Development process Custom Antibody Services
Development Monoclonal
Mouse Monoclonal Antibody Development Service
The following phases are the basic steps of the process we follow to develop custom antibodies. Each project is treated as unique and we work with our Customers to create a protocol that works best for them to provide our Customers with a tailor made antibody.
Service Description:
Pre-immunization Phase – on request (4-6 weeks)
- Antigen Design
- Peptide Synthesis
- Peptide Conjugation for immunization purposes
Phase I: Immunization and Screening (~10 weeks)
- Multiple (3-5) immunization of eight (8) FcRn overexpressing transgenic BALB/C mice
- Collection of test bleeds and determination of antigen-specific antibody titer by ELISA
- Test bleed data will be evaluated jointly to determine the immune response within antisera. Serum samples can be shipped on request.
- Identify 1-2 mice for fusion purposes
Phase II: Fusion (2-3 weeks)
- Fusion (approximately 9 weeks after initial immunization 2 selected mice will be given a final boost for cell fusion)
- Parental clones are screened by ELISA (up to 1000 hybridoma clones) to select antigen-specific IgG producer clones
- Supernatants are saved for further testing
- ELISA data will be provided and customer selects up to 12 supernatants (0.5-1 ml/parental clone) for shipment if requested
- After primary screening up to 5 positive parentals specific to the immunogen will progress to subcloning
Phase III: Selection, Subcloning and Expansion of Hybridomas (2-3 weeks)
- Subcloning of selected microcultures
- All subclones generated in this phase will also be evaluated by direct ELISA against the antigen.
- Only positive reacting subclones specific to the immunogen with sufficiently high titers will be carried forward to the Customer for additional examinations.
- Includes cryopreservation of parental clones (2 vials per clone) and subclones (up to 5 vials per clone).
Phase IV: Antibody Production (on a 1 liter basis) and Purification (up to 100 ml of concentrated supernatant) (2-3 weeks)
- Clones isolated in Phase III will be selected for antibody production
- Mycoplasma testing of selected cell lines (per clone) will be performed prior to the production of antibodies
- Antibody produced (max 2mg) will be purified by Protein A/G chromatography (followed by SDS-PAGE analysis of the protein using COOMASSIE stain - charged separately)
- Antibody concentration will be determined for the purified monoclonal antibody using conventional methods and will be reported in tabular form
Rabbit Monoclonal Antibody Development Service
Service Description:
Phase I: Immunization (~10 weeks)
- Multiple immunization with properly designed antigen
- Collection of test bleeds and determination of antigen-specific antibody production by a suitable method (ELISA, WB, FCM, etc.). Serum samples can be shipped on request.
- Identification of the best responding rabbit(s) for recombinant antibody production
Phase II: Recombinant antibody production (~4 weeks)
- Single cell sorting of antigen-specific B cells from the spleen
- Amplifying the heavy and the light chain variable regions from the sorted cells
- Integrating the variable regions into expression vectors, which contain the appropriate constant sequences.
- Producing recombinant monoclonal antibodies in a mammalian expression system.
- Testing the specificity of the recombinant antibodies with a suitable method (ELISA, WB, FCM). The best clones will be chosen for further examination (sequencing etc.). Samples can be shipped on request.
Development Polyclonal
Rabbit Polyclonal Antibody Development Service
The following phases are the basic steps of the process we follow to develop custom antibodies. Each project is treated as unique and we work with our Customers to create a protocol that works best for them to provide our Customers with a tailor made antibody.
Service Description:
Pre-immunization Phase – on request (4-6 weeks)
- Antigen Design
- Peptide Synthesis
- Peptide Conjugation for immunization purposes
Development Phase: Immunization, Screening and Production (8-10 weeks)
- Multiple immunization (3-5) of three (3) FcRn overexpressing transgenic rabbits
- Collection of animal pre-immune serum
- Collection of test serum bleeds
- ELISA-based antibody titer determination
- Terminal bleed collection
- Cryopreservation and storage of terminal bleed (n=1) until purification
Purification Phase (~1 week)
- Purification of 50 ml of immune sera (2-7 mg purified polyclonal antibody expected)
- Purification using Protein G OR peptide affinity purification including column preparation
Additional Information About CAS
The animals are kept and immunized in compliance with Institutional Animal Care and Ethics Committee-approved protocols (22.1;601 /000/2009).
These projects provides research use antibodies.
