Hrp conjugated anti m13

For cell screenings, endothelial cells (HUVEC and HMEC-1) were seeded in 96-well cell culture plates, 10,000 cells/well in 100–200 µl of growth medium, and cultured for 1–4 days. After two times wash in PBS, the plates were either used directly or dried at 37 °C and frozen at −80 °C. As background controls, 100 µl/well of the appropriate growth medium was added to fresh 96-well cell culture plates and incubated overnight. After two times wash in PBS, the medium plates were used directly.
Coated plates were blocked in 2% M-PBS 1–2 hrs, washed 2–3 times in PBS and incubated with 50 µl phage solution and 50 µl MPBS 1–2 hrs. Plates were washed 2–3 times in PBS-T and incubated with horseradish peroxidase (HRP)-conjugated anti-M13 (GE Healthcare) diluted 1/5000 in MPBS for 1 hr. After 2–3 times wash in PBS-T, plates were developed using TMB (tetra-methylbenzidine, Invitrogen). Reaction was stopped with 1 M H₂SO₄ and absorbance was read at 450 nm with 655 nm as reference.

Phages were produced and Phage antibody ELISA was roughly performed as outlined in the Tomlinson (I+J) protocol. Recombinant vimentin (NeoMarkers) was coated in a 96-well NUNC ELISA plate in decreasing amount ranging from 0.1 μg to 10⁻⁹ μg. Alternatively, 1 μg vimentin and 1 μg Laminin-1 (Sigma-Aldrich) were coated. The plate was blocked 1 hr at RT in 2% MPBS. The phages were incubated in 100 μL 2% MPBS holding 10¹⁰ phages/mL for 2 hrs. HRP-conjugated anti-M13 (GE Healthcare) was used 1:5000 and incubated 1.5 hrs at RT. For a chemiluminescent reaction, 100 μL TMB (Invitrogen) was added. The reaction was terminated using 50 μL 1 M H₂SO_4. The absorbance was measured at OD 450 nm with 655 nm as reference in a plate reader (BIORAD, model 550).

To determine the binding specificity of isolated antibody fragments displayed on phage particles or of whole antibodies, direct binding ELISA against DENV rNS1 was performed. 200 μL of purified rNS1 from DENV1-4 at 3 μg/mL in phosphate buffered saline (PBS) was coated on a MaxiSorp plate (NUNC) overnight at 4°C. The unbound protein was discarded and followed by 3 consecutive washes with PBS containing 0.1% Tween20 (PBS-T). The plate was blocked using PBS with 2.5% milk powder (M-PBS). The blocking buffer was discarded and 200 μL of 3 μg/mL antibody or phage diluted in the M-PBS blocking buffer was added to relevant wells. Following one hour incubation and three washes with PBS-T, the plate was probed with 200 μL of HRP-conjugated anti-M13 (GE Healthcare) or HRP-conjugated goat anti mouse [H+L] IgG (Life Technologies) or HRP-conjugated goat anti human IgG [γ chain specific] (Sigma) diluted to 0.1 μg/mL in PBS with casein. Three final washes with PBS-T were performed then 100 μL TMB substrate (Sigma Aldrich) was added to develop the chromogenic signal. The reaction was stopped with 2 N sulphuric acid and the absorbance at 450 nm was measured using the Spectramax microplate reader (Molecular Devices).