Production Of Diagnostic Reagents Research Articles

Corning HYPERFlask® for viral amplification and production of diagnostic reagents

Viral preparations are essential components in diagnostic research and development. The production of large quantities of virus traditionally is done by infecting numerous tissue culture flasks or roller bottles, which require large incubators and/or roller bottle racks. The Corning HYPERFlask® is a multilayer flask that uses a gas permeable film to provide gas exchange between the cells and culture medium and the atmospheric environment. This study evaluated the suitability of the HYPERFlask for production of Lassa, Ebola, Bundibugyo, Reston, and Marburg viruses and compared it to more traditional methods using tissue culture flasks and roller bottles. The HYPERFlask produced cultures were equivalent in virus titer and indistinguishable in immunodiagnostic assays. The use of the Corning HYPERFlask for viral production is a viable alternative to traditional tissue culture flasks and roller bottles. HYPERFlasks allow for large volumes of virus to be produced in a small space without specialized equipment.

The autoimmune diseases manifested by production of autoantibodies: the autoantigens identified by random peptide library.

Phage-displayed random peptide libraries (RPL) provide a powerful technique for identification, structural and functional analysis of ligands for many different target molecules, including, antibodies, receptors or other proteins. This strategy has been verified to be an effective tool for research in immunology and successfully has been used to determine the target sequence for monoclonal and polyclonal antibodies. The peptide library approach provides great promise for characterization of ligands with no prior information concerning antibody specificity. This would allow the recognition of candidate antigens involved in initiation or perpetuation of autoimmune diseases. This technology also offers the potential for new therapeutic opportunities, production of diagnostic reagents, or even development of effective new vaccines. This review focuses on studies regarding the identification of autoantigens recognized by antibodies in autoimmune diseases using phage-display peptide libraries.

De-glycosylation of Pichia pastoris-produced Schistosoma mansoni cathepsin B eliminates non-specific reactivity with IgG in normal human serum

Production of diagnostic reagents in the yeast Pichia pastoris is particularly attractive since this organism is capable of expressing complex eukaryotic proteins in their correctly folded form and is amenable to large-scale fermentation at low cost. The potential of Schistosoma mansoni cathepsin B as a diagnostic antigen for human schistosomiasis has been previously established using both native and E. coli-derived recombinant proteins. However, when produced in P. pastoris we found that recombinant wild-type cathepsin B was preferentially secreted as a heterogeneously glycosylated molecule that migrated at 39 kDa, 41 kDa and a smear of > 50 kDa on SDS-PAGE, and was susceptible to treatment with Endo H and PGNase F. The addition of yeast sugars to the cathepsin B caused it to react with IgG in the serum of both normal (non-infected) and schistosome-infected humans in immunoblotting and enzyme linked immunosorbent assays (ELISA). To avoid this non-specific reactivity, a non-glycosylated mutant form of cathepsin B, engineered by disrupting its potential glycosylation site, was produced. The non-glycosylated recombinant cathepsin B migrated as a single band of 39 kDa on SDS-PAGE. Most importantly, the molecule was not reactive with IgG in normal sera and, hence, could be employed in immunoblots or ELISA to specifically detect antibodies in schistosome-infected patients. Addition of oligosaccharides by P. pastoris is a potential drawback that needs to be considered before using P. pastoris-produced proteins as diagnostic reagents.

The award of medals by the President, Sir George Porter, at the Anniversary Meeting, 30 November 1989

The Copley Medal is awarded to Dr C. Milstein, F. R. S., in recognition of his outstanding contributions to immunology, in particular to the discovery of monoclonal antibodies and to the understanding of the role of somatic mutations in the maturation of the immune response. The thrust of Dr Milstein’s work has been to understand, at a molecular level, how the diversity of antibodies arises. With G. Brownlee he first proved that the variable and constant regions of antibody light chains are encoded in one messenger RNA molecule. His studies with G. Kohler on certain cell hybrids led to the immortalization of antibody secreting cells, and hence monoclonal antibodies, for which discovery they shared the Nobel Prize for Physiology and Medicine. He has applied these methods to the production of diagnostic reagents and the isolation of cell surface antigens, and has extended the technology to the production of bispecific antibodies, the first example of antibody engineering. Most recently he has developed an approach to studying steps in the antibody response, and has demonstrated the critical role of hypermutation in the maturation process. In all his work, Dr Milstein has shown an unusual gift in making major discoveries by unconventional routes, a gift that he continues to apply in his current work in immunology.

The award of medals by the President, Sir George Porter, at the Anniversary Meeting, 30 November 1989

The Copley Medal is awarded to Dr C. Milstein, F. R. S., in recognition of his outstanding contributions to immunology, in particular to the discovery of monoclonal antibodies and to the understanding of the role of somatic mutations in the maturation of the immune response. The thrust of Dr Milstein’s work has been to understand, at a molecular level, how the diversity of antibodies arises. With G. Brownlee he first proved that then variable and constant regions of antibody light chains are encoded in one messenger RNA molecule. His studies with G. Kohler on certain cell hybrids led to the immortalization of antibody secreting cells, and hence monoclonal antibodies, for which discovery they shared the Nobel Prize for Physiology and Medicine. He has applied these methods to the production of diagnostic reagents and the isolation of cell surface antigens, and has extended the technology to the production of bispecific antibodies, the first example of antibody engineering. Most recently he has developed an approach to studying steps in the antibody response, and has demonstrated the critical role of hypermutation in the maturation process. In all his work, Dr Milstein has shown an unusual gift in making major discoveries by unconventional routes, a gift that he continues to apply in his current work in immunology.