1-19 Immunoassays Flashcards
variable V region contains…
of immunoglobulin molecule contains amino acid sequences coding for the antigen binding site
framework residues
not directly invovled with antigen binding, essnetial in producing the correct folding of the variable region
hypervariable regions
portions of V region with great deal of variability in sequnce composition which provude the sequence of the very specific antigen binding site.
the heavy and light chain contain _______ regions each
3 hypervariable regions each
relative locations of the hypervariable regions?
distant on primary sequence, on separate chains, close together on antigen binding site after folding.
epitope
site on complex antigen that is bound by a single antibody
antibody affinity
the sum of the interactions between a single binding site and an epitope. monoclonal
Describe the basic mechanisms by which antibody binds antigen
The variable region of the antibody binds the epitope of the antigen. The epitope can be a few amino acids, carbohydrates, or modified amino acids. Antibody affinity (the sum of the interactions between a single binding site and an epitope) and avidity (the sum of strengths of the bindings of multivalent antiserum to a multivalent antigen) are used to describe how strong the binding is.
avidity
the sum of strengths of the bindings of multivalent antiserum to a multivalent antigen
Explain the concept of monoclonal antibody and how they are used
Antibodies formed to only react with antigens of interest, as opposed to other contaminants in an immunization mixture.
Originally produced by fusing immunized mouse spleen cells with tumor cells, then selecting for hybrids of the tumor cells and B-cells, then cloning the hybridomas so individual cells can develop on their own, then selecting for the clone that produces the antibody of interest.
The resulting antibodies were mouse antibodies. Now molecular biology techniques allow for the production of purely human monoclonal antibodies. If a drug name ends in “mab” then it is a monoclonal antibody.
Enzyme-Linked ImmunoSorbant Assay (ELISA)
Most widely used assay
- Antibody is adhered to the bottom of a well.
- Antigen to that antibody is added and allowed to incubate, then excess antigen is washed away.
- A second antibody for that antigen (which is also bound to an enzyme) is added and allowed to incubate, then excess of the second antibody is washed away.
- Finally, a chemical that changes color in the presence of the enzyme is added.
- The degree of color change is used to assay the amount of the antibody present.
This assay can be used to assay determine the presence and concentration of antigen within a sample.
Fluorescence Activated Cell Sorter (FACS) Flow Cytometry
Machine uses lasers to sort immunofluorescently labeled cells by amount of fluorescence (FACS) and cell size flow cytometry).
Some machines use multiple lasers, allowing for multiple fluorescent labels to be used in identifying multiple cellular antigens at once.
Western Immunoblot
- A cell lysate or serum is run electrophoretically, and the separated proteins are bound to nitrocellulose paper.
- Antibody to the protein of interest is reacted to the nitrocellulose and binds.
- Unbound antibodies are washed away, and bound antibody is detected as in ELISA.
- Both quantitative (amount of antigen determined) and qualitative (molecular weight of antigen determined).
Immunofluorescence
- Antiserum specific for a cell marker or pathogen is added to a tissue or cell sample.
- Unbound antiserum is washed away.
- A second antibody (with a UV fluorescent molecule attached) specific for the first antibody is added.
- Unbound second antibody is washed away, and UV light is used to visualize the sample.
Can be used to identify specific cell types, cellular structures, or pathogens within cells or tissues.
Chimeric
oldest form. variable regions from mouse monoclonal. constant regions are human
