2.1.1 Hybridoma cell culture
2.1.1.1 Growth and maintenance
Hybridoma cells producing monoclonal antibodies (MAbs) to Koi herpesvirus (KHV) were kindly provided by Dr. Sven Bergmann (Friedrich Loeffler Institut (FLI), Greifswald, Germany). Hybridoma cells producing MAbs to Infectious salmon anaemia virus (ISAV) and rainbow trout (Oncorhynchus mykiss) or Atlantic salmon (Salmo salar) immunoglobulin M (IgM) were developed at the Aquatic Vaccine Unit, Institute of Aquaculture, University of Stirling, Stirling, Scotland. Cells were cultured in Dulbecco’s Minimum Essential Medium plus additive (DMEM+) (Sigma-Aldrich, St. Louis, USA), containing 10% (v/v) foetal calf serum (FCS) (Sigma-Aldrich), 2.5 mL penicillin streptomycin (PenStrep) 1250 units (U) (10,000 U penicillin; 10 mg mL-1 streptomycin (Sigma-Aldrich)), 5 mL L-glutamine (200 mM) (Sigma-Aldrich), and 5 mL sodium pyruvate (100 mM) (Sigma-Aldrich). The cells were cultured at 37°C in 5% CO2. Cells were expanded from 25 cm2 culture flasks containing approximately 8 mL to 75 cm2 (30-50 mL) then finally 150 cm2 flasks (100-125 mL) in order to obtain 1 L of cell supernatant from which the MAbs were concentrated and purified.
Hybridoma cell suspension was harvested when > 90% of cells had died after approximately 10 days of culture. Forty-five mL of lysed cell suspension was aliquoted into 50 mL centrifuge tubes (VWR International, Radnor, USA) and centrifuged at 1912 x g in a Sigma 4 K 15 centrifuge for 10 min at 4°C in order to pellet cell debris, and the supernatant containing the MAbs was pooled and retained. The supernatant stocks were kept at either 4°C for short term storage or -20°C for long periods of time.
2.1.1.2 Concentration of MAbs
MAbs were concentrated through either (1) Amicon Ultra centrifugal filters (Millipore, Cork, Ireland) for small scale production, or (2) a Pall LV Centramate for larger scale production according to the manufacturer’s instructions.
(1) The 10 Kilodalton (kDa) membrane of the Amicon Ultra centrifugal filters provides a simple method for concentrating MAbs, as high molecular weight immunoglobulins are retained within the filter and concentrated. Approximately 5 mL of hybridoma sample was added to the tubes, which were then centrifuged at 2,000 x g for 10 min at 4°C in a Sigma 4 K 15 centrifuge. The eluent was discarded and more sample added. This procedure was repeated to concentrate the MAb samples to a volume of 1-2 mL.
(2) The Pall L V Centramate concentrator consists of a peristaltic pump and membrane system and is more suitable for larger volumes of supernatant. After attachment of the cassettes and gasket to the concentrator, the system was sanitised with 0.1M NaOH at a pressure of 3-5 pounds per square inch (psi), then washed with H2O prior to equilibration with phosphate buffered saline (PBS), pH 7.2. Approximately 500 mL of hybridoma supernatant was fed through the system at 10 psi providing 25 mL of concentrated supernatant. The system was washed through with 0.5M NaOH pre-heated to 37°C at 25-30 psi between runs with different samples.
2.1.1.3 Purification of MAbs
The concentrated MAb hybridoma supernatant was made up to 50 mL with binding buffer (20mM sodium phosphate, pH 7). The solution was filtered through a 0.66 µm filter step followed by a 0.22 µm nitrocellulose membrane (Millipore). All buffers including binding buffer, elution buffer and Tris-HCl were filtered through a 0.45 µm nitrocellulose membrane
before undertaking the procedure in order to eliminate contaminating particles that could affect the efficiency of the system and purity of the sample.
Purification was undertaken by affinity chromatography through pre-packed 1 mL Affinity Purification High Trap Protein-G columns (GE Healthcare) containing Protein G sepharosefor binding Immunoglobulin G. The ÄKTA prime liquid affinity chromatography system (Amersham Biosciences) was used for purification of the MAbs.
After fitting the column to the system, it was washed with ultrapure H2O and equilibrated with binding buffer prior to use. Filtered samples containing MAbs were passed through the system at a rate of 1 mL min-1 to allow IgG to bind to the column. Unbound proteins were washed through the column, monitored by UV spectrophotometry at 280 nm. Once the column was cleared of contaminating proteins, IgG bound to the column was eluted as 1 mL fractions with Glycine-HCl, pH 2.7. The eluted fractions were neutralised with 100 µL Tris-HCl, pH 9. Fractions containing purified MAbs, determined from their absorbance at 280 nm were pooled and dialysed against PBS. Dialysis tubing cut into 20 cm lengths was activated by boiling for 5 min in 5mM EDTA, 200mM sodium bicarbonate then rinsed thoroughly in deionised H2O. This was repeated before autoclaving the tubing in deionised H2O. After extensive washing, the dialysis tubing was tied at either end after having added the purified MAb to the tubing. Three x 4 L buffer changes of PBS were carried out during the dialysis procedure before harvesting the sample. The concentration was then determined using a protein assay as described in the section below.
2.1.1.4 Determining the concentration of the purified MAbs
The Pierce BCA protein assay kit (Thermo Scientific, Rockford, USA) was used to determine the concentration of the affinity purified MAbs. The BCA kit is a colorimetric detection and quantification method for proteins, and is based on the reduction of Cu+2 to Cu+ in the
presence of protein in an alkaline medium, which can then be detected by chelation of molecules of bicinchoninic acid by the cuprous ion (Smith et al., 1985). This reaction is spectrophotometrically analysed at 562 nm and the concentration of unknown samples can be determined from a calibration curve of absorbance for known Bovine Serum Albumin (BSA) standards of known concentration. For this, BSA diluted in PBS was used to establish the standard curve according to the manufacturer’s instructions. Briefly, 25 µl of standard or unknown samples, in replicate, were mixed with 200 µl (1:8) of working reagent which was composed of 50 parts of solution A to 1 part of solution B and added to 96-well plates (Sterlin, Fisher Scientific, Newport, UK). The plates were covered with foil, shaken for 30 sec at 600 shakes per sec (sps) on a plate shaker (Minishaker IKA) and incubated at 37°C for 30 min. The plate was then read on a spectrophotometer (CECIL CE 2021) at 562 nm.
Protein concentrations were calculated from a standard curve of absorbance values from protein standards of BSA ranging from 0-2,000 µg mL-1.