Anti human igg fc hrp conjugated antibody

ELISA plates were coated with rhMPL (R&D Systems) or bovine serum albumin (BSA; negative control; BD Biosciences, Franklin Lakes, NJ, US) at 4 °C overnight. Each well was washed with PBS and blocked with MPBS (5% skim milk in PBS) at 37 °C for 1 h. Next, the phages were added, and incubated at 37 °C for 2 h. After washing five times with PBS, the bound phages were detected using anti-M13 phage-HRP-conjugated antibody (1:2000; Sino Biological Inc, Chesterbrook, PA, US). Absorbance was measured at 450 nm using a VersaMax™ Microplate Reader (Molecular Devices, San Jose, CA, US). The 2R13 was detected using anti-human IgG Fc-HRP-conjugated antibody (1:2000; Abcam, Cambridge, MA, US).

The scFv-Fc constructs were prepared as described by Sokolowska-Wedzina et al. [54 (link)]. Briefly, sequences encoding secretion signal peptide (SSP) and the HindIII and Kpn2I restriction sites were introduced into the scFv DNA sequences in two PCR reactions. In the next step, amplified scFv DNA and pLEV113-Fc expression vector (LakePharma, Belmont, CA, USA), encoding the Fc domain of human IgG1 [52 (link)], were digested using HindIII and Kpn2I restriction enzymes (Thermo Fisher Scientific), and then the PCR product was ligated with the vector with the use of T4 DNA ligase (Thermo Fisher Scientific). The resulting construct, inserted into a pLEV113 expression vector, was used to stably transfect CHO-S cells. scFv-Fc fusion proteins preceded with a secretion signal peptide were expressed and purified using affinity chromatography following the procedure described for the ECD_FGFR-Fc proteins [52 (link)]. The proteins were analyzed by SDS–PAGE, Western blotting using anti-human IgG Fc-HRP-conjugated antibody (ab97225, Abcam) and mass spectrometry.