Tuesday - Dirty D - Immunologic tests

Question 1

HAT Medium

Answer 1

Hypoxanthine – part of scavenger pathway for producing nucleotides
o Can produce ATP and GTP through non-classical nucleotide production pathway
o Requires hypoxanthine phosphoribosyl transferase (HGPRT) to go through rescue pathway to produce GTP (myeloma cells lack HGPRT)

Aminopterin – poison to myeloma cells, blocks synthesis of nucleotides by blocking activity of DHFR
o Blocks classical pathway from producing nucleotides

Thymidine – rescues cells by supplying thymidine back (can make TTP)

Question 2

passive VS direct agglutination assay

Answer 2

Direct - example is putting a drop of antibody into a drop of blood and looking to see agglutination with naked eye

passive - think plastic beads example. antibody constant domains are bound to the beads

Question 3

Hemagglutination inhibition reaction test used to detect if you have antibodies against a virus

Answer 3

each well has serum. The higher number well, the more dilute the serum.

each well has the same amount of virus (introduced synthetically) and RBCs

If your serum has antibodies against the virus, then the blood will not agglutinate and it will look more diffuse in the well instead of sticking to the bottom in a little disc

(the way he explains this is so much more confusing that it should be)

Question 4

Hemagglutination inhibition reaction test used to detect if you have a virus in the blood

Answer 4

each well has virus (assuming you are sick and have it in your serum), the higher number well, the more dilute the virus

each well has the same amount of Ab and RBCs

If you have a ton of virus in the first couple wells, then the virus will agglutinate the blood and it will pool at the bottom into a small disc

Question 5

what is a neutralization assay used for

Answer 5

used to see if you have antibodies in your serum that can neutralize a toxin

Question 6

describe a positive and negative complement fixation assay

Answer 6

positive: the patient has antibodies that bind to complement, so the complement is all used up and cant help hemolysin lyse the cells
negative: the patient doesn’t have antibodies that bind complement, so the complement helps hemolysin LYSE the cells

Question 7

Direct ELISA

Answer 7

Looking for antigens in Pt serum

Antibodies are sitting on the plate

Patient serum introduced to plate

If serum has antigens, they bind.

Enzyme linked antibody introduced to plate - this binds antigen - introduce chemicals that are broken down by enzyme - detect this

Question 8

Indirect ELISA

Answer 8

Looking for antibodies in Pt serum

Antigens are sitting on the plate

Patient serum introduced to plate

If serum has antibodies, they bind.

Enzyme linked antibody introduced to plate - this binds antibodies - introduce chemicals that are broken down by enzyme - detect this

Question 9

Competitive ELISA

Answer 9

Plate has antigen

Introduce serum, Introduce antibody

If serum has antigen, it binds antibody before it can bind to plate - positive result: get no sign that antibody bound plate

if serum doesn’t have antigen, then antibody binds to plate - negative result

Question 10

purpose of ELISPOT

Answer 10

antibodies bind to cytokines produced by cells growing on a plate

Question 11

flow cytometry - forward vs side scattered emissions

Answer 11

forward = cell size; side = granularity

Question 12

ratios of lymphocytes seen in the blood in normal pple

Answer 12

Ratios: 2:1 T cells vs all other lymphocytes

2: 1 CD4+ vs. CD8+
3: 1 B cells vs. NK cells