Cell suspension harvested from cell cultures was clarified by filtration using 0.45-µm PVDF membranes (Merck Millipore) and used to perform either ultrafiltration/diafiltration (UF/DF) or affinity chromatography (AC) experiments. Concentration/diafiltration (90-fold concentration by volume) was achieved by UF/DF using centrifugal devices based on cellulose membranes with 100-kDa cut-off (Merck Millipore). Affinity chromatography was carried out in an Äkta Purifier system (Cytiva) using a 5-mL StrepTrap XT affinity chromatography column (Cytiva) following manufacturer's instructions. Protein concentration, purity and identity in the eluted fractions were confirmed by NanoDrop (Thermofisher), silver-stained SDS-PAGE and Western blot analyses, respectively. For Western blots, a pool of serum of SARS-COV-2 convalescent patients (1:1000) was used as primary antibody, followed by incubation with anti-human IgG (Fc) HRP-labeled antibody (Sigma, #SAB3701282) and then chemiluminescent ECL reagent (BioRad).
Anti human igg fc hrp labeled antibody
Manufactured by Merck Group
Sourced in United States
The Anti-human IgG (Fc) HRP-labeled antibody is a laboratory reagent used for the detection of human immunoglobulin G (IgG) antibodies. It is a conjugate of an anti-human IgG antibody and horseradish peroxidase (HRP), which serves as a reporter enzyme for immunoassays.
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2 protocols using anti human igg fc hrp labeled antibody
1
SARS-CoV-2 Antibody Purification Protocol
2
SARS-CoV-2 Spike Protein ELISA Protocol
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The S-UFRJ ELISA was performed using the trimeric spike protein of SARS-COV-2 produced in stable recombinant HEK293 cells as described in Alvim et al. (2022) (link) and it was provided by the Cell Culture Engineering Laboratory—LECC from the Universidade Federal do Rio de Janeiro—UFRJ. The secondary antibodies concentration used was 1:200 goat anti-human IgM (Fc) HRP-labeled antibody (#MFCD00162459; Sigma, St. Louis, MO, USA), anti-human IgG (Fc) HRP-labeled antibody (#SAB3701282; Sigma, St. Louis, MO, USA), anti-human IgE (Fc) HRP-labeled antibody (#A18793; Invitrogen, Waltham, MA, USA), and anti-human IgA (Fc) (#PA1-74495; Invitrogen, Waltham, MA, USA), incubated for 1 h at room temperature. The reaction was read at 450 nm in a Thermo Scientific Multiskan Sky microplate reader. The results were expressed either as optical density units (OD) or as the ratio of sample OD/cut-off value. The pre pandemic healthy donors (blood samples before 2019) were selected as negative controls. Then, the cut-off was made by means of O.D. of the samples plus three times the O.D. Standard deviation (Alvim et al., 2022 (link)).
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