#conjugated

Choosing the right antibody reagents for flow cytometry is crucial. A poor choice can cause a loss of sample material, money, and time, resulting in incorrect and unreliable results. Researchers need to consider plenty of factors, including antibody format and the antigenic target’s nature, for the success of flow cytometry. Another crucial factor they need to focus on when selecting an antibody is whether to use an unconjugated or directly conjugated product for flow cytometry.

Compared to unconjugated antibodies, directly conjugated antibodies are preferred by researchers for flow as it allows simultaneous use of multiple antibodies in the same species. It also simplifies the assay protocol and reduces the risk of potential mistakes during sample preparation. Read on to know about the conjugated antibodies for flow cytometry.

Conjugated antibodies are monoclonal or polyclonal antibodies with an attached molecule, used for creating a detectable signal by fluorescence, color-generation, or other signals. Besides flow cytometry, conjugated antibodies are used in various applications, like immunocytochemistry, and immunohistochemistry, to mention a few.

Direct and indirect are two types of antibody detection methods. The direct detection method uses a primary antibody conjugated directly to a label. Researchers can directly detect the target by conjugating primary antibodies, eliminating the need for secondary antibodies. It makes the use of multicolor staining easier and eliminates the problems related to the non-specific binding of secondary antibodies.

On the other hand, the indirect detection method uses an unconjugated primary antibody or conjugated secondary antibody, specifically for the primary antibody’s host species. Researchers prefer this method when the target of interest has low expression levels, and it allows signal amplification as secondary antibodies carrying multiple labels bind to primary antibodies. The indirect detection method has higher sensitivity levels and generates more intense signals.

Using fluorescently conjugated antibodies, researchers can detect multiple targets simultaneously. Fluorochromes, used in fluorescent dyes, have absorption and emission wavelengths. The emission of a photon at one wavelength excites another in the presence of light. Since overlapping of emission is common when performing co-staining, taking note of excitation and emission spectra is crucial when using multiple dyes. Fluorescently conjugated antibodies are used in varied techniques, including flow cytometry.

When antibodies are conjugated to enzymes such as alkaline phosphatase and horseradish peroxidase, they react to substrates and localize an insoluble colored product to antigen expression sites. Chromogenic labeled antibodies are majorly used for immunohistochemistry and Western Blot. Biotin conjugated antibodies are used if the target expression is low.

While researchers widely use primary conjugated antibodies, secondary antibodies are preferred as they make it easier to choose different conjugates irrespective of the primary antibodies. Using secondary antibodies reduces the need for chemically labeled primary antibodies, which are a bit costlier.