Infinite m200 elisa reader

Monoclonal Antibodies were diluted to 20ug/ml (final concentration after addition of virus: 10ug/ml) and diluted 10-fold over 4–8 wells in a 96-well plates in duplicates. An equal volume of DENV-1 at MOI 0.01 (DENV-1-WestPac74, accession Nr. U88535.1) or DENV-2 at MOI 0.1 (TSV01, accession Nr. AF013774) was added to each well and incubated for 1hr at 37°C with 5% CO₂. 100μL of the antibody-virus mixture was then added to Vero cells, seeded at a density of 20,000 cells per well the day before, and incubated for 1hr at 37°C - 5% CO₂. 100μL of 10% FCS RPMI medium with 1% Penicillin/Streptomycin was added in each well and the plate was incubated for 4 days at 37°C - 5% CO₂. On day 4, cells were fixed with 3.7% formalin, permeabilized with 0.1% Triton X-100, and blocked by adding 100μL of 10% FCS RPMI medium. Mouse anti-DENV E antibody (4G2) was incubated at 1μg/ml in each well for 2hr at 37°C. Cells were washed before adding secondary antibody goat-anti-mouse-HRP antibody for 1hr at 37°C. Finally, TMB substrate was added for 5 min and the reaction was stopped by adding HCl. OD was read at 450nm using an Infinite M200 Elisa reader (TECAN).

The cytotoxicity of the peptide-drug conjugates was determined by measuring the mitochondrial enzyme activity, using a commercial XTT assay kit. PANC-1 cell line was cultured in RPMI medium containing 10% heat-inactivated Fetal Bovine Serum (FBS), 2 mM glutamine, 1% penicillin and streptomycin; and cultured at 37 °C in a humidified incubator with 6% CO₂. Cells were initiated in microplate wells at a concentration of 2–4 × 10⁴ and allowed to adhere for 24 h. Then the cells were washed with PBS pH 7.4 buffer twice, then PBS buffer containing 2.5% DMSO and different concentrations of the 5-CLB, CLB and OctA (1.6, 3.1, 6.2, 12.5, and 25 μM) were added and the cells were incubated for an additional 10 min. The solvent was removed; all the wells were washed with PBS and cultured for 24 h in fresh medium without drug substances. The cells were washed again, a fresh medium containing the XTT reagent was added, and the cells were incubated for 2 h. The absorbance in the wells were measured with a TECAN Infinite M200 ELISA reader at both 480 nm and 680 nm; the latter being the background absorbance. The difference between these measurements was used for calculating the percent of Growth Inhibition (GI) in test wells compared to the cells that were exposed only to the medium with 2.5% DMSO. All the tests were done in triplicate (Fig. S4†).

Recombinant monomeric E protein from DENV-1 or DENV-2 (E80 provided by Merck) and UV-inactivated virus from DENV-1 (strain DENV-1-D1/SG/05K2916DK1/2005 (EU081234.1)) or DENV-2 (strain DENV-2-TSV01 (AY037116.1)) were used as antigens to coat 96-well half-area plates (675061, Greiner bio-one) overnight at 4°C. Virus was concentrated by PEG precipitation and UV-inactivated for 10min using a germicidal UV light source (UV lamp VL-206.G 2 × 6 X – 254 nm tube, Vilber Lourmat) . Plates were washed and saturated with PBS 1X + 0.05% Tween-20 + 3% skim milk and incubated either with serially diluted plasma (1/500, 1/2500, 1/7500, 1/22500, 1/67500, 1/337500) or supernatant from B cell culture (volume added normalized to total IgG: 4ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml or 0.25ug/ml) for 1.5hrs at room temperature. Plates were then washed and an anti-human-IgG-HRP was added for 1h at room temperature. Finally, 3,3′,5,5′ TetraMethylBenzidine (TMB) substrate was added into the plates for 5 min and the reaction was stopped by adding hydrochloric acid (HCl). Optical density (OD) was read within 30 min at 450nm (reference 570nm) using Infinite M200 Elisa reader (TECAN).