SDS-PAGE analysis showed purity of >95%. Its functionality was verified by its ability to form the stable C3-convertase [47]. The VCP http://www.selleckchem.com/products/epacadostat-incb024360.html specific mAbs were generated by immunizing 5–6 week old BALB/c mice with the rVCP. In brief, mice were immunized with 20 μg of rVCP in Freund’s complete adjuvant, followed by two boosts 15 days post prime at weekly intervals with the same dose, but in Freund’s incomplete adjuvant. Following immunization, spleen was removed and the spleen cells were fused in-house with myeloma cells as per established protocols [48] and [49]. The clones from the fusion were screened by ELISA and subcloned to isolate the individual clones.

Antibody isotyping was performed by an ELISA-based hybridoma isotyping kit (BD Biosciences, San Diego, CA, USA). The IgG mAbs were purified by capryllic acid precipitation method or by Hi-Trap affinity protein G column (GE Healthcare Bio-Sciences, Sweden). Homogeneity of mAbs was assured by SDS-PAGE analysis. VACV pathogenicity studies were performed in rabbits using skin lesion model [36]. In brief, 104 pfu of VACV-WR strain in sterile PBS in a total volume of 100 μl were injected intradermally with or without the mAb on the shaved backs of two New Zealand White GDC-0941 molecular weight rabbits (age 6–7 months) in duplicate and lesions formed (scabs) were measured after every 24 h using calipers. The mean of four measurements was used for graphical representation

of individual time point per site. To study the role of complement during infection, similar experiments were also performed in two additional rabbits depleted of complement by administering 100 U/kg of cobra venom factor.

All the results were grouped and statistically evaluated by performing Mann–Whitney Rank Sum test (SigmaStat). The experimental protocol was approved by the Institute’s Animal Care and Use Committee. The ELISA plates were coated overnight at 4 °C with rVCP or VCP mutants (CCP 1–3, CCP 2–4, CCP 1–2, CCP 2–3, CCP 3–4; 200 ng/well), blocked by adding 5% milk and incubated with mAbs (1 μg/well) for 1 h at room temperature. Binding was probed by adding 1:2000 diluted anti-mouse HRP conjugate (Biorad, Hercules, Tolmetin CA) and detected with 2,2′-Azino-bis (3-ethylbenzthiazoline) 6-sulfonic acid (ABTS) (Roche, Mannheim, Germany) at 414 nm. Inhibition of factor I cofactor activity of VCP by mAbs was determined as described below. rVCP (0.5 μg) was mixed with 3 μg of mAb and incubated for 15 min at 37 °C. Thereafter, 3 μg of C3b or C4b and 0.1 μg of factor I was added to the reaction mixture and the volume was adjusted to 20 μl using PBS. It was then further incubated at 37 °C for 2 h. The reaction was stopped by adding SDS-PAGE sample buffer containing DTT and C3b/C4b cleavages were analyzed on a 10% SDS-PAGE gel [40]. Inhibition of the classical pathway decay-accelerating activity of VCP by mAbs was determined by utilizing a hemolytic assay [42] and [50].