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Primary and Secondary Antibodies
[edit]Antibodies
[edit]Antibodies, also called immunoglobulins, are large Y-shaped proteins whose function is to identify and help remove foreign antigens. Different antibodies recognize different foreign antigens. This specificity is due to the presence of the two unique tips on the “Y” of each antigen, allowing different antibodies to bind differently on foreign antigens.[1]
The main structures of a Y-shaped antibody are the variable region, constant region, light chain, heavy chain, and antigen-binding site.
Primary Antibodies
[edit]Primary antibodies are immunoglobulins that bind to a particular protein or other biomolecule. They are developed as polyclonal or monoclonal antibodies using a mouse, rat, rabbit, goat, and/or other animal species as hosts.
A primary antibody can be very useful for the detection of biomarkers for diseases such as cancer, diabetes, Parkinson’s and Alzheimer’s disease [2].
Secondary Antibodies
[edit]Secondary antibodies bind to the primary antibody to aid the process of detecting and identifying the target antigen. Secondary antibodies must have specificity for the primary antibody being used to enable detection. In immunolabeling, the secondary antibodies bind to the primary antibodies. Initially primary antibodies bind to their target proteins exposing their Fc domains. Then, secondary antibody's Fab domain binds to the primary antibody's Fc domain. Thus, secondary antibodies are used for indirect detection of target antigens. This process reduces the cost by labeling only one type of secondary antibody, rather than labeling different types of primary antibodies.[3][4]
There are a number of reasons a secondary antibody could be used, including: 1. If there is no conjugated primary antibody available. 2. The primary antibody isn’t couple/conjugated to the required fluorochrome/enzyme. 3. The need to increase the sensitivity of the staining procedure.[5] Increased sensitivity can be achieved by using further secondary antibody layers. Amplification systems can also be added such as the biotin/streptavidin couple. [6]
The Use of Antibodies as Research and Diagnostic Tools
[edit]Antibodies are used extensively as diagnostic tools in a wide array of different analyses. Immunoassays are used to detect and quantitate the presence or absence of a particular antigen or antibody in a test fluid.[7]
Some of the common tools are:
- Affinity Chromatography: it is a separation technique that relies on the binding of an antibody to antigen held on a solid matrix. This type of chromatography separates molecules on the basis of their affinity to one another.[8]
- Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and competitive inhibition assay: RIA and ELISA are direct binding assays for antibody or antigen. RIA are commonly used to measure levels of hormones in blood and tissue. ELISA assays are mainly used to detect the presence or absence or viruses (viral diagnostics); an example would be the usage of ELISA to detect human immunodeficiency virus (HIV). In RIA, the antibody against the antigen is radioactively labeled. Whereas in ELISA an enzyme is chemically linked to an antibody that allows for color change once bound to an antigen. For safety and simplicity, ELISA is usually preferred over RIA when running a direct-binding assay. Safety measures include avoiding the hazard of radiation when running an RIA assay.[8]
For example, in order to detect antigen A using ELISA, an antibody that is specific to that antigen needs to be added. Upon binding of this antibody to antigen A, a wash is then added to remove any outcast antibodies or antigens residing in the wells. Binding, in this case, is detected by a reaction that converts the colorless substrate into a colored reaction product.[9]
- Hemagglutination and Blood Typing: ABO and Rh(D) blood typing is one of the most important examinations that are performed prior to blood transfusion. Hemagglutination is used to determine the ABO blood group of blood donors and transfusion recipients. Hemagglutination allows for the detection and measuring of antibodies in the serum.
- Western Blotting: commonly used to detect diseases such as Lyme disease and could also be used for confirming infection with human immunodeficiency.[10]
- Immunohistochemistry: Two families of antibodies are commonly used in immunochemistry are: polyclonal antibodies and monoclonal antibodies. The antibody (monoclonal and polyclonal) used to recognize an antigen into the tissues is called the primary antibody.[11]
- Immunostaining
- Immunofluorescence
- ^ "Antibodies". About.com Education. Retrieved 2016-03-27.
- ^ "DPDx - Laboratory Identification of Parasitic Diseases of Public Health Concern". CDC. November, 2013.
{{cite web}}: Check date values in:|date=(help) - ^ www.rockland-inc.com. "Secondary Antibodies". www.rockland-inc.com. Retrieved 2016-03-27.
- ^ "Secondary antibodies (Resources)". www.antibodies-online.com. Retrieved 2016-03-27.
- ^ "Secondary Antibodies". www.thermofisher.com. Retrieved 2016-03-27.
- ^ Price, Christopher P.; Newman, David J. (1991-11-25). Principles and Practice of Immunoassay. Springer. ISBN 9781349112340.
- ^ King, David J. (1998-11-27). Applications And Engineering Of Monoclonal Antibodies. CRC Press. ISBN 9780748404223.
- ^ a b Murphy, Kenneth (July 24, 2011). Immunology Biology. Garland Science. pp. 717–731. ISBN 9780815342434.
{{cite book}}: CS1 maint: year (link) - ^ Bancroft, John D.; Gamble, Marilyn (2007-10-18). Theory and Practice of Histological Techniques, 6e (6 edition ed.). Churchill Livingstone. ISBN 9780443102790.
{{cite book}}:|edition=has extra text (help) - ^ Stanley, Jacqueline (2002-01-01). Essentials of Immunology and Serology. Cengage Learning. ISBN 076681064X.
- ^ Hammond, Constance (2014-12-30). Cellular and Molecular Neurophysiology. Academic Press. ISBN 9780123973221.