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Monoclonal Antibody Production Modules
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
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Fluorescence labeling-
antibody conjugation with FITC, Rhodamine, TRIIC, and others; removal of
unconjugated fluorescence dye; determination of labeling efficiency;
documentation
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Phycobiliprotein
labeling- conjugation of phycobiliprotein to antibody (1:1), FPLC
chromatographic purification, determination of labeling efficiency, detailed
documentation
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Peroxidase (POD)
labeling- thioether linkage by heterobifunctional crosslinker, FPLC
chromatographic purification, determination of labeling efficiency,
documentation (please inquire for other enzymes)
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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)
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Biotin or LC-biotin
labeling- conjugation of biotin or LC-biotin to antibody, removal of
unconjugated biotin, documentation
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