Sanja Selak of Intercell AG, Vienna, Austria, presents at the ProImmune Antigen Characterization and Biomarker Discovery Summit, January 2011.
Intercell develops vaccines for the prevention and treatment of infectious diseases
8. Principle of candidate antigen and epitope selection by ELISA using human sera Patients (group A) Patients (group B) Healthy individuals Peptide screening with human Abs (ELISA) Gene accession number: ORF# X ORF X -01 ORF X -02 ORF X -03 ORF X -04 ORF X -05 ORF X -06 ORF X -07 ORF X -08 An example of the “candidate antigen” selected by genomic library screening Epitope – the antibody binding site on an antigen (5-30 aa)
23. OPK assay using human granulocytes (HL60) Opsonization (40min, RT) Human HL60 cell line differentiated into granulocytes Incubation 20min at 37C = uptake of bacteria Continue incubation for additional time points at 37C to determine intracellular killing rate (30min, 1hr, 2hr and 3hr) Lysis after 20min incubation with HL60: plated out released bacteria to determine the uptake Lysis and plating of the released bacteria STEP 1: STEP 2: S.pyogenes + + Complement (4% baby rabbit serum) ASSAY SETUP WITH RABBIT COMPLEMENT Antibiotic killing 40min at 4C ( 2% or 20 µg/ml Pen/Streptomycine) Antibodies
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25. OPK assay using mouse macrophages TESTING UPTAKE AND KILLING KINETICS PAGE Non-specific (pre-existing) Ab S.pyogenes-specific Ab S.pyogenes + Mouse antibodies OPSONIZATION INCUBATION 1hr , 37C =uptake of bacteria Continued incubation for additional 6hr at 37C to determine intracellular killing Lysis and plating released bacteria. UPTAKE: KILLING: Antibiotic killing of extracellular bacteria 40min at 4C (Pen/Streptomycine) Lysis after 1hr incubation with MFs: plating out released bacteria to determine the uptake.
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27. OPK assay using mouse macrophages TESTING EFFECT OF HUMAN PLASMA ON UPTAKE AND KILLING OF S. PYOGENES PAGE Non-specific (pre-existing) Ab S.pyogenes-specific Ab Pre-incubation with human plasma Without human plasma + Mouse antibodies OPSONIZATION *binding of both specific and non-specific Abs S.pyogenes Non-immune human plasma + Y Y Bacteria coated with human plasma proteins OPSONIZATION Mouse Abs bound in the correct orientation S.pyogenes + Mouse antibodies UPTAKE KILLING