Final Report Summary - CAPRI (Establishment of high-throughput monoclonal antibody production and hybridoma bank)
Over the last 20 years, monoclonal antibodies (mAbs) against several thousand proteins have become commercially available. When considering the number of proteins predicted in the human and other genomes, this number is still far from meeting the current demand to perform comprehensive proteome analyses of biological systems and it therefore represents a bottleneck to further development of proteome research. In view of general needs in the area of biomedical research, a high throughput development and production of mAbs is considered one of the most promising approaches to build up a research and biotechnology unit. This was a strong argument for the establishment of the Centre for Monoclonal Antibody Production at the University of Rijeka.
The major objective of this project was to support the development of the Centre for Antibody Production Rijeka as a core of the Centre for Proteomic into a centre of excellence capable of developing mAbs to selected sets of proteins in a high-throughput manner, and of establishing a hybridoma and mAbs bank.
For the vast majority of varicella-zoster virus (VZV) proteins, the CAPRI team managed to generate sufficient quantity and purity for immunisation and screening. For each of them fusions were performed in order to generate hybridoma cell lines which were subsequently cloned. After mAb generation, we proceeded with the screening of specific mAbs using different techniques. The basic screening technique was ELISA, which was followed by Western blot (WB) on the protein produced in E. coli and used for the immunisation. In parallel, confocal microscopy (IF) and WB was performed on the cells infected with VZV.
We noticed that significant portion of mAbs, although positive against recombinant fusion protein, failed to react to their natural antigens from VZV infected cells. For that reason, it was necessary to establish eukaryotic expression system in order to preserve glycosylation of recombinant protein and provide more likely conditions to generate mAbs against epitopes expressed on proteins in infected cells, and to generate mAbs that could be useful in a variety of techniques.
We applied two different strategies: the first one was the fragmentation of the original open reading frames (ORFs) into smaller pieces thus avoiding potential hydrophobic and transmembrane regions, and the second approach was the implementation of the baculovirus expression system. For some of the most difficult targets we have also generated peptides for the immunisation.
On October 2006, a three-day meeting which was devised as a summer school was held. In addition to the CAPRI staff, several other experts in this field have been invited, including Dr Elisabeth Krammer and Dr Marius Ueffing, both from GSF-Institute for Molecular Immunology, and Dr Thomas Joos, NMI (Natural and Medical Science Institute), University of Tubingen, Germany.
Another formal meeting took place in March 2007, when CAPRI and Prof. Juergen Haas hosted several Israeli scientists to discuss potential collaboration on the monoclonal antibody production not only to viral proteins, but also to cellular antigens. We presented it as a summer school 'Innate Immunity to viral infection' and it was dedicated to use the CAPRI's expertise to generate new reagents: monoclonal antibodies against innate immunity receptors as well as viral antigens against antibodies.
One scientist from CAPRI spent one week at the monoclonal antibody facility at the GSF in Munich under the supervision of Dr Elisabeth Kremmer in order to adopt some techniques and protocols important for the technology of antibody production. Our partner Juergen Haas at the MvP took over the education of one scientist and one laboratory engineer who spent six weeks working in his group in order to transfer the technology of GATEWAY cloning and protein expression and purification. One scientist from MEFMO spent six months at CAPRI learning basic techniques.
A database / laboratory management software 'webLAB' was designed, which provides detailed information about proteins, hybridomas, mAbs, and is being updated on regular basis. This information includes clone names, isotypes, growing media, storage and other details. It is fully in function and all the data regarding the production, purification, storage of hybridoma and antibodies, as well as the quality control information are already in the system, except for those that are under production.
The major objective of this project was to support the development of the Centre for Antibody Production Rijeka as a core of the Centre for Proteomic into a centre of excellence capable of developing mAbs to selected sets of proteins in a high-throughput manner, and of establishing a hybridoma and mAbs bank.
For the vast majority of varicella-zoster virus (VZV) proteins, the CAPRI team managed to generate sufficient quantity and purity for immunisation and screening. For each of them fusions were performed in order to generate hybridoma cell lines which were subsequently cloned. After mAb generation, we proceeded with the screening of specific mAbs using different techniques. The basic screening technique was ELISA, which was followed by Western blot (WB) on the protein produced in E. coli and used for the immunisation. In parallel, confocal microscopy (IF) and WB was performed on the cells infected with VZV.
We noticed that significant portion of mAbs, although positive against recombinant fusion protein, failed to react to their natural antigens from VZV infected cells. For that reason, it was necessary to establish eukaryotic expression system in order to preserve glycosylation of recombinant protein and provide more likely conditions to generate mAbs against epitopes expressed on proteins in infected cells, and to generate mAbs that could be useful in a variety of techniques.
We applied two different strategies: the first one was the fragmentation of the original open reading frames (ORFs) into smaller pieces thus avoiding potential hydrophobic and transmembrane regions, and the second approach was the implementation of the baculovirus expression system. For some of the most difficult targets we have also generated peptides for the immunisation.
On October 2006, a three-day meeting which was devised as a summer school was held. In addition to the CAPRI staff, several other experts in this field have been invited, including Dr Elisabeth Krammer and Dr Marius Ueffing, both from GSF-Institute for Molecular Immunology, and Dr Thomas Joos, NMI (Natural and Medical Science Institute), University of Tubingen, Germany.
Another formal meeting took place in March 2007, when CAPRI and Prof. Juergen Haas hosted several Israeli scientists to discuss potential collaboration on the monoclonal antibody production not only to viral proteins, but also to cellular antigens. We presented it as a summer school 'Innate Immunity to viral infection' and it was dedicated to use the CAPRI's expertise to generate new reagents: monoclonal antibodies against innate immunity receptors as well as viral antigens against antibodies.
One scientist from CAPRI spent one week at the monoclonal antibody facility at the GSF in Munich under the supervision of Dr Elisabeth Kremmer in order to adopt some techniques and protocols important for the technology of antibody production. Our partner Juergen Haas at the MvP took over the education of one scientist and one laboratory engineer who spent six weeks working in his group in order to transfer the technology of GATEWAY cloning and protein expression and purification. One scientist from MEFMO spent six months at CAPRI learning basic techniques.
A database / laboratory management software 'webLAB' was designed, which provides detailed information about proteins, hybridomas, mAbs, and is being updated on regular basis. This information includes clone names, isotypes, growing media, storage and other details. It is fully in function and all the data regarding the production, purification, storage of hybridoma and antibodies, as well as the quality control information are already in the system, except for those that are under production.