Block A - 2 Flashcards
ouchterlony test ?
Ouchterlony test it uses antibodies to bind antigens which is visualised using a gel matrix, you can see the lines of precipitation. This is relatively primitive and is not a test that relies on the conjugation of a label to the antibody.
example of antibody based assay which allows using the antibody without further development ?
Some antibody-based assays use the antibody without further development for example antibodies that recognise the antigens and binding to the red blood cells , agglutination occurs which is visualised. This formed the basis of testing the blood groups of people to allow for donation.
what is antibody conjugation ?
However, often require conjugation of an enzyme or fluorochrome to allow detection of the antibody.
things to consider when preparing antibodies for use?
In order to detect the specific binding of an Ab, need a way to read-out.
Various different things can be conjugated to antibody, to aid detection
example of enzyme conjugation ?
Enzymes (eg. horseradish peroxidase)
- used to catalyse a reaction, often resulting in colour change can be measured using a spectrophotometer
conjugation radiation ?
Radiation
- can link/incorporate radioactivity emitters with the Ab . - subsequently detect the radiation (alpha, beta or gamma) as appropriate.
fluorescence conjugation ?
Fluorescence
- increasingly popular as avoids hazards associated with radiation. BUT - can require expensive equipment to detect
serology ?
To detect or quantify antibodies made in response to microbial infections. This can be called serology - measure antibody levels in serum.
when are antibodies produced ?
Antibodies are produced during infections and can be used to determine if a person has experienced an infection.
antibodies and strain of microbe ?
In some cases it can even determine the strain of microbe with which a person has been infected (sometime referred to as a peptide ELISA).
type of antibody produced reveal ?
The type of antibody detected can inform you if the person has a historic infection or a recent infection - IgM recent or IgG historic.
toxoplasma Gondii infection (igG positive)
Then she is very unlikely to pass disease vertically to her fetus and will not normally be treated.
toxoplasma gondii infection (IgM positive )
then she is has a high chance of passing the disease vertically to her fetus and will normally be treated or elect for a theraputic abortion.
ELISA for IgG response ?
An Elisa can be used to measure the response of IgG. The reaction is stopped with an acid normally and this causes a stable colour change to occur which is measured on a plate reader or spectrophotometer.
why are samples serially diluted ?
to allow the endpoint to be determined
This is because of the large range of concentrations of Abs found in serum and that these are not all on a the linear portion of a graph of absorbance versus Ab concentration .
It is more consistent between assays from day to day than using a simple absorbance which allows a more accurate assessment of antibody present. The sample is diluted until you can no longer see any antibody present.
immunohistochemistry
The well of an ELISA plate can be swapped to a microscope slide with a tissue section present is called an immunohistochemistry.
western blot ?
A membrane containing proteins that are separated on a gel is called a western blot can be done for radioactivity.
polyclonal antibodies ?
These antibodies can be polyclonal (raised and purified in an animal).
monoclonal antibodies ?
monoclonal (made by a clonal plasma cell that has been immortalised).
why would pre culture need to be used ?
pre-culture is necessary to increase numbers to a detectable level.
ELISA for a pathogen ?
sandwich ELISA as the plate is precoated with antibodies rather than antigen. The pathogen is then put onto the Elisa pate and if the pathogen is present it binds to the antibody, a secondary antibody is then used and this will also recognise the pathogen, the antibody is then conjugated to an enzyme that drives the colour change.
ELISPOT assay ?
uses nitrocellulose membrane on the bottom and there is a capture antibody on the bottom of the plate and the cells of interest are put on top. If these cells are producing, then this will allow the appropriate antibodies to be used for capture. Normally used in the context of measuring the number of cells producing a specific cytokine. Then a secondary antibody is used to detect those antibodies. On the petri dish graphic below, each coloured dot corresponds to a cell producing that product being tested.
quantification of cytokine-producing cells.
simplest way of measuring fluorescence ?
Simplest way of measuring is using a fluorescence microscope, in order to achieve a fluorescent signal the fluorescent probe needs to be stimulated to capture the light at the appropriate spectrum wherever it is emitted.
what is fluorescence ?
Fluorescence is the stimulated emission of light from a substance which has absorbed radiation (light) of another wavelength. It is useful for immunoassays by conjugating fluorochromes onto antibodies. Some commonly used fluorochromes include Green Fluorescent Protein (GFP), fluorescein (FITC), phycoerythrin (PE).
is there just one colour of fluorescence ?
Lots of fluorochromes now available, allowing lots of different parameters to be measured in one assay by coupling each monoclonal antibody to a different colour
basis of fluorescence microscope ?
Can use microscopy to visualise thin slices of biopsies, tissue, etc .
There is an excitation light and a detection system
Stain with enzyme-linked of fluorescence-linked antibodies to detect the cells or molecules of interest
Visualise under light microscope to see colour changes (enzymes) or with laser-scanning fluorescence microscope
light microscopy ?
Two different antibodies with different specificities and labels.
- Different enzymes on each
- Results in different coloured products on substrate
confocal microscopy ?
This is an adapted form of light microscopy which allows the 3D structure of a sample.
flow cytometry ?
Measure’s fluorescence of single cells in a suspension, passes cells through a flow cell with a laser which is used to excite the fluorescence of the cells in the sample, then the light from each individual cell is captured individually and stored on a computer for analysis in real time.
forward scatter ?
light emitted at narrow angles from the laser
Diffracted light
-I.e. shadow of the cell
related to cell size
side scatter ?
Side Scatter
Light approximately perpendicular to the laser
Reflected light
- I.e. light reflecting from cellular components
Related to cell granularity and complexity , surface topography
how are flow cytometry results displayed ?
Flow cytometry results displayed as distribution histograms (single parameter studies) or 2D dot plots (multiple parameter studies). The characteristics of sub populations are possible by carefully selecting or ‘gating’ on the those cell subsets of interest.
single parameter ?
Horizontal axis: level of fluorescence - brighter cells further right
Vertical axis: number of events per channel number
Allows to look at level of expression of marker
two parameter dot plot ?
example CD3 and CD8
One axis shows first colour
Second axis shows second colour
Allows to look at individual populations of cells.
fluorescence ?
Fluorescence is the ability of flow cytometry to look at 1000’s to 1000000’s of particles is good for measuring many parameters.
eg. proportion/number of CD4+ T cells in population
eg. amount of cytokine being produced by cells
eg. upregulation of surface receptor.
what does flow cytometry not show ?
But location, location, location is not shown in flow cytometry and how cells interact.
