Antibodies as Tools in Research and Medicine Flashcards
Antibodies have many uses in the clinic, research laboratories and diagnostic services.
How are they produced, and what are they used for?
Antibodies can be produced by immunising animals (typically rabbits, mice or goats) or by cells called hybridomas that grow in tissue culture.
The antibodies can be purified and used for detecting and quantifying the antigens that they recognise.
Provide examples of diagnostic uses of antibodies.
- detection of pathogens (e.g., ,bacteria, viruses, toxins) in patient samples,
- detection of antibodes in blood that indicate exposure to an antigen (e.g., Coronavirus test),
- detection and measurement of hormone levels (e.g., thyroid hormones, pregnancy tests).
- analysis of blood cells and immune cells (e.g., blood and tissue typing, enumeration of cell types),
- as therapeutic medicines for certain medical conditions (e.g., treatment for certain cancers, Crohn’s disease, psoriasis).
What is serum?
The liquid portion of blood that remains after the blood has been allowed to clot.
How can antibodies be isolated from an animal’s serum?
An animal (rabbit, goat, etc.) can be immunised with an antigen (e.g., a virus, snake venom) and antibodies can be isolated from the animal’s serum.
The serum will contain a mixture of antibodies that recognise different epitopes on the antigen that was injected.
What is a polyclonal antiserum?
Most antigens have more than one epitope and will activate several different B cells, each recognising a different epitope.
Each activated B cell will proliferate to give rise to a clone of cells, and some clones will differentiate to become plasma cells that secrete antibodies that recognise that a specific epitope.
Since there are many different types of plasma cell clones secreting different antibodies (i.e., antibodies that recognise a different epitope on the antigen) into the serum, the mixture of antibodies purified from the serum is called a polyclonal antiserum (plural = antisera).
Explain why a monoclonal antiserum is more desirable than a polyclonal antiserum.
Although polyclonal antisera are important tools in medical and scientific research, they are not a standardized reagent and there will be a limited supply of the polyclonal antibody.
Sometimes it is necessary to detect only one epitope on a pathogen. For example, if you want to detect the presence of a mutant virus strain that differs from the wild type virus strain in only one epitope, polyclonal antiserum would not be helpful.
A polyclonal antiserum that recognised all the epitopes on the virus wouldn’t be able to distinguish the mutant virus from the wild type virus.
Therefore, for a variety of reasons, it is often desirable to have an infinite supply of identical antibodies that recognise only one epitope: a monoclonal antiserum.
What is the process of producing a monoclonal antibody?
The process of producing a monoclonal antibody begins by repeatedly immunising a mouse with an antigen in order to get a good secondary immune response.
B cells specific for different epitopes on the antigen will become activated, proliferate, and differentiate into clones of antibody-secreting plasma cells.
What is a myeloma cell?
The myeloma is a plasma cell tumour, but has lost the ability to make its own antibodies.
How are the antibody-secreting plasma cells from a lab mouse immortalized?
After removing the mouse’s spleen, the antibody-secreting plasma cells are immortalized by fusing them with a type of myeloma cell that can grow indefinitely in culture.
B cells are fused to myeloma cells using a chemical called polyethylene glycol to create a hybridoma; a cell that has characteristics of both the B cell (e.g., the ability to make antibody and the ability to grow in the presence of certain drugs), and the myeloma (e.g., immortality).
How long can Plasma B cells live in a culture?
How long can a hybridoma live in a culture?
Plasma B cells only live (and secrete antibodies) in culture for a few days before they die, whereas the hybridoma can grow and divide indefinitely in tissue culture dishes.
Since the plasma cells that were fused to the myeloma cells were a mixture of different B cell clones, you now have a mixture of immortal hybridomas making antibodies against different epitopes.
How are hybridoma clones making antibodies against the desired epitope selected?
To obtain clones of the hybridoma that is making antibody to the desired epitope, a single cell is placed in each well of a 96 well tissue culture plate.
The cells in each well will divide and form clones of identical cells.
Hybridoma clones that are making antibodies against the desired epitope are then selected.
This is achieved by taking a little bit of the culture medium from each well, and using an ELISA assay to see if the antibodies that were secreted into the medium by the hybridoma cells bind to the epitope of interest.
How are large amounts of monoclonal antibodies produced?
A selected hybridoma clone can be grown indefinitely in culture and can be used to make large amounts of the monoclonal antibody.
The antibody secreted into the culture medium can be collected and purified and the hybridoma cells can also be frozen for long term storage.
Describe the production of monoclonal antibodies.
What is a hybridoma?
The technique involves fusing myeloma cells that cannot produce antibodies (but multiply indefinitely) with plasma cells which product antibody (but are short-lived).
HGPRT is an enzyme that allows cells to grow on a medium containing HAT (or hydroxanthine, aminopterin, and thymidine).
The resulting hybrid cells, called hybridomas, grow at the rate of myeloma cells but also produce large amounts of the desired antibody.
Only hybridomas can live in the HAT medium; unfused myeloma cells, lacking HGPRT, die in the medium.
Unfused plasma cells also die in the medium because they are naturally short-lived.
What is ELISA?
What is it used for?
Enzyme-linked immunosorbent assay.
It is a very sensitive method for detecting the presence of antibodies or antigens in fluids and determining their concentrations.
How is ELISA used to screen a population of hybridomas to see if any of them make an antibody to the antigen of interest?
The procedure starts by the coating of an ELISA plate with the antigen you want an antibody against. The coating is possible because the plastic of the ELISA plate has been specially treated so that proteins will bind to it.
A small volume of culture medium from hybridoma is added to the wells of the ELISA plate. After an incubation, the ELISA plate is washed. If antibodies that bound to an epitope on the antigen were present, they remain bound to well. Antibodies against other antigens will be washed away so that no antibody will be bound in those wells.
The next step is to determine which wells have antibodies bound to the antigen. This is done by using a secondary antibody – that is, an antibody that recognizes the heavy chain of another antibody. This is possible because the heavy chains of antibodies are species specific.
The antibodies produced by the hybridomas are mouse IgG antibodies since the hybridomas are derived from mouse B cells. To detect the mouse IgG bound to the wells, you use a secondary antibody, one that recognises the constant region of the antibody you want to detect.
Rabbits (or sheep or goats) are immunised with purified mouse IgG to produce the anti-(mouse IgG) antibodies. The secondary antibodies are then covalently linked to an enzyme (hence the “enzyme-linked” in ELISA), which allows you to detect the presence of the antibodies bound to the antigen in the well.
The type of enzyme that is linked to the secondary antibodies turns a colourless substrate into a coloured product.
Add the secondary antibodies to the wells, incubate 1 hour, pour out the fluid from the plate and wash out the wells. The unbound material is washed away and the secondary antibody will only bind to wells in which mouse IgG from the hybridoma medium had bound to the antigen (which is bound to the ELISA plate and coated onto the well).
If there were primary antibodies bound to the antigen, there will be some secondary antibody bound. If there were no primary antibodies bound to the antigen, none of the secondary antibody will remain (it does not bind to the antigen).
A colourless substrate is added, and if the secondary antibodies are present, the enzyme converts the substrate into a coloured product.
Thus, only the wells that had antibodies bound to the antigen will show colour.
How can ELISAs be used to screen a population of hybridomas for the presence of antigen?
ELISAs can also be used to screen for the presence of soluble proteins, such as cytokines or hormones.
Research laboratories use this type of ELISA to measure the concentrations of proteins secreted by cells into the surrounding medium.
In our example, we will measure the concentration of IL-4 (antigen) in a patient’s serum. IL-4 is a cytokine made by helper T cells which causes activated B cells to divide.
The procedure begins by using an unlabelled primary antibody to coat the wells of the 96 well ELISA plate. In our example, it involved coating the wells with antibodies against IL-4.
The next step is to add known amounts of the sample (IL-4) to some wells, and diluted patient serum to other wells. During incubation, the IL-4 binds to the antibody coated onto the wells. After 1 hour, dump out the fluid and wash the wells.
After the plate is washed, add an enzyme-linked antibody (secondary antibody) that recognises a different epitope on then IL-4 than the antibody bound to the well (primary antibody).
Having each antibody recognise a different epitope ensures that the two antibodies are not competing for the same spot on the antigen and have ample room to bind.
After 1 hour, dump out the fluid and wash the wells, disposing of secondary antibodies that have not bound to the antigen. If there were no antigens present in the sample, the secondary antibodies would have nothing to bind to, and would be washed away.
Add the substrate for the enzyme. The amount of coloured product produced is proportional to how much IL-4 was bound. The more IL-4 bound to the well, the more enzyme-linked antibody that binds and the more substrate that can be turned into a coloured product. Use the automated ELISA plate reader to determine numerical values for the amount of coloured product in each well.
To determine how much IL-4 was in the sample, the readings for the wells with the known amounts of IL-4 are used to create a standard curve. The standard curve is used to convert the absorbance reading in the ELISA assay (how much coloured product was generated) into picograms of IL-4.
Describe immunofluorescence.
In immunofluorescence, antibodies that are covalently linked to small fluorescent molecules (fluorescent-labeled antibodies) are used to detect proteins on cells.
The basic concept of immunofluorescence is to allow fluorescent antibodies to bind to cells.
The antibodies will only bind if the cells bear the antigen (epitope) that the antibody recognises.
After adding the antigen-specific antibodies (linked to fluorescent molecules) to a sample, the sample is rinsed to wash away the unbound antibodies.
The next step is to shine light onto the sample.
If the light is of the correct wavelength, it will cause the fluorescent molecules to give off light of a different wavelength.
Describe how the FACS sorts cells.
The following step would be to put the cells into the FACS instrument. The FACS instrument creates a stream of cells with one cell per drop.
A laser shines light on the stream of cells that excites the fluorescent molecules. If the cell has the fluorescent-coupled anti-CD3 bound to it or fluorescent-coupled anti-CD19, it will emit light. A detector determines whether each drop (cell) is emitting light or not. The computer counts the number of cells emitting light. The FACS instrument can analyze 10,000 cells in a few seconds.
How are cells labelled for fluorescent microscopy?
The cells are labeled incubated with a primary antibody that is tagged with a fluorescent compound, or incubated with a primary antibody and an tagged secondary antibody.
The slide is viewed with a fluorescent microscopy with a specific wavelength.
If two antibodies are used (say to different epitopes), they are labeled with different fluorescent compounds.
The slide is viewed with the first wavelength of light and an image is captured.
The slide is then viewed with the second wavelength of light and an image is captured.
For analysis, the two images are merged together to create the final image.
Describe the basic function of the fluorescence microscope.
The basic function of a fluorescence microscope is to irradiate the specimen with a desired and specific band of wavelengths, and then to separate the much weaker emitted fluorescence from the excitation light.
In a properly configured microscope, only the emission light should reach the eye or detector so that the resulting fluorescent structures are superimposed with high contrast against a very dark (or black) background.
How is the data from FACS presented?
Represented in an XY plot if one antibodies was used, or a four quadrant plot if two antibodies were used.
