Production Of Specific Monoclonal Antibodies Research Articles

The design of new vaccines

The prospects for vaccine development have been transformed by the advent of hybridoma technology for the production of specific monoclonal antibodies and recombinant DNA technology which permits the in vitro expression of genes coding for protective antigens of pathogenic organisms. These techniques are leading to the refinement of existing vaccines and the production of new vaccines against organisms which either cannot be cultured in vitro at all, e.g. hepatitis-B, or can be cultured only on a limited scale, e.g. plasmodial species. The techniques allow for the novel production of both live and dead vaccines. New live vaccines are produced using either site-directed mutagenesis or insertion of genes into hosts such as vaccinia virus, which is a potential carrier of polyvalent vaccines. New subunit vaccines are produced by expression of appropriate genes in E. coli, yeast cells or mammalian cell lines and some, e.g. hepatitis-B surface antigen (HBsAg) are at an advanced stage of development. Recombinant subunit vaccines are also being produced against the sporozoite and blood stages of malaria. Problems, in regard to the development of malaria vaccines, concern the antigenic plasticity of plasmodia and the need to induce both antibody and cell-mediated immunity; nevertheless, the work shows considerable promise of success.

7] The use of conventional antisera in the production of specific monoclonal antibodies

We have shown here that conventionally produced antisera aids in the production of specific monoclonal antibodies. The complex of immunoglobulin plus antigen acted as an effective immunogen in both rats and mice. Stable cell lines producing anit-human alpha 2M monoclonal antibodies to numerous antigenic sites on the alpha 2M molecule have been generated by this method. This method has also been used to produce monoclonal antibodies to human complement components. This procedure of immunizing mice with an immunoprecipitated product derived from conventionally produced antisera is a unique approach in that the antigen is conveniently enriched without tedious and time-consuming biochemical purification. In addition, the use of the immunoprecipitated product in conjunction with the immunoprecipitating antisera allows for rapid screening of the hybridomas in the initial stages when cell growth and maintenance is critical. This method thus further simplifies the task of obtaining a specific monoclonal antibody from a complex mixture. A final consideration is the wide availability of conventionally produced antisera to numerous proteins and other biological substances that could be used in the production of monoclonal antibodies, and consequently these antibodies could be used in more detailed studies of these same molecules.

Antibody production by human × human hybridomas in serum-free medium

Four human × human hybridomas were adapted to growth in serum-free medium consisting of RPMI 1640 supplemented with bovine serum albumin and transferrin (BSA/Tf medium). Production of specific monoclonal antibodies was maintained for more than 2 months. Although the maximal cell density achieved was lower than that in serum-supplemented medium, immunoglobulin production was similar or higher when results were expressed on a per viable cell basis. Thus it is feasible to grow human × human hybridomas in serum-free culture and it is possible that this will become the method of choice for large scale production of human monoclonal antibodies.

Single shot intrasplenic immunization: an advantageous procedure for production of monoclonal antibodies specific for human fibrin fragments.

We describe the use of the single shot intrasplenic immunization technique as a particularly effective procedure for the production of specific monoclonal antibodies against a high molecular weight antigen. We found that with this technique several different, completely specific monoclonal antibodies could be produced against high molecular weight crosslinked fibrin degradation products. These results contrasted with those obtained using conventional multidose immunization, which only produced monoclonal antibodies that were cross-reactive with fibrinogen and/or noncrosslinked fibrin degradation products.

The use of conventional antisera in the production of specific monoclonal antibodies

Monoclonal antibodies specific for human α-2-macroglobulin ( α 2M), a plasma glycoprotein, have been produced using a novel immunization method. Commercially available antisera to human α 2M was used to precipitate the antigen from whole serum. Immunization of animals with this immunoprecipitate resulted in the production of hybridomas with a specificity for human α-2-macroglobulin as confirmed by immunoprecipitation.

Production of specific antisera and monoclonal antibodies to choline acetyltransferase: characterization and use for identification of cholinergic neurons.

Choline acetyltransferase (ChAT) has been purified from pig brain to greater than 95% homogeneity (purification factor: 646 000, specific activity of the purified enzyme: 128 mumol acetylcholine formed/min/mg). Gel electrophoresis of the purified enzyme in the presence of sodium dodecylsulphate and beta-mercaptoethanol revealed a single protein band at 68 000 daltons. Immunoprecipitation and double immunodiffusion tests showed that antisera raised against this protein specifically recognize ChAT. A monoclonal antibody prepared against the enzyme specifically binds a protein from crude pig brain supernatants which has a mol. wt. of 68 000 and a specific activity of 153 mumol/min/mg. This antibody shows no species cross-reactivity. The specificity of the immunohistochemical localization of ChAT has been established by comparing the labeling of pig retina using the antiserum with that obtained using the monoclonal antibody. Both probes specifically identify the same retinal structures: labeled cell bodies are found in the inner nuclear layer and the ganglion cell layer, while a double band is stained in the inner plexiform layer. In rat spinal cord, the antiserum labels the motoneurons and the preganglionic sympathetic neurons, located in the intermedio-lateral nucleus, the intercalated region, and the central autonomic area.

Monoclonal antibodies to polioviruses. Production of specific monoclonal antibodies to the Sabin vaccine strains.

Murine lymphocyte hybridomas which produce neutralizing or non-neutralizing monoclonal antibodies to different type 1, 2 or 3 poliovirus strains were isolated. The majority of these monoclonal antibodies reacted with antigenic determinants present on different poliovirus strains of the same type. However, hybridomas producing monoclonal antibodies specific for each of the three Sabin vaccine strains were also generated.