Antibody Guide To Flow Cytometry

 

What is Flow Cytometry?

When it comes to high-throughput measurement of antigens on live cells, there is no better technique than flow cytometry. Flow Cytometry uses fluorescent markers on antibodies or other proteins in order to quantifiably detect changes in protein expression via excitation of the various fluorescent markers. Flow cytometry can be used for detecting numerous targets either on the surface or within cells in the same sample. A countless number of interesting Flow cytometry experiments can be designed including subtle stages of the cell cycle, various apoptotic states, or organelle integrity. The ease and speed with which cells are sorted, coupled with a high rate of cell viability for use in follow-up assays, make flow cytometry the method of choice for cell and tissue biologists.

Antibodies for Flow Cytometry

Antibodies allow scientists to detect a specific antigen, making them useful for characterizing the proteins on the surface of live cells. These antibodies can be directly labeled with a fluorescent marker or can be visualized by binding to a secondary antibody that is labeled with a fluorescent marker. This allows flow cytometry to sort cells based on more than one color, each representing a different antigen that is bound by a different antibody.

Polyclonal vs. Monoclonal for Flow Cytometry

Polyclonal antibodies are comprised of a mixture of antibodies that bind to different epitopes of the same antigen. The fact that polyclonal antibodies bind multiple regions on an antigen makes them great for quick and cost-effective detection of cell surface markers. An advantage of monoclonal antibodies (mAbs) compared to polyclonal antibodies is that they provide cleaner data. Since mAbs are produced by clones of one hybridoma cell of origin and bind to the same epitope on an antigen, they produce less non-specific background noise. In order to do complex experiments detecting multiple antigens, or just to get a primary antibody that is compatible with certain fluorescently labeled secondary antibodies, the proper host species for each antibody must be considered. Our antibody selection guide makes it easy to find the right antibody for your application and desired specificity.

Single Domain Antibody Advantage

An alternative to monoclonal antibodies are single domain antibodies (sdAbs). In contrast to mAbs, sdAbs are much smaller in size (~15 kDa vs. ~150 kDa). A major advantage of sdAbs over mAbs is that they have less non-specific binding. sdAbs do not contain an Fc region found on traditional immunoglobulins. As such, sdAbs do not bind to all of the immune cells that have Fc receptors on their surface, minimizing background. mAbs are commonly used to detect carcinoembryonic antigen (CEA or CEACAM5) on cancer cells. A common problem is cross-reactivity of the mAb for nonspecific cross-reacting antigen (NCA or CEACAM6), which is expressed on granulocytes. sdAbs have been shown to detect CEA via flow cytometry without cross-reactivity for NCA, allowing for higher specificity and the ability to sort CEA positive cancer cells apart from the abundant granulocytes in an in vivo live cell extract[1].

SdAbs also have less potential for unwanted cross-linking when conjugated to larger molecules. Standard antibodies, such as mAbs, have heavy chains that can cross-link with each other or other molecules during the conjugation process. The heavy chains also affect the orientation of how the mAb binds to the particle to which it is conjugated. This random orientation can hinder the active site of the mAb, thus lowering detection efficiency. sdAbs have other unique characteristics, including increases stability, pH and temperature resistance. Once mAbs denature due to heat, they cannot refold into functional antibodies. sdAbs, however, are able to refold after denaturation and still exhibit binding capacity, giving them more potential for subsequent detection and quantification.

Single Domain Quantum Dot Imaging for Flow Cytometry

A versatile emission spectrum and higher detection rate gives Quantum Dots (QD) a number of advantages over antibodies that detect antigens using fluorescent molecules. When conjugated to QDs, sdAbs have been shown to be superior to conventional antibodies for detecting the breast cancer cell surface marker HER2[2]. HER2 is an EGFR-family receptor that is overexpressed in certain breast cancer subtypes and is a target of antibody therapies. The simpler structure of sdAbs allows for a higher rate of optimal orientation when they are conjugated to QDs. This makes it possible to apply the high-sensitivity detection of QDs to a cell by targeting surface antigens using the specificity of sdAbs.

References

1. Behar et al. “Llama single-domain antibodies directed against nonconventional epitopes of tumor-associated carcinoembryonic antigen absent from nonspecific cross-reacting antigen.” FEBS Journal, 2009, 276, pp 3881–3893.

2. Rakovich et al. “Highly Sensitive Single Domain Antibody–Quantum Dot Conjugates for Detection of HER2 Biomarker in Lung and Breast Cancer Cells.” ACS Nano, 2014, 8(6), pp 5682–5695.