Development and Characterization of Antibodies Against Avian Influenza: Application in Research and Clinical Diagnosis Influenza (Orthomyxoviridae)Life CycleInfluenza typesVariationWhat is “Bird Flu”Host range of FluWhy InfluenzaCurrent H5N1 statistics Why develop new antibodies?Uses of AntibodiesMethod for producing antibodiesMethod for producing antibodiesAnalysis of clonesWhat is HemagglutinationHemagglutination Inhibition AssayMonoclonal Antibody ResultsNext stepsHow to produce Ab in massConclusions ThanksDr. Manoj PasteyDepartment of Biomedical Sciences Development and Characterization of Antibodies Against Avian Influenza: Application in Research and Clinical DiagnosisInfluenza (Orthomyxoviridae){ Enveloped{ Negative strand RNA genome (8 segments){ Neuraminidase{ Hemagglutinin{ PB1, PB2, PA{ M(1-2), NS(1-2), NPLife Cycle{ Entry{ Nuclear import{ Replication{ Releasehttp://www.learner.org/channel/courses/biology/archive/animations/hires/a_infect8_h.htmlInfluenza types{ Typesz A,B,C{ Sub-types (Influenza A)z 16 HA and 9 NA types{ Strainsz Each type or sub-type may have several forms that vary slightly in sequence and structureVariation{ Antigenic Driftz Due to point mutations in HA or NAz Seasonal flu{ Antigenic Shiftz Due to reassortment of HA or NAz Cells infected with more than one strainSegments of nucleic acid can beexchanged, resulting in a novelpathogenANTIGENIC SHIFTNew Variant VirusWhat is “Bird Flu”{ Birds are the natural host of all flu viruses.{ Occasionally, a zoonosis occurs (virus crosses a species barrier){ Recently, “Bird Flu” refers specifically to H5N1, sometimes called HPAIHost range of FluWhy Influenza{ Influenza kills{ 250,000 to 500,000 die each year (WHO, 2003).{ Frequent epidemics and pandemicsCurrent H5N1 statistics{ Total humans:230/132{ 2006:86/56{ 153 Captive large cats{ House cats{ Hundreds of Millions of poultryWhy develop new antibodies?{ New virus subtypes.{ Few monoclonal antibodies (mAbs) exist for HPAI.{ Urgent need for new and better mAbs. { Enhanced detection in domestic and wild birds.{ Timely detection of interspecies transmission.Uses of Antibodies{ Research potential z Antigenic analysisz Serological diagnosisz Epidemiological surveysz Evaluation of AIV vaccine or antiviral drugs { Types of assaysz Western blotz ELISAMethod for producing antibodies{ Three Mice groupsz Five mice with H5N1 virusz Five mice with H7N2 virusz Five control mice{ Injections at 2-week intervals{ Final boost on day 59{ Serum titers were measured using ELISA on days 32 and 47.Method for producing antibodiesAnalysis of clones{ Tested supernatant for reactivity using ELISA{ Concentrated supernatants using VIVASPIN ultra filtration column.{ Retested using ELISA{ Tested Ab for ability to inhibit hemagglutinationWhat is HemagglutinationHemagglutination Inhibition Assay{ Place 25uL of 4HA virus in wells of a 96 well round bottom plate.{ Place same volume of antibody in wells (n=2).{ Mix and incubate.{ Add 50uL chicken RBCs to each well.{ Mix and incubate.Monoclonal Antibody ResultsH5N1 Ab H7N2 AbH5N1 no Ab Control H7N2 no Ab ControlNext steps{ Characterization using Western Blot{ Producing Ab at higher titer{ Test efficacyHow to produce Ab in mass{ Grow cells in culture and concentrate supernatant{ OR{ Inject clone cell line into mice and allow tumor to develop and collect ascites.Conclusions{ Vaccination of mice was successful in producing antibodies against H5N1 and H7N2 viruses.{ Antibodies produced are reactive with viruses in ELISA assays.{ Develop rapid virus detection tests.Thanks{ Christopher Lupfer{ Adam Klomp - Michigan State University, CVM{ Veterinary Diagnostic Laboratoryz Rockey Bakerz Wendy Blackz Donna Mulrooney{ Merck-Merial and Oregon State University, College of Veterinary Medicine for funding contributions{ USDA for grant to continue