Custom Monoclonal Antibodies

Primm has a very efficient monoclonal antibody production service using mice immunized with antigens either provided by the customer or produced by Primm.

Monoclonal antibodies are important for their high value, for their monospecificity in fact they have affinity for the same epitope and specificity for a unique region of the protein. They could block peculiar protein function and they are very useful tools in biochemistry, molecular biology and medicine.

As antigen the best choice are proteins. Proteins can be supplied by the costumer or produced by Primm as HIS-Tag protein fusion (either full length or domains), Primm also uses synthetic peptides even if they are not the ideal immunogen, because they are too small and they wouldn’t allow to generate a large number of monospecific clones.

On average 2-3 mg of antigen are necessary for immunization and analysis.

This service includes the generation of monoclonal antibodies through somatic fusion and the possibility to choose either 2 or more hybridoma after the first screening. We can provide both the cell lines and the purified monoclonal Abs. Upon request a cell banking service is available for the storage of the desired clones.

Monoclonal Antibody Generation:

• Antigen choice
• Immunization to obtain polyclonal response
• Splenectomy with spleen cells isolation (lymphocytes)
• Splenocytes fusion with myeloma cells
• Selection of positive hybridoma cell lines and isolation of mother clones
• Expansion and sub-cloning by limiting dilution
• Isolation of final clones
• Production (in vivo / cell culture)
• Immunoaffinity-purification of Mabs

FAQs

Which are the characteristics of our service?
Primm provides a complete service assuring the quality of the results expressed in terms of generation of antibodies able to recognize the antigen used for the immunization. The complete service is based on different phases: preparation of the antigen (if required peptides and recombinant proteins), immunization of either mice or rats, splenectomy and fusion of splenocytes with myeloma cells, selection of hybridomas by ELISA, cloning and sub-cloning by limiting dilution of positive clones and production of purified antibodies (mgs) in “cell culture” or from induced ascitic fluid. 

Which are the specifications of the monoclonal antibodies?
The antibodies pass the quality control specs if the hybridomas are able to recognize the antigen used for immunization at a dilution of their supernatants not lower than 1:10. Then customers can test these positive hybridomas (supernatants) and choose 2 or more of them to go on with cloning and sub-cloning phases. 

Is Primm ready to modify the protocol if required by the customer or if necessary for the success or improvement of the results?
Yes, Primm is available to start the immunization schedule with a protocol suggested by the customer. A specific quotation will be issued in this case. Also, in the event that the standard immunization scheme does not yield antibodies passing the established specifications, Primm may prolong or change the immunization protocol. 

How does Primm test the quality and quantity of the monoclonal antibodies?
First serum collected from the immunized animals and then supernatants of positive hybridomas are tested by ELISA. According to Primm procedure, the antigen used for immunization is coated on a microtiter plate and allowed to react with the specific serum and supernatants. The reaction is followed by labelled-secondary antibodies able to recognize the specific immunoglobulins bound to the antigen. The intensity of the resulting label signals gives a quantitative measure of the antibodies. In case of antibodies generated against synthetic peptides, the peptide conjugated to a different carrier protein (from which used for immunization) is coated on the microtiter plate. 

What type of antigen does Primm use?
Primm suggest to make monoclonal antibodies against recombinant or natural proteins, either supplied by the customer or produced by Primm (as His-tag fusion proteins). Synthetic peptides are not ideal because of they are too small and there is low possibility to generate a large number of monospecific clones Anyway in case of synthetic peptides, they are previously conjugated with a carrier protein which in general is either Ovalbumin or KLH. The conjugation of peptides with large proteins is necessary in order to render the peptides immunogenic enough to provoke the desired immunological response which leads to the generation of specific antibodies.

What about the antigen send by the customer?
We recommend sending 2-3 mg of the protein antigen (useful for immunization and screening test). We recommend 0.4 mg/ml as a minimum protein concentration and welcome higher concentrations. Please ship the antigen in a Styrofoam box with dry ice via courier. 

What will be delivered to the customer?
Primm delivers 2 or more hybridoma cell lines – single clones (selected by the customer), each able to produce a single MAb The cell lines are permanent sources. Upon cell cultivation they produce Abs in the culture supernatant. They are stable cell lines and, if requested, some aliquots can be stored at our facility. 

Does Primm offer production and purification of MAbs?
Yes, Primm offer production both in cell culture (in vitro) and by liquid ascetic production. If requested, milligrams of monoclonals will be purified by Immunoaffinity chromatography (IgG and IgM). We will deliver quantity as requested by the customer.

How does Primm test the quality and quantity of the purified monoclonal antibodies?
Primm quantify the purified monoclonals by UV detection. To verify the quality we test the clone productivity with ELISA against the antigen.

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