Antibody-Antigen Interactions (Immunoassays) Flashcards
The basic mechanisms by which antibody binds antigen
- the variable regions are really variable
- some parts of the variable regions are hypervariable and these are the parts that bind antigen
- the variable region can be placed in front of any constant region
- the light chain has its own variable region (from its own genes)
- two light chain constant region types are possible (kappa and gamma)
- variable regions and light chains are not tied to one distinct constant region type
Antibody affinity
-the expression of the sum of all the interactions between an antibody binding site and its homologous antigenic determinant
Antibody avidity
the strength of of binding of multivalent antiserum to multivalent antigen
Antibodies are heterogeneous
- polyclonal
- many B cells respond to antigen
- antibodies to many different antigens in serum
- multiple specificities and affinities
Use of Antibody in the Lab
- antibodies are specific
- antibodies are used to measure many biologic parameters
- many different immunoassays exist
Specific Antibodies
- affinity purify
- adsorb non-specific antisera
- monoclonal antibody
Affinity Purify Antibody
- makes the antibody more specific by purifying the antibody you want
- have the antibody stick to the specific protein you want (protein covered bead)
- the other antibodies wash away
- then you can specifically detached desired antibody and collect
Absorb the cross-reacting antibody
- makes the antibody more specific by removing the contaminating antibody
- have undesired proteins and antigens
- wash through desired antibodies
Monoclonal antibodies
- made from a single cloned B cell
- one specificity
- once produced the clone can survive forever
- huge amounts can be produced
- less cross reactivity
- lower affinity and little avidity
Preparation of Monoclonal Antibodies
1) Immunize an animal (mice are commonly used)
2) Isolate spleen cells from the immunized animal
3) Fuse spleen cells to plasmacytoma tumor cells
4) Select for only those cells that are hybrids of Tumor cells and B cells- by growing in drug-containing medium
5) Clone the hybridomas so that each single cell grows up independently
6) Select the individual clone with the specificity that you are interested in
Serum sickness
- serum sickness is a type of hypersensitivity reaction. Specifically, it is an immune system reaction to certain medications, injected proteins used to treat immune conditions, or antiserum
- symptoms (fever, rash swollen lymph nodes) usually do not develop until 7-21 days after the first dose of antiserum or exposure to the medication. However, people may develop symptoms in 1-3 days if they have previously been exposed to the substance
Four types of therapeutic monoclonal antibody
- murine monoclonal antibodies (suffix -omab)
- chimeric (constant regions are human) monoclonal antibodies (suffix -ximab)
- humanized (only the points of contact with the antigen remain mouse) monoclonal antibodies (suffix- zumab)
- fully human monoclonal antibody (suffix- umab)
Enzyme-Linked ImmunoSorbant Assay (ELISA)
- antigen is stuck to the bottom of a well or tube
- antibody is added, allowed to incubate, and the unbound antibody washed away
- a second antibody that will bind to the first antibody is then added
- this second enzyme has an enzyme molecule covalently bound to it
- after another incubation period the unbound second antibody is washed away
- the amount of second antibody is detected by adding a chemical reagent that turns color in the presence of the enzyme bound to the second antibody
- could also do a sandwich ELISA
Immunofluorescence
- tissue or cells are reacted with antisera specific for a cell marker or pathogen
- the unbound antiserum is removed by washing
- a second antibody specific for the first antibody is added and allowed to bind
- attached to the second antibody is a molecule that is fluorescent, that is will emit visible light when exposed to UV light
Flow Cytometry
- immunofluorescence can be automated by using a device called a fluorescence activated cell sorter or flow cytometer
- these machines scan large numbers of cells for immunofluorescence as well as cell size
- using highly focused lasers tens of thousands of single cells are analyzed in a few minutes
- cell sorters can analyze and sort cells by fluorescence while flow cytometers are only capable of analyzing cells
