Immunodiagnostic tests or immune mediated disease

Question 1

Explain an ELISA

Answer 1

• Antigen immobilised on a solid support either non specifically (indirect) or specifically (via capture by another antibody specific to same antigen (sandwich)
• Antigen is recognised by specific antibody
• Antibody is recognised by second antibody which has an enzyme attached
• Substrate reacts with enzyme to produce product (normally colour)

Question 2

What are the uses of ELISA in vet med?

Answer 2

• Disease detection: e.g. canine distemper virus, canine/ feline heartworms, FELV
• Detection of illegal drugs
• Detection of hormones (PD kits)

Question 3

What is the role of an indirect ELISA?

Answer 3

To measure antibody concentration in whole blood/ plasma/ serum

Question 4

What is the role of a sandwich ELISA

Answer 4

• To measure Ag concentrations in various different sample types

Question 5

What is a competitive ELISA?

Answer 5

ELISA technique used for detection of small antigens with only 1 epitope (meaning sandwich ELISAs wouldn’t work)

• A known amount of tagged Ag will compete with unknown amount of Ag in the sample for binding the capture Ab
• The amount of bound, tagged Ag is inversely related to conc of Ag in test sample

Question 6

What must you consider when interpreting ELISA results?

Answer 6

• Limit of detection (minimal conc that the test can detect)
• Sensitivity (ability to ID + results)
• Specificity (ability to ID - results)
• Seroconversion (development of detectable specific Ab to microbes in blood serum as a result of exposure, infection, immunisation)

Question 7

What must you consider when interpreting ELISA results?

Answer 7

• Limit of detection (minimal conc that the test can detect)
• Sensitivity (ability to ID + results)
• Specificity (ability to ID - results)
• Seroconversion (development of detectable specific Ab to microbes in blood serum as a result of exposure, infection, immunisation)

Question 8

Describe the Coombs test

Answer 8

• Direct Antiglobulin test
• Identified presence of non-agglutinating Ab on surface of particles e.g. bacteria or erythrocytes.
• RBCs covered in antibodies
• Add antiglobulin and agglutination will occur

Question 9

What is radial immunodiffusion?

Answer 9

Method to demonstrate precipitation of antigen by antibody
(-Antigen added to agar with specific antiserum
-Ring of precipitation forms around antigen well
-Area of ring proportional to amount of antigen in the well)

Question 10

What is a direct fluorescent antibody test?

Answer 10

Identifies presence of antigen in a cell or tissue sample by direct binding

Question 11

What is an indirect fluorescent antibody test?

Answer 11

• Identifies presence of either antigen in a cell or tissue sample or antibody in serum

Question 12

What is an anti-nuclear antibody test (ANA)?

Answer 12

Tests for development of autoantibodies against antigens located within cell nucleus

• Cultured cell/ frozen sections on a microscope slide= used as antigen source
• Dilutions of patient’s serum applied
• Incubation with fluorescin labelled antibodies specific to patient species e.g. dog
• Analyse with microscope
• Positive= produced nuclear antibodies with fluorescent nuclei