Monoclonal antibodies can be developed against a variety of antigens including proteins and haptens (i.e. peptides conjugated to a carrier protein). For anti-protein projects Covalab guarantees at least one positive and specific clone against the used immunogen. Covalab guarantees to return at the end of the project.

Each project is discussed before the start of the protocol to ensure that the customer's requirements are met. Covalab offers advice and technical expertise and works closely with customers throughout the project. A detailed report is supplied to the customer at the end of the project.

Our monoclonal antibody production service has been composed of several phases. At the end of each phase, results will be discussed with the customer before beginning the next step. If a phase should meet with limited success, the project can be terminated.

• Immunisation

4 Balb/C female mice will be immunised with a series of injections containing the mixture of the immunogen and Freund’s adjuvant. The first intraperitoneal injection will consist of 0,1ml protein in PBS mixed in 1/1 ratio with 0,1ml of complete Freund’s adjuvant. The other series of injections with incomplete Freund’s adjuvant will replace the complete one. Preimmune serum is taken on day 0. Sera will be collected during the protocol and titers will be determined by ELISA in order to select the best responding mouse after. If the antibody titer is too low after the last injection, you may request additional injections. If the ELISA test remains negative, the immunisation should be terminated because the probability of success is too low. If the antigen is a hapten, it will be coupled to an appropriate protein carrier. Antigen preparation supplied by the customer

• Development of screening method for antibody detection

Antigen will be coated to microtiter plate using Covalab technologies. This procedure is often used for the screening of antiserum. The remaining free sites on the solid support will be blocked in order to avoid the non-specific interaction of the antiserum for the ELISA test; several parameters will be optimized such as: antigen concentration, serum dilutions, buffers and solvent, incubation time, temperature of the immunoreactions, dilutions of anti-mouse peroxydase conjugate. The immunoreactivity of the antiserum will be controlled by the customer.

• Fusion and screening

• Final boost: At 3 to 4 days before the fusion, the mouse exhibiting the highest antibody titer will be injected with antigen as a final boost. This will allow most of the circulating antibodies to be cleared from the blood stream by the mouse.
• Fusion of murine myeloma cell line (Sp2/O-Ag-14 ) and spleen cells from the best responding animal using PEG (polyethylene glycol)
• The HAT mixture of drugs will be used to select against the growth of the myeloma cells. The cells will then be dispensed in 96-well microtiter plates and placed at 37°C in a CO2 incubator. 5 to 7 days after, cells are fed by adding 1/2 volume of HAT medium to each well. Visual control is made over 3 weeks for growing colonies and these will be identified for antibody secretion.
• Screening: The supernatants from the wells containing hybridomas will be removed and the immunoreactivity of the antibodies is assessed by ELISA as described in the previous paragraphs.
• Amplification of positive colonies: three vials of every hybridoma will be stored in liquid nitrogen. At the end of the screening phase, we send you 5 to 10 ml of supernatant of the best hybridomas for confirmation and additional tests in your laboratory. This will ensure that we will clone only those antibodies that suit your purpose.

• Cloning and isotyping

After confirmation of positive hybridomas, the next step will be the cloning of antibody-producing cells. The original positive well often contains more than one clone of hybridoma cells and some have an unstable assortment of chromosomes. Single cell cloning ensures that cells which produce the antibody of interest are monoclonal and that the secretion of this antibody can be maintained in a stable way. Different approaches to cloning are used but usually cloning is performed by limiting dilution. Within three weeks of cloning, supernatants from wells containing hybridomas will be screened to determine the clone which produces the desired antibody. Positive wells which are likely to be of monoclonal origin will be expanded. Class and subclass isotyping of all selected clones will be determined. Remark: Cloning and isotyping of 3 hybridomas selected at the previous step, Freezing of hybridomas secreting specific immunoglobulins at clonal stage: 3 hybridomas at maximum, 5 vials/hybridoma, 3 different freezing dates.

• Cloning by limiting dilution and expansion of cells
• 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

• Keys stages

The success of Mab projects depends upon the successful immunization and selection of animals. The Mab technique in itself can not produce an antibody (clone) that is not present in an immunized animal (in polyclonal serum).

Hence, it is very important to analyze 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 absolute necessity 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 Mab. Covalab can’t guarantee that the Mab 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 mice
• Purification of antibodies
• Labelling of antibodies

At the end of the contract, Covalab proposes to store the hybridomas in vials frozen in liquid nitrogen. Minimum storage period: 1 month.

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