4 Balb/C mice will be immunized following a protocol developed by Covalab. Screening methods for antibody detection will be developed for each different project according to the specificity of the antigen. Each bleeding will be tested following this screening procedure and send to customer to confirm the immunoreactivity in their experiment.

After agree of customer, fusion of spleen cells from chosen mouse will be performed using myeloma cell line (Sp2/O-Ag-14) as fusion partner.
Mixture of hybrydoma and non-fused cells will be treated with culture medium containing Hypoxanthin Aminopterin Thymidin (HAT). After treatment, just hybridoma will survive and will be screened by the specific procedure previously mentioned.
Three positive hybridomas at the most will be selected. If the total number of positive hybridomas exceeds 3, Covalab will freeze at the maximum 7 other positive hybridomas (5 vials/hybridoma, 3 different freezing dates). Thus the client could request later, the cloning of these hybridomas.

For each selected hybrydoma following step will be done:

• Cloning by serial dilution to get one cell by well
  
• Screening and amplification of positive clones
• Isotyping of clones and determination of Ig subclass
• Storage of selected hybridomas in liquid nitrogen
• The final clones are sent to the customer for testing and confirmation

The success of monoclonal antibody projects depends upon the successful immunisation and selection of animals.

Hence, it is very important to analyse the polyclonal antibodies (mouse serum) for the presence of antibodies that will be used in a particular technique. For example, if antibodies are intended for Western blotting and / or Immunochemistry, it is absolutely necessary to confirm that the polyclonal serum does recognize the corresponding antigen from appropriate tissues or cell extracts.

This is particularly important when making anti-peptide monoclonal antibody . Covalab cannot guarantee that the monoclonal antibody produced will work in a given technique. Antibody secreting clones are selected by using antigen-coated plates (ELISA). The users must do further testing, according to the application envisaged. It is very important to fully discuss the project with us. Covalab must coordinate with the user for the timely testing of mouse serum, hybridoma medium, or ascites at various stages of development.

Covalab undertakes:

• Not to divulge any information concerning the immunogen and the antibodies to a third party without the written consent of the client.
• To return to the client at the end of the contract, any immunogen remaining, corresponding anti-sera, antibodies and all the hybridomas in its possession.
• The client owns all rights including intellectual property rights to the hybridomas developed by Covalab.

• Production of monoclonal antibodies in vitro
• Production of monoclonal antibodies in vivo
• Purification of antibodies