US Veterinary Immune Reagent Network Project Directors Cynthia Baldwin University of Massachusetts Amherst Eva Bengten University of Mississippi Medical Center Erin Bromage University of Massachusetts Dartmouth ID: 770087
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US Veterinary Immune Reagent Network Project DirectorsCynthia Baldwin, University of Massachusetts Amherst Eva Bengten, University of Mississippi Medical Center Erin Bromage, University of Massachusetts DartmouthJohn Hansen, WFRC-USGS-Biological Resources Division Joanna LaBresh, Kingfisher Biotech Hyun Lillehoj, USDA-ARS Beltsville Joan Lunney, USDA-ARS Beltsville Bettina Wagner, Cornell University Melanie Wilson, University of Mississippi Medical Center www.vetimm.org Funded by: USDA-CREES 2006-2009; USDA-NIFA 2010-2014
Objective A major obstacle to advances in veterinary immunology and disease control is the lack of sufficient immunological reagents specific for ruminants, swine, poultry, equine and aquaculture species. The goals of the project are to develop sets of reagents to begin to address this dearth.
Targets of US-VIRN Cytokines and chemokines: bioactive recombinant proteins as well as Abs to themAb to cytokines & chemokines: mAb pairs for ELISA, multiplex and ELISpot assays (in some cases this may include polyclonal Abs); single mAb for intracellular staining for cytokinesmAbs to Igs isotypes and IgG subclassesmAb anti-CD molecules and T cell receptors: mAb reactive with native molecules and used to stain viable cells for flow cytometric analyses including cell sorting to purify cells by flow cytometry or magnetic beads; for anti-cytokine receptors mAb that block function and signaling
Kingfisher Biotech Presented by Joanna LaBresh
Kingfisher Biotech 14 SpeciesBovine, Canine, Catfish, Chicken, Dolphin, Equine, Feline, Guinea Pig, Mouse, Ovine, Rabbit, Rat, Swine, TurkeyProduct TypesProteins (>175), Antibodies(>140), ELISA Pairs, (>50) ELISA Sets (25)
Proteins Expressed in yeast because of the advantages of yeastExpressed into mediaProperly foldedPost-translationally modifiedNo endotoxinWe never use tags
Proteins expressed in yeast Note: not all targets in list were requested for all species Those check-marked in Red have ELISAs developed by KingfisherBiotech called VetSetsCytokine/ chemokine Cattle Swine Horse Poultry IL-1β & α √ √ turkey IL-2 √ √ √ IL-4 √ √ √ √ IL-5 √ √ IL-6 √ √ √ √ IL-8 √ √ √turkey IL-10 √√ √ IL-13 √ √ √ In progressIL-15 √ √ √ IL-16√ IL-17A √ √ √IL-17FIn progress√ √IL-18 √ √ IL-21 √ √IL-22 √ √ IFNα √ √ √IFNβ √ √ √ IFNγ √ √ √ √ CCL2 √ √ √ CCL3 √ √ CCL4 √√ √ CCL5 √ √ √ CCL20 √ CCL11 & CXCL9√ √ CXCL10 √ √ √ CXCL11 √ √ TNFα √ √ √ √ (TNF-SF) GM-CSF √ √ Progress US-VIRN target
Example: ELISA kit results
New Products Our pipeline is dynamicWe receive a couple of requests a week. We are happy to hear what products our customers would like to further their research
Catfish Presented by Mel Wilson
For catfish, the highest priority reagents have been and are mAbs that recognize B and T cell subsets and macrophages. Antibodies to inflammatory cytokines have also been requested.Our goal is to prove specificity by FACS, western blotting, and immunoprecipitation with peptide sequencing.Peer review publications: 8Abstracts: 16Oral presentations:17VIRN: Catfish Species Report
Catfish Progress & Planning
Anti-IgD (IgG 1k) and Anti-IgL s (IgMk) mAb Available mAbsAnti-IgM Anti-IgDAnti-IgL k F Anti-IgL sAnti-IgL k GB cell subpopulationsIgM+/IgD+IgM+IgD+
Anti-TCR alpha (IgG 1k) mAbT cell line TS32.15PBL CRP 1419 %20 %PBL CRP 16Anti-TCR alphaAnti-TCR alphaAnti-TCR alpha
Trout Presented by Erin Bromage
IgT CD79 α CD8 TCR β CD4rel CD4 CD80 CD83 IgD CD3e LCK Molecule Full length cDNA Protein produced Mice Immunized Polyclonal sera reactivity mAb Fusion mAb reactivity Assay development Current Position (April 2011) Years 3-4Seeking alternative external funding or collaborationsIL1B-2CXCR4Trout Progress & PlanningIgL3IgL2IgL1secIgMTCRβ C3dIL6IgL4
Anti –IgD mAbs
Anti-CD3e mAbs
Anti-IL1B-2 mAb
Chicken
Molecule Full length cDNAProtein producedMice ImmunizedPolyclonal sera reactivitymAb FusionmAb reactivityAssay developmentIL-2IL-1β IL-6 IL-7 IL-10 IL-12p35 IL-12p40 IL-15 IL-16 IL-17F IL-17A IL-18 IL-21 IL-22 CD80 CD25 CD83 CD86 IL-13 Current Position At the end of Mar 2013 At the end of April 2014 IL-4IL-8Chicken Progress & Planning
Chicken Progress & Planning CCL20CCL4CXCL14 GMCSF IFN- γ LITAF LT MIF TL1A IL-10R- β IL17R IL21R IL1R1 IL-7R VEGF Β -defensin8 Molecule Full length cDNA Protein produced Mice Immunized Polyclonal sera reactivitymAb FusionmAb reactivityAssay developmentIL-5RNK lysinIFN-αTGFB4Current Position At the end of Mar 2013At the end of April 2014Seeking external funding
Reagents Completed through to mAbs 11 mAbs to chicken protein - IL1β, IL2, IL6, IL8, IL15, IL18, CD25, CD80, CD83, CD86, IFN-γTest methodWestern: IL1β, IL2, IL6, IL8, IL15, IL18, CD25, CD80, CD83, CD86, IFN-γFlow cytometry: CD25, CD80, CD83, CD86Tissue staining: CD25, CD80, CD83Neutralizing functional assay: IL1β, IL2, IL6, IL8, IL15, IL18, CD25, CD80, CD83, IFN-γ23
148 98 6450 IL-1β14898 64503622 Western IL-8 148 98 64 50 36 22 IL-6 IFN- γ mAb #1 #2 IL-18 ( kDa) 148 98 64 503616 Flow cytometry CD25 CD83CABFlow cytometric analysis of mAbs to chCD25 (A), CD80 (B) and CD83 (C) Using CHO-chicken cells
Tissue staining with mAbs to CD25, CD80 & CD83 Immunolocalization of chCD25 in tissues. Bursa of Fabricius (A), spleen (B), and intestinal duodenum (C)Expression of chCD80 on LPS-stimulated dendritic cells. Immunolocalization of chCD83 in lymphoid organs. (A) Caecal tonsil, (B) bursa of Fabricius, and (C and D) spleen
Neutralization of IL-18 bioactivity by chIL18 mAbs. Inhibition by mAb to CD25 on IL-2-dependent splenocytes proliferation.Effect of anti-chCD80 mAb on IL-2 driven lymphoblast cell proliferation.Functional assays with mAbs (anti-IL-18, CD25, CD80 & IL-1)26Functional activity of chIL-1β on thymus lymphocyte proliferation and its mAb’s neutralization effects.
Horse
Used both yeast and mammalian-expressed cytokines & chemokines for immunizationCOMPLETED:IL-2IL-4 (mamm)IL-5IL-6IL-10 (mamm)IL-17AIFN- (mamm)CCL2CCL3CCL5CCL11GM-CSFCXCL9 CCL11 CCL5 CCL3 CCL2 IL-15 IL-12 IL-8 IL-6 IL-13 IL-1 IL-5 IL-4 IL-2 Molecule Full length cDNA Protein produced Mice immunized mAb Fusion mAb reactivity Assay developmentCurrent Position (1/2012)Continued during this funding periodCXCL10IL-17AHorse Progress & Planning: Cytokines & ChemokinesExpression vector/TransfectantIL-18TGF-Mabs to yeast protein did not react with native cytokineNo mAbs with E. coli or yeast expressed proteinsYeast protein development/ mAb development not planned (antibodies exist)IFN-IFN-
Completed:CD14 CD23CD25CD16CD40FcRITCRMoleculeFull length cDNAProtein producedMice ImmunizedmAb FusionmAb reactivityflow cytometryProtein purified Current Position (1/2012) Procedure repeated CD16 CD23 Foxp3 IgD CD28 CD25 Fc RI CD40 TLR2 IFNAR TCR TCR TCR CD19 CD34CD105NCR2NCR3Continued Horse Progress & Planning: Cell Surface MoleculesDone by collaborator lab
Characterized and published antibodies (available through Cornell): Anti-equine IL-4 Anti-equine IL-10 Anti-equine IFN- Anti-equine CD14 Anti-equine CD23 Characterized antibodies (available soon): Anti-equine IL-2 Anti-equine CCL2 Anti-equine CCL3 Anti-equine CCL5 Anti-equine L-17A Anti-equine CD25 Anti-equine CD16 http://courses2.cit.cornell.edu/wagnerlab/research/reagents.htm http://www.vetimm.orgCharacterization ongoing: anti-IL-5, anti-CCL11Equine cytokine multiplex assay (IL-4, IL-10, IL-17, IFN-, IFN-) available through the Animal Health Diagnostic Center at Cornell (ahdc.vet.cornell.edu) Monoclonal antibodies to equine cytokines, chemokines and cell surface molecules developed at Cornell
Anti-equine CD14 mAbs (Kabithe et al. 2010, Vet Immunol Immunopathol)Magnetic cell sorting, CD14 mAb 105CD14 mAb 59 (lane 2)Mammalian expressed equine CD14 was used for immunization
Anti-equine CD25 mAbPBMC, 48h, medium CD4 CD25PBMC, 48h, PWM CD4 CD253 mAb clones, tested on PBMC of various horses in 2011, given to two equine labs outside US-VIRN for additional specificity testing who confirmed our resultsMammalian expressed equine CD25 was used for immunizations
Anti-equine IL-17A mAbs Equine PBMC, non-stimulated or stimulated with PMA/ionomycin, stained and analyzed by flow cytometryAnti-equine L-17A mAbs were made using yeast expressed IL-17A
Applications for anti-equine cytokine mAbs produced Anti-IL-17A mAbs have been also tested now and work well for ELISA, Multiplex assay and FACS.
Swine
Expanded Swine cytokine Fluorescent Microsphere Immunoassay (FMIA) (magnetic bead assays)Lab developed FMIA* 6-plex detected IL-1b, IL-4, IL-8, IL-10, IL-12, IFNa; As a result of US Toolkit efforts** have recently added CCL2 to multiplex* Lawson S, Lunney JK, et al. Vaccine 32: 5383-5391, 2010.**Wagner, Lunney et al. in preparation
Increased levels of serum CCL2 were associated with pigs which had higher serum PRRS virus burden Souza, Araujo, Lunney et al. in preparation PP<0.05 repeated measures combined with 0-21 dpi viral load [low vs high]
Cattle
IL-1 βIL-15IL-12p35IL-10IL-8IL-7IFN- IL-2 IL-5 IL-4 IFN- β IFN- α CXCL11 CXCL9 CCL5 CCL2 CXCL10 Molecule Full length cDNA Protein produced Mice immunized mAb Fusion mAb reactivity Assay development Current Position ContinuingWith new funding IL-17AIL-6IL-17FCattle Progress & Planning: Cytokines & ChemokinesExpression vector/TransfectantIL-12p40IL-13TNFαBioactivity determinedCCL11 IL-18Commercially available
Cattle Progress & Planning: Cell Surface Molecules IL-23RMoleculeFull length cDNAProtein producedMice ImmunizedmAb FusionmAb reactivityProtein PurifiedCurrent Position Continuing in current grantWith new funding IL-10R TCR γ-C3 TCRδ –V1 TCR α CCR7 TCR β CD107a CD207 TCR δ -V2 TCR δ -V3 TCR δ-V4TCRγ-C5
Monoclonal antibodies to bovine IL-17F using mammalian-expressed protein – 3 parent clones available
Monoclonal antibodies made to bovine IL-17A using mammalian-expressed protein: Subclones of 6F2
Mouse titers to Yeast (KF)-expressed bovine CCL2
WHERE CAN I GET THESE REAGENTS? GO TO www.vetimm.org FOR EMAIL ADDRESS OF SPECIES COORDINATORSKINGFISHER BIOTECH @ www.kingfisherbiotech.comFrom Cornell University Animal HealthDiagnostic Cntr http://ahdc.vet.cornell.edu/sects/Serol/
http://ahdc.vet.cornell.edu/sects/Serol/ The Animal Health Diagnostic Center is the NY State Veterinary Diagnostic LaboratoryEquine 5-plex cytokine assay