After washing with EB buffer, elution was carried out with 0,1 M of citric acid pH 3. Elution drops containing the HAH5 protein were immediately collected in 50 mM Tris–HCl pH 8. Purity was estimated by densitometric analysis of SDS-PAGE gels stained with a Coomassie blue R-250 solution at 0,05% using the software TDI’s 1D Manager, version 2.0. The detection and quantification of the HAH5 protein was accomplished as described by MK-1775 cell line [8]. Briefly, polystyrene high binding microtiter plates (Costar, USA) were coated with 2,5 μg/mL of the monoclonal antibody anti-HA2 (Sancti-Spíritus, Cuba) overnight at 4 °C. Plates were washed with PBS

plus 0,05% Tween 20 (PBST) and blocked with 1% of bovine serum albumin (BSA) (Sigma, USA) in DMEM medium for 2 h at 37 °C. After washing with PBST, standard curve (for quantification) and culture samples were added for 2 h at 37 °C. Standard curve was performed using values from 100 to 1,56 ng/mL of the HAH5 protein purified by IC and culture samples were diluted 1:64 in DMEM plus 0,5% BSA. Plates were washed with PBST and the monoclonal antibody anti-HA3 conjugated to horseradish peroxidase (Sancti-Spíritus, Cuba) diluted 1: 20 000 was added. After 1 h at 37 °C, plates were washed Fulvestrant with PBST and visualized with 0,04 M of 3,3′,5,5′-tetramethylbenzidine (Sigma, USA) in dimethyl sulphoxide using hydrogen peroxide as substrate. Reaction Cyclin-dependent kinase 3 was stopped

with 3,5% of sulfuric acid and absorbance was measured in a microplate reader model SUNRISE-BASIC TECAN (Austria) at 450 nm. The concentration of the HAH5 protein in the supernatant was calculated by extrapolating the optical density (OD) values of samples into the standard curve OD. Polystyrene high binding microtiter plates (Costar, USA) were coated overnight at 4 °C with 2,5 μg/mL of the HAH5 protein obtained from SiHa or CHO cells. The HAH5 protein was used in a pure state or directly from the culture supernatant of both cell lines. Blocking and wash were performed as above. Serum samples were diluted from 1/500 to 1/32 000 in DMEM plus 0,5%

BSA and added to coated plates for 2 h at 37 °C. The antibody detection in sera of chicken immunized with the HACD protein was performed at a dilution of 1/1000. After washing with PBST, the monoclonal antibody anti-IgG (Y) of chicken conjugated to horseradish peroxidase (Sigma, USA) diluted 1/30 000 in DMEM plus 0,5% BSA was added. After 1 h at 37 °C, plates were washed with PBST. Absorbance measurement was performed as above. The statistical procedure was done using the statistical software GraphPad Prism v.4.02 (GraphPad, USA). A Kruskal–Wallis test and a Dunn post-test were performed to compare the OD values of the HAH5 protein in different clones of CHO-HAH5. The HAH5 production by different batches of the clone CHO-HAH5 78 was compared by the ANOVA test and the Tukey post-test.