Lecture 18 - Immunodiagnostics

Question 1

What does ELISA stand for?

Answer 1

Enzyme-linked immunosorbent assay

Question 2

True or False

Antigen/Antibody binding is covalent

Answer 2

FALSE

Antigen/Antibody binding is reversible and goes to equilibrium.

Question 3

Learning Objective 1

List the basic principles of antigen/antibody binding used in immunodiagnostics.

Answer 3

Ag/Ab binding is non-covalent. Goes to equilibrium and is reversible.

Binding is influenced by pH and salt levels. Note that this is generally only a factor in the laboratory (requires extreme conditions to see an effect).

Binding at optimal proportions can form a visible precipitate.

Question 4

How would you go about producing an antibody to canine IgG?

Answer 4

Inject canine IgG into a rabbit. Rabbit will produce rabbit anti-dog IgG.

Question 5

What is a conjugate in terms of antibody labeling?

Answer 5

A molecule covalently bound to the antibody.

Examples include enzymes that act on a substrate and fluorescent dyes.

Question 6

Describe the function of chromogen substrate in an immunoassay.

Answer 6

The enzyme conjugate bound to your antibody acts on the chromogen, causing a color change.

A color change in your assay indicates that the antibody has successfully bound to the desired antigen/antibody/whatever.

Question 7

Describe an “antibody sandwich” in an immunoassay.

Answer 7

Antibody is adhered to the well and will capture antigen.

Antigen in the sample binds to the antibody in the well.

Antibody labeled with an enzyme is added and binds to the antigen.

Adding substrate to the well at this point will create a color change because of the enzyme action on the substrate.

Question 8

Describe an antigen sandwich in an immunoassay.

Answer 8

Antigen is bound to the well.

Antibody from the serum binds to the antigen.

Inactivated antigen covalently bound to an enzyme is added and will bind to the antibody.

If substrate is added to the sandwich, the sample will change color because of the enzyme’s action on the substrate.

Question 9

Learning Objective 2

Draw an ELISA to detect an antigen (e.g. a virus). Properly label all reagents and explain a positive result and a negative result. You must give the name of your conjugate.

Answer 9

1. If you want to detect antigen, an antibody to the antigen should be at the base of your well. Add serum to the well.
2. Antigen binds to antibody. Wash to remove excess material.
3. Add antibody covalently bound to enzyme. Wash.
4. Add chromogen substrate.

Positive result - if assay changes color, antigen has bound to antibody in the wells, so antigen was present in the serum.

Negative result - no color change. No antigen in serum.

Question 10

Learning Objective 2

Draw an ELISA to detect an antigen (e.g. a virus). Properly label all reagents and explain a positive result and a negative result. You must give the name of your conjugate.

Answer 10

Question 11

Learning Objective 3

Draw an ELISA to detect antibody. Properly label all reagents and explain a positive and negative result.

Answer 11

1. If you want to detect antibody, antigen should be at the base of your well. Add serum to the well.
2. Antibody binds to antigen. Wash.
3. Add antigen conjugated with enzyme. Wash.
4. Add chromogen substrate.

Positive result - assay will change color when the enzyme acts on the chromogen, indicating that antibody was present in the serum.

Negative result - antibody was not present in serum.

NOTE: negative antibody test does not mean infection is not present! Some antibodies like IgE are seldom present in serum.

Question 12

Learning Objective 3

Draw an ELISA to detect antibody. Properly label all reagents and explain a positive and negative result.

Answer 12

Question 13

True or False:

It is important to know the vaccination history of a cat for FeLV because the vaccination could potentially interfere with an antigen test.

Answer 13

FALSE

The test tests for antigens, not for antibodies. Only antigens in the serum would result in a positive test. Antibody production does not affect the result of the test.

Question 14

True or False:

It is important to know the vaccination history of a cat for FIV because the vaccination could potentially interfere with an antibody test.

Answer 14

TRUE

A vaccinated animal will produce antibodies to the antigen, which could potentially interfere with the antibody test. Antibodies present in the serum will produce a positive test.

Question 15

Describe the process of an indirect ELISA to detect antibody.

Answer 15

1. Antigen is bound to the well. Add serum.
2. Antibody in serum binds to antigen. Wash.
3. Add enzyme labeled antiglobulin (antibody to the target antibody). Wash.
4. Add chromogen substrate. Enzyme on antiglobulin will produce a color change in the assay.

Note: the difference between this assay and the direct antibody detection assay is that instead of an enzyme-labeled antigen in step 3, we added an enzyme-labeled antiglobulin.

Question 16

What is a DIVA (marker) vaccine?

Answer 16

DIVA - Distinguishing Infected from Vaccinated Animals

Vaccine will produce different antibody titers than a wild type infection.

Question 17

Describe how a DIVA (marker) vaccine works.

Answer 17

The virus in the vaccine has a portion deleted (usually delete a gene encoding for a protein).

The animal will produce antibodies to all of the viral proteins except for the one that has been deleted.

An animal with a wild type infection will produce antibodies against viral proteins, including against the one that was deleted in the vaccine.

An assay well coated with the key viral protein will pick up antibodies from an animal with a wild type infection.

Question 18

Learning Objective 4

Draw an immunofluorescent or immunohistochemistry assay to detect the canine autoantibody to the skin desmoglein molecule in a skin biopsy from a dog with pemphigus foliaceus.

Answer 18

Immunofluorescence assay

1. Take a skin biopsy. If the animal has the disease, it will have antibody to desmoglein bound to the skin.
2. Add rabbit anti-canine IgG.
3. Add anti-rabbit IgG conjugated with fluorescein. This step helps to amplify the fluorescent signal.
4. Look at sample with a UV light microscope to detect fluoresence. Fluoresence indicates a positive result.

Question 19

Learning Objective 4

Draw an immunofluorescent or immunohistochemistry assay to detect the canine autoantibody to the skin desmoglein molecule in a skin biopsy from a dog with pemphigus foliaceus.

Answer 19

Question 20

Learning Objective 5

Define precipitation and agglutination and explain the difference.

Answer 20

Precipitation - the clumping of soluble antigen and antibody.

Agglutination - the clumping of particulate antigen and antibody. The term agglutination usually refers to cellular antigens.

Both produce a visible line in an assay.

Question 21

What is the zone of optimal proportions?

Answer 21

The concentration of antigen and antibody that will produce precipitation/agglutination.

Question 22

Learning Objective 6

Explain what the precipitate is in a precipitation assay and what the equivalence zone means. Describe why there is no precipitate when there is antibody excess or antigen excess.

Answer 22

Precipitate is a cross-linking of antigen and antibody to the point where they fall out of solution. This happens at the zone of optimal proportions.

If antibody is in excess, there will be no cross-linking. If the antigen is in excess, all of the antibody’s binding sites will be occupied and there will be no cross-linking.

Question 23

How does a serial dilution for antibody titer work?

Answer 23

Start with antibody in solution. Dilute the solution in a series (1:10, 1:20, 1:40…) and add antigen to each of the solutions.

The smallest concentration at which the solution forms a precipitate when antigen is added represents the titer of the antibody.

Question 24

If your antibody solution forms precipitates with antigen at dilutions 1:10, 1:20, and 1:40 but not 1:80, what is your antibody titer?

Answer 24

1:40

Question 25

Learning Objective 7

Define titration and explain how a titer is determined.

Answer 25

The measurement of a specific antibody in serum based on serial dilutions. The highest dilution that is positive for precipitation is the titer.

Note: titers can only be compared with one another if the dilution procedure is done in exactly the same way.

Question 26

Learning Objective 8

Explain what makes the red line in a positive Heska Solo Canine Antigen test and what a positive result means

[Note: this is a test for *Dirofilaria immitis *antigens]

Answer 26

1. Antibody to worm uterine antigen is covalently bound to red beads (AB-R) and embedded in the test pad. Some free-floating blue beads are also present.
2. Add the sample. Mixes with AB-R. If antigen is present, it will be bound by AB-R. AB-R, antigen, and blue beads wick through the pad.
3. There are two test lines at the end of the pad. The first contains antibody to the worm antigen covalently bound to the pad. The second contains antibodies to the blue beads (acts as a control).
4. If antigen is present, it will be captured by the line of covalently-bound antibodies. The red beads will form a red line, indicating a positive test.

Question 27

Learning Objective 8

Explain what makes the red line in a positive Heska Solo Canine Antigen test and what a positive result means

[Note: this is a test for Dirofilaria immitis antigens]

Answer 27

Question 28

Learning Objective 9

Compare and contrast the meaning of a positive antibody test and a positive antigen test

Answer 28

Antigen tests are easy to interpret. A positive antigen test indicates that antigen was present in the serum.

Antibody tests are harder to interpret. A positive test indicates that antibody was present in the serum. However, a negative test does not necessarily mean antibody is not being produced. Some antibodies are not found in the serum (think IgE).

Question 29

Learning Objective 10

Give an explanation for why test kit reagents should be at room temperature before running the tests.

Answer 29

Antigen/Antibody binding is temperature-dependent. Tests are designed to have a specefic incubation period. A cold temperature from a test that just came out of the fridge could prolong the incubation period.