After washings, the plates were incubated with substrate-chromogen solution (OPD 0.75 mg/mL, hydrogen peroxide 0.015%, in citrate–phosphate buffer, pH 5.5) for 15 min. The reaction was stopped 3-Methyladenine concentration by adding 2 M sulphuric
acid and the absorbance read at 492 nm in a BioRad microtiter plate reader. Inhibition of VEGF/KDR-Fc interaction was calculated according to: inhibition % = 100 − (A492 nm immune serum/A492 nm pre-immune serum). Monkey IgG was purified from sera of pre-immune and immunized animals using affinity chromatography (PROSEP-G Spin Columns; Millipore), as suggested by the manufacturer. IgG was quantified by ELISA: a 96-well plate was coated with 3 μg/mL of anti-human kappa light chain antibodies (Sigma), and 50 μL of the test or control samples were added per well. After incubating 16 h at 4 °C, the reactions were developed using anti-human IgG gamma chain antibodies, conjugated with alkaline phosphatase (Sigma) diluted 1:5000 for 1 h at 37 °C. β-Nitrophenyl phosphate was employed as substrate. A standard curve of serial 1:2 dilutions, starting at 30 ng/mL, of a humanized anti-EGF receptor IgG1 antibody (TheraCIM®, CIMAB S.A., Havana) was included in order to quantify the amount of IgG present in Selleck Obeticholic Acid the samples. Animals were sedated with intramuscular
ketamine chloride (10 mg/kg) prior to invasive or direct manipulations. DTH was done in all monkeys after the second booster immunization of the maintenance phase. Test antigens included P64K-hVEGFKDR− and hrVEGF. Saline buffer was used a control. The back of the monkeys were shaved and 100 μg of the test antigens were injected intradermally in the middle of circles marked with indelible ink, using 0.5 mL insulin
syringes fitted with 29 gauge needles. After 48 h, the injection sites were independently assessed by two experienced readers unaware of animal treatment. Induration diameter was measured with a digital caliper and results were expressed as the group geometric mean area  and . Erythema and swelling were not considered Ketanserin in the measurement. Due to caliper characteristics, the lower measurable limit of a detectable reaction was 0.5 mm in diameter. For geometric mean calculations, measurements below 0.5 mm were considered to be 0.5 mm. Results are presented according to the score: ++ positive = >5 mm2 of geometric mean; + positive = between 0.5 and 4.99 mm2 of geometric mean; − = no detectable reaction. Four millimeter punch biopsies were made at selected sites 48 h after DTH induction. Paraffin embedded sections (5 μM) were stained with hematoxylin and eosin and reviewed by a veterinary pathologist unaware of group assignment or test antigen. At least two sections from each biopsy were examined. For each sample, the general nature of the dermal infiltrate was evaluated in terms of the presence of mononuclear cells, neutrophils, or eosinophils.