Journal: medRxiv

Article Title: DIFFERENTIAL SARS-COV-2 ANTIGEN SPECIFICITY OF THE HUMORAL RESPONSE IN INACTIVATED VIRUS-VACCINATED, CONVALESCENT, AND BREAKTHROUGH SUBJECTS

doi: 10.1101/2022.07.01.22277165

Figure Lengend Snippet: Five hundred nanograms (500 ng) of SARS-CoV-2 recombinant proteins (ORF1a, ORF3a, ORF8, NSP1, NSP8, NSP9, NSP10, NSP14, E (envelop), ME (membrane)) were immobilized in nitrocellulose membranes using 2 uL of proteins prepared in 8M Urea denaturant buffer. The membranes were blocked with 10% BSA and incubated overnight at 4°C with a sera pool obtained from control naïve individuals, convalescents (PCR (+) + 4w), CoronaVac-vaccinated with 2nd dose + 2 weeks, and breakthrough (PCR (+) + 2 weeks) subjects (dilution 1/250). As positive controls, all proteins (500 ng) were incubated with an anti-HisTag antibody conjugated with biotin. After incubation with sera, the membranes were treated with anti-human IgG-HRP. Finally, the membranes were incubated with an enhanced chemiluminescence Western blot detection system (Femto, ECL).

Article Snippet: Proteins evaluated included ORF1a polyprotein (Invitrogen, #RP-87701), ORF3a (LSBio, #LS-G145920), ORF8 (LSBio, #LS-G145919), NSP1 (R&D systems, #10666-CV), NSP8 (R&D systems, #10633- CV), NSP9 (R&D systems, #10631-CV), NSP10 (R&D systems, #10630-CV), NSP14 (R&D systems, #10667-CV), E (Sino Biological, #40609-V10E3) and ME (Sino Biological, #40598-V07E).

Techniques: Recombinant, Membrane, Incubation, Western Blot