Q: Which are the characteristics of our service?
A: 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), 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”.
Q: Which are the specifications of the monoclonal antibodies?
A: 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. The best 10 mother clones will be taken for the subcloning phase, by limiting dilution of positive clones and production of purified antibodies (mgs) in “cell culture”.
Q: Is Primm ready to modify the protocol if required by the customer or if necessary for the success or improvement of the results?
A: 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. Variations are not granted for the following phases.
Q: How does Primm test the quality of the monoclonal antibodies?
A: 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 use of pre-immune sera allows to establish the specificity 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.
Q: What type of antigen does Primm use?
A: Primm normally makes monoclonal antibodies against recombinant or natural proteins and synthetic peptides. In case of synthetic peptides, the peptide is previously conjugated with a carrier protein which in general is either Ovalbumin or KLH.
The synthetic peptides can be made directly at Primm laboratories or may be given by the customer. Also recombinant proteins can be made at Primm labs.
The conjugation of peptides with large proteins such as Ovalbumin or KLH is necessary in order to render the peptides immunogenic enough to provoke the desired immunological response which leads to the generation of specific antibodies.
Of course, in the event that the antigen is already a protein, there is no need of conjugation to carriers.
Q: What about the possible antigens provided by the customers? Is there any limitation?
A: As general principle, Primm can start the immunization scheme with any antigen provided by the customer, knowing, however, that the resulting antibodies will be consequence of the antigen used. For instance, if the antigen is a mixture of different proteins, very likely we will generate different monoclonal antibodies against the different proteins present in the mixture.
Q: For peptide antigens, can Primm help the customer with the choice of the peptide sequence?
A: Yes. Primm will highlight the most antigenic regions of a protein thus allowing us to choose the most immunogenic peptides. This help is provided at no additional charge and it is based on a computer analysis of the protein sequence using algorithms that take into consideration the hydrophobicity and the knowledge of the tridimensional conformation of the different amino acid residues.
Q: What about the possibility that antibodies generated against synthetic peptides will be able to efficiently recognize the intact protein?
A: At a frequency of 10-15%, it may happen that antibodies generated through the use of synthetic peptides are indeed able to recognize the synthetic peptide used for immunization but they do not recognize the intact protein from which the peptide sequence was derived. This could be observed particularly in experiments of immunoprecipitation or immunohistochemistry.