Immunology in the Clinic and Research Lab Flashcards
Describe the structure of antibodies.
Antibodies are fairly complex. They are made up of two heavy chains and two light chains held together by disulphide bridges.
We can split the antibody into two parts: the Fab region that binds to the antigen, and the Fc region which interacts with various aspects of the immune system to modulate various processes.
The Fab region can bind to specific epitopes on antigens via the complementarity determining regions (CDRs), also called the hyper-variable regions.
The Fc region, as mentioned previously, can interact with various aspects of the immune system to modulate processes like antibody-dependant cell-mediated cytotoxicity (ADCC), or antibody-dependant cellular phagocytosis (ADCP), or fixation of compliment. It is also related to the half-life of your antibody.
Briefly, describe the polyclonal antibody response.
We have B-cells in our body that have specificities to particular antigens. The binding of the epitope on the pathogen to the B-cell activates it, causing it to proliferate. They form clonal B-cells that will then secrete the antigen-specific antibodies.
Describe the method to making monoclonal antibodies.
We take the antigens (to which we want to make antibodies) and inject them into a mouse. After about a week or two, we harvest the B-cells that make the antibody.
We then take those B-cells and fuse them (in polyethylene glycol) with immortal myeloma cells. The myeloma cells are derived from a B-cell tumour but do not produce antibodies themselves. They lack the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene.
After this fusion process, we have a mixture of cells. We’ll have unfused B-cells and myeloma cells, then we have the fused cells called hybridomas.
We want to select these cells and get rid of the unfused cells.
We use Hypoxanthine-Aminopterin-Thymidine (HAT) selection.
HAT basically stops cells making DNA using the normal pathway. What can happen is that cells that contain the HGPRT gene can still make DNA using a different pathway.
The myeloma cells don’t have this gene so they can’t make DNA and die off. The B-cells do have the gene and can make DNA, but they are naturally short-lived, so they die off after a short time. The only cells that survive are the hybridomas.
We take the mixture of hybridoma cells and dilute them out to a single cell, then we culture that cell individually. The assay will then divide to form clones of the original cell. These cells will now produce an antibody of only one specificity. Those are called monoclonal antibodies.
What are some features of monoclonal antibodies?
- Hybridomas can be stored indefinitely and grown to produce monoclonal antibody when required.
- Antibody genes can be cloned from the hybridomas which allows antibodies to be engineered for different applications.
- Polyclonal or monoclonal antibodies can be produced which bind to Fc regions of particular antibody classes e.g. to IgG’s, IgA’s etc. These are called anti-isotypic antibodies.
Define immunoassays.
Using the antibody-antigen interaction (one of which is “labelled” or “tagged” to allow its detection), we measure the amount/concentration of antibody or antigen.
It is very specific and sensitive, hence, it is widely used in research and analytical labs.
Immunoassays use polyclonal or monoclonal antibodies.
What labels are used for immunoassays?
They were originally radioactive (radioimmunoassay, RIA), but now the antibodies are normally coupled to an enzyme (for e.g., horseradish peroxidase or alkaline phosphatase) which which convert substrate into a coloured substance. This can be detected using colourimetry.
This is called ELISA (enzyme-linked immunosorbent assay).
Other alternatives are luminescent.
What are the two types of solid phase immunoassays, such as ELISA?
DIRECT/INDIRECT: often used to quantify an antibody
SANDWICH (CAPTURE): often used to quantify or detect an antigen
Using these assays, the concentration of analyte (antibody or antigen) in the sample can be calculated by comparison to analyte standards of known concentration.
Describe how to perform a direct ELISA.
With a direct ELISA, we take an ELISA plate and we add antigen onto the plate. This is immobilised on the bottom of the well.
We then add our test antibody solution, which may be linked to an enzyme (like the ones mentioned previously), and we then add the enzyme substrate.
If the antibody binds to the antigen, when we add our substrate, the substrate will become coloured. We can then measure the amount of colour using a spectrophotometer.
What are some uses for the direct ELISA?
- screen hybridoma supernatants
- detect exposure to infectious agent
Describe how to perform an indirect ELISA.
With direct ELISA, it is similar to indirect ELISA, but there is one more step.
When we add the antigen onto the ELISA plate, we first add the primary antibody which is not labelled. Then, we add the secondary antibody, which is the one we are trying to detect. The secondary antibody binds to the Fc region of primary antibody.
Secondary antibody is often polyclonal and so may bind to different epitopes on a primary antibody. This allows multiple secondary antibodies to bind to the same primary antibody thereby amplifying the signal and increasing the sensitivity of the test
The amplification of the secondary antibodies is very helpful if you have a low amount of antibody that you are trying to detect.
Describe how to perform an Elispot immunoassay.
Another assay used in the labs, and is based on the ELISA, is called Elispot. It is used to detect cytokines that are given off by cells.
We have antibodies that are specific for the cytokine we are trying to detect, and they have been immobilised on the bottom of an ELISA plate. We add activated T-cells to the plate, so the T-cell will then secrete the cytokine. If it is the correct one, it will bind to the antibody.
We can then come in with a second antibody conjugated to an enzyme to detect a certain colour.
Describe how to perform an SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) immunoassay.
We start off by taking the sample with the protein that we are trying to detect. Typically, we will boil it with sodium dodecyl sulphate. This binds to the protein and gives it a net negative charge.
We will then run that protein in a polyacrylamide gel. The protein will run through the gel according to its size. We will then take the gel and blot the protein onto nitrocellulose, which is like paper.
We can probe that nitrocellulose with an antibody that is linked to an enzyme that gives it a coloured product. Thus, when we add the substrate, we can see a band form on the nitrocellulose.
We will then compare the result to protein standards of known size. We can also tell, if there are smaller bands underneath where the protein should be, that it has been degraded into those smaller bands.
Compare using ELISA and SDS-PAGE/WB (western blotting).
SDS PAGE/Western blotting often used alongside ELISA (to give us as much information as possible about protein).
In WB, protein concentration can be measured by comparing intensity of band we are detecting to band from a protein standard of known concentration.
If the protein is degraded, it may be more useful to use WB to calculate the protein concentration, as some of degradation fragments may contribute to the signal in ELISA if both coating and detecting antibody are able to bind to them.
How can you use antibodies to purify immune cells?
The antibodies are covered with microscopic magnetic beads, and they will bind to certain proteins on the surface of the immune cell that you’re trying to isolate.
Once bound, you put the cells into a machine that has an iron wall mesh. When you apply a magnetic field, only the bound cells will stick to the iron mesh; the other cells will be washed out.
We can then remove the magnetic field, releasing the cells and collect them.
Describe how the antibody-antigen interaction is used in flow cytometry.
Individual cells within a mixed population are tagged by treatment with monoclonal antibodies which bind to surface molecules and are labelled with fluorescent dyes.
Mixed cells are then forced through a nozzle to form stream of single cells.
Individual cells pass through a laser beam which scatters light and causes dye to fluoresce and provides information on bound antibody and cell surface protein.