• Agglutination reactions• DAT• IAT• Immunofluorescence• ELISA• Western blot• Flow cytometry
Immunologic Lab Tests Outline
• What does it measure?• Where does the Ag-Ab interaction occur?• How is the Ag-Ab complex detected?
Things to Remember About Each Test
• Detection of Ag or Ab in patient specimen• Examples:
• testing for antibodies to infectious agents• testing for Hemophilus influenzae type B capsular
antigen in CSF
Agglutination Reactions: Purpose
• Use particles coated with Ag or Ab• Add patient’s serum (containing Ab or Ag)• See if particles clump
Agglutination Reactions: Method
• Detection of Ab (or complement) on patient’s red cells
• Also called the direct Coombs Test• Performed in patients with hemolytic anemia
DAT: Purpose
• Use patient’s red cells (coated with Ab)• Add anti-human globulin (AHG) (Coombs reagent)• Look for agglutination
DAT: Method
• Detection of antibodies to red cell antigens• Also called the indirect Coombs Test• Performed as part of pre-transfusion testing
• antibody screen• cross-match
IAT: Purpose
• Use patient serum (containing Ab)• Add donor RBCs (coated with Ag)• Add anti-human globulin (Coombs reagent)• Look for agglutination
IAT: Method
patient serum(without red cell
Ab)
AHG
reagent RBC(with red cell Ag)
patient serum(with red cell
Ab)
AHG
reagent RBC(with red cell Ag)
ANTIBODY SCREENING
no agglutination(negative test)
agglutination(positive test)
• Detection of a specific antigen or antibody in a histologic specimen
• Examples: • detection of bacterial organisms• detection of antigen-antibody complexes
Immunofluorescence: Purpose
• Fix specimen on slide
• Add antibody specific for the desired antigen
• Look for fluorescence
• Fix specimen on slide
• Add antibody specific for the desired antigen
• Add second antibody
• Look for fluorescence
Direct Indirect
Immunofluorescence: Methods
• Detection of antibodies in patient specimen• Examples:
• home pregnancy tests• HIV tests• tests for some coagulation factors, cytokines,
and autoantibodies
ELISA: Purpose
• Add patient specimen to well coated with ligand• Add AHG with enzyme attached• Add substrate• Measure color change
ELISA: Method
Sandwich immunoassay• detects antigen (not antibody)• coat well with antibody• rest is like ELISA
Radioimmunoassay• detects antibody or antigen• detector is a radioactive substance• otherwise like ELISA or sandwich immunoassay
ELISA: Variations
• Agglutination reactions• DAT• IAT• Immunofluorescence• ELISA• Western blot
Immunologic Lab Tests Outline
• Make a protein suspension of the target of the antibody you’re looking for (e.g., HIV)
• Electrophorese the suspension onto a little gel strip
• Apply the patient’s specimen (containing antibodies) to the strip
• Add AHG that has an enzyme attached
• Add substrate and look for bands
Western Blot: Method
• Agglutination reactions• DAT• IAT• Immunofluorescence• ELISA• Western blot• Flow cytometry
Immunologic Lab Tests Outline
• Characterization of cell size, complexity, antigens• Examples:
• diagnosis of leukemia and lymphoma• determination of CD4/CD8 counts in patients
with HIV
Flow Cytometry: Purpose