Custom Antibody Production
The different steps
The production of monoclonal antibodies involves the following steps and this activity is ISO 9001:2015 certifyed.
Immunization of animals and analysis of their immune response
Four BALB/c mice are immunized over several weeks and their immune response is analysed (intermediate bleeds will be performed after the 4th immunization). A special adjuvant is used to ensure a strong immune response in the injected animals.
Cell fusion and production of hybridomas
Spleen cells of an immunized animal are fused with a Sp2/0-Ag 14 myeloma cell line, using polyethyleneglycol as fusion agent. Hybridomas are selected using HAT selective medium.
Screening of hybridomas
Hybridomas are screened by ELISA using suitable antigens. 10 ELISA plates will be used. For custom production of monoclonal antibodies, it is necessary to provide the Company with suitable antigenic material. A polyclonal antiserum directed against the antigen, produced in rabbits or other animals, may also be required.
Cell cloning and characterization
Monoclonal antibodies are obtained from hybridoma cells culture. Subcloning of these cultures is necessary to insure the monoclonality of the cell culture. Monoclonal antibody will be isotyped.
Production of monoclonal antibodies
The specific hybridomas are grown by cell culture and the cell lines are stored in liquid nitrogen. Normally, monoclonal antibody will be produced by in vitro cell culture (collection of cell culture supernatant) at an approximate concentration of 5-20 µg/ml. Maintenance of hybridomas and production of large amounts of antibodies can be undertaken on request.
Purification and enzymatic coupling of antibodies
Monoclonal antibodies (IgG class) can be purified by affinity chromatography using protein G column. Antibodies of other isotype (IgM) can be purified using other methods. Purified antibodies can be linked to biotin, alkaline phosphatase or peroxidase. The reactivity of the labelled antibodies is tested by ELISA.
Calibration of ELISA test
The quality of a detection method depends on three principal factors : sensitivity of the technique, specificity of detection, and experimental conditions for routine use. All three factors are taken into account.
Validation of the reagents
Reagents need to be validated to develop reference ELISA tests. This can be done using different strains of the virus from different parts of the world. Reagents showing the largest detection spectrum will be selected.