Immunoassays Flashcards
1
Q
points about antibodies
A
- variable regions are really variable
- some parts of the variable regions are hypervariable and these parts bind the antigen
- the variable region can be placed in front of any constant region
- the light chain has it’s own variable region
- the light chain constant region can be kappa or lambda
- variable regions and light chains are not tied to one distinct constant region type
2
Q
affinity
A
- the sum of all the interactions b/n antibody binding site and its homologous antigenic determinant
- only precise in a monovalent antigen-antibody system and antibody should be monoclonal
- affinity(K) =[AbAg]/[Ab][Ag]
3
Q
avidity
A
-sum of all affinities of antibodies since they are multivalent- can bind to many different parts of an antigen (epitopes)
4
Q
antibodies are heterogeneous
A
- polyclonal
- many B cells respond to antigen
- antibodies to many different antigens in serum
- multiple specificities and affinities
5
Q
use of antibodies in the lab
A
- antibodies are specific
- used to measure many biologic parameters
- many different immunoassays
6
Q
cross reactivity
A
- antiserum reacts with many antigens
- may be due to impurities
- immunizing agent (animal) is not pure
- impurities may be better antigens-antibody against second antigen
- due to common or similar structures on antigens
7
Q
specific antibody
A
- even most pure prep will induce antibodies with cross reactivity
- other antibody in blood of immunized animal-highly antigenic other proteins or homology
- may be ok for some immunoassays
- however may mask results, give false results, or lower sensitivity
- absorption and affinity chromatography eliminate cross reactivity, leads to more specificity but not a multiclonal antibody
8
Q
affinity chromatography (purity)
A
- makes antibody more specific by purifying the one you want
- put protein on agarose (CD4)
- pass antiserum over CD4 agarose
- only anti CD4 will bind (red in picture)
- other antibody will flow through (blue)
- add salt to get antibody off
-can also have antibodies bound to beads, add solutes, wash away unbound and then elute the antigen you want
9
Q
Absorption
A
- makes the antibody more specific by removing the contaminating antibody (the cross reacting one)
- if you have mixture of antiserum that binds B and T cells, but you only want the T cell one, you add bunch of B cells til they are all bound then centrifuge the B cells away and only the antibody for T cells will be left
10
Q
monoclonal antibodies
A
- made from a single cloned B cell
- one specificity
- once produced the clone can survive forever
- huge amounts can be produced
- less cross reactivity
- but lower affinity and little avidity
- since clone of one B cell is produced by fusing that cell to a tumor cell and isolating the clone with the antibody specificity of interest
11
Q
normal growing of anbibodies
A
- if you wanted CD4 antibodies from a rabbit, you would get T cells, Lymphocytes, and other antigens on all human cells because each antibody has a different specificity
- hundreds or thousand of B cell clones respond to one protein and its different parts leading to antibodies with different specificities- react to the protein you want and any other molecules in the preparation, wouldn’t detect the one you want
12
Q
production of monoclonal antibodies
A
- Immunize animal and raise B cells to antigen
- Isolate spleen cells from immunized animal
- Fuse the spleen cells to plasmacytoma tumor cells (myeloma)
- Select for only those cells that are hybrids of tumor cells and B cells (growing on a drug containing media-B cells will die anyway and it will kill tumor cells)
- Select for the antigen specific hybridoma
- clone the selected cells
13
Q
serum sickness
A
- when using mouse monoclonals, humans have a reaction
- hypersensitivity reaction
- immune system reaction to meds, injected proteins used to treat immune conditions or antiserum
- symptoms (fever, rash, swollen nodes) usually develop 7-21 days after, but then need second injection-1-3 days can die
14
Q
four types of therapeutic monoclonal antibody
A
- mouse
- chimeric- puts our constant regions on the monoclonal variable region
- humanized- takes out framework too-only points of contact with antigen remain mouse
- human-totally made with molecular biology
15
Q
source of antibody in drug name
A
- murine-oamb
- chimeric-ximab
- humanized-zumab
- fully human-umab
16
Q
ELISA
A
- Enzyme Linked ImmunoSorbant Assay
- antigen stuck to bottom of well or tube
- antibody is added and incubated, unbound antibody is washed away
- second antibody binds to first antibody
- second antibody has an enzyme molecule covalently bound to it
- another incubation period
- unbound second antibody washed away
- amt of second antibody detected by chemical reagent that turns color in presence of the enzyme
17
Q
fluorescently labeled antibody
A
- used to identify cells and their structures
- tissue/cells reacted with antisera specific for a cell marker or pathogen
- wash unbound antiserum
- second antibody with fluorescent molecule added
- special microscope
18
Q
Flow cytometry
A
- counts types of cells via lasers
- binds fluorescent antibodies to molecules of choice with different colors
- sends through a tube
- laser beam hits molecule and refracts light of a certain color to photomultiplier tube and computer counts number of certain types of cells
19
Q
Fluorescence activated cell sorting
A
- puts a charge on the molecule based on its color
- charge takes it to opposite charge plate
- red could be pos and go to neg
20
Q
reading cytometry
A
- can do one dimension or two
- 1st quadrant is both, 2nd is one cell, 3rd is neither, 4th is other cell
- see pictures
21
Q
western immunoblot
A
- determine apparent molecular weight and concentration of an antigen
- proteins separated electrophoretically
- bound to nitrocellulose paper
- antibody to protein of interest is reacted and unbound washed away
- detected like ELISA
- quantitative and qualitative
- amount of antigen, MW, and different forms
