Nunc maxisorp plate

The following variants of sera were used for the ELISA analysis: (1) convalescent sera, designated as R (12 samples); (2) archival sera collected in 2018 (before the pandemics), designated as naïve (10 samples); and (3) paired sera of people vaccinated with the Sputnik V vaccine on day 0 (d0) and day 42 (d42) (12 samples each). The paired sera samples were obtained from volunteers who gave their informed written consent. The project was approved by the ethics committee.
The total protein concentration within the inactivated preparations was measured using a Qubit fluorometer with supplied reagents (Thermo Fisher Scientific). All inactivated antigen preparations, at a concentration of 10 µg/mL, were adsorbed onto Nunc MaxiSorp plates (#442064) at +4 °C overnight. The antigen preparations were then removed, the plates were washed three times, and a blocking buffer containing 5% milk powder was added for 2 h at RT. Afterwards, all sera were diluted at a ratio of 1:100 in 5% milk powder and incubated with plates for 1 h at RT. Each serum was analyzed in duplicate. Anti-human IgG HRP (Sigma #A0170-1ML) was diluted at a ratio of 1:5000 and used as detecting antibodies, a TMB reagent (Biolegend 421101) was used as a substrate, and 2N H₂SO₄ was used as a stop solution. Absorbance was calculated by measuring an optical density (OD) at 450 nm (Multiscan SkyHigh, Thermo FS).

All ethical approval for the study was obtained from the institutional (AIIMS, New Delhi) ethics committee (IESC/OT-02/02.07.10). All methods were carried out in accordance with the approved guidelines of the AIIMS ethics committee. Also it is confirmed that an informed consent was obtained from all the subjects before enrolment into the study.
Individuals who had naturally recovered from hepatitis B but were on routine check-up in the Liver clinic of All India Institute of Medical Sciences (AIIMS) were recruited. The selection criteria included chance detection cases with positivity for anti-HBc and anti-HBs antibodies but with no detectable amounts of HBsAg or HBV-DNA and no history of therapy for infection. 10 ml blood collected from all the individuals (n = 7), was used for serum separation as well as peripheral blood lymphocyte (PBL) isolation (using Histopaque-1077). For serum ELISA, serum (1:100) was incubated with the antigens immobilized on Nunc maxisorp plates (Nunc, 442404) at 37 °C for 1 hour and the bound IgG antibodies were detected using anti-human IgG-HRP conjugate (1:5000) and substrate OPD.

sCD59 concentrations in serum were determined via ELISA (USCN Life Science Inc., China) according to manufacturer’s instructions. Serum samples were thawed, diluted 1:400 in PBS, and incubated on NUNC maxisorp plates (NUNC, Roskilde, Denmark) coated with a mouse anti-human CD59 monoclonal antibody for 2 hours at 37 °C. The plates were washed and incubated with biotin-conjugated rabbit anti-human CD59 polyclonal antibodies for 1 hour at 37 °C. Plates were washed again and incubated for 30 minutes with streptavidin-horseradish peroxidase (HRP). Bound biotinylated antibodies were visualized with TMB substrate for 15 minutes at 37 °C. The reaction was terminated with H₂SO₄. Optical density of the wells was measured at 450 nm with a Multiskan EX Microplate photometer (ThermoScientific, IL). OD450 values were compared to standard concentrations of recombinant CD59 and are expressed in pg/ml. The minimal detectable dose of human sCD59 is 6.7 pg/ml. Intra- and inter-assay coefficients of variation are <10% and <12% respectively. Furthermore, the recovery rate of the ELISA in our hands was 87% (range 83–91%), which was in concordance with the manufacturers description (average recovery rate in serum 87%, range 80–94%). All samples were measured in duplicate.

His-DDX3X^414-582 protein fragment was coated onto 96-well Nunc MaxiSorp plates (NUNC, Denmark) at a final concentration of 0.01 µg/µl in 100 mM sodium carbonate buffer (pH 9.6) at 4°C overnight. Unspecific binding sites were blocked with 5% FCS in PBS. Biotinylated CK1δ- and CK1ε-derived peptides (1 µg/well) were added and incubated for 2.5 h at room temperature. DMSO (0.35%) and water were used as negative controls. Detection of the bound biotinylated peptides was performed by incubation with HRP-streptavidin (1:8000 in 0.5% FCS in PBS). Finally, plates were incubated with ABTS detection reagent [50 mM potassium phosphate buffer, pH 5.7, 5% ABTS solution (1 mg/ml), 0.05% H₂O₂]. Absorption was measured at 405 nm after incubation for 40 min.

For phage binding assays, 1 μg of purified IgG was immobilized on Nunc Maxisorp plates (Nunc, Wiesbaden, Germany) overnight at 4°C. After blocking wells with PBS containing 0.1% Tween-20 and 1% non fat dry milk (NFDM) for 2h at room temperature (RT), we added 10⁸ TU of either amplified phage clones or control phage for 2h at RT. For inhibition assays, increasing amounts of corresponding GST-fused peptides or wild-type (negative control) GST were pre-incubated for 30 min at RT prior to addition of phage. Unbound phage were removed by washing 5 times with PBS containing 0.1% Tween-20 and 1% NFDM. Bound phage were recovered with 200 μl log-phase E. coli K91 and plated in serial dilutions (triplicate plates for each dilution).