Immunology in Infection and Diagnosis Flashcards
How can you diagnose HIV?
via ELISA
Western Blot
PCR
Flow Cytometry
What three antigens do you need to diagnosis HIV on a western blot
gp120
gp41
p24
(P17-> matrix antigen is also often detected)
If you want to see if someone is infected what do you look for? If you want to see if someone has ever been infected, what do you look for?
IgM
IgG
What are the general principes for immunoassays?
decide if you want to measure antigen or antibody
decide upon system of detection
notice how antigen-antibody complexes are separated
establish controls, positive, and negative
Only (blank) anti-HIV will be diagnostic for infection of the baby
IgM
Maternal (blank) anti-HIV will cross the placenta and persist for months
IgG
At what age do babies lack all maternal antibody and has to start making its own?
6-9 months
How does an ELISA work?
antigens are stuck onto a plastic surface, a sample is added and any antibodies for the disease we are testing for will bind to the antigens. Next a second antibody with a marker is added and a positive reaction is detected by the marker changing colour when an appropriate substrate is added. If there are no antibodies in the sample, the second antibody will not be able to stick and there will be no colour change.
What is a sandwich ELISA?
For an antigen ELISA, antibodies are bound to a plastic surface, a sample is added and if antigens from the virus we are testing for are present they will stick to the antibodies. This test then proceeds in the same way as the antibody ELISA
When do you use ELISAs?
for detection of IgG, IgM,
What would you want to detect IgM for?
current infections such as H. pylori, EBV, Rotavirus
What is this:
True positives/ (true positives + false positives)
sensitivity
What is this
False positive/ (true positive + false positive)
specificity
Is the P24 EIA a sensitive and specific test for HIV
yes!!! 100% sensitive and 99.75% specific
How does a western blot work to find HIV?
multiple Ag proteins (virus and cell protein)
use SDS to give proteins negative charge.
utilize electrophoresis.-> proteins are stained -> proteins are blotted from gel onto filter paper-> human serum is added -> enzyme conjugated goat antibody to IgM is added and use to detect bound (human) antibody-> bands of insoluble product from substrate indicate where ab’s have been bound to different HIV ag’s
On western blot, what do you need to have to prove that someone is HIV positive?
minimum of 2 of 3 major HIV proteins (P24, Gp41, Gp120/Gp160)
When are western blots used?
when the stakes are high for a correct diagnosis
e.g HIV, Hep C, lyme diseae
How can you use flow cytometry to detect AIDS? How?
you look at the CD4 T cells to see if there is depletion. Take mouse anti-CD4 and CD8 which are coupled with flouresecents
What color will a CD8 T cell flouresce?
red
What color will a CD4 T cell flouresce?
green
How does flow cytometry work
you put markers on cels and thne place them in a single file stream to be excited by lazors and separated out based on color that fluoresces
Besides CD4 T cells what else can HIV infect?
monocytes and macrophages
CD4 Ts below (blank) =AIDS
200 per microliter
In AIDs, CD4 Ts decrease and C8 Ts stay constant for a long time. What is the normal ratio of CD4:CD8?
2 to 1
What are the unstained cells for in flow cytometry?
they are your negative controls
Flow cytometry is often expressed how?
in a histogram
What is the MFI?
median fluorescent intensity and it is used to describe the extent to which cells are labeled with antibodies
What are some clinical applications of flow cytometry in medicine?
identify T and B leukemias
monitor CD 4 T cells in AID
check for immunlogical changes in diseases, such as NK cells in Chronic fatigue syndrome
What is FITC?
What is PE?
IgD
IgM
