Immunologic Tests Flashcards
How do we make monoclonal antibodies?
- purify antigen
- inject antigen into a mouse
- allow time for the antigen to reach spleen in dendritic cells and ativate B cells
- Harvest spleen and make a suspension of cells
- Take the cells and fuse them with myeloma cells - making them immortal
- Inducate them in a HAT medium to select for the cells that successfully fused - the hybridomas
- Use an ELISA set-up to select for the hybridomas that are making antibody against the antigen of interest
- THen two options; let those hybridomas grow in culture more and then harvest antibodies OR inject the hybriomas into a mouse, which will grow a tumor that will crank out IgG which you can later harvest
How does HAT select for the hybridomas?
Aminopterin blocks DHFR
However, normal cells are able to overcome this block because they can synthesize TTP if thymidine is supplied (the T in HAT) and GTP if hypozanthine is supplied (the H in HAT)
Myeloma cells, however, have a mutated HGPRT, so it can’t use the hypoxanthine - so it has neither GTP or TTP and it dies
And the B cells that didn’t fuse just die because they’re not immortal
How does an immunoprecipitation-based assay work
Using an agarose gel, you drill out wells and place the patient’s serum in one of them. In the others you place the antigens of the various species you’re looking for.
If the antibodies are present in the serum, as they diffuse out from the serum well, they will come in contact with the antigens diffusing form the other wells
If the concentration is right (equivalence), the antibody will bind antigen in a complex that precipitates out.
You can tell the concentration of the antibody in the patient’s serum by how far the antigen was able to diffuse before it was precipitated out - if it gets closer to the serum well, hen antibody concentration is low
If no precipitation occurs - the patient doesn’t have antibody to the antigen (or you did it wrong)
How do we use agglutination reactions to do blood typing?
- We take the patient’s blood cells and apply antibody to different antigens and look for which one causes agglutination
or - We take the patient’s serum and apply it to RBCs of a known blooc type and watch for agglutination
What is passive agglutination?
When you can’t see the agglutination with the naked eye, so you tag the antibodies with something you can see like latex bead
What virus do we often use hemaglutination inhibition assays for?
influenza - good for typing
What does agglutination look like on a hemaglutination inhibition assay?
The RBCs are in a suscpension, so when agglutination happens, the antibodies bind to the RBCs and hold them up to the surface, so you see them all across the top instead of grouped together in the center
If a hemaglutination inhibition assay keeps the RBC and antibody against the flu constant, but varies the concentration of flu virus, What’s the reaction that occurs and what is conidered a positive result?
When the flu virus is at a greater concentration than the antibodies, then the flu virus doesn’t get bound by antibody and is free to interact with the RBCS and cause agglutination
If the patient does have antibody in high concentration, then it will bind the flu virus before it has a chance to interact with the RBCs
So a positive result is agglutination occuring down in the high concentraiton on the left, which decreases as you go to the right
If a hemaglutination inhibition assay keeps the RBC and flu virus constant, but varies the amount of patient serum (aka, varies concentration of antibody), what reaction occurs and what is considdred a positive result?
If there’s antibody in the patient’s serum, it will block the virus, blocking its ability to interact with RBCs and cause agglutination
If there is NO antibody in the patient’s serum, the virus will be free to bind RBCs and cause agglutination
So here, you see the agglutination with the lwo concentration on the right and no agglutination with th ehigh concentration on the left
Describe a neutralization assay
- Take a test cell and add it to patient’s serum
- add a toxin
- if the patient has no antibody to the toxin, the toxin will kill the test cell
- if the patient does have antibody to the toxin, the cell will survive
What do complement fixation assays take advantage of?
complement’s ability to lyse cells
How does a complement fixation assay work?
- Use patient’s serum and add test RBCs
- Add hemolysin, which will bind to the RBCs
- Add the antigen you want to know if the patient has antibodies for
- Add complement
- If the patient does not have antibodies to the antigen, the complement will bind the hemolysin on the RBCS and lyse them
- IF the patient DOES have antibodies to the antigen, the complement will preferentially bind to the antibody-antigen complexes and the RBCs will survive
What are the two general types of microscopy we use to identify immunological functions?
- immunohistochemistry
2. immunofluorescence
What’s the difference between direct immunofluorescence and indirect immunofluorescence?
direct: the antibody that is specific to the antigen of interest is labeled with a fluorescent tag
indirect: you use a tagged antibody that’s specific to the constant region of the antibody that’s specific to the `antigen of interest - this is cheaper and more versatile
Describe a DIRECT ELISA assay.
- start with ANTIBODY on a plate - specific tot he antigen of interest
- Use a positive and negative and compare the patient’s serum
- let it incubate a bit, then wash off any unbound antigen
- Use the same antibody you had on the bottom, only with a tag now
- visualized
THIS IS A SANDWICH ELISA - you DIRECTLY look for antigen in the patient’s blood
If the patient does have antigen - then it will bind the antibody and fluoresce
Describe an indirect ELISA assay.
- start with ANTIGEN on the surface of the well
- Use a positive, negativ control and patient serum
- Was off anyting unbound
- Then add a tagged IgG specific for the Fc of the antibody you’re interested in
If the patient has antibody, then it will bind the antigen and then fluoresce with the tag
You INDIRECTLY look for antigen in the blood by looking for antibody to the antigen
How does a competitive ELISA work?
- start with ANTIGEN on the surface of the plate
- Mix patient serum with a known concentration of antibody specific to the antigen of itnerest
- place this mix in the wells
If the patient has the antigen in their serum, the loose antibody will bind the antigen in the serum and no antibody will be left over to react to the antigen that’s bound to the plate and you won’t have anything tagged
So a positive result here is NO result
Which is more sensitive - an indiret or competitive ELISA?
comeptitive is more sensitive
How does an ELISPOT assay work?
- coat the bottom of a plate with antibody specific to an antigen - usually a cell product
- Plate cells (like T cells) onto the plate and let them grow
- Then wash the cells off
- If the T cells were making what you expected them to make (usually IFN gamma), the antibodie son the bottom of the plate owuld have bound to that product
- Then you add a second tagged antibody specific for that product and see if you can visualize
How does a Western blot work
- take PROTEIN from cell sample
- Denature it in buffer and head to linearize it
- Load the protein in a gel and run via electrophoresis
- proteins will be separated out by size - big at the top, small at the bottom
- Transfer the proteins to a nylon membrane
- Treat with a tagged antibody specific to the protein of interest
- VIsualize the nylon under fluorescence and see if the protien you’re interested in is there
Give an example of why a western blot wouldn’t work even though th eprotein is there?
the antibody might not be able to bind to it unless it’s in it’s specific conformation
How do you overcome that problem?
You can incubate the protein with the antibody BEFORE you denature it
How does flow cytometry work?
You take a single cell suscpension and tun them through a capillary tube that makes them go single file
then you run them past a detector which will tell you how big a cells is (forward scatter) and how many granules are insid ethe cell (side scatter)
You can also prelabel them and the detector will count out the differnt populations
