Antibody technologies

Methods

• Antigen and protein preparation for immunisation:
  • Spectroscopic analysis of the immunisation proteins and transfer into a buffer suitable for immunisation
  • MS analysis of the antigen for quality assessment
  • For low-molecular antigens: covalent bond to corresponding carrier proteins (KLH, BSA)
• Analysis using immunogenicity tests to assess the general immune response in mice in the framework of the generation of anti-bodies
• Immunisation, splenetic cell preparation, cell fusion
• Epitope-specific serum titre (using the enzyme-linked immunosorbent assay (ELISA) or, upon request, surface plasmon resonance (SPR))

• Fusion of isolated murine splenetic cells with SP2/0-Ag14 cells for hybridoma generation
• Screening methods (standard ELISA, if desired: SPR) to isolate hybridoma cells which generate specific antibodies
• Additional ELISA or SPR screenings to exclude cross-reactivity during the separation of the hybridoma cell clones
• Quality control, e.g. mycoplasma tests
• Transfer of cryogenic stocks to the project partner or long-term storage

• Cultivation and expansion of hybridoma cells producing antibodies according to the desired quantities of antibodies
• Isolation of antibodies from the cell culture supernatant, e.g. using protein G/A affinity chromatography 

• Characterisation of monoclonal antibodies in terms of their bonding behaviour and affinity to the epitope considered with the help of various methods, e.g. Western Blot, ELISA and SPR
• Determination of subtypes / isotypes of the heavy and light chain of the antibody candidate
• X-ray structure analysis of antibody Fab fragments for targeted changes to the AB-epitope bond:
  • Separation of antibodies for generating the Fab fragment 
  • Co-crystallisation of the Fab-epitope complex using random matrix screening
  • Crystal optimisation and X-ray diffraction analysis
  • Structure calculation and modelling

• In-silico analysis of variable domains of the heavy and light chain of the mouse antibody regarding the CDR (complementary determining region) and other amino acid residues with possible involvement in the antigen bond (Vernier residues)
• Selection of corresponding human AB candidates for the targeted transfer of previously identified sequence areas or amino acids (grafting)
• Cloning in the expression vector for recombinant expression in eucaryotic cell systems and subsequent protein purification
• Subtype exchange during recombinant expression
• Characterisation of the bonding properties compared to the parental mouse antibody using various methods, such as ELISA, SPR, immunohistochemical staining 

• Development of specific enzyme-linked immunosorbent assays (ELISA) for the quantitative detection of the analyte using antibodies produced in-house or provided by the project partners
• Demand-driven functional testing of the developed ELISA: From simple quality testing to GLP-compliant ELISA validation in compliance with the applicable FDA (Food and Drug Administration) and EMA (European Medicines Agency) guidelines