Immunological Diagnostics

Question 1

When are serological assays used for immuological diagnostics?

Answer 1

When pathogen is difficult, dangerous, or impossible to culture

Question 2

What must be available for serological assays for immunological diagnostics?

Answer 2

The antibody or antigen in pure, detectable form

Question 3

What are the 4 most important characteristics of antibody binding used in serological assays?

Answer 3

Specificity
Amount
Isotype
Affinity

Question 4

____________ is an important property of an immunodiagnostic test referring to the ability to detect small quantities of Ab or Ag

Answer 4

Sensitivity

Question 5

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

Answer 5

C. False negatives

[lack of detection when Ab or Ag is present]

Question 6

What can you conclude about disease status if the sensitivity of an immunodiagnostic assay is HIGH?

Answer 6

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]

Question 7

__________ is an important property of an immunodiagnostic test referring to the ability to distinguish the immunogen from all other similar antigens

Answer 7

Specificity

Question 8

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

Answer 8

A. False positives

[detects something other than the immunogen of interest]

Question 9

What can you conclude about disease status if the specificity of an immunodiagnostic assay is HIGH?

Answer 9

You can rule IN disease

Question 10

What type of serological assay results in the formation of a complex that visibly precipitates?

Answer 10

Precipitin reactions

Question 11

What type of serological assay results in cells/particles bound together through antibody-antigen interactions?

Answer 11

Agglutination reactions

Question 12

What type of serological assay results in antibody covalently-linked to a label?

Answer 12

Labeled antibody methods

[antibody bound to Ag is detected by presence of the label]

Question 13

What is the difference between direct and indirect serological assay methods?

Answer 13

Direct detects ANTIGEN

Indirect detects ANTIBODY

Question 14

What are some other terms used for antibody?

Answer 14

Antiserum
Serum
Antitoxin
Antivenin

Question 15

What is the direct fluorescent Ab test?

Answer 15

Identifies Ag in tissue by labeling monoclonal Abs specific to the epitope you are looking for

Question 16

In the direct fluorescent Ab test, where on the Ab does the label stick?

Answer 16

The Fc region of IgG

[if this Ab binds Ag of interest, you will see fluorescence on microscopy]

Question 17

What is the indirect fluorescent Ab test?

Answer 17

Identifies Ab in patient

[make test Ag that patient’s fluorescent-labeled Abs will bind to]

Question 18

In precipitin reactions, precipitation occurs when _________ antigens and antibodies ______-______, forming large molecular aggregates

Answer 18

Multivalent; cross-link

Question 19

For precipitin reactions, what is the term for the point at which precipitation occurs?

Answer 19

Zone of equivalence (or equivalence point)

Question 20

Prior to reaching the zone of equivalence in a precipitin reaction, what is in excess - antibody or antigen?

Answer 20

Antibody

Question 21

Once you are past the zone of equivalence in a precipitin reaction, what is in excess - antibody or antigen?

Answer 21

Antigen

Question 22

What serological test is used to determine if two antigens contain overlapping epitopes?

What is this test also used for?

Answer 22

Ouchterlony double diffusion

[also used to determine concentrations of antigens]

Question 23

Agglutination reactions are similar to precipitin reactions, but what is the difference in agglutination reactions?

Answer 23

In agglutination reactions, the antigen is part of a whole cell or cell-sized particle

Question 24

Describe the degree of sensitivity and speed in an agglutination reaction

Answer 24

Very sensitive

Very fast reaction - occurs in minutes

Question 25

_____________ is the agglutination of erythrocytes and is used for blood typing

Answer 25

Hemagglutination

Question 26

What test uses anti-human immunoglobulins to precipitate agglutination of RBCs bound to anti-Rh antibodies?

Answer 26

The coombs test

Question 27

What does the direct Coombs test detect?

Answer 27

Detects anti-Rh antibodies found on fetal RBCs

Question 28

What does the indirect coombs test detect?

Answer 28

Detects anti-Rh antibodies in mother's serum

Question 29

Which of the following best describes the Coombs test? A. Precipitin reaction B. Agglutination reaction C. Labeled antibody method D. Labeled antigen method

Answer 29

B. Agglutination reaction

Question 30

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.

Answer 30

Solid Small; Fc Indirect

Question 31

What test involves labeled antibodies (linked to enzyme) added to wells coated wtih different antigens?

Answer 31

ELISA [antibodies specific to Ag will bind, while those that do not recognize Ag will be washed away]

Question 32

How is Ag:Ab binding detected in ELISA tests?

Answer 32

Measuring color change, mediated by enzyme-dependent conversion of a colorless substrate to a colored reactant

Question 33

What type of blotting technique is sometimes used following a positive ELISA to confirm the findings?

Answer 33

Western blot (looks for protein)

Question 34

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

Answer 34

SDS; PAGE Immunoglobulins

Question 35

What technique is used as a tool for defining and enumerating lymphocytes using antibodies specific for cell-surface proteins?

Answer 35

Flow cytometry [the flow cytometer detects and counts individual cells passing in a stream through a laser beam]

Question 36

Flow cytometers are equipped to identify what aspect of cells?

Answer 36

Their cell-surface antigens (it sorts them accordingly)

Question 37

Which type of T lymphocyte is more numerous, CD4 or CD8?

Answer 37

CD4

Question 38

What technique is used for detection of lymphocyte subpopulations using specific antibodies?

Answer 38

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