Immunochemical_Methods

Immunochemical_Methods - Immunochemical Methods in the...

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Unformatted text preview: Immunochemical Methods in the Clinical Laboratory Roger L. Bertholf, Ph.D., DABCC Chief of Clinical Chemistry & Toxicology, UFHSC/Jacksonville Associate Professor of Pathology, University of Florida College of Medicine ASCP/Bertholf Name The Antigen Early theories of antibody formation Paul Ehrlich (1854-1915) proposed that antigen combined with pre-existing side-chains on cell surfaces. Ehrlichs theory was the basis for the genetic theory of antibody specificity. The Template theory of antibody formation Karl Landsteiner (1868-1943) was most famous for his discovery of the A/B/O blood groups and the Rh factor. Established that antigenic specificity was based on recognition of specific molecular structures; he called these haptens; formed the basis for the template theory of antibody formation. Aminobenzene Sulphonate, a Hapten NH 2 NH 2 NH 2 SO 3 SO 3 SO 3 Ortho Meta Para Classification of immunochemical methods Particle methods Precipitation Immunodiffusion Immunoelectrophoresis Light scattering Nephelometry Turbidimetry Label methods Non-competitive One-site Two-site Competitive Heterogeneous Homogeneous Properties of the antibody-antigen bond Non-covalent Reversible Intermolecular forces Coulombic interactions (hydrogen bonds) Hydrophobic interactions van der Waals (London) forces Clonal variation Antibody affinity Ag Ab Ag Ab + ] ][ [ ] [ Ag Ab Ag Ab K a = Precipitation of antibody/antigen complexes Detection of the antibody/antigen complex depends on precipitation No label is involved Many precipitation methods are qualitative, but there are quantitative applications, too Factors affecting solubility Size Charge Temperature Solvent ionic strength Zone of equivalence The precipitin reaction Precipitate Antibody/Antigen etc. Single radial immunodiffusion Ag Single radial immunodiffusion ] [ Ag r r Double immunodiffusion rjan Ouchterlony Developed double immunodiffusion technique in 1948 Double immunodiffusion (Ouchterlony) Quantitative double immunodiffusion S 1 S 2 S 3 S 4 S 5 P Electroimmunodiffusion Why would we want to combine immunodiffusion with electrophoresis? SPEED Specificity Carl-Bertil Laurell (Lund University, Sweden) Laurell Technique (coagulation factors) Rocket electrophoresis Electroimmunodiffusion +- Immunoelectrophoresis Combines serum protein electrophoresis with immunometric detection Electrophoresis provides separation Immunoprecipitation provides detection Two related applications: Immunoelectrophoresis Immunofixation electrophoresis Immunoelectrophoresis Specimen -human serum +- Immunoelectrophoresis P C P C P C +- Immunofixation electrophoresis SPE IgG IgA IgM Particle methods involving soluble...
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Immunochemical_Methods - Immunochemical Methods in the...

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