Journal: Biomedicines

Article Title: New Monoclonal Antibodies Specific for Different Epitopes of the Spike Protein of SARS-CoV-2 and Its Major Variants: Additional Tools for a More Specific COVID-19 Diagnosis

doi: 10.3390/biomedicines11020610

Figure Lengend Snippet: WB analysis of anti-S1 and anti-S2 mAbs. ( A ) Supernatants of SARS-CoV-2-infected Vero E6 cells subjected to SDS-PAGE and WB. Polyclonal antibody against SARS-CoV N protein was used as a positive control. One experiment representative of three is shown. ( B ) WB analysis of anti-S1 mAbs (S12 and S157), anti-S2 mAb (S3), and anti-Flag, the latter as a control of transfection; SDS-PAGE of cell lysates from HEK293T-expressing SARS-CoV spike protein followed by WB is shown. One experiment representative of three is shown. ( C ) Sandwich ELISA with S71–S79 pair of mAbs for the detection of scalar quantities of SARS-CoV-2 (PFU/well). Error bars indicate the standard deviations of technical duplicates from a representative experiment repeated twice.

Article Snippet: Membranes were incubated in blocking solution containing 3–5% skim milk (Millipore, Burlington, MA, USA) in Tris-buffered saline (25mM Tris-HCl pH7.4, 0.5 mM NaCl, TBS) for 2 h. In order to detect the S proteins, the following commercial antibodies, in TBS containing 0.05% Tween (TBST), were used: (1) polyclonal SARS-CoV-2 S antibody, Rabbit Pab, Sino Biological; (2) polyclonal SARS S Protein Antibody NB100-56578T, Biotechne; (3) monoclonal Tetra HIS Antibody (Qiagen).

Techniques: Infection, SDS Page, Positive Control, Transfection, Expressing, Sandwich ELISA

Journal: Biomedicines

Article Title: New Monoclonal Antibodies Specific for Different Epitopes of the Spike Protein of SARS-CoV-2 and Its Major Variants: Additional Tools for a More Specific COVID-19 Diagnosis

doi: 10.3390/biomedicines11020610

Figure Lengend Snippet: Isolated S-specific mAbs recognize the major SARS-CoV-2 Variants of Concern. ( A ) WB analysis of all of the generated mAbs specific for the S1 and S2 domains in SDS-PAGE supernatants of Vero E6 cells infected with SARS-CoV-2 Wuhan, Alpha, Beta, and Delta VOC. The signal was detected by HRP chemiluminescent substrate except for the S200 and S136 mAbs, which were revealed by TMB colorimetric substrate. ( B ) Representative WB of anti-N and anti-M polyclonal Abs (pAbs) detected by colorimetric substrate. ( C ) WB analysis of all of the generated mAbs specific for S1 and S2 proteins in SDS-PAGE supernatants of Vero E6 cells infected with SARS-CoV-2 Omicron VOC. WB of anti-N polyclonal Ab (pAb) was performed as a control on the same strip of S106 mAb. ( D ) WB analysis of a mixture of the two representative mAbs specific for the S1 domain (S71 and S79) in SDS-PAGE nasal swabs from a positive (Covid+) or negative (Covid−) individual. One experiment representative of three is shown.

Article Snippet: Membranes were incubated in blocking solution containing 3–5% skim milk (Millipore, Burlington, MA, USA) in Tris-buffered saline (25mM Tris-HCl pH7.4, 0.5 mM NaCl, TBS) for 2 h. In order to detect the S proteins, the following commercial antibodies, in TBS containing 0.05% Tween (TBST), were used: (1) polyclonal SARS-CoV-2 S antibody, Rabbit Pab, Sino Biological; (2) polyclonal SARS S Protein Antibody NB100-56578T, Biotechne; (3) monoclonal Tetra HIS Antibody (Qiagen).

Techniques: Isolation, Generated, SDS Page, Infection, Stripping Membranes