Mabtrap kit

First, a β₁-AAB-positive animal model was established by actively immunizing rats with the second extracellular loop antigen peptide of β₁-adrenergic receptor (β₁-AR-ECII), as described in the previous studies.^{9 (link)} Then, animal sera from the β₁-AAB-positive group (actively immunized) and the control group were collected and extracted using MAbTrap Kit (GE Healthcare, 17-1128-01, Uppsala, Sweden) for affinity and purification of IgG.

In order to obtain large concentrations of mAbs, for further studies, ascitic fluids of monoclonal antibodies BURK24 and BURK37 were generated produced in 2,6,10,14-tetramethyl pentadecane i.e., Pristane (Sigma, India) primed retired breeder BALB/c mice, by injecting BURK24 and BURK37 hybridomas as described by R B Westerman et al.[33] (link) and purified using mAbTrap kit and HiTrap IgM Purification HP (GE Healthcare, UK) respectively as per the manufacturer's instructions. The purified mAbs were lyophilized, quantified and reconstituted in sterile 1× PBS according to the required concentrations of each study.

BALB/c mice were immunized subcutaneously twice with the purified SFTSV rN protein emulsified in TiterMAX Gold (TiterMax USA, Inc., Norcross, GA, USA). Hybridomas were produced by fusion of myeloma cells with the splenic cells, obtained 4 days after the last immunization, using ClonaCell-HY Hybridoma Kit (Stem Cell Technologies) according to the manufacturer’s instructions. The culture supernatants of hybridoma cells were screened for the presence of antibodies against SFTSV antigen by IgG ELISA as described below. Positive hybridoma cells were cloned by limiting dilution. The isotypes of the MAbs were determined using Mouse Monoclonal Antibody Isotyping Kit (AbD Serotec, Kidlington, UK). The MAbs were purified from mouse ascitic fluid (Unitech Co. Ltd., Chiba, Japan) or from the culture supernatant by protein G column chromatography (MAbTrap Kit, GE Healthcare UK Ltd., Buckinghamshire, UK) according to the manufacturer’s instructions. The concentration of each purified MAb was determined using the Pierce BCA Protein Assay Reagent. Two hybridoma clones (designated as 2D11 and 9D3) producing MAbs reactive to SFTSV N protein were obtained. MAb 9D3 and MAb 2D11 were isotypes of IgG₁ and IgG_2a, respectively. The light chain of these MAbs was κ-type.

An anti-Eap polyclonal antibody was raised in a rabbit against purified Eap (Sugimoto et al., 2013 (link)) (Scrum, Tokyo, Japan). The anti-Eap IgG was purified with a MAbTrap Kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's instructions. A rabbit anti-CsgA polyclonal antibody was raised against the CsgA peptide (LDQWNGKNSEMTVKQFGGGN) (Loferer et al., 1997 (link)) with the C-terminal carrier cysteine residue (Medical and Biological Laboratories, Aichi, Japan). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and Cy3-labeled goat anti-rabbit IgG were purchased from Bio-Rad Laboratories (Hercules, CA, USA) and GE Healthcare respectively. WGA-Alexa488, SYTO9 and PI were purchased from Life Technologies.

38–2 [17 (link)], 3S9 [24 (link),25 (link)], and 2H9 hybridoma cells [24 (link),25 (link)] were inoculated into the peritoneal cavity of BALB/cSlc-nu/nu mice (Japan SLC Inc.) pretreated with pristane (Sigma). Abs in their ascites were then purified using a MAbTrap Kit (GE Healthcare) according to the manufacturers’ instructions.

Two litres of infected culture fluid (ICF) was harvested from cultures of Vero cells 5 days after inoculation with RVFV (Smithburn strain). ICF was purified by sucrose-gradient ultracentrifugation at 50,000 x g for 14 hrs at 4 °C. The purified virus (0.25 mg/mL) was inactivated with 1% (final concentration) formalin for about 2 days at 4 °C. Two 3-month-old New Zealand white rabbits were immunized once with formalin-inactivated virus mixed with an equal volume of Freund’s complete adjuvant (0.125 mg/shot/rabbit), and after 1 week boosted thrice with formailin-inactivated virus mixed with an equal volume of Freund’s incomplete adjuvant at intervals of 1 week. Serum was collected and the rabbit anti-virus IgG was purified by using MAb Trap Kit (GE Healthcare) according to the manufacturer’s instructions.