Keeping pigs healthy and productive is a major goal for producers and researchers. Preventing disease, using biosecurity and planned vaccinations, are hallmarks of well-managed swine facilities. In the case of disease outbreaks, diagnostic laboratories use an array of tests to quickly identify underlying infections and causative pathogens; once diagnosed veterinarians can prescribe therapies to treat the infections and propose vaccinations to prevent disease outbreaks in other pigs. All of these disease associated functions require reagents that can quantitate the infection and identify and quantitate swine immune responses, specifically serum antibodies or immunoglobulins (Igs) that affirm pathogen exposure. Additionally most vaccine programs require anti-swine Ig reagents to detect IgG antibody levels that confirm the success of virus eradication programs. In mammals there are numerous IgG subclasses, which have different activities. Currently most investigators rely on polyclonal antisera, prepared typically in goats or rabbits, to identify swine IgGs. However these polyclonal antisera are tedious to prepare, lack immortality, vary between batches, and usually exhibit reactivity for multiple swine IgG subclasses. 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. Our goal was to address swine reagent needs by producing mAb reagents that uniquely identified each IgG subclass for more effective swine immunity quantitation.
Our first objective was to “Identify all immunoglobulin-G (IgG) subclass genes; express Ig proteins for each swine IgG subclass gene.” This was completed using molecular genetic approaches at the Univ. of IA. All IgG subclass cDNAs were cloned and sequenced. These clones were then provided to our collaborator in Belgium to express quantities of each specific IgG protein. This was perfomed using their camelid-porcine Ig expression system. The expressed proteins prepared in Belgium were shipped to the US where they were used to address objectives B “Characterize the reactivity of known anti-swine Ig monoclonal antibody (mAb) reagents with each Ig gene product” and C “Develop new mAbs that are specific for each of the expressed IgG subclass proteins.” For Obj. B previously developed hybridoma cell lines expressing mAb reactive with swine Igs were collected at BARC from labs worldwide. Hybridoma supernatants containing mAb were prepared, purified and sent to Iowa for use in their tests of specific reactivity with the expressed swine IgG subclass. As expected none of the currently available anti-swine IgG mAbs was specific for just one IgG subclass. Thus Obj. C had to be pursued. Mice were separately immunized with each swine IgG subclass protein, as specific camelid-porcine IgG. All hybridoma fusion supernatant mAb produced were tested for their reactivity with swine Igs and the camelid-swine IgG subclass proteins. Several fusions were performed but unfortunately to date no mAb has been developed that reacted against just one swine IgG subclass protein. [It should be noted that as part of a separate funding initiative these immunizations and hybridoma fusions are continuing.] Comparative analyses of ELISA data for every new mAb were performed, and compared to known standards and control IgGs.
Our first objective was to “Identify all immunoglobulin-G (IgG) subclass genes; express Ig proteins for each swine IgG subclass gene.” This was completed using molecular genetic approaches at the Univ. of IA. All IgG subclass cDNAs were cloned and sequenced. These clones were then provided to our collaborator in Belgium to express quantities of each specific IgG protein. This was perfomed using their camelid-porcine Ig expression system. The expressed proteins prepared in Belgium were shipped to the US where they were used to address objectives B “Characterize the reactivity of known anti-swine Ig monoclonal antibody (mAb) reagents with each Ig gene product” and C “Develop new mAbs that are specific for each of the expressed IgG subclass proteins.” For Obj. B previously developed hybridoma cell lines expressing mAb reactive with swine Igs were collected at BARC from labs worldwide. Hybridoma supernatants containing mAb were prepared, purified and sent to Iowa for use in their tests of specific reactivity with the expressed swine IgG subclass. As expected none of the currently available anti-swine IgG mAbs was specific for just one IgG subclass. Thus Obj. C had to be pursued. Mice were separately immunized with each swine IgG subclass protein, as specific camelid-porcine IgG. All hybridoma fusion supernatant mAb produced were tested for their reactivity with swine Igs and the camelid-swine IgG subclass proteins. Several fusions were performed but unfortunately to date no mAb has been developed that reacted against just one swine IgG subclass protein. [It should be noted that as part of a separate funding initiative these immunizations and hybridoma fusions are continuing.] Comparative analyses of ELISA data for every new mAb were performed, and compared to known standards and control IgGs.
The final objective was “Distribute mAbs and develop reference standard sera.” We worked with the USDA Animal and Plant Health Inspection Service (APHIS) National Veterinary Services Laboratories (NVSL) facilities in Ames IA to establish a resource for veterinary reagents; NVSL has made two anti-swine reagents, anti-IgM (M160) and anti- IgA (1459), available to researchers. It is hoped that this will be just the first step in NVSL making a broader panel of immune reagents available to veterinary researchers. The development of the USDA APHIS NVSL resource is a major accomplishment for this grant.
In summary, researchers require IgG subclass specific reagents to determine Ig function; diagnostic laboratories use them to measure Ig levels and specific antibody responses. This NPB project (#06-043 and #05-015) attempted to develop a full panel of well-characterized new mAb reagents that uniquely recognized swine each IgG subclass protein. It affirmed the reactivity of previously produced mAb but was unsuccessful at producing new mAb reagents. Once produced such mAb reagents will refine swine disease diagnostic tests and enable scientists to more accurately compare results among labs, thus opening up new understanding of disease control mechanisms and pathologies, as well as better characterizing swine vaccine responses.
