Block A - 2

Question 1

ouchterlony test ?

Answer 1

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.

Question 2

example of antibody based assay which allows using the antibody without further development ?

Answer 2

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.

Question 3

what is antibody conjugation ?

Answer 3

However, often require conjugation of an enzyme or fluorochrome to allow detection of the antibody.

Question 4

things to consider when preparing antibodies for use?

Answer 4

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

Question 5

example of enzyme conjugation ?

Answer 5

Enzymes (eg. horseradish peroxidase)

  - used to catalyse a reaction, often resulting in colour change can be measured using a spectrophotometer

Question 6

conjugation radiation ?

Answer 6

Radiation

  - can link/incorporate radioactivity emitters with the Ab .
  - subsequently detect the radiation (alpha, beta or gamma) as appropriate.

Question 7

fluorescence conjugation ?

Answer 7

Fluorescence

  - increasingly popular as avoids hazards associated with radiation.  BUT 
  - can require expensive equipment to detect

Question 8

serology ?

Answer 8

To detect or quantify antibodies made in response to microbial infections. This can be called serology - measure antibody levels in serum.

Question 9

when are antibodies produced ?

Answer 9

Antibodies are produced during infections and can be used to determine if a person has experienced an infection.

Question 10

antibodies and strain of microbe ?

Answer 10

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).

Question 11

type of antibody produced reveal ?

Answer 11

The type of antibody detected can inform you if the person has a historic infection or a recent infection - IgM recent or IgG historic.

Question 12

toxoplasma Gondii infection (igG positive)

Answer 12

Then she is very unlikely to pass disease vertically to her fetus and will not normally be treated.

Question 13

toxoplasma gondii infection (IgM positive )

Answer 13

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.

Question 14

ELISA for IgG response ?

Answer 14

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.

Question 15

why are samples serially diluted ?

Answer 15

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.

Question 16

immunohistochemistry

Answer 16

The well of an ELISA plate can be swapped to a microscope slide with a tissue section present is called an immunohistochemistry.

Question 17

western blot ?

Answer 17

A membrane containing proteins that are separated on a gel is called a western blot can be done for radioactivity.

Question 18

polyclonal antibodies ?

Answer 18

These antibodies can be polyclonal (raised and purified in an animal).

Question 19

monoclonal antibodies ?

Answer 19

monoclonal (made by a clonal plasma cell that has been immortalised).

Question 20

why would pre culture need to be used ?

Answer 20

pre-culture is necessary to increase numbers to a detectable level.

Question 21

ELISA for a pathogen ?

Answer 21

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.

Question 22

ELISPOT assay ?

Answer 22

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.

Question 23

simplest way of measuring fluorescence ?

Answer 23

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.

Question 24

what is fluorescence ?

Answer 24

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).

Question 25

is there just one colour of fluorescence ?

Answer 25

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

Question 26

basis of fluorescence microscope ?

Answer 26

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

Question 27

light microscopy ?

Answer 27

Two different antibodies with different specificities and labels.

• Different enzymes on each
• Results in different coloured products on substrate

Question 28

confocal microscopy ?

Answer 28

This is an adapted form of light microscopy which allows the 3D structure of a sample.

Question 29

flow cytometry ?

Answer 29

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.

Question 30

forward scatter ?

Answer 30

light emitted at narrow angles from the laser

Diffracted light

-I.e. shadow of the cell

related to cell size

Question 31

side scatter ?

Answer 31

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

Question 32

how are flow cytometry results displayed ?

Answer 32

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.

Question 33

single parameter ?

Answer 33

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

Question 34

two parameter dot plot ?

Answer 34

example CD3 and CD8

One axis shows first colour

Second axis shows second colour

Allows to look at individual populations of cells.

Question 35

fluorescence ?

Answer 35

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.

Question 36

what does flow cytometry not show ?

Answer 36

But location, location, location is not shown in flow cytometry and how cells interact.