Maxisorp 96 well plates nunc

MaxiSorp 96-well plates (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) were coated with 0.25 μg/well anti-M13 antibodies (Sigma-Aldrich, St. Louis, MO, USA) in 100 mM carbonate buffer (pH 9.0) at 4 °C overnight, and washed with PBS, containing 0.1% Tween 20 (pH 7.4). This washing was performed after each step. The wells were then blocked with 5% dry milk in carbonate buffer and incubated for 1 h at 37 °C. The plates were incubated with analyzed bacteriophages diluted in PBS containing 0.05% Tween 20 and 0.5% dry milk at 37 °C for 1 h; then, respective monoclonal HRP-conjugated antibodies were added in dilution 1:3000 (anti-HA-HRP, Sigma-Aldrich, USA) or 1:5000 (anti-flag-HRP; anti-M13-HRP, GE Healthcare, Amersham, UK), and the plates were incubated again at 37 °C for 1 h. After washing, tetramethyl benzidine (Amresco, Solon, OH, USA) was added to each well, and the plates were placed in the dark for 10 min. The reaction was stopped by adding 10% phosphoric acid. The A₄₅₀ values were measured on a Varioskan Flash microplate reader (Thermo Fisher Scientific).

Indirect ELISA was used to determine specific anti-S antibody levels in sera from immunized mice. Nunc 96-well MaxiSorp plates (Thermo Fisher Scientific) were coated with 150 ng/well of purified RBD by overnight incubation at 4 °C. Coated wells were subsequently blocked by incubation with PBS containing 0.1% Tween 20 (PBST) and 3% dried milk for 1 h at RT, and then washed three times with PBST. For antibody determination, sera were diluted in PBST 1% dried milk and incubated in the wells for 1h at RT. After three washes anti-mouse IgG-HRP secondary antibody (Dako cat. number P0447) diluted 1:2000 in PBST or anti-mouse IgG-HRP secondary antibody (cat. number AP128P, Sigma-Aldrich, St. Louis, MO, USA) diluted 1:1000 was added to the wells and incubated for 1 h at RT. After washing three times with PBST, the reaction was developed with 50 μL of substrate solution TMB (Sigma-Aldrich) and finalized by addition of the same volume of 3 NH₂SO₄. Results were expressed as optical density (OD) measured at 450 nm.

Optiprep density gradient medium, Proteinase K and Phorbol-12-myristate-13-acetate were purchased from Sigma Aldrich (Burlington, MA, USA). THP-1 cell line was procured from NCCS, Pune, India. Pierce BCA Protein Assay Kit, RPMI 1640 media, Fetal Bovine serum and Penicillin–streptomycin, Nunc™ Lab-Tek™ II Chamber Slide™, Lysotracker Green and Prolong Gold Antifade DAPI Mountant, BD cytofix/cytoperm Fixation/Permeabilisation Kit, MaxiSorp Nunc 96 well plates were all from Thermo Fisher Scientific (Waltham, MA, USA).
All oligonucleotides used in the current study were purchased from Integrated DNA Technology (IDT, Coralville, IA, USA). PCR master mix was procured from Takara, Japan. Nuclease free water was obtained from HI Media Laboratories, India. For all buffers/solutions preparation, milliQ water was used. The aptamers population was cloned in a TA cloning vector (pTZ57R/T vector) from Thermo Fisher Scientific, USA, using InsTA Clone PCR cloning Kit. 7H9, 7H11 and OADC were purchased from BD (Franklin Lakes, NJ, USA) and Luria Bertiani media was purchased from HI Media Laboratories, Mumbai, India. Most chemical reagents were purchased from either Sigma Aldrich, USA or HiMedia Laboratories Pvt. Ltd., Mumbai, India.