Phosphorylated non-phosphorylated peptides for each target are designed at Abwiz using the human sequence of the target protein and by choosing residues surrounding the site of phosphorylation. Peptide synthesis is outsourced and peptide sequences are validated by mass spectrometry. Peptides are typically conjugated to an immunogenic carrier protein such as KLH to elicit a robust immune response.
The peptides are then used to immunize two New Zealand White rabbits (typically four injections with two-weeks intervals) for 42 days. One week after the fourth injection, the serum titers are checked by appropriate assays (e.g. ELISA, Western blot, flow cytometry) for specificity to the phospho-peptide and for reactivity to cellular phosphorylated antigen. The rabbit that elicited the most robust immune response is chosen for library construction.
Total RNA is extracted from bone marrow/spleen of the selected rabbit and messenger RNA is purified. First strand cDNA is synthesized and engineered through our Abwiz proprietary method. The two major factors that differentiate our cloning method and allow us to make superior quality libraries are (1) unbiased, uniform amplification of antibody genes captures the true antibody gene diversity present in the rabbit and (2) a highly-optimized rabbit gene primer set captures more antibody genes than any other primer set reported to date. The antibody fragments are amplified and cloned into our proprietary phage display vector.
Phage Display Antibody Selection
Rabbit Fabs are typically toxic in E. coli, but our phage display vector has been engineered to allow rabbit Fab expression in phage display format without modification to the Fab. Our robust antibody selection platform is based on the power of the phage display system to link genotype to phenotype. The phage panning process is optimized for each antigen. The highly diverse antibody libraries typically go through 3 to 4 rounds of selection on the specific antigen and the selection often includes soluble non-phospho-peptide as a competitor to eliminate non-specific peptide binders.
The proprietary Abwiz vector has been optimized for both Fab-phage production and soluble Fab expression, allowing for a seamless transition from selection to screening. In this step, the output phage from the final round of panning are used to infect E. coli and titrated on agar plates to obtain single clone colonies. Randomly selected colonies are then picked and soluble Fab are expressed in 96-well format. Periplasmically-targeted expression causes Fab to leak into the culture supernatant, allowing for easy screening from the crude culture medium following cell pelleting. Each 96-well Fab culture plate is then screened using ELISA to test each clone for (1) expression, (2) phospho-peptide, and (3) non-phospho-peptide binding. Additional screening can be performed at this stage as well. When screening by Western blotting, polyclonal mixtures of Fab clones can be combined prior to incubation with the membrane. Using this strategy of successively focusing on smaller pools of polyclonal mixtures, an entire 96-well plate can be screened using as few as four standard mini-gel Western blots. Alternatively, 96-well flow cytometry can be used to test each Fab for binding to both positive (e.g. stimulated) and negative (un-stimulated or treated with an inhibitor) control cells in as little as five hours. Following screening, every single clone can be sequenced for heavy and light chain analysis. Using our in-house software that clusters clones with similar CDR sequences, we can rapidly identify unique lead antibody candidates and can ensure that no clone is overlooked.
Recombinant Production of IgG antibodies
Once the heavy and light chains of selected clones are cloned into our full-length IgG antibody expression vectors, they are co-transfected in mammalian cells such as HEK293 suspension cells. The culture is then analyzed over a time course for expression efficiency then harvested approximately 7-days post transfection. The culture media are recovered by centrifugation and filtration. The rabbit IgG antibodies are then small-scale purified with protein A/G affinity chromatography. The purified IgG antibodies are then analyzed for purity by PAGE and for concentration by spectrophotometry, then further validated with Flow Cytometry and Western Blot. Once validation is complete, the clone(s) showing the best expression, and adequate activity in Flow Cytometry and Western Blot, is/are selected, and the relevant plasmid(s) are again amplified in E. coli, on a large scale for large-scale batch culture transfection of the same HEK293 suspension cell line. The culture media are again harvested, filtered, buffer-exchanged, and 10x concentrated after ~7 days, then purified and fractionated with protein A + G affinity chromatography. The fractions are collected and analyzed for purity and concentration. The appropriate fractions are then combined and concentrated, or diluted (if needed) to the appropriate concentration per volume. The large-scale IgG antibody lot is again analyzed for concentration and purity, and again validated with Flow Cytometry and Western Blot.
Antibody Validation and Quality Assurance
At Abwiz Bio we are fully committed to ensuring that our antibodies recognize the intended target and that each product lot exhibits high activity and reproducibility. We have incorporated antibody quality controls into nearly every stage of development and production. Biochemical as well as immunological techniques are used to examine the specificity and sensitivity of antibodies. This includes testing by ELISA, flow cytometry and Western blot: rabbit sera post-immunization, crude bacterial supernatant in Fab format, and each monoclonal IgG antibody product lot. For phospho-specific antibodies, we use specific inhibitors as well as stimulators of signal transduction pathways to validate antibody specificity.