Chapter 17 Topics Phenotypic Genotypic Immunological Method

Phenotyic Methods Phenotype – appearance of a gene (expression of a genotype) Subtypes: Microscopic observations (gram stain) Macroscopic observations (colonies) Physiological/Biochemical (SIM, carbohydrate fermentation) Chemical Composition (chemical analysis of cell wall or the cell structures)

Genotypic Methods Genotype – the combination of two alleles (one from mother, one from father) that creates a trait. (With microbes, analysis of the Genes) Polymerase Chain Reaction (PCR) Fast and more precise Restriction Fragment Length Polymorphism (RFLP) Long but more specific G + C composition Percentage of Guanine and Cytosine

Immunological Methods Basic principle Agglutination Precipitation Western blot Complement fixation Immunofluorescence Common assays

Basic principle Serology In vitro method Antibodies have specificity for antigens Detect Identify Quantify Sensitive

Serological identification of disease and serotyping are two methods that can be used in serology testing. Fig. 17.8 Basic principles of serological testing using antibodies and antigens.

Serological tests are specific and sensitive. Fig. 17.9 Specificity and sensitivity in immune testing

Agglutination Antigens are whole cells (eg. RBC or bacteria) Antibodies cross-link the antigens Determine serum titer Types of tests Rapid plasma test (syphilis) Cold agglutinin test (mycoplasma) Weil-Felix test (rickettsial) Latex agglutination test (pregnancy)

Agglutination reactions involve whole cell antigens, while precipitation reactions involve soluble antigens. Fig. 17.10 Cellular/molecular view of agglutination and Precipitation reactions that produce visible Ag-Ab complexes.

Precipitation Soluble antigens Binding of antibody to antigen, makes the complex insoluble or visible Types of test Tube precipitation Ouchterlony (double diffusion) Immunoelectrophoresis

An example of the tube precipitation test and the Ouchterlony method. Fig. 17.11 Precipitation reactions.

Immunoelectrophoresis enables the identification of specific antibody classes that bind antigen from a serum sample. Fig. 17.12 Immunoelectrophoresis of normal human serum.

Western blot Electrophoretic separation of protein (antigen) sample Specific antibodies bind to protein sample Specific and sensitive Counterpart of the Southern blot (DNA test)

An example of human serum being tested for antibodies against HIV antigens using the Western blot method. Fig. 17.13 The Western blot procedure.

Complement fixation Antibody (lysin), antigen, complement, and sensitized sheep rbc are required Complement is fixed to a Ab-Ag-complex Fixed complement cannot participate in rbc lysis = positive reaction or identification

An example of the complement fixation test. Fig. 17.14 Complement fixation test.

Immunofluorescence A monoclonal antibody labeled with a fluorescent dye Direct testing Indirect testing

An example of direct and indirect immunofluorescence testing. Fig. 17.15 Immunofluorescence testing

Common assays Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA Horseradish peroxidase or alkaline phosphatase Enzyme releases dye (chromogen) Types of test Indirect Capture or Sandwich

An example of the indirect and capture ELISA methods. Fig. 17.16 Methods of ELISA testing.

Different methods commonly used to detect and identify viral infections. Fig. 17.18 Summary of methods used to diagnose Viral infections.