Transcript Chapter 36
36 Clinical Microbiology and Immunology 1 Copyright © McGraw-Hill Global Education Holdings, LLC. Permission required for reproduction or display. The Clinical Microbiology Laboratory • Clinical microbiologist – major function is to isolate and identify microbes from clinical specimens rapidly • Clinical specimen – portion or quantity of human material that is tested, examined, or studied to determine the presence or absence of specific microbes 2 Microbiology Risk Groups 3 Biosafety Levels • Recommended guidelines for additional precautions reflect the laboratory’s biosafety level (BSL) 4 Immunological Techniques • Detection of antigens or antibodies in specimens – especially useful when cultural methods are unavailable or impractical or antimicrobial therapy has been started – serology is the study of blood, namely serum, to identify antibodies and other blood components – often referred to as a titer – a measurement of the antibodies or antigen based on a method that utilizes serial dilution 5 Immunofluorescence • Process in which fluorescent dyes are exposed to UV, violet, or blue light to make them fluoresce • Dyes can be coupled to antibody molecules without changing antibody’s ability to bind a specific antigen • Can be used as direct fluorescent-antibody (FA) technique or indirect fluorescentantibody (IFA) technique assay 6 7 Agglutination • Agglutinates – visible clumps or aggregates of cells or particles • e.g., Widal test – diagnostic for typhoid fever • e.g., latex agglutination tests – pregnancy test – viral hemagglutination • e.g., antibody titer measurements 8 9 10 Complement Fixation • Binding of complement to an antigen-antibody complex • Basis of diagnostic tests that determine if antibodies to an antigen are present in patient’s serum • Very sensitive, measure extremely small amounts of antibody 11 12 Enzyme-Linked Immunosorbent Assay (ELISA) • One of the most widely used serological tests – direct test can be used to detect antigens in a sample – indirect test can be used to detect antibodies in a sample • Reaction visualized by addition of chromogen 13 14 15 Immunoblotting (Western Blotting) • Procedure – proteins separated by electrophoresis • transferred to nitrocellulose sheets – protein bands visualized with enzyme-tagged antibodies • Sample uses include – distinguish microbes – diagnostic tests – prognosis of infections 16 Immunoprecipitation • Detects soluble Ag reacting with Ab (precipitins) • Binding of Ab to Ag forms lattice that precipitates • Lattice formation occurs only in optimal ratio of Ag to Ab 17 Immunodiffusion • Precipitation reaction that occurs in agar gel medium • Two commonly used techniques – single radial immunodiffusion (RID) assay (antibody in agar, antigen diffuses) • quantitates antigen – double diffusion agar assay (Ouchterlony technique) • antibody and antigen both diffuse • identifies antigens 18 Immunoelectrophoresis • Antigens first separated by electrophoresis according to charge • Antigens visualized by precipitation reaction • Has greater resolution than immunodiffusion assays 19 Flow Cytometry • Detects organisms in clinical samples • Detection based on cytometric parameters or by use of fluorochromes – fluorochromes often bound to antibodies or oligonucleotides • Flow cytometer – forces suspension of cells through laser beam and measures amount of light scattering of fluorescence – can detect heterogeneous microbial populations with different responses to antimicrobial treatments 20 Radioimmunoassay (RIA) • Purified antigen labeled with radioisotope competes with unlabeled standard for antibody binding • Amount of radioactivity associated with antibody is measured 21