Notesale: Turn your study into money

Document Preview

Extracts from the notes are below, to see the PDF you'll receive please use the links above

---

Immunoassay
Immunoassays (IA) are analytical techniques based on the specific and high affinity binding
of antibodies to a particular target antigen
...
The conjugated enzymes are selected on the basis of their ability to catalyze the
conversion of a substrate into a coloured, fluorescent, or chemiluminescent product
...
ELISAs
can be used qualitatively to detect the presence of antibody or antigen
...

Indirect ELISA
Indirect ELISAs can detect, or measure the concentration of, specific antibodies present in
serum
...
free Ab2 is washed away
...
The amount of
coloured, fluorescent, or luminescent reaction product that forms after incubation of the
enzyme with its substrate is measured using a plate reader
...

Sandwich ELISA
Sandwich ELISAs can be used to detect, or measure the concentration of, a specific antigen
...
Serum
containing unknown amounts of antigen is added to the well, the antigen binds to the
antibody
...
A second enzyme-linked antibody specific for a different
epitope on the antigen is added and binds to bound antigen
...

Competitive ELISA
Competitive ELISAs are highly sensitive assays used to detect, and determine the
concentration of, small amounts of antigen
...
The antigen-antibody mixture
is then added to an antigen coated well
...
Washing removes unbound antibodies
...
In the competitive
assay, the higher the concentration of antigen in the original sample, the lower the final
signal (ie
...

ELISPOT
ELISPOT assays allow the quantitative determination of the number of cells in a population
that are producing a particular type of molecule, such as a cytokine
...
For example, in a ELISPOT assay for
IFN-gamma, assay plates are coated with a monoclonal anti-IFN-gamma antibody
...

A known number of cells is then added to each well of the coated plated and incubated with
stimulating antigens
...
The plate is then washed to remove the cells and any excess reagent
...
The plate is washed
...
Substrates are colourless but when acted on by their
cognate enzyme they produce a solid, coloured product that precipitates out of solution,
leaving a coloured spot wherever the enzyme-conjugated antibody bound
...
The number of
spots are counted, and the fraction of cells in the original population that secreted the
cytokine of interest can be determined
...
If the emitted light
has a wavelength in the visible region of the spectrum, the fluorescence dye can be used to
detect any molecules bound by that dye
...

Other protocols utilize the affinities of easily obtained proteins (which can be readily
conjugated with fluorescent dyes), to bind to biologically important molecules
...

Modern instruments use combinations of several filters and mirrors that allow the investigator
to detect light emitted at multiple different fluorescence wavelengths
...

Cytometry
Cytometry allows for the localization of antigens
...

Flow cytometry
Flow cytometry is analytical technique that quantifies the frequencies of cells that are made
fluorescent by binding to fluorescent antibodies or to probes
...
The amount of light scattered
in a forward direction, that is, the same direction of the laser bean, provides a rough
measure of the size of the cell that caused the light scattering, the magnitude of forward
scatter is directly proportional to the size of the cell
...
When a flow cytometer is adapted to sort cell
subpopulations on the basis of their fluorescence and light scattering properties, it is referred
to as a fluorescence-activated cell sorter (FACs)
...

Advantages of flow cytometry include; high sensitivity, rapid as many cells can be analysed
in a short time frame, allows for the discrimmination of cell subtypes, and allows multiple
parameters to be assessed simultaneously
...
Cells passing through the flow cell are interrogated by the
laser
...
there, the induced voltages are digitized and represented
by the software in graphical form
...
Cells entering the flow cell are focused by
the encircling sheath fluid and exposed to the laser beam one cell at a time
...

Flow cytometry applications
The flow cytometer can be used to detect and to classify leukaemias in order to inform
treatment
...
when the number of CD4+ T cells in the blood of a patient with
AIDS falls below a certain level, the patient is at high risk for opportunistic infections
...
A transport inhibitor, such as brefeldin A, is added to block the secretion of the
produced cytokines
...

Bromodeoxyuridine-based assays
Bromodeoxyuridine (BrdU) assays are used to assess cell division/proliferation
...
cells that divide following BrdU
incorporation can then be identified using fluorescently labelled antibodies to BrdU
...
The greater the fluorescence intensity, the greater the proportion of anti-BrdU
bound, the greater the concentration of BrdU, thus the greater the rate of proliferation
...
The uncharged acetyl groups of the CFSE molecule enable CFSE to
enter a cell and are then cleaved by intracellular esterases, so that CFSE remains trapped in
the cytoplasm
...
The amount of fluorescence emitted is cut in half each time a CFSE labelled cell
divides
...




---