20 Experimental Systems and Methods Flashcards
In antibody generation, polyclonal antibodies are secreted by…
… multiple clones of antigen-specific B cells
What are the Pros of antibody generation? Cons?
Pros: A mixture of Ab directed toward a variety of epitopes are formed, good for agglutination and immunoprecipitation
Cons: May produce cross-reactivities against other Ag, Different bleed points may yield different degrees of affinity
What is a monoclonal antibody?
the product of a single stimulated B cell by fusing a B cell and immortalized myeloma cell; produces mass quantities of single-specificity Ab
When can immunoprecipitation be performed?
In a solution when Ab/Ab concentrations are roughly equal which leads to the formation of large complexes to purify antigen molecules or remove antigens from a solution
Hemagglutination reactions can be used to
detect any Ag conjugated to the surface of red blood cells. Antibodies that can bind a surface Ag will cross-link the RBCs, forming a large clump that doesn’t settle
When are agglutination reactions used?
Routinely used in blood typing; can be adapted to measure Ab levels in sample directed against any Ag that can be attached to the surface of a Recombinant Glycoprotein
Antibody assays based on molecule binding to solid-phase supports
- Radioimmunoassays (RIAs): measure concentrations of biologically relevant proteins/hormones in body fluids
- Enzyme-linked immunosorbent assay (ELISA) assays: use antibodies/antigens covalently bound to enzymes (Indirect ELISA: detects presence and cx of Ab in sample)
ELISPOT assays
measure molecules secreted by individual cells
Western blotting
IDs and quantifies (roughly) specific protein in a mixture of proteins
Methods to determine the affinity of antigen-antibody interactions
Equilibrium dialysis: measure antibody affinity for antigen
Surface plasmon resonance (SPR): measurements of antibody affinity can be made
What is the practice of creating microscopic visualizations of cells and subcellular structures?
Immunocytochemistry and immunohistochemistry through techniques such as
Immunoelectron microscopy (gold beads to visualize Ab-bound Ag)
Immunofluorescence-based imaging (fluorescence use to visualize cells/molecules)
Confocal fluorescence microscopy (images w/ extraordinary clarity), for greater resolution, multiphoton fluorescence is a variation of this
Intravital imaging: observation of immune responses in vivo
3-D Fluorescent In Situ Hybridization (FISH): fluorescent dye-labeled RNA
What is flow cytometry?
scattered and fluorescent light from cells flowing past a laser beam are measured with a flow cytometer with cells “stained” using fluorescently-tagged mAb. Used to sort cell populations based on user-defined parameters
Magnetic activated cell sorting
Cells are incubated with tagged mAb, then passed across a metal mesh with a magnetic field applied; mAb-labeled cells will adhere to the mesh, while non-labeled cells wash through
