Lecture 3 - Immunoassays

Lecture 3 - Immunoassays - IMMUNOASSAYS Edwin Oaks, Ph.D....

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EVO IMMUNOASSAYS Edwin Oaks, Ph.D. George Mason University Fall 2006
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EVO Immunoassays ELISA (enzyme linked immunosorbant assay) Western blot (already covered in lecture 2) ASC (antibody secreting cells) Neutralization assays CTL (cytotoxic lymphocytes) Proliferation assays
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EVO Immunoassay Controls Negative controls Non-immune serum or cells From subject not exposed to antigen or pathogen Naïve subject Normal Negative Positive controls are obtained from subject that has been infected with the pathogen or immunized with the antigen in question Serum Contain antibodies to antigen used in assay Cells Immune cells - react with target cells
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EVO ELISA Measures reaction between antibodies and antigens using an enzyme as a reporter molecule Antigen Protein Lipopolysaccharide Polysaccharide Whole bacteria or bacterial extracts Viruses Antigen must be bound to support matrix Matrix is usually plastic (polystyrene or polyvinylchloride)
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EVO ELISA Antigen is coated onto plastic well of a 96-well plate Open space on matrix is “blocked” With protein filler (red line)
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EVO ELISA Incubate with antibody (primary antibody) wash Incubate with antibody-enzyme conjugate that reacts with primary antibody wash
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EVO ELISA Incubate with substrate and measure color change or Fluorescence Use spectrophotometer substrate Amount of color is proportional To amount of primary antibody
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EVO ELISA Titer: the last dilution of antiserum that gives a positive reaction Compare pre-immunization or pre-infection titers with post- immunization of post-infection titers
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Lecture 3 - Immunoassays - IMMUNOASSAYS Edwin Oaks, Ph.D....

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