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Elise Notes
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An#bodies produced in B cells
• An#bodies are produced in B cells, which are a type of lymphocyte.
• B cells are produced in the bone marrow. • Each B cell is gene#cally programmed to display a specific membrane-‐bound receptor.
• When a B cell encounters an an#gen (Ag) that binds to its receptor, it becomes an ac#vated B cell.
Ac#vated B cells
• Ac#vated B cells divide rapidly and differen#ate into two types of cells: – Plasma cells are short-‐lived, and produce millions of an#body (Ab) molecules, which are soluble versions of the membrane-‐bound receptor specific to that B cell.
– Memory B cells produce only low levels of Ab, but they con#nue to grow and divide, and allow for a rapid response upon re-‐exposure to the Ag.
Polyclonal Ab • An#bodies that are raised in response to an exposure to a par#cular Ag will all bind to the Ag in slightly different ways.
• This popula#on of Ab is known as polyclonal an#bodies and will all recognize different epitopes (binding sites for Ab) on the same an#gen.
• Within the popula#on, some will bind with higher specificity or strength.
Monoclonal Ab
• OKen, it can be more useful to work with a single Ab whose binding is uniform.
• In that case, we need to produce monoclonal an#bodies, which are all iden#cal copies.
• This needs to be done in cell culture, fusing B cells and lymphoma cells (cancerous) to produce immortal hybridoma cells that produce the Ab of interest.
ELISA
• Enzyme-‐linked immunosorbent assay • Uses an#bodies to detect molecules that are present in such low concentra#ons that they cannot be measured directly.
• Most forms of ELISA are qualita#ve, not quan#ta#ve, so we only know if our target is present or not. We don’t know how much there is.
Direct ELISA
Simplest form of ELISA, a labeled primary Ab (1° Ab*) binds to the an#gen coated on the well of a plate. We can do a detec#on reac#on to see if molecule is present or not. This is not commonly used.
Indirect ELISA
More common form of simple ELISA, an unlabeled 1° Ab binds to Ag coated on well, then labeled 2° Ab binds to 1°. Detec#on reac#on shows which samples have the molecule. Also called an#body capture assay.
An#gen capture An#gen is sandwiched between two 1° Ab, one labeled and one unlabeled, which is bound to the well. This is used only for assays that are repeated oKen, like pregnancy tests. Also called two-‐site capture assay or sandwich assay. Ag needs to be large enough to allow binding to two Ab at once.
Compe##on ELISA
This is the only form of ELISA that can give you data about how much of your an#gen is present.
Quan#fying your assay
• Along with your sample, run a series of standards with known amounts of unlabeled an#gen.
• Set up a standard curve with the absorbance in the standard wells (aKer the detec#on reac#on) vs. log [Ag].
• Use this informa#on to determine the amount of an#gen in your unknown samples.
Reac#on Zone Test Zone
Control Zone
At RZ: unbound mouse monoclonal an#-‐hCGβ Ab*
At TZ: bound mouse monoclonal an#-‐hCGα Ab
At CZ: bound an#-‐mouse IgG Ab
Pregnancy Tests
Urine travels from this end of the filter paper, through reac#on zone, to test and control zones.