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The HuCAL PLATINUM® library is based on the human IgG1 Fab format, which consists of the first two domains of the antibody heavy chain (VH and CH1) plus the complete light chain (either kappa or lambda). After phage display selection, the enriched antibody Fab genes are cloned into an E. coli expression vector, which determines the format of the purified antibody – either monovalent Fab or bivalent mini-antibody (functionally equivalent to F(ab’)2 fragment), with a wide choice of epitope tags.
Fig. 1. Schematic of the main antibody formats available. Fab-FH: monovalent Fab (shown with two tags). Fab-dHLX-MSx2: bivalent Fab, dHLX domains shown as blue cylinders (with two tags). Fab-A-V5Sx2: bivalent Fab formed by dimerization of bacterial alkaline phosphatase (blue/green) with two tags. F= DYKDDDDK tag; H= His-6 tag; M= c-myc tag; Sx2= two extended Strep-tags; V5= V5 tag.
Selection of the optimal antibody format is an important step in HuCAL® antibody generation projects, and is necessary for ensuring success in your desired research application. Antibody formats can also be changed later by subcloning the genes into a new expression vector. Consult our technical manual for advice on how to choose the best format and tag combination and view a complete list of options.
Once you have received and evaluated your initial HuCAL recombinant antibodies, you may need more for your assay development or manufacturing. We offer two options - we carry out the antibody production for you, or you bring production of your antibody into your own lab.
HuCAL Fab antibodies can be converted into full length immunoglobulins or chimeric antibodies (human-mouse or human-rat) in a range of isotypes. This may be necessary if the Fc region is needed for binding or agglutination reactions. In addition, HuCAL immunoglobulins offer significant advantages as human calibrators and fully human standards in immunogenicity testing.
In many applications it is recommended to use a negative control, matching the format of the custom HuCAL Fab antibody. This enables controlled validation of antibody functions in the experimental model being used. The table below shows the negative controls available for HuCAL Fab formats; they are specific to green fluorescent protein (GFP), which has no known reactivity with mammalian proteins or other antigens.