HuCAL® Antibody Technology
HuCAL® (Human Combinatorial Antibody Library) Antibody Technology is a unique and innovative concept for the in vitro generation of highly specific and fully human antibodies.
HuCAL PLATINUM® – Cutting Edge Technology
In the HuCAL PLATINUM® library, the structural diversity of the human antibody repertoire is represented by 7 heavy chain and 6 light chain variable region genes, giving rise to 42 frameworks in the master library. Highly variable genetic cassettes (CDRs – Complementarity Determining Regions) are then superimposed on these frameworks to mimic the entire human antibody repertoire (Fig 1). More than 45 billion functional human antibody specificities in Fab format are already prefabricated and available in the HuCAL PLATINUM® phage library.
Selecting Your Antibodies from 45 Billion Candidates
Your antigen enters the automated selection process (AutoPan®), where it is immobilized in microplates or on magnetic beads for 3 rounds of selection against antibody-displaying phage. For standard HuCAL® projects, it is only necessary to provide 0.25 to 0.5 mg of protein antigen. CysDisplay® technology (Fig 2) provides for the simple elution of high-affinity binders and reduces the number of potential antibody candidates to hundreds or thousands of sequences.
Screening and Producing Your Antibodies
370 of the monoclonal antibody candidates are screened in a robust 384-well ELISA against the antigen(s) (AutoScreen®). Positive clones are automatically forwarded for sequencing and further validation. Up to 10 unique antibody candidates from the screening process are expressed in bacteria and affinity purified on a small scale for QC and specificity testing. We screen all purified antibodies against the antigen plus several unrelated proteins, or upon request, against closely-related proteins.
Customers receive 250 µg of up to 10 purified antibodies for functional testing. At any time, customer selected antibodies can be purified and delivered in larger scale.
Antibody Formats – the Choice is Yours
The basic antibody format is a monovalent Fab fragment, which can be easily converted into a bivalent mini-antibodies format (Fig 3). While monovalent Fab fragments are smaller, mini-antibodies show up to 10-fold higher avidity than monovalent Fabs in some applications, mimicking the bivalent binding behavior of IgGs. Fab fragments and mini-antibodies are ideal for use in most applications and offer significant advantages over full-length antibodies due to their smaller size and lower cross-reactivity. However, if full-size antibodies are required, HuCAL® Fabs can easily be converted into various immunoglobulin isotypes and expressed in mammalian cell lines.
Figure 1 – HuCAL® Antibody Library Construction
Highly variable CDRs are combined with 7 heavy and 6 light chain variable region genes to create more than 45 billion antibody specificities in vitro.
Figure 2 – Cys Display®
Phage display is a technique which physically links antibody DNA with antibody protein, i.e genotype with phenotype. CysDisplay® is an improved version of phage display allowing specific elution of antibody displaying phage binding to antigens through the disruption of a disulfide bridge between phage and antibody. This ensures that all specific binders are eluted independent of their binding affinity to the antigen.
Figure 3 – Schematic Drawing of the Main Antibody Formats
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Fab-MH Fab (with two tags); Fab-dHLX-MH Bivalent Fab; dHLX domains shown as blue cylinders (with two tags); Fab-A-FH Bivalent Fab formed by dimerization of bacterial alkaline phosphatase (blue/green) with two tags; full IgG. MH = Myc:His-6 tag; FH = FLAG®:His-6 tag.
