Immuno maxisorp

ELISA assays were performed with Nucleoprotein (N) proteins made in house, as described below. Briefly, recombinant N proteins were coated on 96 well flatbottom immunosorbent plates (Nunc Immuno MaxiSorp) at a concentration of 500 ng/mL, in 100 μL coating buffer (PBS with 53% Na₂CO₃ and 42% NaHCO₃, pH 9.6) at 4°C overnight. An additional plate coated with a non-specific protein (blocking buffer, PBS with 5% fetal bovine serum (FBS)) was used to measure the background binding of each plasma sample. Following FBS blocking and thorough washing, diluted plasma samples (1:100) were bound for 2 hours, further washed and then detected by an anti-human IgG secondary antibody labelled with HRP (Invitrogen), and absorbance detected at 450nm on a spectrophotometer (Wallac).

1-KGP-P or 4-KGP-P (50 µg ml⁻¹) in carbonate--bicarbonate buffer (pH 9.6) was coated on 96-well Nunc-Immuno MaxiSorp microtiter plates overnight at 4°C. The coating solutions were removed and 200 µl well⁻¹ of 4% (w/v) milk protein in phosphate-buffered saline (MP/PBS) was added to block the plates for 2 h at room temperature to prevent non-specific binding. The plates were washed and then 100 µl well⁻¹ of the primary antibody (LM10, LM 15 and LM 21) was added at a 1 : 50 dilution in MP/PBS. Following incubation for 1.5 h, the plates were washed and the wells were incubated with anti-rat IgG coupled to horseradish peroxidase (HRP) for an additional 1.5 h. After washing with PBS, 100 µl well⁻¹ of freshly prepared HRP-substrate (18 ml of deionized water, 2 ml of 1 M sodium acetate buffer, pH 6.0, 200 µl of tetramethylbenzidine and 20 µl of 6% (v/v) hydrogen peroxide) was added. The reaction was stopped after 5 min by the addition of 100 µl well⁻¹ of 2 M H₂SO₄. Antibody binding was determined by measuring the absorbance at 450 nm in a micro-plate reader (Bio-Rad, USA) [18 (link)].

Plates (Nunc-Immuno MaxiSorp) were coated with anti-E7 antibody, each of the vaccine preparations were added at a concentration corresponding to 60 ng/mL of E7 protein. Following washing and incubations, anti-Qβ monoclonal antibody was added followed by secondary detection antibody goat anti-mouse IgG-HRP and TMB. Data expressed in OD450.

Supernatant from hybridoma culture was screened by indirect ELISA as described previously [38 (link)]. Briefly, assay plates (Nunc Immuno MaxiSorp, Thermo Electron LED) were coated with recombinant FLT3‐Fc, FLT3s‐Fc, FLT3‐V5H6, FLT3s‐V5H6 or relative control protein in carbonate/bicarbonate buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate and 3 mM sodium azide; pH 9·6) and incubated overnight at 4 ºC. Plates were washed in PBS containing 0·1% (v/v) Tween 20 (PBS‐T) and blocked with 0·5% (w/v) casein/PBS at RT for 1 h. Neat supernatant of hybridoma culture or purified mAb was added to the plate and incubated at RT for 1 h. After three washes with PBS‐T, plates were incubated with goat anti‐mouse IgG‐horseradish peroxidase (HRP) at RT for 1 h. After a further three washes, plates were visualized by 3,3’,5,5'‐tetramethylbenzidine (TMB) substrate (Invitrogen, Thermo Fisher Scientific), and the reaction was stopped by 2N H₂SO₄. Plates were read at 450 nm in a SpectraMax 250 microplate spectrophotometer system (Molecular Devices, Sunnyvale, CA, USA).

Nunc-Immuno MaxiSorp™ plates were coated with RSV (10⁵ plaque-forming units/mL) overnight. Test samples were diluted and incubated in microtiter wells for 45 min alongside mouse IgA standards, using IgA ELISA (Life Diagnostics, #IGA-1). Horseradish peroxidase conjugate was added and incubated for 45 min, followed by incubation with TMB reagent for 20 min at room temperature. Color development was stopped by adding the stop solution, and optical density was spectrophotometrically measured at 450 nm. The IgA concentration was derived from a standard curve with a range of 0.93–30 ng/mL.