Immunologic Tests- Diebel Flashcards
What, in general, is a hybridoma?
Fused Myeloma cells (immortal) and B cells from a spleen in a mouse that were exposed to antigen……produce monoclonal antibodies used for experiment/testing stuff
What is HAT? What is the significance of the different chemicals?
HAT is the media for making hybridomas
Hypoxanthine- scavenger pathway, need HGPRT, NOT found in Myeloma cells….so they die
Aminopterin- blocks DHFR
Thymidine- rescue cells by supplying thymidine
Why do plasma cells die in HAT media?
They die because of lack of growth factors, they need to be fused with the myeloma cells to be immortal.
Why do myeloma cells die in HAT media?
Because myeloma cells don’t have HGPRT to process hypoxanthine
What type of immunodiagnostic assay is the most sensitive?
ELISA
What is a very important principle with immunopricipitation assays?
You need the correct ratio of Aby and Ag for lattice to form and precipitation to take place…..this is why you often see titration reactions
After making hybridomas what do they do to get the antibody of interest?
Isolate hybridomas, one per cell in plate
Add antigen and select cell/antibody with best binding
How do agar diffusion tests work?
Take an agar plate, punch out some holes, add antigen or antibody into punch holes….these diffuse and precipitate
What is the difference between direct and passive (indirect) agglutination?
DIRECT: Add aby to sample and you can see agglutination with naked eye (Ex: Blood typing)
PASSIVE: Add aby with something tagged on it so you can see the agglutination (usually use beads)
What is the difference between hemagglutination testing looking for viruses and antibodies?
Viruses- constant RBC, constant aby, decreasing amount of patient virus
Button = Positive
RBC floating on surface = negative
Antibodies- constant RBC, dec. [serum] L to R
Button = negative
RBC floating on surface = positive
How do neutralization assays work?
o Cell + toxin molecules –> cell damage
o Toxin + antitoxin (purified Ab) = neutralized toxin + cell –> cell not damaged
Gauges whether you have neutralizing antibodies in patient’s serum against a particular toxin
How do complement fixation assays work?
Cells lysing = negative result
Cells don’t lyse = positive result……patients antibody is outcompeting complement so it’s not binding to RBC and killing them
What is so cool about immunohistochemical microscopy?
You can stain specific things like CD4, CD8 etc. and then check it out under the microscope….
What is the difference between direct and indirect immunofluorescent microscopy?
Direct has a specially synthesized antibody with fluorescent tag on it…..one antibody to make pretty colors
Indirect has an antibody that binds a target, then another generic fluorescent labeled antibody is added…..Two antibodies to make pretty colors…this option is used more often because it’s cheaper
Describe the direct ELISA process in general terms
looking directly for antigen
- Antibodies bound to wells of microtiter plate
- Add patient sample
- Wash with buffer
- Add antibody containing conjugated enzyme
- wash with buffer
- Add substrate for E and measure amount of colored product
Quantitation = amount of colored product produced is proportional to amount of antigen
Describe indirect ELISA in general terms
looking for body’s immune response for specific antigen
- Microtiter wells coated with antigen preparation
- Add patient serum sample, specific Ab bind to antigen, other Ab do not bind
- wash with buffer
- Add anti-IgG antibodies conjugated to enzyme
- wash with buffer
- Add substrate for enzyme and measure amount of colored product
Quantitation = amount of colored product is proportional to antibody concentration
What is an advantage of indirect ELISA compared to direct?
Secondary antibodies can be specific enough to look for IgM vs. IgG vs. IgA = benefit of indirect test
So, what is competitive ELISA?
Start with antigen coated wells
Mix serum with antibody, put in plate
Positive result is no color change because antibody right away binds the antigen in the sample
Pretty dumb test, don’t think it’s really ever used
What are ELISPOT assays?
- Growing cells over a plate (bottom of plate has been coated with Ab for specific cytokines)
- If cells are producing cytokines –> will bind the Ab
- Cells producing cytokine that antibodies on the plate bound
- Wash off cells –> add antibody with enzyme attached to it –> colorimetric change in that area of the plate (just like a direct ELISA)
- Takes live cells growing and secreting cytokines to get results
Describe Western Blotting….
- *Gives you size and relative amount of protein present**
- *Used for HIV diagnosis**
Run protein mix through SDS-gel electrophoresis to separate proteins by size
Run current to transfer proteins to membrane
Wash solutions containing aby of interest over membrane
Bind antigen of interest with enzyme linked aby (Indirect, 2 abys), get some color
What are the two big things flow cytometry detects in general? Why are these good things to know?
Size (Forward)
Scattering or granulation (Side)
**Can distinguish different cell types with these characteristics (Neutrophils, Basophils, lymphocytes, etc)
How can you use flow cytometry to distinguish between very similar looking cells like CD4+ and CD8+?
• Fluorescence emitted from stained cell detected looking for surface proteins
o Ratios: 2:1 T cells vs all other lymphocytes, 2:1 CD4+ vs CD8+, 3:1 B cells vs. NK cells
Cells are stained for specific surface markers so that when they pass by the laser you can distinguish between CD4+ or CD8+ cells
Can also see double negative/double positive CD8+ CD4+ cells
What percentage of lymphocytes are NK cells and CD3+ cells in a normal blood count?
NK cells: 1/3
CD3+ cells: 2/3
What is the ratio of CD4+:CD8+ cells?
2:1
