Purification of antibodies is carried out to concentrate and enrich antigen-specific antibodies and reduce the background by removing non-specific proteins.
A well known method for purifying antibodies from serum, ascites, and cell culture supernatant is to utilize the strong natural binding affinity of the Fc part of IgG to protein A from Staphylococcus or protein G from Streptococcus. The binding strengths of protein A and protein G for immunoglobulins depend on the source species and the subclass of the particular immunoglobulin.
For small scale purification and screening of antibodies, in the µl to ml range, there is now the option to use commercially available magnetic separation techniques. These procedures utilise protein A and protein G affinity ligands immobilised on magnetic particles/beads and have several advantages including easy handling of samples and parallel antibody purification. Additional the equipment to prepare the samples using magnetic beads is a simple to use magnetic device that gives low investment cost.
Method - simplicity and speed
GE Healthcare has developed Protein A Mag Sepharose Xtra and Protein G Mag Sepharose Xtra magnetic beads, which are designed for efficient, high capacity small-scale purification/screening of monoclonal and polyclonal antibodies from various species. Antibody purification is carried out in a single tube, where the samples are incubated with the magnetic beads equilibrated with binding buffer. This enables the antibodies to be captured by the high specificity protein A or protein G ligands on Mag Sepharose resin. The tube is then placed in a magnetic rack, where the beads are attracted to the magnet within seconds. This allows easy removal of the supernatant whereas the magnetic beads remain in the tube, ready for further processing such as wash out unbound sample and elution of the antibody. The entire purification procedure can take place in less than 40 minutes.
The magnetic property of the beads means that they respond to the magnetic field but do not retain magnetic properties when the field is removed (paramagnetic). This ability to become magnetized permits efficient extraction. The bead format also has other excellent properties for small scale experiments including high density, which allows rapid capture by magnetic devices while the visibility of the beads ensures reliable collection of the antibodies in the purification procedure. This separation technique provides flexible purification allowing a wide range of sample volumes and easy scaling up by adjusting the bead quantity. Additionally a large number of samples can also be screened in parallel with high throughput on a robotic device.
Antibody binding capacity
In addition to Protein A Mag Sepharose Xtra and Protein G Mag Sepharose Xtra several different magnetic beads for small scale antibody purification are available commercially. These include: PureProteome Protein A and PureProteome Protein G from Millipore, BioMag Protein A and BioMag Protein G from Qiagen and Dynabeads Protein A and Dynabeads Protein G from Invitrogen.
An internal benchmark study was carried out, in GE Healthcare laboratories, to investigate the relative binding capacity for antibodies using these different magnetic beads. The capacity for each one was determined by purifying an excess of human IgG and rabbit IgG according to the relevant manufacturer's instructions. Comparison of this internally generated data showed that both Protein A Mag Sepharose Xtra and Protein G Mag Sepharose Xtra had considerably higher binding capacities for human IgG and rabbit IgG compared to the other corresponding products. Approximately 380µg of purified human IgG and 780µg of rabbit IgG were obtained in a single run with both Protein A Mag Sepharose Xtra and Protein G Mag Sepharose Xtra.
These amounts were approximately double those achieved with the PureProteome Protein A and G (approximately 230µg of purified human IgG and 550 µg of rabbit IgG) and four-times greater than with the other commercially beads.
One of the key advantages of magnetic bead purification is the ability to use different volumes of sample and bead slurry. In this study, low expressed monoclonal mouse IgG2b in 50ml diluted hybridoma cell supernatant (0.07mg Ab/ml) was successfully purified and concentrated using 1.75ml Protein A Mag Sepharose Xtra. The sample load was 20 mg/ml sedimented medium and the experiment was performed in duplicate. The results, illustrated in Fig. 2, showed high specificity according to SDS-PAGE analysis and recoveries of ~70 per cent. The purified mouse IgG2b was concentrated from 50 ml to 3.5 ml.
Repeatable antibody purification
To show the repeatability of Protein G Mag Sepharose Xtra, six replicate purification runs were performed. The load was half of the total binding capacity of the medium. The yield and purity of the eluted fractions were determined via absorbance measurements and SDS-PAGE analysis respectively. High repeatability, as shown in Fig.3, was demonstrated for both yield and purity using Protein G Mag Sepharose Xtra (RSD <2 per cent).
In summary, protein A or protein G magnetic beads for antibody purification provides a simple, easily-handled tool for rapid separations that is scalable across the µl to ml range. A benchmark study demonstrated that GE Healthcare's Protein A Mag Sepharose Xtra and Protein G Mag Sepharose Xtra had considerably higher binding capacities for both human IgG and rabbit IgG than other commercially available products.
Furthermore, experiments demonstrated that antibodies can be reproducibly purified with high recovery and purity.
Ann Bergh, Fredrik Borg, Stefan D Eriksson, Gunnar Glad, Therese Granér, Helena Hedlund and Ulf Hellberg, GE Healthcare Bio-Sciences AB, Uppsala, Sweden. www.gelifesciences.com