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- can bind to antigens
- every antibody has the same constant region (at end of heavy chain)
How do you form antibodies?
inject protein into rabbit--> not recognized--> antibodies form--> circulate through bloodstream and search for antigens
cross-reaction of antigens occurs in that one epitope on a particular antigen may recognzie similar epitope on another antigen--> complication
Because of the complication of polyclonal antibodies, what do you do?
use monoclonal instead
How do we form monoclonal antibodies?
inject the protein to form antibodies (in the spleen)
- take spleen cells out
- Find one or two thata re making antibodies unique for the epitope of interest. Because they only do this for a short period of time, we fuse the to myeloma cells--> lives forever as long as it has nutrients
Grow the cells in culture and separate one at a time.
One by one, find the exact cell that is synthesizing our antibody taht is specific for our epitope
With what method can we use antibodies to find target proteins?
- indirect: find the antibody
- sandwich: find the antigen
Explain indirect ELISA.
enzyme-linked immunosorbet assay
used for find antibody and determine whether they are producing antibodies against a particular antigen
In a small well, coat the bottom with antigen. Take blood sample from the animal and incubate it into the well with buffer
If they are producing antibodies for that antigen, it will bind and be attached.
Washing the solution to get rid of unbound proteins is the next step. Although it is bound, we cannot see it. As a result, we add a second antibody with marker molecule (or enzyme) attached
The two most common enzymes are peroxidse adn alkaline phosphatase. This enzyme does not alter the function of the antibody.
This second antibody with enzyme attached will bind to the first antibody
At this point, wash it again. Inject a substrate that will react with the enzyme, causing a product to form and a change in color of the solution. Yes, they are attached (This is called colorometric assay)
How do we get the second antibody to stick to the first?
we cut off the constant region of the primary antibody, inject it into the host so that the body builds up antibodies against the primary antibody. Afterwards, draw the blood and centrifuge, creating an antiserum with the secondary antibodies.
Explain sandwich ELISA.
test for presence of an antigen
To capture the antigen, we need a monoclonal antibody that binds the antigen.
Take the blood sample, put it in the solution. If the antigen is present, it will bind--> we won't see because it's colorless.
Add a secondary enzyme bound antibody that is specific for the antigen (but binds at a different epitope).
The antigen is sandwiched in. Substrate is then added and the color determines whether it is present or not
Explain Western blotting briefly.
done with antibodies
combines SDS-PAGE with the use of antibodies to detect proteins
Steps of Western Blotting
take protein sample and run through gel, separating it by size.
--> In our sample, we want to know whether our protein is present and, of the tissues, which has the protein.
- Proteins are seParated in the gel and they contain the antigen
- --> Take the gel and put in a contraption with filter paper. The paper will be placed onto the gel and an applied current will cause the proteins to pull away from the gel onto the surface of the membrane. This creates a polymer sheet that is a mirror image of the bonds in the gel.
- Now that the protein is more accessible, we put it in a buffer and shake
- --> An antibody is added. If an antigen is there, the antibody will bind to it.
- --> Still colorless
- We label a second antibody that contains a tag and is specific for the primary antibody and rinse with this second antibody . A tag on the antibody enables the identification of the model. Or, the antibody can have an enzyme that reacts with a substrate to form a colored product.
- The product will reciitate where the band was located. A bigger band means more protein