Nunc maxisorp 96 well elisa plate

Nunc Maxisorp 96-well ELISA plates (Thermo Fisher Scientific) were coated with 100 ng/well of the antigen overnight at 4 °C. Standard blocks and washes were applied. Heat-inactivated murine sera were serially diluted and incubated in precoated plates for 1 h at RT. Following washes, antibody binding was detected using antimouse IgG, IgG1, or IgG2a-horseradish peroxidase conjugates (Thermo Fisher Scientific), according to the manufacturer’s instruction. Plates were then developed with 3,5,3′5′-tetramethylbenzidine (TMB) (KPL) substrate, and the reaction was stopped with 1 N sulfuric acid. The resulting signals read at OD_450/650 were used to calculate endpoint titers, defined as the dilution that emitted an optical density exceeding a 4× background (secondary antibody alone). EC₅₀ titers were determined as the serum titers using a log (agonist) vs. normalized response (variable slope) nonlinear function in GraphPad Prism v8.

ELISA plates were incubated overnight at 4 °C with 100 ng per well of purified protein in PBS: Pierce nickel-coated 96-well ELISA plates (Thermo Fisher Scientific, USA) were coated with p67ΔTM and Nunc MaxiSorp 96-well ELISA plates (Thermo Fisher Scientific, USA) were coated with BLV Gag. Wells were washed thrice with PBS and blocked with blocking buffer (5% (w/v) skim milk in PBS) for 1 h at room temperature. Plates were washed thrice with PBS and incubated with sera diluted 3-fold in 2.5% (w/v) skim milk in PBS. These were washed thrice with PBST (PBS with 0.1% (v/v) Tween 20), then incubated with goat anti-mouse-IgG conjugated to horseradish peroxidase (HRP) (ab97023, Abcam, UK), 1:10,000, for 1 h at room temperature. Plates were washed thrice with PBST, incubated with tetramethylbenzidene (Abcam, UK) for 7 min (p67ΔTM) or 10 min (BLV Gag) and the reactions stopped with 1 N H₂SO₄. Readings were taken at 450 nm subtracted by 540 nm to remove background (VersaMax ELISA Microplate Reader, Molecular Devices, Silicon Valley, California, USA). Endpoint titres for each mouse were determined by taking the reciprocal of the highest dilution that had a reading 2-fold greater than that of the average background PBS control group sera at the lowest dilution. A one-way ANOVA with a post-hoc Bonferroni test was conducted using Prism version 5.0 (GraphPad, San Diego, California, USA).

Mouse anti-Gal d 2 monoclonal antibodies were generated by immunizing female BALB/c mice (Janvier Labs, Le Genest-Saint-Isl, France) with ovalbumin (Sigma-Aldrich, Deisenhofen, Germany) denatured by incubating for 30 min at 60 °C in 8 M urea. The immunization experiments were approved by the State Animal Care and Use Committee (Landesdirektion Sachsen, Leipzig, Germany, V 07/14) and were carried out in accordance with the European Communities Council Directive (86/609/EEC) for the Care and Use of Laboratory Animals. Splenocytes were isolated from the mouse with the highest antibody titer specific for fully denatured ovalbumin containing both variants (ovalbumin and S-ovalbumin [18 (link)]) and were fused to X63.Ag8.653 myeloma cells (ACC 43; DSMZ, Braunschweig, Germany). Hybridoma supernatants diluted 1:50 in culture medium were screened by indirect ELISA on flat-bottom Nunc MaxiSorp 96-well ELISA plates (Thermo Fisher Scientific, Darmstadt, Germany) coated with fully denatured Gal d 2 (10 µg/mL) or biotinylated Gal d 2-specific peptides.