PID+Immunodiagnostics+Fall+23 (2).pdf

BASIS OF DIAGNOSTIC TESTING & IMMUNODIAGNOSTICS PEDRO BITTENCOURT, DVM, MSC, PHD PRINCIPLES OF INFECTIOUS DISEASES HOW DO WE KNOW WHAT IS GOING ON? DIAGNOSTIC TESTS Learning Objectives Define serol...

BASIS OF DIAGNOSTIC TESTING & IMMUNODIAGNOSTICS PEDRO BITTENCOURT, DVM, MSC, PHD PRINCIPLES OF INFECTIOUS DISEASES HOW DO WE KNOW WHAT IS GOING ON? DIAGNOSTIC TESTS Learning Objectives Define serology Differentiate between Direct & Indirect tests Understand and interpret serological titers Understand the basis and applications of selected immunodiagnostic techniques Immunodiagnostics Serology Study of antigen-antibody interactions in vitro One the most commonly diagnostic tools Infectious diseases, hormones, cancer, autoimmunity The basis of several different techniques Most commonly performed on serum (blood) samples Serology Direct Detects the presence of the antigen in the sample Positive result means infection* Indirect Detects the presence of antibodies or other products of the immune response in the sample Positive result means exposure* Serology Titers Expresses concentration of antibodies/antigens Correlates with the highest dilution at which antibodies/antigens are still detectable Serial dilutions and antigen antibodies is te amount of It AAntigenInftion A antihadExpose  En Highest newTvs 1 is of tone Titely Serology Understanding antibody production dynamic and how it correlates with the disease is important for a correct diagnosis Choice of target antibody Time of testing Paired serology  When to test and which Ig to target? 19 11 4 a I IgG V Antibody Levels quern IgM I Deensins 0 7 14 21 28 recorder to hit Time (days) like is saying  Which cut-off to use? IgG Antibody Levels IgM Moresensitid Cut-off 1 Cut-off 2 Move specificity 0 7 14 21 28 Time (days)  Maybe both? IgG Antibody Levels IgM + Cut-off Inconclusive - Cut-off 0 7 14 21 28 time at all itis Time (days)  Dont she sent Paired serology know te increase will better 1st Test 2nd Test xyyou test option hate Antibody Levels IgG + Cut-off of - Cut-off isfi get 0 7 14 21 28 Thy Time (days)  need to we but Tgis Enzyme-Linked Immunosorbant Assay ELISA Uses enzyme labeled antibodies or antigens to, upon addition of a substrate, detect and/or measure the concentration of an analyte There are several variations of this technique DIRECT ELISA INDIRECT ELISA 1 4 5 2 antibody Antibodyagainst 3 0 SANDWICH ELISA - DIRECT 1 4 2 5 3 Lateral or Bidirectional Flow Assays ELISA-based Qualitative or semi-quantitative Point-of-care testing Fast results No lab needed Lateral or Bidirectional Flow Assays Immunofluorescence Fluorescent-labeled antibodies Tissue or cells, on microscope slides Requires a fluorescent microscope to read results Direct or Indirect Immunofluorescence Y Y Y Primary Ab Y Y Secondary Ab Antiglobulin Fluorochrome Direct Indirect Epitope Immunofluorescence Immunofluorescence Immunofluorescence Immunofluorescence Immunohistochemistry Used principally on frozen or formalin-fixed tissues In situ results Can detect tissue proteins or pathogen antigens Immunohistochemistry Growth hormone Rabies virus Agglutination Reaction between Antibody + Particulate antigen Antibody + Soluble antigen coated latex beads Immunoprecipitation assays Precipitation of antigen-antibody complexes Exploits the concentrations of antibody and antigen (zone of equivalence) to measure their relative quantities (titers) Solution based Gel based Solution-based Immunoprecipitation Turbidity = Precipitation of Immune complexes Line of precipitation on the gel indicates the zone of equivalence Example: Coggins test for Equine Infectious Anemia QUESTIONS? THANK YOU FOR ATTENDING! ©2021 Ross University School of Veterinary Medicine. All rights reserved.