Hrp conjugated goat anti mouse igg antibody

The anti-HPV16 L1 IgG titers of mouse sera were measured as described [13] (link), [14] . Briefly, 96-well ELISA plates were coated overnight with 100 ng of purified HPV16 L1 VLPs per well and blocked with 5% skim milk in PBST. The plates were reacted with three- or four-fold serial dilutions of mouse sera for 1 h at 37°C. The anti-HPV16 L1 IgG bound to the coated HPV16 L1 VLPs was detected using HRP-conjugated goat anti-mouse IgG antibody (Bethyl), and color reactions were developed as described above.

The ELISA was carried out as described [13] (link). Briefly, a 96-well ELISA plate (Greiner Bio One, Germany) was coated overnight with 400 ng of purified HPV16 L1 VLPs per well and blocked with 5% skim milk in PBS containing 0.05% Tween 20 (PBST). The plate was incubated with 250 ng/mL of anti-HPV16 neutralizing monoclonal antibodies (Mabs), H16.V5 or H16.E70, for 1 h at 37°C. The Mabs bound to VLPs were detected using HRP-conjugated goat anti-mouse IgG antibody (Bethyl Laboratories, USA). Color reactions were developed with o-phenylenediamine (Sigma, USA), and optical density was measured at an absorbance of 492 nm.

The assay was run according to the protocol from Petrucci and co-workers [20 ] with slight modifications. All the steps were conducted at RT. Briefly, microtiter plates (Maxisorp 96well, Nunc #442404) were coated with human recombinant PF4 (10 μg/ml, ProSpec-Tany TechnoGene, Rehovot, Israel) in phosphate buffered saline (PBS) for 2h followed by the addition of either vehicle, heparin (0.002–200 μg/ml, Sigma-Aldrich, Darmstadt, Germany) or ONs (0.001–3 μM). After an overnight incubation, the plates were washed 3x with 0.1% Tween-20 and blocked with 3% BSA in PBS for 2 h. After washing with 0.1% Tween-20 plates were incubated with KKO antibody (0.1 μg/ml in 0.005% Tween 20, ThermoFisher Scientific, Waltham, MA, USA) for 1h. The plates were washed with 0.1% Tween-20 and further incubated for 1 h with the HRP conjugated goat anti-mouse IgG antibody (1:3000 dilution in 0.005% Tween-20, Bethyl Laboratories). Plates were washed with 0.1% Tween-20 and incubated with the HRP substrate BM Blue POD (100 μl/well, Roche Diagnostics GmbH) and further incubated until color development (10–30 min). The reaction was stopped by the addition of stop solution (1N H₂SO₄) and absorbance was detected at 450nm on a plate reader (EnSpire, Perkin Elmer).