Define a qualitative immunodiagnostic test.
Indicates yes/no as to presence of pathogen without indicating strength of immune response.
In some cases, just knowing that an immune response was developed to pathogen is all clinical info needed.
Define a quantitative immunodiagnostic test.
Measures the level of the causative agent of the disease or the level of antibodies against a certain pathogen.
Important because the level of causative agent correlates with pathogenesis and influences whether no, mild, or severe disease produced.
Describe the basic common methods used for immunodiagnostic testing.
Primary antigen/antibody Interaction – non-covalently binding of specific antibody to its matching epitope
- Radioimmunoassay, ELISA, Immunofluorescence Assay, Flow Cytometry, Immunoelectrophoresis/Western blotting, Immunohistochemistry, Antiglobulin test, and monoclonal antibody testing.
Secondary antigen/antibody interaction - multiple antigen–antibody bindings cross-link many molecules/cells
- Rocket electrophoresis, Immunodiffusion, Complement fixation test, Agglutination, Immunoelectrophoresis
Define an antibody titer and what it represents.
Antibody titer is the highest dilution at which agglutination occurs
Ex: Dilutions tested: 1:2, 1:4, 1:8, 1:16, 1:32; Agglutination seen up to 1:16, but not at 1:32 → Titer = 16
It tells that the patient has been exposed to the antigen at some point.
Interpretation includes:
* ≥4-fold rise over 1–3 weeks → recent/active infection
* Stable/low titer → past exposure or immunity
Interpret single titer results.
Low or absent titer → may indicate no prior exposure or infection.
High titer (in isolation) → could indicate past exposure and immunity or recent/current infection.
Paired Samples (taken 1–3 weeks apart)
What does a fourfold rise in titer indicate?
Strong evidence of recent or ongoing infection.
Paired Samples (taken 1–3 weeks apart)
What does a stable titer suggest?
Past exposure or immunity rather than active disease.
Contextual Interpretation
Does presence of antibody equal a positive infection?
No, antibodies may persist long after infection has resolved.
Low specificity → ______________.
Low specificity → risk of false positives.
Low sensitivity → risk of ____________.
Low sensitivity → risk of false negatives.
What is the relationship between diagnostic sensitivity and specificity?
There is an inverse relationship; improving one often reduces the other.
What is diagnostic sensitivity calculated from?
Calculated from the number of false negatives given by the test.
- A 100% sensitive test has zero false negatives.
What is diagnostic specificity calculated from?
Calculated from the number of false positives given by the test.
- A 100% specific test has zero false positives.
Difference between primary and secondary antigen/antibody binding?
The primary antibody to antigen interaction is a direct interaction (serology) visualized through conjugate-substrate reaction, fluorescence or radiolabeled antiglobulin.
Primary
* Measures direct Ab–Ag binding with labels (enzyme, fluorescent, radioactive)
Secondary
* Measures effects of binding: precipitation, clumping, lysis
How does the detection of antibodies in serum assist in the diagnosis of
infectious diseases and noninfectious disorder?
Infectious disease
* Exposure/active infection (e.g., ELISA; Coggins = immunodiffusion for EIA)
Noninfectious disorders
* Hormones by RIA (prolactin, progesterone, testosterone, LH)
* Autoimmune disease (Coombs for IMHA)
- In infectious disease, antibody detection shows whether an animal/human has been exposed, is actively infected, or has immunity.
- In noninfectious disorders, antibody detection identifies abnormal immune responses such as autoimmunity or allergies.
What is Radioimmunoassay?
Labeled and unlabeled antigen compete for limited antibody binding sites.
Radioactivity measured reflects how much unlabeled antigen is present.
What is ELISA?
Enzyme Linked Immunosorbent Assay. Enzyme is chemically linked to antibody or antigen. Detects antibodies or antigens in serum/other fluids.
Direct:
A target protein (or a target antibody) is immobilized on the surface of microplate wells and incubated with an enzyme-labeled antibody to the target protein (or a specific antigen to the target antibody
Indirect:
A target protein is immobilized on the surface of microplate wells and incubated with an antibody to the target protein (the primary antibody), followed by a secondary antibody against the primary antibody. After washing, the activity of the microplate well-bound enzyme is measured. Although indirect ELISA requires more steps than direct ELISA, labeled secondary antibodies are commercially available, eliminating the need to label the primary antibody
What is Immunofluorescence (IFA)?
Fluorescent dye is linked to the antigen/antibody.
- Reagents need to be in pure form
- Need immunofluorescence microscope to read
Direct: detect antigen. Labeled antibody is incubated with antigen of the micro-organism to be diagnosed, wash, and check for fluoresence
Indirect: detect AB or antigen. Uses antiserum.
What is Immunochromatography?
Sample flows along a strip by capillary action, and if the target antigen (or antibody) is present, it binds to labeled antibodies to form a visible line through specific antigen–antibody interactions. Can be read by naked eye.
Example: covid rapid antigen test/pregnancy test
What is Flow cytometry (FACS)?
Counts, examines, and sorts cells based on fluorescence activation. For example, you want CD4+ cells. You label the machine to find them, and it will fluoresce those cells. This also allows you to detect a fluorescent monoclonal antibody to a specific cell surface antigen.
What is Immunoelectrophoresis?
Perform gel electrophoresis on a protein mixture (run the mixture on a gel and based on fragment size, the smallest size will travel the furthest).
What is Western blotting?
Follows immunoelectrophoresis.
You then transfer the bands created from the gel over to nitrocellulose paper (blotting paper on top of the gel with sponges in between in a buffer).
- Visualize results using an enzyme immunoassay or radioimmunoassay.
- This is useful to identify antigens in microorganisms or parasites.
What is an Antiglobulin Test (Coombs Test)?
Lab test that identifies antibodies that can bind to the surface of RBCs or platelets and destroy them. This is used to diagnose blood disorders where they make antibodies that are destroying their own cells.
- Generally, a supportive diagnostic test for immune-mediated hemolytic anemia (IMHA)
- Commonly performed in dogs, cats, and horses
What is Monoclonal antibodies testing?
Antibodies produced in a lab to recognize one single epitope of an antigen/pathogen. This is useful to find the precise identification of an organism.
- Monoclonal antibodies can be used in different tests (like every quantitative immunodiagnostic test we discussed)
- Used against bacterial and fungal infections of livestock
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Plasma Proteins (Zimmerman)32
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Anatomy & Organization of the Immunohematologic System (Walling)8
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Immunology & Overview of the Immune System (Satti)17
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White Blood Cell Microscopy Lab (Thomas)37
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Lymphocyte Development and Central Tolerance (Satti)44
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Antigen Processing & Presentation: Clonal Selection & T-cell Response (Satti)20
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Red Blood Cell & Platelet Lab (Zimmerman)48
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T-cell Response to Antigens, APCs (Satti)31
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PVC/TS Lab - Hematology Methods (Zimmerman)25
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Erythrogram - Automated Hematology Methods (Zimmerman)43
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Lymphatic Drainage Patterns (Walling)30
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Hematopoiesis, Marrow Collection & Assessment (Lucidi)38
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B-cell Responses to Antibody, Structure & Functions (Satti)40
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Live Dog Anatomy (Walling)8
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Immunodiagnostic Tests (Satti)45
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Leukogram & Thrombogram (Thomas)21
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Mucosal Immunity (Satti)45
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Hemostasis 1 (Thomas)91
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Hemostasis 2 (Thomas)44
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Vaccines & Their Production/Use (Satti)60
