Resolution of an immunodiagnostic dilemma: Heavy chain chimeric antibodies for species in which plasmocytomas are unknown

Authors: BUTLER, JOHN - University Of Iowa; WERTZ, NANCY - University Of Iowa; SUN, X - University Of Iowa; Lunney, Joan; MUYDERMANNS, SERGE - Free University Of Brussels

Submitted to: Molecular Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/21/2012
Publication Date: 8/21/2013
Citation: Butler, J.E., Wertz, N., Sun, X.Z., Lunney, J.K., Muydermanns, S. 2013. Resolution of an immunodiagnostic dilemma: Heavy chain chimeric antibodies for species in which plasmocytomas are unknown. Molecular Immunology. 53:140-148.

Interpretive Summary: A major goal of our research is the development of new vaccines and infectious disease treatments to improve pig health and well-being and monitor responses and recovery. To achieve this goal requires an understanding of the basic immune system and tools to measure responses. Unfortunately we have limited reagents available to characterize the cells and immune proteins, e.g., cytokines and antibodies, involved in controlling normal development as well as responses to various pathogens or vaccines. This paper presents our strategy for preparing monoclonal antibodies (mAb) to swine immunoglobulin-G (IgG) subclass antibodie. It uses a novel Ig heavy chain chimeric antibody technology based on camelid Ig heavy chains that are unique in that they do not require light chains for expression or binding activity. Specifically, it involves the in vitro synthesis of chimeric porcine-camelid heavy chain antibodies (HCAbs) using a single mammalian transfection vector. The expressed chimeric HCAbs are comprised of the camelid antigen binding (VHH) domain encoding specificity for lysozyme and the constant hinge regions (CH2 and CH3) domains of the different porcine IgGs. constructs have been expressed for most of the 11 swine IgG heavy chain genes. Each HCAb has the binding region that recognizes lysozyme and can be used for purifying each expressed HCAb. The purified HCAbs are then used as immunogens for developing and screening mAb reactive with the different swine IgG heavy chains. These immunizations are underway. Characterization of the specificity of previously produced mAb was probed using the panel of expressed HCAbs. The engineered binding region also assures specificity so that as the specificity of anti-swine IgG antibodies can be tested on each HCAb directly or immobilized in native configuration as it binds lysozyme. In summary, these HCAbs are now available for use in hybridoma production and mAb selection as well as for use as reference standards for evaluating the specificity of currently available anti-swine IgG antibodies.

Technical Abstract: The immunoglobulin (Ig) genes of many vertebrates have been characterized but IgG subclasses, IgD and IgE proteins are only available for three species in which plasmacytomas occur. This creates a major problem in the production and specificity verification of diagnostic anti-Ig reagents for the vast majority of mammals. We describe a novel solution using the swine system with its eleven different variants of IgG. It involves the in vitro synthesis of chimeric porcine-camelid heavy chain antibodies (HCAbs) that do not require light chains and therefore only a single transfection vector. The expressed chimeric HCAbs are comprised of the camelid VHH domain encoding specificity for lysozyme and the hinge, CH2 and CH3 domains of the various porcine IgGs. These HCAb retain their antigenic integrity and their ability to recognize lysozyme. The engineered specificity assures that these HCAb can be immobilized in native configuration when used for testing the specificity of anti-swine IgG antibodies. Comparative data to illustrate the importance of this point are provided. These are now available for use in hybridoma selection and as reference standards for evaluating the specificity of currently available anti-swine IgG antibodies.