Storkbio's complete hybridoma development service is custom designed to produce hybridoma cell lines that secrete monoclonal antibodies specific to your antigen. We will review your requirements and antigen details and define a strategy for immunization, screening and assaying, which meets best your specific needs. The whole protocol is divided into 4 phases: antigen preparation, immunization, fusion and screening and finally clone selection, antibody production and purification.
We will select 5 parental hybridoma clones and customer will get from each of them:
Antigen preparation (1-2 weeks)
To get well working monoclonal antibodies, antigen used for production must be right and serve the purpose of later antibody applications. Monoclonals at Storkbio can be made with customer provided antigen (~1 mg) or with newly, at Storkbio's facility synthesized peptides. We can assist customers by preparation and purifications of their own batch of antigen or make bioinformatical analysis, to propose suitable peptides from customer's protein sequences. The analysis may increase the probability of an immunogenic response, but Storkbio does not make any expressed or implied guarantees that such antigens will yield an antibody for use in any immunochemical procedure.
Little antigens, like peptides and antigens with probably weak immunogenecity will be conjugated to carrier proteins. As standard we use KLH-conjugates for immunization and BSA-conjugates for screening.
Immunization (10-12 weeks)
After the antigen is ready, 5 female Balb/C mice will be immunized 4 times and test bleeds are taken, to determine titer by ELISA (or Western Blot or other specified assay, if requested). Animal(s) with best titer will get an additional boost before hybridoma production.
Fusion and screening (4-6 weeks)
Splenocytes (cells at spleen) from the immunized mice with best titer will be fused with Sp2/0 myeloma cells. Fused clones are screened usually 10 days after the fusion by ELISA (or Western Blot or other specified assay, if requested) to select positive antibody-secreting clones. Positive hybridomas are then expanded for next phase and cells cryopreserved for back-up.
Clone selection, antibody production and purification (4-6 weeks)
Monoclonal selection is performed with best 5 parental clones by limiting dilution cloning usually on 96-well plates. This step ensures monoclonality, specifity and viability of the hybridomas. Screening is made by ELISA (or Western Blot or other specified assay, if requested). Subclones with high specifiy are then expanded and cells cryopreserved. In the same time, supernatant is collected and antibodies purified from the medium using protein A/G method. Finally, antibodies are concentrated to 1-2 mg/ml using ammonium sulfate precipitation.
Duration: 6-6,5 months
All custom work is completely confidential.