Custom Monoclonal Antibodies Service
We provide custom antibody production, from antigen design to antibody validation with more than 25 years of experience.
Our service encompasses antigen design, immunization of rats and mice, screening and establishment of custom monoclonal antibodies. We can generate antibodies against all non-toxic types of target molecules, including difficult targets such as transmembrane proteins, post-translationally modified proteins, small molecules and cell surface proteins, virus-like particles or extracellular vesicles.
Using the hybridoma technology, we generate thousands of clones per immunized animal which are then analyzed on an automated high-throughput flow cytometry screening platform. We design ELISA assays to identify high-affinity binders or neutralizing antibodies in the primary screening. Thorough antibody validation is performed in collaboration with our customers. Specific hybridoma clones are subcloned to obtain stable monoclonal cell lines which are cryo-conserved to guarantee an unlimited antibody source.
We provide custom antibody production, from antigen design to antibody validation with more than 25 years of experience.
Our service encompasses antigen design, immunization of rats and mice, screening and establishment of custom monoclonal antibodies. We can generate antibodies against all non-toxic types of target molecules, including difficult targets such as transmembrane proteins, post-translationally modified proteins, small molecules and cell surface proteins, virus-like particles or extracellular vesicles.
Using the hybridoma technology, we generate thousands of clones per immunized animal which are then analyzed on an automated high-throughput flow cytometry screening platform. We design ELISA assays to identify high-affinity binders or neutralizing antibodies in the primary screening. Thorough antibody validation is performed in collaboration with our customers. Specific hybridoma clones are subcloned to obtain stable monoclonal cell lines which are cryo-conserved to guarantee an unlimited antibody source.
Project Workflow
Biorender | Dr. Katharina BräuerOverview and timeline for our customized three-step antibody manufacturing workflow
Helmholtz Munich | Matthias BalkCore Facility Monoclonal Antibodies
Projects start with the discussion of the specific antibody requirements, the selection of the antigen for immunization, and discussion of the antibody validation strategy.
The antigens are provided by the customer. These can be the most commonly used antigens such as peptides or proteins, but also any immunogenic and non-toxic substance such as cells, extracellular vesicles, virus-like particles and haptens coupled to carrier proteins can be used.
Proteins are a good choice for immunization because they can result in multiple antibody clones specific for different epitopes of the protein. Approximately 1mg of protein is required for immunization and subsequent screening tests. Tagged proteins or fusion proteins are ideal for the screening process.
Peptides can be used when purified protein is not available or for antibodies specific to mutation sites or against post-translational modifications. Immunization with different peptides of the same protein increases the chance of successful antibody generation. The Core Facility assists in identifying suitable immunogenic peptides and can also arrange for peptide synthesis. Ovalbumin-coupled peptides are generally used for immunization and biotinylated peptides for screening.
Helmholtz Munich | Matthias BalkImmunization
Routinely, we immunize three animals (rats and/or mice) per antigen. For the generation of antibodies against mouse extracellular antigens, we can also use appropriate knock-out mice for immunization, if available.
The antigen is emulsified together with CpG adjuvant in Freund’s incomplete adjuvant. For peptide immunizations, we perform immunizations with a peptide mixture. Immunization is followed by a booster injection approximately 6-10 weeks after immunization.
Fusion
Three days after the last booster injection, splenocytes from immunized animals are collected and prepared for fusion with the mouse myeloma cell line P3X63-Ag8.653. The fused cells are plated in 96-well plates in HAT selection media to grow hybridomas for subsequent screening.
Screening
Hybridoma supernatants are screened for the presence of IgG antibodies directed against the antigen in an automated high-throughput flow cytometry analysis (TECAN Fluent and iQue Screener Plus) 10 days after selection. Cells from selected positive wells are expanded and cryopreserved to allow sufficient time for further validation of antibody supernatants. At this stage, we determine the specific IgG isotype and the antibody titer and send 4ml of antibody supernatant along with appropriate isotype-specific secondary antibodies to our customers for further validation in the desired applications.
Helmholtz Munich|Matthias BalkAntibody validation is one of the most important steps during antibody production to ensure the selection of the best antibody clones(s) and to prove affinity, specificity and reproducibility. We deliver 4ml of selected supernatants (primary supernatants) together with appropriate isotype-specific secondary antibodies for further validation in the desired application. The antibody supernatants can be tested e.g. in Western blot (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunocytochemistry (ICC), ELISA, immunoprecipitation (IP) or chromatin immunoprecipitation (ChIP) on specific biological test specimen. We are happy to provide expert advice on antibody validation strategies.
Helmholtz Munich | Matthias BalkCore Facility Induced Pluripotent Stem Cells
Selected hybridomas are subcloned by at least two rounds of limiting dilution cloning to establish chromosomally stable monoclonal antibody-producing cell lines. Hybridoma clones are tested for mycoplasma and are stored in dedicated liquid nitrogen tanks for safe long-term storage. Furthermore, we provide 200ml of antibody supernatant by default, but different quantities or formats are available upon request.
Antibody supernatant is suitable for most in vitro applications such as Western blot, immunofluorescence, immunohistochemistry, immunoprecipitation, and flow cytometry.
