The reaction between an antibody and an antigen is a so specific process that it can always be observed under microscope for various reasons. This is made possible particularly given that the reaction take place only between an antigen and its corresponding group of antibodies. For this reason, antibody labeling is done to observe the reaction process for localization, quantification or even detection purpose.
In order to visualize enzyme label under a light microscope, enzyme histochemical methods are employed through chromogenic reaction. Visualizing fluorophore label on the other had can be done directly by using fluorescent microscope. When colloidal gold are used, the appropriate viewing media is an electron microscope. Biotin label however can be visualized under all the three types of microscopes in addition to ABC technique.
A researcher has a choice between covalent method of labeling and the non-covalent method. With covalent method, the whole process is expensive and calls for very high level of expertise. The non-covalent alternative on the other hand is much cheaper and with moderate expertise level.
Covalent method involves introduction of labels into the antibodies through chemical means. In order to achieve this process, a number of functional groups of antibodies are targeted using a group-specific reagent. This requires a large quantity of purified antibodies which is definitely expensive. In addition to this, the technicians involved must be knowledgeable in dialysis method of conjugate purification, how to use the desalting spectra and absorbance spectra taking.
SBA stabilizer has an impact of reducing efficiency during reaction. Any attempt to remove SBA however leads to the loss of the antibodies. One of the ingenious solutions to this complication lies in the LinkTM conjugation procedure which makes production of high quality substrate under a high concentration of SBA attainable. Covalent method also requires that the user is well versed conjugate purification by the use of dialysis method, the use of desalting spectra and taking of absorbance spectra.
The now common method which is more economical to covalent procedure is the non-covalent method. A bridge monovalent Fab fragment is used in vitro to label primary antibodies with the reporter molecules. The labeling complex if formed through a simple mixing of Fab fragments of monovalent and some primary antibodies that are unconjugated.
The success recorded in the localization, observation, recording, measurement and even tracing the reaction paths of both the antibody and the antigen largely depends on the substrate used and the procedure followed. The substrates current available in the market that can achieve this purpose include m-periodates, glutaraldehyde and the maleimide derivatives which replaced many other substrates that had been in use for long.
The labels to choose from are so many largely depending on the microscope to be used and the procedure to be adopted. For a success process of antibody labeling however, there are many other factors that greatly matters. These include the level of experience of the researchers, how well the laboratory is equipped, the reagents used among others. It is however important to understand that some methods allows multiple labeling of the targeted antibodies in a single incubation and sample.
In order to visualize enzyme label under a light microscope, enzyme histochemical methods are employed through chromogenic reaction. Visualizing fluorophore label on the other had can be done directly by using fluorescent microscope. When colloidal gold are used, the appropriate viewing media is an electron microscope. Biotin label however can be visualized under all the three types of microscopes in addition to ABC technique.
A researcher has a choice between covalent method of labeling and the non-covalent method. With covalent method, the whole process is expensive and calls for very high level of expertise. The non-covalent alternative on the other hand is much cheaper and with moderate expertise level.
Covalent method involves introduction of labels into the antibodies through chemical means. In order to achieve this process, a number of functional groups of antibodies are targeted using a group-specific reagent. This requires a large quantity of purified antibodies which is definitely expensive. In addition to this, the technicians involved must be knowledgeable in dialysis method of conjugate purification, how to use the desalting spectra and absorbance spectra taking.
SBA stabilizer has an impact of reducing efficiency during reaction. Any attempt to remove SBA however leads to the loss of the antibodies. One of the ingenious solutions to this complication lies in the LinkTM conjugation procedure which makes production of high quality substrate under a high concentration of SBA attainable. Covalent method also requires that the user is well versed conjugate purification by the use of dialysis method, the use of desalting spectra and taking of absorbance spectra.
The now common method which is more economical to covalent procedure is the non-covalent method. A bridge monovalent Fab fragment is used in vitro to label primary antibodies with the reporter molecules. The labeling complex if formed through a simple mixing of Fab fragments of monovalent and some primary antibodies that are unconjugated.
The success recorded in the localization, observation, recording, measurement and even tracing the reaction paths of both the antibody and the antigen largely depends on the substrate used and the procedure followed. The substrates current available in the market that can achieve this purpose include m-periodates, glutaraldehyde and the maleimide derivatives which replaced many other substrates that had been in use for long.
The labels to choose from are so many largely depending on the microscope to be used and the procedure to be adopted. For a success process of antibody labeling however, there are many other factors that greatly matters. These include the level of experience of the researchers, how well the laboratory is equipped, the reagents used among others. It is however important to understand that some methods allows multiple labeling of the targeted antibodies in a single incubation and sample.
About the Author:
You can visit the website www.columbiabiosciences.com for more helpful information about Antibody Labeling: The Concept And Methods