IMMUNIZATION
- Mouse strain: BALB/c (please inquire for other strains)
- 3 booster injections within 3 months
- Screening and evaluation of serum titer by ELISA
- Selection of the animal with the best response against the antigen for first fusion (if more than 1 mouse is used)*
- Additionally, immunized animals can be kept longer for monthly booster injections and further fusion.
*If no animal produces sufficiently high titers due to low immunogenicity of the antigen, the production process can be ended here without further costs.
FUSION
- Spleen cell preparation, fusion with mouse myeloma cell line P3-X63-Ag8.653
- Distribution of hybridoma cells into 96-well plates
- Selection of hybridomas
ANALYSIS
- Detection of specific antibody in culture medium of 96 supernatants by ELISA with 1 antigen (immunization antigen)
- Cryopreservation of all antigen-positive culture supernatants
- Delivery of 20 supernatants with the best response against the antigen
- Delivery of 5 vital or cryopreserved positive clones
OPTIONAL:
- Detection of specific antibodies in culture medium of 96 supernatants by ELISA with 2 antigens (immunization and control antigen)
- Cryopreservation of all antigen-positive culture supernatants
- Delivery of 20 supernatants with the best response against the antigen
- Delivery of 5 vital or cryopreserved positive clones
- Storage of one cryopreserved clone in liquid nitrogen
- Revitalization and cultivation of one clone
- Isotope analysis
SUB-CLONING
- Revitalization and cultivation of one cryopreserved clone
- Sub-cloning
- Detection of specific antibody in culture supernatant by ELISA with immunization antigen
- Delivery of vital or cryopreserved positive clone with the best response against the antigen
PRODUCTION OF ANTIBODY CULTURE SUPERNATANT
Vital or cryopreserved clones provided by the customer are cultured. The antibody production rate of the clone can be determined and may be optimized.
- Production of antibody culture supernatant (ACS)
PURIFICATION OF MONOCLONAL ANTIBODIES
- Purification of monoclonal antibodies from culture supernatant
- Ammonium sulfate precipitation of culture supernatant (production rate > 5 μg/mL), Protein A or G affinity chromatography, concentration of eluate, dialysis, determination of protein amount, purification analysis by FPLC, detailed documentation
- SDS-PAGE gel electrophoresis- 2 mini gels with 1 standard and up to 8 probes each; Coomassie staining, digital print and preserved original PAGE gel
LABELING OF MONOCLONAL ANTIBODIES
- Fluorescence labeling- antibody conjugation with FITC, Rhodamine, TRIIC, and others; removal of unconjugated fluorescence dye; determination of labeling efficiency; documentation
- Phycobiliprotein labeling- conjugation of phycobiliprotein to antibody (1:1), FPLC chromatographic purification, determination of labeling efficiency, detailed documentation
- Peroxidase (POD) labeling- thioether linkage by heterobifunctional crosslinker, FPLC chromatographic purification, determination of labeling efficiency, documentation (please inquire for other enzymes)
- Alkaline phosphatase (AP) labeling- thioether linkage by heterobifunctional crosslinker, AP > 7000U/mg, FPLC chromatographic purification, determination of labeling efficiency, documentation (please inquire for other enzymes)
- Biotin or LC-biotin labeling- conjugation of biotin or LC-biotin to antibody, removal of unconjugated biotin, documentation
For technical support or to place an order for antibody production services, please email us at antibody@crl.com.