#05-015

Complete

Date Full Report Received

05/08/2007

Date Abstract Report Received

07/18/2008

Investigation

Institution: , ,
Primary Investigator:

Swine disease and vaccine research will be advanced by the development of sophisticated tools to measure swine antibodies or immunoglobulins (Igs). Swine produce antibodies, or Igs, in response to infection or vaccination. But not all Igs are equal. Scientists measure infectious disease exposure and vaccine efficacy by quantitating Ig levels in serum or mucosal secretions. We know for porcine reproductive and respiratory syndrome virus (PRRSV) infections that there is a well-characterized antibody response as measured by the IDEXX ELISA. However, the more relevant test is whether infected or vaccinated pigs produce neutralizing Igs against the virus. These Igs may be a specific class of Igs or a subclass of IgGs. Neutralizing Igs are known to take longer to develop but are essential for recovery from PRRSV infection.
Our NPB project’s goal was to expand the immune toolkit for pigs, by developing and characterizing reagents that identify and quantify the major classes of swine Igs, IgA and IgM, and the subclasses of IgGs. This grant was aimed at developing a broader panel of anti-swine Ig reagents to determine exactly which Ig classes, in particular IgG subclasses, are critical for vaccine and disease responses. Reseachers require such reagents to determine Ig function; diagnostic laboratories use them to measure Ig levels. Currently most investigators rely on polyclonal antisera to identify the swine Ig subclasses. These are tedious to prepare, are rarely class specific, lack immortality, and vary between batches. Thus we proposed to produce mouse monoclonal antibody (mAb) reagents made by hybridoma technology. MAbs recognize only one epitope, thus the name monoclonal. It is generally stated that a mAb can “recognize a needle in a haystack” whereas a polyclonal recognizes “the needle and the haystack”. Furthermore, mAb provide a sustainable and renewable resource, so data from laboratories around the world can be compared.
For this project we started by first using molecular techniques to express the 5 known swine IgG genes. [In the process we actually found cDNA evidence for several new swine IgG genes; these are being further characterized and will be used for our renewal grant’s expression work.] We worked with a collaborator, Dr. Serge Muyldermans, in Belgium to express each of the 5 previously known swine IgG subclass cDNAs as swine-camelid IgG proteins in vitro using his novel camelid-swine Ig expression system. With this system he expressed each of the 5 specific swine IgG heavy chain genes as camelid-swine Ig constructs. Once developed and purified these constructs were shipped to BARC. Portions of these constructs were shipped to Univ. IA and are now being used to characterize the IgG binding specificity of known mAb. In our next grant we will use them to immunize mice to prepare new IgG subclass specific mAb.
Our overall goal is to have a full panel of well-characterized mAb that react specifically with each swine Ig isotype and IgG subclass so scientists will be able to compare accurately the functions of each swine Ig isotype and subclass. For our second objective, we characterized the reactivity of the currently available mAb anti-swine IgA and IgM as well as anti-IgGs. We identified mAb with excellent specificity for swine IgA and IgM and have worked with USDA APHIS to start to develop a repository for such hybridoma lines and mAb reagents. The anti-IgG mAb are being tested now on the HCAbs, as noted above. Overall, we expect that these reagents will help to expand our understanding of disease control mechanisms and pathologies, as well as serve as improved tools for characterizing swine vaccine responses.
Contact information: Joan Lunney, BARC, ARS, USDA, Bldg.1040, Beltsville, MD 20705; Phone: 301-504-9368, email: Joan.Lunney@ars.usda.gov; John Butler, University of Iowa, Dept. of Microbiology, Iowa City, IA 52240, Phone: 319-335-7776, email: john-butler@uiowa.edu.