HEXT Core Facility Project > Antibody Facility
The integration of the Antibody Core Unit in the Institute for Immunology and in the study group Molecular Immunology gives you access to the scientific and technical know-how in the area of antibody production, especially against proteins in their native conformation by cDNA-immunization.
Aim of the established facility is to assist scientists in developing suitable immunization strategies, creating new antibodies as well as producing poly- and monoclonal antibodies, which fit specific scientific demands.
Service includes cDNA preparation needed for immunizing the animals, immunization of rabbits, rats or mice as well as processing and initial testing of the received immune serum.
In case of rats and mice, service also covers the production of monoclonal antibodies (somatic cell fusion, screening and subcloning of hybridoma cell lines, as well as isotyping of monoclonal antibodies and the conjugation to different fluorochromes or to a carrier matrix).
Within the core unit a technical assistant is responsible for realizing the planned projects.
Prof. Friedrich Nolte
Institut für Immunologie
Tel: 7410 53612
Technische Angestellte Antibody Core Unit
Tel.: 7410 54669
Production of polyclonal antibodies
For the production of polyclonal antibodies, cDNA must be delivered in a suitable vector. After immunization of animals with the provided cDNA, the titer of the immune serum is determined (cell culture tests). 18 weeks later, aliquots of the preimmune- and immune serum can be delivered for testing. During the following three weeks the user decides together with the core unit whether animals should be used for hydrodoma cell lines or for bleeding.
The facility delivers aliquots of preimmune serum (ca. 0.5 ml from rabbits, ca. 0.1 ml from rats) and after final bleeding (ca. 30 ml from rabbits, ca. 3 ml from rats).
Production of monoclonal antibodies
Four days before fusion, animals will get a boost immunization, preferentially with recombinant proteins or with HEK cells, which are transiently transfected with the provided cDNA construct. Spleen cells are then extracted from the animals and fused with SP2/0 myloma cells. Four aliquots of these fused cells are deep-frozen in RPMI/20%FCS/10%DMSO. Each aliquot corresponds to approximately 200-2.000 growing hybridoma cells.
288 clones (one aliquot of fused cells) are tested for their reactivity with CHO cells, which are transiently transfected with the construct used in the immunization process. The four best clones are delivered (14 ml living hybridoma cells).
After subcloning by limited dilution 15 growing subclones are picked. Supernatants are tested for their reactivity with CHO cells, which are transiently transfected with the construct used in the immunization process. The two best clones are delivered (14 ml living hybridoma cells).
An aliquot of hybridoma supernatant is used to identify Ig-Subclasses by ELISA.
A strong promotor should control the delivered cDNA (for example: CMV, pgk). For membrane proteins and secreted proteins the success rate is a lot higher than for intracellular proteins. However, in most cases intracellular proteins and/or defined subdomains can be transmuted into membrane proteins by cloning the cDNA in a "Display"-Vector (e.g. between a leaderpeptide and a transmembrane domain). The Antibody Core Unit can provide a suitable vector with an N-terminal FLAG-tag to verify the successful expression in transiently transfected cells (if you are interested please fill out the attached MTA).
The rate of success for immunization should amount to:
- about 80% in case of membrane and secreted proteins
- about 50% in case of cytosolic proteins
- about 30% in case of a "blind" immunization with a not yet characterized expression vector, which contains the predicted complete open reading frame.
Regarding the immunization of animals, the facility performs one prime and three boost immunizations.
For the immunization reaction the supplied plasmids are conjugated to gold particles. Together with an adjuvant they are then injected into the animal by ballistic transfection.
Specificity and titer of the received serum ís tested by its reactivity in immune fluorescence analyses with transient transfected CHO. Therefore, cells are co-transfected with a cDNA construct that comprises a nuclear located GFP (Green Fluorescent Protein). Serum is regarded as specific if it stains GFP transfected cells but not controls.