Hugid: Immunology in Infection & Diagnosis

Question 1

What are 3 ways to diagnose HIV?

Answer 1

1. ELISA
2. Western Blot
3. PCR

Question 2

What is one way to diagnose AIDS?

Answer 2

flow cytometry

Question 3

What 3 proteins are needed for a Western blot confirmation of HIV?

Answer 3

1. gp41
2. gp120
3. p24 core antigen

**p17 often detected, too

Question 4

What are the major antigenic proteins for the HIV virion?

Answer 4

1. gp41
2. gp120
3. p24

Question 5

Which anti-HIV antibody would be diagnostic for infection of a baby?

Answer 5

IgM

Question 6

How do you perform an ELISA?

Answer 6

1. Antigens from the sample are attached to a surface.
2. Then a specific antibody is applied over the surface so it can bind to the antigen.
3. This antibody is linked to an enzyme.
4. In the final step, the enzyme’s substrate is added.
5. The reaction produces a detectable signal, most commonly a color change in the substrate and you can detect if the Ab was present.

Question 7

What are two important things to consider when performing an ELISA?

Answer 7

1. pay attention to + and - controls

2. wash carefully!

Question 8

What is the equation for sensitivity?

Answer 8

true positives / true positives + false negatives

Question 9

What is the equation for specificity?

Answer 9

true negatives / true negatives + false positives

Question 10

Are true positives HIV infected? Are false negatives HIV infected?

Answer 10

yes; yes

Question 11

What is this:

If a person has a disease, how often will the test be positive (true positive rate)?

Put another way, if the test is highly sensitive and the test result is negative you can be nearly certain that they DON’T have disease.

Answer 11

sensitivity

*SENSITIVE - RULE OUT

Question 12

What is this:

If a person does not have the disease how often will the test be negative (true negative rate)?

In other terms, if the test result for a highly specific test is positive you can be nearly certain that they actually have the disease.

Answer 12

specificity

*SPECIFICITY - RULE IN

Question 13

What are the steps in a Western blot?

Answer 13

1. you start with multiple antigen proteins
2. you use SDSO4- to give them a negative charge
3. Run gel electrophoresis (smaller proteins move farther)
4. Proteins are stained in gel
5. Proteins are blotted from gel to filter paper
6. Add human serum
7. Use an enzyme-conjugated goat-antibody to human IgM to detect if the human has bound antibody
8. Where bands appear, it indicates that ANTIBODY has bound to the antigen

**bottom line: if specific bands show up on a Western that are specific for the antigen you are testing (ex: HIV), then the person is infected because they have antibodies to that antigen

Question 14

When is it appropriate to perform a Western blot?

Answer 14

when the stakes are high

ex: HIV infection, Hep C infection, Lyme disease

Question 15

What is flow cytometry useful for in the case of AIDS?

Answer 15

it counts CD4 and CD8 T cells in order to monitor CD4 T cell depletion during HIV infection

Question 16

What is one advantage of flow cytometry?

Answer 16

You can analyze 100s of cells per second!

Question 17

Besides AIDS, what else can flow cytometry be useful for?

Answer 17

identifying T and B cell leukemias

looking at immunological changes in disease such as NK cells in Chronic Fatigue Syndrome