hek293 human ace2 stable cell line  (ACROBiosystems)

Journal: Nature Communications

Article Title: De novo protein sequencing of antibodies for identification of neutralizing antibodies in human plasma post SARS-CoV-2 vaccination

doi: 10.1038/s41467-024-53105-8

Figure Lengend Snippet: A Separation of the polyclonal antibody on a native gel. Initially, the enriched polyclonal antibody was digested with IdeS, and the resulting sample was fractionated into 12 fractions and further digested with trypsin. The separation was carried out once with IdeS and once without IdeS, respectively. Separation without Ides resulted in fewer fractions (see Source Data file). B Quantitative profiling of various peptide “contigs” across those 12 fractions, where contigs were grouped based on their similarity to assemble chains and pair heavy-light (HL) chains (more information on the contig sequence can be found in SI Table ). C top: shows an example of the sequence coverage resulting from EThcD analysis of trypsin and pepsin digestion. Peptides resulting from pepsin digestion were labeled with Arginine methyl ester. Bottom: displays EThcD spectra acquired to resolve specific Isoleucine/Leucine assignments; in that case, the generation of w-ions under EThcD fragmentation allows the assignment of w8 to an isoleucine. Additionally, in this case, a positive charge (i.e., Methyl Arginine as a y1 ion) was added to the C-terminal end of the non-tryptic peptide to facilitate the C-terminal fragment to be detectable in MS/MS mode. D Expressed candidates and their affinities in ELISA Assay, Angiotensin-converting enzyme 2, ACE2 competition binding assay and in vitro neutralization assay (Using either the Receptor-Binding Domain, RBD or the protein S1). Source data are provided as a Source Data file.

Article Snippet: For the in vitro, cell-based neutralization assay, HEK293/Human ACE2 Stable Cell Line (Acro Biosystems) was cultured in complete DMEM medium (Shanghai BasalMedia Technologies) supplemented with 10% fetal bovine serum (VivaCell) at 37 °C with 5% CO2.

Techniques: Sequencing, Labeling, Tandem Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Binding Assay, In Vitro, Neutralization

Journal: Nature Communications

Article Title: De novo protein sequencing of antibodies for identification of neutralizing antibodies in human plasma post SARS-CoV-2 vaccination

doi: 10.1038/s41467-024-53105-8

Figure Lengend Snippet: The Materials and Methods section describes how the various ELISA procedures were performed. A Conventional ELISA assay comparing recombinant antibodies with the natural polyclonal antibody PD124 affinity enriched in this study as a benchmark measure. The red dashed arrows are the IC50 for the best recombinant antibody (R2 at approx. 3 nM) versus the natural polyclonal antibody (PD124 at approx. 4 nM). Nonbinding recombinant antibodies (r3, r7, r8, r9, r10) were eliminated from further comparisons. Due to its restricted availability, the natural polyclonal antibody PD124 was excluded from other investigations. Figure 4B illustrates an ACE2 competitive binding assay conducted on the recombinant forms found in ( A ), utilizing the RBD domain. C ACE2 competitive binding assay that targets the S1 protein as the bait in the test. For A - C , although these ELISA assays focus on different aspects, they all utilized the horseradish peroxidase assay with TMB (3,3′,5,5′ tetramethylbenzidine, TMB, resulting in the measurement outcomes at 450 nm across the three different assays. D In vitro neutralization test that utilizes SARS-CoV-2-Spike/Flu-GFP pseudovirus to neutralize a positive standard S1N-M122. The assay measures the ratios of Absolute IC55. The positive control in B , C is AS35, an Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG1, while in ( D ), it is the S1N-M122, a known anti-Spike RBD Neutralizing Antibody, Chimeric mAb, Human IgG1 (AM122), both sourced from Acro Biosystems. Source data are provided as a Source Data file.

Article Snippet: For the in vitro, cell-based neutralization assay, HEK293/Human ACE2 Stable Cell Line (Acro Biosystems) was cultured in complete DMEM medium (Shanghai BasalMedia Technologies) supplemented with 10% fetal bovine serum (VivaCell) at 37 °C with 5% CO2.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Competitive Binding Assay, In Vitro, Neutralization, Positive Control