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Hybridoma Development Hybridoma Development

GeneMay offers highly successful and affortable hybridoma development service to accelerate your research. Our staff has extensive experience with many monoclonal antibody development projects. It usually takes approximately 4-6 months to produce a hybridoma from the antigen of your choice.

STAGE I
Immunization Ten virus antibody-free mice will be immunized with your antigen. The mice will be housed in an isolator during the immunization period. At the sixth week, following the primary immunization and the first boost, the mice will be tested for antibody titre by ELISA assay unless an alternative assay is requested.
If additional boosts are required, they will be administered and the mice will be retested for antibody titre. Once immunization is complete, the mice will be held for three to four weeks before fusion. Three to five days before the fusion, a mouse will be selected for the pre-fusion boost.

 
STAGE II
Fusion The spleen from the mouse will be harvested and all spleen cells will be fused with a myeloma cell line. We use six to ten 96-well panels for plating. Cells will be grown in HAT selection medium. A maximum of three separate fusions will be performed.

Screen and Selection All 96-well panels will be screened by ELISA for antibody positive wells. Fifty to one hundred positive wells will be expanded and retested three to four days later by ELISA. The standard production will be screened with a single antigen on ELISA. Additional antigens may be added as an option. A maximum of 30 positive wells will be frozen as back-up and 0.2ml of tissue culture supernatant/clone can be sent to the customer for further testing.
 
STAGE III
First Cloning   A maximum of 15 positive wells will be selected for cloning by the limiting dilution method. Isolated colonies will be tested by ELISA, and positives selected to expand for the next cycle of cloning. Cells from the clones will be frozen as back-up.
 
Final Cloning  Approximately 12 positive clones from the first cloning cycle will be selected for the second cycle. Isolated colonies will be tested by ELISA and positives selected to expand. Several cycles of cloning may be required in order to obtain true, stable clones. Cells from these positive clones will be frozen as back-up.
 
Selection for Non-HT Requirement  A maximum of ten selected clones will be expanded and a small stock of each will be frozen. Each clone will be check for it's isotype.

Frozen Stock and Ascites
  The final ten clones selected will be expanded and a small stock of each will be frozen. Each clone will be isotyped. In addition, several mice will be inoculated with each clone for ascites production. Ascites fluid and/or vials of cells will be shipped to the customer for evaluation.
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