Antibody conjugates are used in histological and enzymatic antigen detection assays to locate bound antibodies. They are physically attached to antibodies to label antigens of interest, allowing for identification and analysis. Primary antibodies are raised against specific antigens, while secondary antibodies are conjugated to label molecules and raised against the primary antibody for cost-effectiveness. In some cases, primary antibodies are labeled to reduce noise in detection signals. Kits can be purchased to label conjugates with preferred antibodies, increasing the cost but reducing protocol steps and possibly increasing the signal-to-noise ratio.
An antibody conjugate is a labeling molecule that can be used in histological or enzymatic antigen detection assays to locate bound antibodies. It works by being physically attached, or conjugated, to the antibody that is used to label an antigen of interest. An antibody that is labeled with an antibody conjugate is a tool often used in research to identify the exact location or amount of the protein or antigen of interest, against which the antibody or antibodies have been raised.
Histological and proteomic tests often rely on antibodies as a reagent in research techniques by which specific proteins can be labeled. By exposing an antigen or protein of interest to an antibody that has been raised against it, antigens or proteins can be labeled. Using an antibody conjugate, that is, an antibody that has been conjugated to a specifically used or synthesized molecule to be easily detected, antibodies can be identified and analyzed against their respective antigens. The most common type of antibody conjugate is a fluorescent molecule, which allows for the identification of antigens in tissue under a fluorescence microscope. Other conjugates include molecules such as horseradish peroxidase, which is used in conjugation with antibodies for further development with diaminobenzidine (DAB).
Often the primary antibodies will be raised against specific antigens, while the secondary antibodies will be conjugated to label molecules and raised against the primary antibody. For example, one might be interested in identifying an antigen called antigen X. One would then use, for example, an antibody raised in a mouse that reacts and attaches to antigen X. To be able to find where they found these primary antibodies mouse and bound to these X antigen molecules, a secondary antibody would then be used, such as one reared in a goat, which would be tagged with a conjugate that makes this secondary antibody detectable in enzymatic or fluorescent microscopic assays. For antigen localization assays using an electron microscope, gold particles are often used as an antibody conjugate because they can be detected using an electron beam.
The reason for using primary and secondary antibodies, instead of using an antibody conjugation where the primary antibody is directly conjugated, is cost effectiveness. Antibody conjugation is expensive in terms of time and reagents. Especially for uncommon antigens, antibody conjugation to primary antibodies is not worth the expense of manufacturing. Rather, by using antibody conjugation to secondary antibodies, large quantities of a conjugated antibody can be synthesized simultaneously and used in assays with a tailored range of primary antibodies to localize many different antigens, making the manufacturing process both cost-effective and cost-effective.
In some cases, however, a primary antibody will be labeled to reduce noise in the detection signal due to increased nonspecific antibody binding that results from using multiple antibodies. Although these expensive and highly sensitive tests will still require this method. Kits can be purchased that allow a researcher to label conjugates with their preferred antibody, increasing the cost of the reagent, but reducing protocol steps and possibly increasing the signal-to-noise ratio in the final antibody detection assay.
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