Maxisorp nunc immunoplates

Blood was collected from mice at specific time points pre- and postchallenge and diluted 1 in 20 in PBS. The samples were centrifuged at 8,928 × g for 5 min, and the supernatant was removed and stored at −80°C. Ninety-six-well MaxiSorp Nunc immunoplates (Nunc) were coated with 10 μg/ml of crude parasite antigen in coating buffer for 2 h at room temperature. Plates were blocked with 5% skim milk in PBS overnight at 4°C. Serum samples were used at a 1 in 20 dilution or titrated using 3-fold dilutions from 1 in 50 to 1 in 109,350. Samples were dispensed at a volume of 50 μl per well and incubated for 2 h at room temperature. For the assessment of memory B cell responses, a dilution of 1 in 1,350 of the titrated sera was chosen as a representation of the data. After five washes with PBS–0.05% Tween 20 (Merck), the plates were incubated with goat anti-mouse IgG/horseradish peroxidase-conjugated antibody (1:3,000 in blocking buffer) (Bio-Rad) for 2 h. Plates were washed a further five times and then incubated with tetramethylbenzidine substrate (BD Biosciences) for 15 min. The reaction was stopped using 1 M sulfuric acid, and the plates were read using a Victor 3 plate reader (Perkin-Elmer) at 450 nM.

Conditioned media from GBM cells were obtained by incubating semi-confluent cultures for 24 h in 0.1% BSA/DMEM medium without FBS. These conditions did not significantly affect cell viability. Supernatants were concentrated at least 10-fold in Centriplus concentrators (Amicon, Beverly, MA). Cells were detached from the flasks with PBS/EDTA. Cytokine secretion values were normalized by the total number of cells.
Quantification of the amount of VEGF-A and PlGF in the conditioned medium was performed using goat anti-VEGF-A or anti-PlGF IgGs (R&D Systems, Abingdon, UK), at a concentration of 10 μg/ml in PBS, to coat Maxisorp Nunc immunoplates (Nunc, Roskilde, Denmark). Detection of the cytokines was performed with biotinylated goat anti-VEGF or anti-PlGF IgGs (0.4 μg/ml; R&D Systems) followed by incubation with streptavidin alkaline phosphatase conjugate (1:10,000) (Roche, Monza, Italy) and alkaline phosphatase reaction. Optical density at 405 nm was measured in a 3550-UV Microplate reader (Bio-Rad, Hercules, CA).

Quantification of PDGF-C binding to NRP-1 was performed on 96-well Maxisorp Nunc immunoplates (Nunc, Roskilde, Denmark) coated with 1 μg/ml PDGF-C in PBS. After blocking of the plates with 2% BSA (w/v) in PBS (hereafter referred to as blocking buffer), 50 μl of NRP-1/hFc chimera (1 μg/ml) were added to selected wells and plates incubated at 37°C for 30 min in blocking buffer supplemented with 1 mM CaCl₂ and MgCl₂. The amount of chimera bound to the cytokine was quantified by incubating wells with an anti-human Fc alkaline phosphatase-conjugated goat IgG (1:2.000) at room temperature for 1 h. After alkaline phosphatase reaction, optical density at 405 nm was measured in a Microplate reader 3550-UV (Bio-Rad, Hercules, CA). The specificity of PDGF-C binding to NRP-1 was investigated by pre-incubating at room temperature for 30 min selected PDGF-C-coated wells with graded concentrations (2.5-10 μg/ml) of a neutralizing antibody (goat anti-human PDGF-C, from R&D Systems) or control normal goat IgG (R&D Systems), before adding the NRP-1/hFc chimera. Background was determined in blocking buffer-coated wells. As positive and negative controls of PDGF-C binding, selected wells were incubated with (1 μg/ml) PDGFRα/hFc or VEGFR-2/hFc chimeras, respectively.

SE36, SE36-1 to -4 recombinants, purified VTN, VTN recombinants (1 μg/mL), or SE36 peptides (0.3 μM) were suspended in 100 μL carbonate/bi-carbonate buffer (pH 9.6) and coated onto flat-bottom 96-well MaxiSorp NUNC-Immuno plates (Nunc, Roskilde, Denmark) overnight at 4 °C. The plates were washed with PBS-0.05% Tween 20 and blocked for 1 h at room temperature with PBS-0.05% Tween 20 containing 5% skim milk. After washing, the wells were incubated with human serum (diluted 1:2000), purified or recombinant VTN (2 μg/mL), or SE36 (2 μg/mL) in PBS-0.05% Tween 20 containing 5% skim milk for 2 h at room temperature. Anti-SE36 mouse serum (diluted 1:1000), anti-VTN mAb (diluted 1:2000), or anti-VTN pAb (diluted 1:5000) was added after the washing steps prior to another incubation for 2 h at room temperature. This was followed by incubation with HRP-conjugated anti-mouse pAb (diluted 1:10000) or anti-rabbit pAb (diluted 1:10000) for 2 h at room temperature. Color was developed using TMB microwell peroxidase substrate (KPL, Gaithersburg, MD, USA). Reactions were stopped by addition of 1 M sulfuric acid. Absorbance was measured at 450/540 nm.

Antibody titers to periodontopathogenic bacteria (P. gingivalis, Prevotella intermedia, Treponema denticola, Tannerella forsythia, and Aggregatibcter actinomycetemcomitans) were measured by enzyme-linked immunosorbent assay (ELISA). The antigens of the fraction of outer membrane proteins from periodontopathogenic bacteria were coated onto 96-well Maxisorp Nunc Immunoplates (Nunc, Roskilde, Denmark) in sodium bicarbonate buffer, pH 9.4, overnight at room temperature (RT). After blocking each well with 1% bovine serum albumin (BSA) in PBS supplemented with 0.05% Tween 20 (PBST) at RT for 1 h, human serum (3200-fold dilution) or mouse serum (100-fold dilution) were applied to each well at RT for 2 h. Wells were washed three times by PBST and incubated with a human or mouse horseradish peroxidase (HRP)-conjugated secondary antibody (2000-fold dilution in PBST) at RT for 1 h. After the final washes, citrate-phosphate buffer, pH 5.0, containing 0.3% hydrogen peroxide and 0.25% o-phenylenediamine was added. The coloring reaction was allowed to continue for 15 min and was stopped by the addition of 25 μL of 2 N sulfuric acid. Absorbance at 405 nm was measured by using a plate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Maxisorp Nunc-immunoplates (Nunc, Roskilde, Denmark) were coated with 1 µg/mL rAg85A in PBS overnight at 4 °C. To reduce unspecific binding, wells were blocked with 1% BSA (w/v) in PBS for 2 h at room temperature (RT). After extensive washing with PBS, serial dilutions of serum ranging from 100–2.2 × 10⁵ were applied in duplicates. After incubation for 1 h at RT and extensive washing, Ag85A-specific antibodies were detected using goat anti-mouse total IgG, IgG1 or IgG2c conjugated to horseradish peroxidase (Southern Biotech, Birmingham, AL, USA) for 1 h at ambient temperature and by developing plates with TMB substrate (3,3′,5,5′-tetramethylbenzidine substrate; (Becton Dickinson, Basel, Switzerland) for 5 min in the dark. Reactions were stopped by adding an equal volume of 1 N sulfuric acid, and the optical density (OD) was measured at 450 nm using an iMARK microplate absorbance reader (Bio-Rad Laboratories, Hercules, CA, USA). IgG2c/IgG1 ratios above unity, calculated from serum antibody titers, were associated with Th1-related immune response profiles.

Quantification of VEGF-A and PlGF binding to VEGFR-1 was performed on 96-well Maxisorp Nunc immunoplates (Nunc, Roskilde, Denmark) coated with 10 μg/ml VEGFR-1/Fc chimera in PBS. After blocking of the plates with 3% BSA (w/v) in PBS (hereafter referred to as blocking buffer), 50 μl of PlGF or VEGF-A solution (50 ng/ml in blocking buffer) were added to selected wells and incubated for 2 h. Detection of the amount of VEGFR-1/Fc chimera bound cytokine was performed with biotinylated goat anti-VEGF-A or anti-PlGF antibodies and streptavidin-alkaline phosphatase-conjugate (1:10,000; Roche). After alkaline phosphatase reaction, optical density at 405 nm was measured in a Microplate reader 3550-UV (Bio-Rad, Hercules, CA). The specificity of VEGF-A or PlGF binding to VEGFR-1 was demonstrated by preincubating the growth factors with neutralizing antibodies, before adding them to the wells coated with VEGFR-1/Fc chimera, and background was determined in blocking buffer coated wells.
The effect of D16F7 mAb on VEGF-A or PlGF binding to VEGFR-1 was evaluated by pre-incubating selected sVEGFR-1 coated wells with 10 μg/ml of D16F7 or control mAb for 30 min at room temperature, before adding the growth factors to the plate.