Single domain antibodies: naturally derived antibody fragments
Whereas conventional antibodies consist of heavy and light chains, animals from the camelidae family (i.e. camels, llamas and alpacas) contain a particular class of antibodies that are devoid of light chains.
These heavy chain-only antibodies (HcAbs) undergo normal selection and maturation by the animals’ immune system. For this reason, HcAbs can exhibit high affinities and serum stability. Single domain antibody fragments (sdAb) are the variable domains of such HcAbs.
In vivo maturation, combined with a small size and uniquely shaped paratope make single domain antibodies ideal targeting molecules for a wide variety of applications, ranging from simple detection to drug delivery. Single domain antibodies are also referred to as single domain antibodies, VHH or Nanobodies (trademark Ablynx Sanofi).
QVQ provides expertise in each step of the single domain antibody development trajectory. The services range from immunizations and generating phage display libraries, through biopanning and screening up to bio-informatics, production, purification and quality control. All lead development services are conduced according to carefully validated standard operating procedures.
In addition, QVQ offers high quality, off-the-shelf single domain antibodies for research. Upon request, all single domain antibodies can be equipped with our proprietary C- direct tag that allows directional conjugation to various labels.
Basic Monoclonal Single Domain Antibody development
Immunization and library construction
QVQ has experience with immunization
and selection on a large variety of antigen types, such as purified natural and recombinant protein and more complex samples like cells and tissues. QVQ collaborates with well-accredited CROs for the immunization of llamas. The immunizations are conducted according to the applicable animal welfare act at place and all immunizations are reviewed by the internal animal experiment commission.
At least 2 animals are scheduled for each immunization to ensure diversity of the selected single domain antibodies and to avert possible non-responder animals.
Four injections with antigen are included in the standard 43-day protocol, which can be extended with additional boost immunizations. After the immunization, titers of antigen-specific antibodies in sera are determined by ELISA. Total RNA is then isolated from a collection of white blood cells and phage display libraries are constructed using proprietary primers and phage display plasmids. QVQ libraries contain on average 10^8 transformants with ~100% correct insert frequency.
Phage display selection and screening
Based on extensive experience and available tools, QVQ can propose strategies for the selection of target binding single domain antibodies by phage display selections. Such strategies are designed together with customers and by taking into account the final application of the lead molecules.
Output from the biopanning are stored as glycerol stocks and are used to generate masterplates with single clones. Outputs can also be subjected to Sanger and/or NGS sequencing followed by detailed sequence analysis. Initial screening of the masterplates involves assessing target binding of single domain antibody in crude extracts by ELISA.
Based on the sequences and screening data, lead clones are selected for small scale production and purification by affinity chromatography. QVQ commits to select at least 6 different single domain antibodies from at least 2 different germline families, based on the V-D-J gene combination.
All the above mentioned steps and all data is collected in the form of a report, which is send to the customer along with 0.5 mg of purified protein of the 6 lead clones.
QVQ acknowledges that customizing lead development projects ensures high success rate and therefore, we’re open to any deviation from this standard project plan.
Production, purification and
QVQ has ample experience with the production and purification of recombinant single domain antibodies from Escherichia coli bacteria and Saccharomyces cerevisiae yeast up to 500 mg scale.
Production is done in shake flask or fed batch fermentation and purification is done by either affinity chromatography or IEX chromatography.
QVQ routinely equips single domain antibodies with a proprietary C-Direct tag at their C-terminus. This low immunogenic tag allows for good production in yeast, provides an affinity tag for purification and contains an unpaired thiol for directional conjugation to a wide range of labels.
QVQ offers labeling services in which tagged single domain antibody are directionally conjugated to fluorescent dyes (e.g. Hilyte, IRDye), chelators (e.g. NOTA) or biotin. Other labels can be requested.
Based on its large sequence database and experience, QVQ can suggest
modifications of single domain antibody sequences to enhance their production
levels, thermo- and enzymatic stability and homology to human VH domains (humanization).