Synaptic Systems offers specialized services providing individual solutions for our customers' specific requirements. A complete service from peptide synthesis or protein expression to monoclonal antibody production and long term storage of hybridoma cells is available.
Phase I / Immunization
For immunization, 3 mg of purified peptide or 1 mg of purified protein is required (2 mg protein if a Western blot screening is requested). As a general procedure, three Balb/c mice or Lewis rats are immunized with 90 to 150 µg each. If the immunogenicity of the antigen is critical (e.g. mouse/rat proteins or peptides), special strains can be used.
Two different immunization strategies are available. In our short program animals are immunized over a period of 17 days every second day and one day before fusion. This in general yields a broad spectrum of monoclonal antibodies directed against different epitopes of the immunogen with moderate to high affinity.
In our long immunization program animals are immunized over a period of three month every third week and shortly before fusion. The longer affinity maturation of B-cells leads to a smaller panel of antibodies with lower variability but in general higher affinity.
Depending on the antigen, different immune stimulating agents are used like Gerbu or incomplete Freund's Adjuvant combined with CpG oligo-nucleotides (Krieg AM 2002. Annual Review of Immunology 20: 709-60).
Phase II / Fusion
After immunization, cells from knee lymph knots and spleen of all three animals are prepared and fused with Ag8 myeloma cells in the presence of PEG 1500. After fusion, cells are plated on 24-well plates resulting in 360 oligo clones each usually consisting of 5 - 10 clones. Cells are cultivated for 10 days in selective and conditioned medium selecting for fused cells only.
Phase III / Screening and subcloning
Within the next two days, all 360 oligo clone supernatants are screened by ELISA for antibody positive wells.
For the next screening step we offer four options:
- Up to 72 oligo clone supernatants are tested in Western blots at Synaptic Systems.
- Up to 360 oligo clone supernatants are tested by immunofluorescence on GFP transfected cells at Synaptic Systems.
- Up to 360 oligo clone supernatants are screened by a specific sandwich ELISA at Synaptic Systems to identify capture or detector antibodies.
- Up to 20 oligo clone supernatants (3 - 5 ml each) are shipped to the customer for screening under their own conditions.
Usually screening is completed within a period of three weeks. After the customer's decision of which oligo clone should be subcloned, the selected oligo clone is subcloned to monoclonality by the limiting dilution method. Subclones are screened according to the options (1 - 4) chosen by the customer, and are expanded for the next cycle of subcloning. Several cycles of subcloning may be required in order to obtain a true, stable clone which is then used to produce 100 ml of monoclonal antibody supernatant.
Positive clones will be frozen as back up and stored for up to 12 months if desired. Long term storage is possible upon request.
It is our goal to provide our customers with monoclonal antibodies that fit to their assays in the best possible way. Hence, close collaboration during the course of the project is our first priority.
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