Goat anti mouse igg alkaline phosphatase

Serum collected from the mice was taken for IgG antibody analysis using ELISA as described previously [27 (link),31 (link),43 (link),44 (link)]. Briefly, whole-cell formalin-killed bacteria (P. gingivalis (1:150), T. denticola (1:30), and S. gordonii (1:60) was used as an antigen and coated onto the 96 well polystyrene plate (Corning Incorporated Costar EIA/RIA Plate, Corning, NY, USA). The coated plates were incubated on the rotator at 37 °C for 3 h and then overnight at 4 °C. The plates were washed thrice with wash buffer the next day, and the unbound bacteria were removed. 100 µL of 1:100 diluted serum of the mice in triplicate was added to the wells, and the plates were incubated for 2 h on the rotator at room temperature. After incubation, the plates were washed with a wash buffer, and 100 µL of goat anti-mouse IgG alkaline phosphatase (Sigma Aldrich, St. Louis, MO, USA) was added, and the plates were incubated for 2 h. After washing, the plates were developed with 200 µL of p-nitrophenylphosphate for 15 min, and the development was stopped using 3 M NaOH. The plates were read at OD 405 nm and analyzed using Gen5 software in Epoch Microplate Spectrophotometer (BioTek, USA, Winooski, VT, USA). IgG antibody concentrations were determined using the standard curve consisting of standard IgG concentrations (Sigma Aldrich, St. Louis, MO, USA).

Western blot analysis was performed using the SNAP i.d. 2.0 system (Millipore, Burlington, MA, USA) according to the manufacturer’s recommendations. Serum of ferret infected with influenza A (H5N8) virus (1:200) (FBRI SRC VB «Vector», Rospotrebnadzor) was used as the primary antibody. Mouse anti-ferret IgG (1:3000) (FBRI SRC VB «Vector», Rospotrebnadzor) and goat anti-mouse IgG-alkaline phosphatase (1:5000) (Sigma) were used as the secondary antibodies. The immune complex was visualized by adding 1-Step™ NBT/BCIP substrate (Thermo Fisher Scientific, Waltham, MA, USA).

PCT was purchased from Huibiao Biological Technology Co., Ltd. Freund’s adjuvant, horseradish peroxidase (HRP), goat anti-mouse IgG-alkaline phosphatase, Bis (p-nitrophenyl) phosphate (BNPP) and Tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich. Mouse anti-HA tag antibody was obtained from Covance. Fast Track 2.0 Kit and ThermoScript RT-PCR Kit was obtained from InVitrogen. Pst I, Not I, Nco I, BstE II and T4 ligase were obtained from NEB (USA). Streptavidin Mutein Matrix was purchased from Roche. 96-well Maxisorp plates were purchased from Thermo Scientific NUNC. DNA markers were provided by Takara. Protein markers were obtained from Vazyme Biotech Co., Ltd and Thermo Scientific. Polyethylene glycol (PEG) 6000 and Biotin were obtained from Shanghai Sangon Biotech. BeaverNano™ Streptavidin Matrix Coated 96-Well Plates were provided by Beaver. Phagemid vector pMECS, VCSM13 helper phages, Escherichia coli (E. coli) TG1 and WK6 cells, plasmids pBAD17 and pBirA were from Prof. Serge Muyldermans’s lab (Laboratory of Cellular and Molecular Immunology, VUB-Vrije Universiteit, Brussel, Belgium). Bactrian camels were provided by “Joint Center for Nanobody Research & Development between SEU and Egens Bio”. Affinity analysis by surface plasmon resonance imaging (SPRi) binding assay was performed on PlexArray ® HT system (Plexera LLC).