Mouse Monoclonal Antibody Development
Your antigen and our skilled technical staff create an ideal combination for your custom antibody requirements. We keep your project in strict confidence. Any hybridoma cell lines that we develop for you remain your exclusive property.
The successful development of hybridoma cell lines producing monoclonal antibodies depends on three factors: 1) the immune system of the host, 2) the antigen, and 3) the assay used to measure antigen-antibody binding.
The project length depends on these factors. Large, highly immunogenic antigens typically result in many hybridoma cell lines from a limited number of immunized mice. Small, highly conserved antigens often require more immunizations, mice, and screening to select an appropriate antibody-producing cell line.
Our hybridoma development process
The four stages of our hybridoma development define critical project milestones. We require your approval before advancing to each subsequent stage. Because we invoice when milestones are reached, you pay only for work completed.
Stage I (Weeks 1-7): Establish project web site. Immunize mice. Screen sera by ELISA for immune response.
Mice receive an initial injection on Day 0 in Complete Freund's adjuvant, and booster injections in Incomplete Freund's on Days 14 and 28. Serum is collected and the titer is determined. Samples are available for testing in your assays at your facility. Booster injections are given until one or more mice have appropriate titers.
Stage II (Weeks 8-11): Fuse splenocytes from high titer host. Screen for specific antibody production by ELISA. Subclone and freeze selected positive parent wells. Our fusions are plated into 4 x 24-well plates; a step that enhances initial growth conditions for these fragile cultures. Screening of supernatants from the 96 parent wells identifies those wells with positive hybridomas. Selected wells with positive hybridomas are frozen for future use and cloned to isolate positive cell lines.
If no positive clones are obtained from the first fusion, the remaining animals receive additional booster injections for another fusion. At this stage the screening assay or antigen may require additional optimization.
Stage III (Weeks 12-15): Cloning by limiting dilution of positive parent wells to obtain monoclonal hybridoma cells. Approximately 10-14 days after cloning, small volumes of media are removed from the wells and tested by immunoassay to identify antibody-producing clones. Positive clones are expanded, retested, and grown to sufficient numbers (5 x 106/milliliter) for freezing and production of antibody in ascites or mass culture. All frozen cell lines are sent back to you for storage.
Stage IV: Produce and purify monoclonal antibody. (Weeks 16-18). Approximately 30 ml of conditioned tissue culture supernatant from each of 5 clones is sent to you as part of the project. Upon your evaluation, you may then select one clone for a 250 ml culture and purification. Additional clones can be done at our standard culture/purification rates.
For project continuation, you may select from either in vitro (roller bottles, spinners, bioreactors) or in vivo (ascites) production of antibody as a continuation of the project. We can grow and purify any amount, including purification by several different methodologies. Additionally, we can label or otherwise modify the antibody specifically for your needs.