topic 22 Flashcards
How are immunoprecipitation RXNs used? How do they work?
They are used for soluble antigens? Basically, you find the right amount of Ab needed to cause precipitation of Ag/Ab complexes without adding too much Ab to where there is no longer precipitate.
How can immunoprecipitation RXNs be used on solids? What are two types? Which is quantitative which is qualitative?
Radial immunodiffusion-Ab diffused into agar and plated. Antigen placed on agar and the distance the Ag diffuses is measured to determine the conc. of Ag (quantitative)
Double immunodiffusion-Similiar but ag/ab are placed next to eachother and if there is a precipate you’ll see. You can use various ag/ab and thus get a qualitative idea of the identity of unknown ag/ab.
What are agglutination rxns used for? How do they work?
They are used for particulate Ag. Can be used especially in blood typing. Basically, certain dilutions of ab are tried out and put into conical wells. If, when the Ag is added, there is a rxn with the Ab, a precipitate will occur and will settle all along the bottom of the conical well. If no rxn occurs, only the very bottom of the well will contain precipitate.
What can be done when a specific Ag-Ab reaction is occuring but agglutination is not occuring?
You could add a secondary antibody that recognizes IgG which will bind to the first antibody which will lead to agglutination.
How do Elisas work? INdirect? direct?
In indirect elisas, a known Ag is used to detect whether or no a specific Ab is present.
An antigen coated well is washed, then specific antibody to be measured is added, then it is washed, then an enzyme conjugated secondary antibody is added, then its washed, then a substrate is added that will react with the enzyme and produce a color which can be measured in order to measure levels of antibody of interest.
In direct elisas, a known Ab is used to detect whether or not a specific Ag is present.
Similiar process, but the well is coated with antibody and the antigen of interest is added.
How does western blot work?
The cell or virus of interest is dissociated and placed on a sds page where it has different bands. This is transferred to nitrocellulose and overlayed with antiserum. The bound antibody is detected with enzyme linked Ig-G which can show colors.
Basically an elisa in solid form.
How are antigenic epitopes visualized on or within cells? Indirect? direct?
Indirectly, an unlabed primary antibody ends up in a cell. then, a flourescently labeled antibody is placed in the cell and binds to the unlabeled one and can then be viewed with a flourescent microscope.
Direct, a labeled primary antibody is placed in the cell then its viewed.
Greater flourescent signalling results in indirect.
How does flow cytometry work?
flourescent-antibody labeled cells are dripped one by one from a container. they are then shot with a laser. Different antigens can be labelled with different colors. Receptors then pick up the coloring of the laser once it passes through and all this info is passed to a computer. The side scatter is debris. The forward scatter are cells. The computer can then say which perentage has this antigen, which has another, which has both and which has neither.
Also, electro magnetism can be used to sort the cells into different containers based on which label they receive.
What method is used to determine if cells can proliferate?
An antigen or mitogen is placed in a solution with cells. Radioactive hydrogen is then placed in the solution. If the cells are proliferating, you’ll be able to measure hydrogen in the cells.
How does a mixed lyphocyte reaction occur? How can t cell recogntion be tested in vitro?
Donor t cells are placed in a solution with recipients t cells. If the donor t cells aren’t recognized by the recipient t cells, no reaction occurs and all is well. If the donor cells do recognize the recipient t cells, they will proliferate and if radioactive hydrogen is added, this will result in radioactivity in the DNA.
What method is used to measure cytotoxic killing activity by CD8 cytolytic cells?
Label target cells with radioactive chromium. Add Cytolytic t cells to the solution. If the CTLs kill the target cells, radioactivity will be released from cells and can be measured.
