HuCAL Technology
The HuCAL (Human Combinatorial Antibody Library) technology is a unique and innovative concept for the in vitro generation of highly specific and fully human antibodies.
HuCAL – Our key technology
In the HuCAL library, the structural diversity of the human antibody repertoire is represented by seven heavy chain and seven light chain variable region genes, giving rise to 49 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 15 billion functional human antibody specificities in Fab format are already prefabricated and available in HuCAL phage libraries.
Selecting your antibodies from 15 billion candidates
Your antigen enters the automated panning process (AutoPan), where it is immobilized in 384-well microplates for screening against antibody-displaying phage. A 0.5 mg antigen sample is usually enough for comprehensive screening. CysDisplay technology (Fig 2) provides simple elution of high-affinity binders and reduces the number of potential antibody candidates to several hundreds or thousands of sequences. These candidates are then screened in a robust 384-well ELISA (AutoScreen). Positive clones are automatically forwarded for further validation, sequenced, and entered into the central database.
Generating your monoclonal antibody
Unique antibody candidates from the screening process are expressed in bacteria and affinity purified on a micro scale for QC and specificity testing. We screen all purified antibodies against the antigen plus several unrelated proteins, or on request, against closely-related proteins supplied by the customer. Micropurified antibodies are then sent to the customer for functional testing in their in-house applications. Selected antibodies are then purified in larger scale. All antibodies can be ordered in large scale at any time.
Antibody formats – the choice is yours
The basic antibody format is a monovalent Fab fragment, which can be easily converted into a bivalent Mini-Antibody 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 antibodies due to their smaller size and lower cross-reactivity. However, if full-size antibodies are required, HuCAL Fabs can easily be converted into IgGs and expressed in mammalian cell lines.
Figure 1 – HuCAL Antibody Library Construction
Highly variable CDRs are combined with 7 heavy and 7 light chain variable region genes to create more than 15 billion antibody specificities in vitro. The 3D structure of the IgG molecule below shows the antibody binding regions formed by the CDRs in gold.
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 that are available.
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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.
