Laboratory Techniques: Antibody Techniques, and Aseptic Techniques & Cell Culture Flashcards
What are antibody techniques used for?
Detection and identification of specific proteins.
What are antibodies?
Antibodies are y-shaped globular proteins produced by B-lymphocytes that will only bind to a specific antigen. B lymphocytes are produced in the spleen and bone marrow.
What is a polyclonal serum?
A serum containing many different antibodies.
What are monoclonal antibodies?
Antibodies which are identical and will bind to excatly the same feature of the antigen.. The generation of monoclonal antibodies is essential for all antibody techniques.
How are monoclonal antibodies produced?
- A mouse is injected with a specific antigen and after a while, B-lymphocytes are taken from the spleen of the mouse.
- In order for the B-lymphocytes to divide in culture they are fused with myeloma (cancer) cells which are immortal using polyethylene glycol (PEG). The cells produced are called hybirdomas.
- Each hybridoma is placed in its own screening well and cultures are grown for the hybridomas producing the antibodies of interest.
- The secreted antibodies are then extracted and purified using centrifugation.
What are monoclonal antibodies used for?
The diagnosis and detection of disease. Can be used to treat cancer and viruses such as HIV.
Describe how immonoassay techniques which use monoclonal antibodies with enzymes atttached such as ELISA work.
The reporter enzyme catalyses a colour change reaction which is used to detect and quantify the presense of an antigen.
- If the antigen is present, the antibody binds which causes the reporter enzyme to produce a coloured product to show the antigen is present.
What is fluorescent-labelled protein blotting?
A technique used for identifying specific proteins that have been seperated using gel electorphoresis.
How does fluorescent-labelled protein blotting work?
The proteins are blotted from the gel onto a nylon filter. This is then flooded with fluorescent labelled monoclonal antibodie which will bind to their target proteins and the excess is then washed away. When it is exposed to certain wavelengths of light the fluorescent labelled antibodies show the precise location of their specific protein.
What is immunohistochemcial staining?
A technique used to study tissues and visualise the distribution of specific cellular components in live cells.
When is bright field microscopy used and when is fluorescence microscopy used?
Bright field microscopy is used to examine whole organisms, parts of organisms or dissected tissues.
Fluorescence microscopy is used to study particular protein structures.
What is cell and tissue culture?
The growth of cells, tissues or organs in an artifical media in a laboratory to produce many genetically identical clones of an initial cell sample.
What is an inoculum?
The starting material you use to grow a culture from - usually growing cells from a previous culture. An inoculum made of plant/animal tissue is called an explant (most animal and plants meed an explant but other cell type just need one cell).
What are aseptic techniques?
A set of precautions taken to prevent contamination. They include washing hand and sterilising equipment and work surfaces.
What are the conditions needed for tissue cultures to work?
- An inoculum
- Aseptic techniques
- A suitable medium containing growth factors such as proteins, salts, vitamins etc which can be provided from an animal serum such as FBS.
- Antibiotics are sometimes needed to prevent spoilage from microorganisms.
