using 3,3′,5,5′-tetramethylbenzidine. BSA, bovine serum albumin; EGF, epidermal growth factor; FXII, factor XII; HC: heavy chain; IgG, immunoglobulin G; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; TBS, Tris-buffered saline.

Article Snippet: Polyclonal antibodies against recombinant mouse EGF (antimouse EGF) were purchased from R&D Systems Inc. (Minnesota, United States), and recombinant mouse EGF was obtained from ProSpec-Tany TechnoGene Ltd. (Ness Ziona, Israel).

Techniques: Binding Assay, SDS Page, Incubation, Recombinant, Nucleic Acid Electrophoresis