Pierce mouse igg1 fab and f ab 2 preparation kit

A polyclonal rabbit antiserum raised against recombinant PfSUB1 (ref. 13 (link)) was used for western blot analysis at a dilution of 1:1,000.
Purification of mAb NIMP.M7 (ref. 4 (link)) was by affinity
chromatography using Protein G Sepharose (GE Healthcare, UK). Immobilized Ficin
was used to prepare Fab fragments from purified NIMP.M7 according to the
manufacturer’s instructions (Pierce Mouse IgG1 Fab and
F(ab′)2 Preparation Kit, Thermo Scientific). Fab fragments were
generated in the presence of 25 mM cysteine and 0.25 ml of the
settled Ficin resin, and the digestion reaction incubated for 4 h at
37 °C, followed by purification on NAb Protein A Spin
Columns. The Fab fraction was further purified on a HiLoad 26/60 Superdex 200
prep-grade column (GE Healthcare, UK) before being used for complex formation
with rPfSUB1_cat–Prod_p9.

Anti-MOG monoclonal Ab clone 8.18C5 was generated by hybridoma cells kindly provided by Dr. Linington (Glasgow, UK). Hybridoma cells were cultured in complete medium (RPMI, 5–10 % fetal calf serum, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM l-glutamine, 1 mM sodium pyruvate, 0.05 mM β-mercaptoethanol) in large-scale flasks (Greiner bio-one, Kremsmuenster, Austria) and Ab was purified using rProtein A/Protein G Sepharose columns (rProtein A/Protein G GraviTrap, GE Healthcare, Little Chalfont, UK), according to manufacturer's recommendations. 8.18C5 IgG was digested by ficin (Pierce Mouse IgG1 Fab and F(ab′)₂ Preparation Kit, Thermo Scientific, Waltham, USA), according to manufacturer’s recommendations; integrity and binding capacity of 8.18C5 Ab and resulting 8.18C5 F(ab′)₂ fragments were verified by 6 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, followed by protein staining with Coomassie brilliant blue G250 (Biorad, Munich, Germany) and competitive anti-MOG enzyme linked immunosorbent assay (ELISA), respectively.

Generation of monoclonal antibodies and mouse sera is described elsewhere³³ . All animal procedures were approved by the Institutional Animal Care and Use Committee of the Georgia Institute of Technology and performed in accordance with relevant guidelines and regulations.
Monoclonal antibody 3A2 was cleaved into F(ab′)₂ and Fab fragments using Pierce Mouse IgG1 Fab and F(ab′)2 Preparation Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Briefly, the two fragments were generated by controlling the specificity of immobilized ficin by cysteine-HCl concentration and incubation time. Immobilized protein A was used to remove uncleaved mAb and Fc fragments. The fragments were concentrated with 500 × buffer exchange against PBS using spin columns with 10 kDa molecular weight cut-off (Amicon, Millipore Sigma). mAbs were diluted to 10/3/1/0.3 µg/mL and the fragments were diluted to corresponding antigen-binding molarities and analyzed as above.
mAb 3A2 was also used as quantification control. For this purpose, purified mAb was spiked into normal human serum at concentrations of 10, 2.5, or 0.5 µg/mL; normal serum was left unspiked as the negative control. The preparations were aliquoted and frozen, and freshly thawed aliquots were used to monitor run-to-run and day-to-day assay performance.

To determine whether Abs in human (HS) or ferret sera (FS) were binding to the HA epitopes recognized by the generated mouse mAbs, a cELISA was performed. Fab fragments from mouse mAbs were generated using a Pierce Mouse IgG1 Fab and F(ab′)₂ preparation kit (Thermo Scientific, USA). Recombinant HA from Vic361(c) was incubated on nickel-coated plates (G-Biosciences, USA) at 4 °C overnight and then washed with 0.1 % (v/v) Tween20 in PBS. Fabs in SEA BLOCK blocking buffer (Thermo Scientific, USA) were added. After 2 h incubation, serum, diluted 1 : 800 in blocking buffer, was added for 1 h. Plates were washed and peroxidase-conjugated Abs, recognizing either ferret Abs (SAB3700801, Sigma-Aldrich, Germany) or human Abs (2044–05, Southern Biotech, USA), were added. After washing, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Sigma-Aldrich, Germany) was added and, after colour development, the reaction was stopped with 0.1 M H₂SO₄. Absorbance was determined by a microplate reader at 450 nm with 620 nm as a reference.