Small Animal In Vivo Imaging Antibody Labeling

Optimal labeling of antibodies for in vivo applications

Antibodies that are fluorescently labeled for use in animals have more stringent requirements than antibodies labeled for other types of applications. The SAIVI™ Rapid Antibody Labeling Kits are uniquely designed to provide the rigorous requirements to control the degree of labeling (DOL). This control is accomplished by limiting the number of fluorescent molecules covalently attached to the antibody (Figure 1). It is crucial to restrict the DOL for antibodies used in vivo compared to other types of antibody applications, in order to maximize the desired signal at its target location while minimizing accumulation in the liver (Figure 2). Antibodies labeled with the SAIVI™ Rapid Antibody Labeling Kit are ideal for in vivo use.

• SAIVI™ Antibody Labeling Kits vs. Others

Non-invasive optical imaging in small animals is best achieved with antibodies or proteins labeled with near-infrared (NIR) fluorophores. AlexaFluor® dyes that emit in the NIR 700-900 nm spectrum are ideal for labeling antibodies to be used for in vivo imaging. Dyes in this range are critical to maximize the penetration of the signal while reducing autofluoresence from nontarget tissues. This highly sensitive detection can often be viewed to 3 mm or more, depending on the instrument used for visualization.

Figure 1. Conjugated antibodies used for in vivo applications have a limited and controlled number of labels attached.

Figure 2. A DOL of 2.4 provides ideal fluorescence intensity of the target while limiting intensity in the liver.

An anti-CEA (Invitrogen) antibody targeting colon cancer xenografts in mice show the optimal DOL for the fluorescence antibody to be 2.4, as imaged with the CRi Maestro™ In Vivo Imaging System.

For a more detailed explanation of the effects of DOL on fluorescent antibodies used in vivo see the Bioprobes 52 article.

In addition to the DOL control reagent, the SAIVI™ Rapid Antibody Labeling Kits include a novel column that has an easy to use pre-measured slurry and is specifically designed for a fast, 10 minute prep time and short wash step. The removal of azide is not required as it is for other columns resulting in additional time savings. This protocol results in high yields of ready-to-use antibody every time (Figure 2). The chart below compares the use of these kits, and shows multiple advantages of using the SAIVI™ Rapid Antibody Labeling Kits for all in vivo applications.

Figure 3.  High yields with 0.5 – 3.0 mg mouse polyclonal IgG-680 conjugated according to kit protocol.