Immunological Diagnostics Flashcards
When are serological assays used for immuological diagnostics?
When pathogen is difficult, dangerous, or impossible to culture
What must be available for serological assays for immunological diagnostics?
The antibody or antigen in pure, detectable form
What are the 4 most important characteristics of antibody binding used in serological assays?
Specificity
Amount
Isotype
Affinity
____________ is an important property of an immunodiagnostic test referring to the ability to detect small quantities of Ab or Ag
Sensitivity
An immunodiagnostic test with poor sensitivity would result in which of the following?
A. False positives
B. True positives
C. False negatives
D. True negatives
C. False negatives
[lack of detection when Ab or Ag is present]
What can you conclude about disease status if the sensitivity of an immunodiagnostic assay is HIGH?
You can rule OUT disease
[if high sensitivity test is positive, may want to confirm with a highly specific test, because it is possible you got a false positive]
__________ is an important property of an immunodiagnostic test referring to the ability to distinguish the immunogen from all other similar antigens
Specificity
An immunodiagnostic test with poor specificity would result in which of the following?
A. False positives
B. True positives
C. False negatives
D. True negatives
A. False positives
[detects something other than the immunogen of interest]
What can you conclude about disease status if the specificity of an immunodiagnostic assay is HIGH?
You can rule IN disease
What type of serological assay results in the formation of a complex that visibly precipitates?
Precipitin reactions
What type of serological assay results in cells/particles bound together through antibody-antigen interactions?
Agglutination reactions
What type of serological assay results in antibody covalently-linked to a label?
Labeled antibody methods
[antibody bound to Ag is detected by presence of the label]
What is the difference between direct and indirect serological assay methods?
Direct detects ANTIGEN
Indirect detects ANTIBODY
What are some other terms used for antibody?
Antiserum
Serum
Antitoxin
Antivenin
What is the direct fluorescent Ab test?
Identifies Ag in tissue by labeling monoclonal Abs specific to the epitope you are looking for
In the direct fluorescent Ab test, where on the Ab does the label stick?
The Fc region of IgG
[if this Ab binds Ag of interest, you will see fluorescence on microscopy]
What is the indirect fluorescent Ab test?
Identifies Ab in patient
[make test Ag that patient’s fluorescent-labeled Abs will bind to]
In precipitin reactions, precipitation occurs when _________ antigens and antibodies ______-______, forming large molecular aggregates
Multivalent; cross-link
For precipitin reactions, what is the term for the point at which precipitation occurs?
Zone of equivalence (or equivalence point)
Prior to reaching the zone of equivalence in a precipitin reaction, what is in excess - antibody or antigen?
Antibody
Once you are past the zone of equivalence in a precipitin reaction, what is in excess - antibody or antigen?
Antigen
What serological test is used to determine if two antigens contain overlapping epitopes?
What is this test also used for?
Ouchterlony double diffusion
[also used to determine concentrations of antigens]
Agglutination reactions are similar to precipitin reactions, but what is the difference in agglutination reactions?
In agglutination reactions, the antigen is part of a whole cell or cell-sized particle
Describe the degree of sensitivity and speed in an agglutination reaction
Very sensitive
Very fast reaction - occurs in minutes
_____________ is the agglutination of erythrocytes and is used for blood typing
Hemagglutination
What test uses anti-human immunoglobulins to precipitate agglutination of RBCs bound to anti-Rh antibodies?
The coombs test
What does the direct Coombs test detect?
Detects anti-Rh antibodies found on fetal RBCs
What does the indirect coombs test detect?
Detects anti-Rh antibodies in mother’s serum
Which of the following best describes the Coombs test?
A. Precipitin reaction
B. Agglutination reaction
C. Labeled antibody method
D. Labeled antigen method
B. Agglutination reaction
Labeled Ab methods detect antibodies bound to a _________ surface.
Antibodies are labeled in _______ amounts to ensure binding to Ag is not interrupted. The label is added to the ____ region of the Ab.
Direct and indirect labeling of antibodies is done, but _______ labeling is more popular.
Solid
Small; Fc
Indirect
What test involves labeled antibodies (linked to enzyme) added to wells coated wtih different antigens?
ELISA
[antibodies specific to Ag will bind, while those that do not recognize Ag will be washed away]
How is Ag:Ab binding detected in ELISA tests?
Measuring color change, mediated by enzyme-dependent conversion of a colorless substrate to a colored reactant
What type of blotting technique is sometimes used following a positive ELISA to confirm the findings?
Western blot (looks for protein)
Western blot is used to confirm diagnosis of HIV. The HIV particle is dissociated by ______ and separated on a _______.
Anti-HIV Abs in the serum of an infected individual will bind to the various HIV proteins. Labeled anti-human ______ are used to detect antibodies bound to the HIV proteins
SDS; PAGE
Immunoglobulins
What technique is used as a tool for defining and enumerating lymphocytes using antibodies specific for cell-surface proteins?
Flow cytometry
[the flow cytometer detects and counts individual cells passing in a stream through a laser beam]
Flow cytometers are equipped to identify what aspect of cells?
Their cell-surface antigens (it sorts them accordingly)
Which type of T lymphocyte is more numerous, CD4 or CD8?
CD4
What technique is used for detection of lymphocyte subpopulations using specific antibodies?
Fluorescence-activated cell sorting (FACs) - a specialized type of flow cytometry that sorts a heterogenous mixture of cells, one cell at a time, based on specific light scattering and fluorescent characteristics of each cell
