Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3

Page Contents

• Features of Zenon Antibody Labeling Technology
• Zenon Labeling Kits and Their Applications
• Ordering Information

Our exceptional Zenon immunolabeling technology provides an easy, versatile and truly unique method of labeling antibodies with Molecular Probes premier dyes, haptens and enzymes. This enabling technology not only eliminates the need for secondary detection reagents in many applications, but also simplifies immunolabeling applications that previously were time consuming or impractical, including the use of multiple antibodies derived from the same species in the same protocol, as well as the detection of antibody binding in tissues when both the antibody and the tissue are derived from the same species. Moreover, Zenon immunolabeling technology permits the rapid and quantitative preparation of antibody complexes from a purified antibody fraction or from a crude antibody preparation such as serum, ascites fluid or a hybridoma supernatant.

The Zenon labeling method takes advantage of the immunoselectivity of the antibody binding reaction by forming a complex between an intact primary IgG antibody and a fluorophore-, biotin- or enzyme-labeled Fab fragment directed against the Fc portion of that IgG (Figure 7.3.1). Simple mixing of the labeled Fab fragment, which is supplied in the Zenon Antibody Labeling Kits, with the corresponding primary antibody quantitatively produces the Fab–antibody complex in under 10 minutes, with no pre- or postlabeling purification required. This labeled Fab–antibody complex can be immediately used to stain cells, tissues and other targets in the same manner as a covalently labeled primary antibody.

Molecular Probes Zenon immunolabeling technology has many outstanding features that open several avenues for research and development. These features, which are discussed in detail later in this section, include:

• Rapid antibody labeling. The Zenon antibody labeling protocol permits rapid and reproducible labeling of submicrogram quantities of a primary antibody. The Zenon complex is ready for use in cell-labeling protocols in less than 10 minutes. In the absence of competing antibodies of the same species, the Zenon complex can also be stored for later use.
• Quantitative antibody labeling. Labeling is quantitative with respect to the primary antibody, and the extent of antibody labeling (and thus the intensity of the fluorophore or activity of a conjugated enzyme) can be adjusted by changing the molar ratio of the dye- or enzyme-labeled Fab fragment to the primary antibody. In flow cytometry, this unique property of the Zenon reagents permits use of the same fluorescent color to label two or more cell populations in the same sample.
• Affinity-purified Fab fragments. The Zenon dye- and enzyme-labeled Fab fragments have been affinity purified during their preparation to ensure their high affinity and selectivity for the Fc portion of the corresponding primary antibody. Furthermore, our procedure for chemical labeling of the Fab fragments protects the Fc-binding site, resulting in more active labeling reagents.
• Function like covalently labeled primary antibodies. The Zenon complexes formed with the Zenon Antibody Labeling Kits can be used to label cells and tissues without introducing nonspecific background, which can often arise from secondary antibody detection. Moreover, these Zenon complexes are much easier to prepare and more versatile than covalently labeled primary antibodies, and they show higher activity because only the Fc portion of the primary antibody is labeled, leaving the antigen-binding sites available for cell and tissue labeling. Furthermore, the Zenon Labeling Kits permit easy and efficient labeling of antibodies that are not commercially available as direct conjugates.
• Many fluorophore and enzyme labels available. Zenon immunolabeling technology makes it very easy to change fluorescent color combinations or detection methodologies by simply using a different dye- or enzyme-labeled Fab fragment from our extensive selection of Zenon Antibody Labeling Kits (Zenon Antibody Labeling Kits—Table 7.7).
  

Figure 7.3.1 Labeling scheme utilized in the Zenon Antibody Labeling Kits. A) An unlabeled IgG antibody is incubated with the Zenon labeling reagent, which contains a fluorophore-labeled, Fc-specific anti-IgG Fab fragment. B) This labeled Fab fragment binds to the Fc portion of the IgG antibody. C) Excess Fab fragment is then neutralized by the addition of a nonspecific IgG, preventing crosslabeling by the Fab fragment in experiments where primary antibodies of the same type are present. Note that the Fab fragment used for labeling need not be coupled to a fluorophore, but could instead be coupled to an enzyme (such as HRP) or to biotin.

Zenon Immunolabeling Is Rapid and Quantitative

Labeling of a suitable primary antibody with the Zenon Antibody Labeling Kits is very fast. The Zenon labeling reagent contains dye- or enzyme-labeled Fab fragments that have been affinity purified to ensure their high affinity for the Fc portion of the corresponding primary antibody. We recommend incubation of the Zenon labeling reagent with the appropriate primary antibody for 5 minutes, followed by a 5-minute blocking reaction of any remaining labeled Fab fragments with excess nonspecific IgG; however, the actual rates of the complex formation and IgG blocking steps are likely to be even faster and may occur within the mixing time of the components (Figure 7.3.2).

Fab–antibody complex formation can be quantitative with respect to the primary antibody (Figure 7.3.3). Furthermore, the degree of labeling (and thus the intensity of the fluorophore or activity of a conjugated enzyme) can be adjusted to some extent by changing the molar ratio of the labeled Fab fragment to the primary antibody. We find that approximately equal weights of a dye-labeled Fab fragment generated from a goat anti-Fc antibody (MW ~50,000 daltons) and the intact mouse primary antibody (MW ~150,000 daltons)—a 3:1 molar ratio of the labeled Fab fragment to the primary antibody—yields an Fab–antibody complex that is suitable for most applications. Thus, the 50 µg of labeled Fab fragments in the Zenon labeling reagent provided in the organic dye–based Zenon Antibody Labeling Kits is sufficient for labeling approximately 50 µg of the corresponding intact IgG antibody; we have defined "one labeling" in all of our Zenon Antibody Labeling Kits as the amount of Zenon labeling reagent required for labeling 1 µg of an intact primary antibody. Use of a 5:1 molar ratio of the Zenon labeling reagent gives an apparent 100% conversion of the primary antibody to the labeling complex (Figure 7.3.3) and a somewhat brighter total fluorescence when compared with the fluorescence of Zenon complexes prepared using a 3:1 ratio.

Increasing the molar (or weight) ratio of the Zenon labeling reagent to the antibody can yield a somewhat brighter conjugate and ensure quantitative utilization of the primary antibody, whereas decreasing the ratio can yield a somewhat less fluorescent complex. The ability to adjust the fluorescence intensity (or enzymatic activity) of a labeled complex is a feature of Zenon technology that is not at all practical with direct chemical labeling of antibodies. This property also permits the researcher to rapidly optimize the best complex for their experiment, rather than depending on the quite variable degree of substitution that is typical of covalently labeled antibodies from different commercial sources or from chemical labeling in a research laboratory. A 3:1 molar ratio of Zenon labeling reagent to primary antibody is also suitable for labeling with the phycobiliprotein- and enzyme-derived Zenon labeling reagents. The Zenon Antibody Labeling Kits containing a phycobiliprotein- or enzyme-labeled Fab fragment include sufficient reagents for labeling ~25 µg of an intact primary antibody; the Zenon Antibody Labeling Kits containing an Alexa Fluor dye–phycobiliprotein tandem conjugate include sufficient reagents for labeling ~10 µg of a primary antibody.

Figure 7.3.2 Formation of antibody–Fab complexes. An anti-biotin mouse IgG1 monoclonal antibody was mixed with the Zenon Alexa Fluor 488 labeling reagent (a component of Kit Z25002) for varying time intervals before the reaction was quenched by the addition of excess mouse IgG blocking reagent. The quenched reactions were then added to a microplate well containing biotinylated bovine serum albumin and incubated for 20 minutes. After washing, the fluorescence of the remaining bound signal was measured. Binding was found to be essentially complete in less than 5 minutes. Three trials are shown, along with a control () where no labeling reagent was added.

Figure 7.3.3 High-performance size-exclusion chromatographic analysis of the Zenon Alexa Fluor 488 labeling reagent (a component of Kit Z25002) binding to a mouse IgG1 antibody. The Zenon labeling reagent peak appears at 38 minutes; the mouse IgG1 peak appears at 33 minutes. When combined at a molar ratio of ~5:1 (Zenon labeling reagent:IgG1), the IgG1 antibody is quantitatively converted to a labeled complex, which appears as a peak at 29 minutes.

Zenon Technology Simplifies the Use of Multiple Antibodies of the Same Isotype in the Same Protocol

Unlike detection with secondary antibodies, Zenon immunolabeling technology allows staining of a cell or tissue sample with multiple antibodies of the same isotype. Our affinity purification of the Zenon labeling reagent (which we perform subsequent to dye or enzyme conjugation) helps to ensure that the formation of the Fab–antibody complex is stable. Reversal of Zenon complex formation by the excess of nonspecific IgG used to capture any uncomplexed Zenon labeling reagent does occur, particularly at temperatures above room temperature. The stability of the Zenon complex, however, is sufficient to allow sequential (or simultaneous) labeling of different targets in cells and tissues with multiple antibody complexes (, , ). Subsequent to staining, an aldehyde-based fixation step can permanently block the transfer of Zenon labels between different primary antibodies and will preserve the staining pattern. Zenon complexes can be used together with other Zenon complexes, with directly labeled primary antibodies, with labeled secondary antibodies to primary antibodies derived from other species, with antibodies that lack an Fc fragment and with avidin–biotin techniques (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) for multiplexed immunolabeling. Some crossreactivity of the Zenon labeling reagent with the Fc portion of antibodies from other species may occur unless the excess Zenon labeling reagent is captured with the soluble nonspecific IgG included in the Zenon Antibody Labeling Kits.

Zenon Immunolabeling Is Reliable, Even with Very Small Quantities of Reagents

Formation of the Fab–antibody complex with the Zenon Antibody Labeling Kits is extremely reliable and reproducible, even with very small (submicrogram) amounts of primary antibody. Successful chemical labeling of submicrogram quantities of an antibody with a succinimidyl ester or an isothiocyanate of a dye is typically not possible; chemical labeling and purification of proteins usually requires at least 100 µg of the carrier-free protein. Because submicrogram amounts of an immunolabeling complex may be all that are required for an experiment, there is absolutely no waste of expensive or difficult-to-obtain antibodies when using the Zenon Antibody Labeling Kits. Additionally, optimization of the degree of labeling by a dye or an enzyme is trivial, as compared with any chemical labeling method. Although we define "one labeling" in the Zenon Antibody Labeling Kits as the amount of Zenon labeling reagent required for labeling 1 µg of the primary antibody, we routinely label about 0.4 µg of the primary antibody dissolved in 2 µL of a buffer.

Unfortunately, many providers of mouse monoclonal antibodies do not indicate how much of the pure antibody they provide in their products. Furthermore, these antibodies are frequently supplied diluted with an albumin or other carrier protein, which precludes estimation of the antibody concentration from the optical density at 280 nm. However, the ease of preparing Zenon complexes makes it practical to rapidly optimize the labeling reagent, even when the amount of primary antibody in the sample is unknown.

Zenon Immunolabeling Does Not Require Antibody Pre-Purification

Unlike chemically modifying antibodies, labeling antibodies with the Zenon Antibody Labeling Kits does not require removal of exogenous proteins such as serum albumin from the antibody. Serum albumin is frequently added to laboratory-derived or commercially supplied antibodies to help preserve the activity of dilute solutions of antibodies. Because the Zenon labeling reagent selectively binds only to the Fc portion of the primary antibody, there should be limited or no effect of exogenous proteins that do not have an Fc fragment. The Zenon labeling reagents function equally well when used to label a purified antibody or a crude antibody preparation such as serum, ascites fluid or a hybridoma supernatant. Zenon labeling is usually quite successful with even dilute solutions of a primary antibody, as well as with antibodies that are dissolved in amine-containing buffers such as Tris.

Zenon Immunolabeling Is Versatile

We currently offer Zenon Antibody Labeling Kits containing a wide selection of different fluorescent labels (Zenon Antibody Labeling Kits—Table 7.7), with spectra that span ultraviolet, visible and near-infrared wavelengths. Zenon immunolabeling technology makes it particularly easy to change fluorescent colors or detection methodologies by simply using a different Zenon labeling reagent from our extensive selection of Zenon Antibody Labeling Kits. There is no need to purchase multiple direct conjugates of the same mouse primary antibody (such as with the cluster of differentiation (CD) antibodies) in order to perform multicolor flow cytometry (Figure 7.3.4) and imaging protocols.

The fluorescence intensity or enzymatic activity of a Zenon complex prepared using our protocol is usually similar to that of the corresponding directly labeled conjugate of the primary antibody, although it is typically somewhat lower than the intensity that can be obtained using a labeled secondary antibody. If necessary, the sensitivity of the assay can be increased through use of a Zenon complex in combination with a signal amplification method such as TSA (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2); see below for a description of our Zenon Alexa Fluor 488 Mouse IgG₁ Labeling Kit enhanced with TSA technology (Z25090).

Figure 7.3.4 Human peripheral blood lymphocytes were stained with the following three antibodies: an anti-CD3 mouse IgG₁ antibody prelabeled with the Zenon Alexa Fluor 647 Mouse IgG₁ Labeling Kit (Z25008), an anti-CD4 mouse IgG₁ antibody prelabeled with the Zenon R-Phycoerythrin Mouse IgG₁ Labeling Kit (Z25055) and an anti-CD8 mouse IgG_2a antibody prelabeled with the Zenon Alexa Fluor 488 Mouse IgG_2a Labeling Kit (Z25102). Panels A and B show that cells can be separated by plotting the orange-fluorescent versus green-fluorescent signal or red-fluorescent versus orange-fluorescent signal, respectively, demonstrating that the Zenon label does not transfer to other antibodies in the same sample. The samples were analyzed on a Coulter Elite flow cytometer using 488 nm excitation for R-phycoerythrin and the Alexa Fluor 488 dye, and 633 nm excitation for the Alexa Fluor 647 dye.

Our wide selection of Zenon Kits can be mixed and matched in the same experimental protocol and even in the same cell-labeling solution, providing the freedom to experiment with multiple dye–antibody combinations in flow cytometry and imaging applications. In addition, we offer a Zenon Mouse IgG₁ Labeling Kit enhanced with TSA technology (Z25090),  which combines the advantages of Zenon labeling technology with the sensitivity of tyramide signal amplification (TSA).

Zenon Antibody Labeling Kits for Mouse IgG, Rabbit IgG, Goat IgG and Human IgG

We offer an extensive assortment of Zenon Antibody Labeling Kits for use with mouse IgG₁, mouse IgG_2a, mouse IgG_2b, rabbit IgG, goat IgG and human IgG antibodies (Antibody Structure and Classification—Note 7.1). Each of these kits contain a Zenon labeling reagent, comprising Fab fragments generated from goat secondary antibodies—or in the case of the Zenon labeling reagents for goat IgG antibodies, from rabbit secondary antibodies—directed against the Fc portion of the corresponding primary antibody and covalently conjugated with a detectable label. These detectable labels include our outstanding Alexa Fluor dyes (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3), as well as phycobiliproteins, tandem conjugates of phycobiliproteins with Alexa Fluor dyes (Phycobiliproteins—Section 6.4), enzymes (horseradish peroxidase and alkaline phosphatase), biotin and some conventional dyes such as fluorescein. Three types of kits are available:

• Zenon Antibody Labeling Kits that contain an affinity-purified Fab fragment of a goat anti-Fc antibody (or, in the case of the Zenon Goat IgG Labeling Kits, a rabbit anti-Fc antibody) that has been conjugated to one of our premier Alexa Fluor dyes, to one of our violet laser–excitable Pacific Blue, Pacific Green or Pacific Orange dyes, or to fluorescein, Texas Red-X dye or biotin.
• Zenon Antibody Labeling Kits that contain an Fab fragment of an anti-Fc antibody labeled with a R-phycoerythrin (R-PE), allophycocyanin (APC) or a tandem conjugate of R-PE or APC with one of our long-wavelength Alexa Fluor dyes.
• Zenon Antibody Labeling Kits that contain HRP or alkaline phosphatase conjugates for use in enzyme-amplified protocols (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2, Phosphatase-Based Signal Amplification Techniques—Section 6.3); see below for a description of the Zenon Alexa Fluor 488 Mouse IgG₁ Labeling Kit enhanced with TSA technology (Z25090).

In addition to these Zenon Antibody Labeling Kits, we offer three different types of Zenon Tricolor Labeling Kits for mouse IgG antibodies, rabbit IgG and human IgG antibodies (Zenon Antibody Labeling Kits—Table 7.7). The Zenon Tricolor Labeling Kit #1 contains 10 µg each of the Alexa Fluor 488, Alexa Fluor 555 and Alexa Fluor 647 Zenon labeling reagents. This kit is designed for optimal triple-antibody staining with confocal laser-scanning microscopes, but it can also be used with any suitably equipped fluorescence microscope. The Zenon Tricolor Labeling Kit #2 contains 10 µg each of the Alexa Fluor 350, Alexa Fluor 488 and Alexa Fluor 594 Zenon labeling reagents, yielding simultaneous blue-, green- and red-fluorescent immunostaining that is useful with almost any fluorescence microscope. The Zenon Tricolor Labeling Kit #3 is especially suitable for flow cytometry applications and contains 10 µg each of the Alexa Fluor 488, R-phycoerythrin (R-PE) and Alexa Fluor 647–R-PE Zenon labeling reagents, which are each excited efficiently with an argon-ion laser and exhibit minimal spectral overlap.

Zenon Antibody Labeling Kit Enhanced with TSA Technology

The Zenon Alexa Fluor 488 Mouse IgG₁ Labeling Kit enhanced with TSA technology (Z25090) provides exceptional target-identification capabilities. This kit provides the necessary reagents from both the Zenon Horseradish Peroxidase Mouse IgG₁ Labeling Kit and the Alexa Fluor 488 TSA Kit for researchers who want both the ease of labeling mouse IgG₁ antibodies with Zenon labeling reagents and the signal amplification afforded by use of TSA technology. TSA technology is an enzyme-mediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP) to generate high-density labeling of a target protein (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2). Each HRP label on the target-bound Fab–mouse IgG₁ complexes can activate multiple copies of Alexa Fluor 488 tyramide to produce short-lived tyramide radicals that are highly reactive with nucleophilic residues near the interaction site, yielding an amplified fluorescent signal with minimal diffusion. The Zenon Alexa Fluor 488 Mouse IgG₁ Labeling Kit enhanced with TSA technology provides sufficient reagents for 25 labelings, including:

• Zenon HRP mouse IgG₁ labeling reagent
• Zenon mouse IgG blocking reagent
• Alexa Fluor 488 tyramide
• DMSO
• TSA blocking reagent
• TSA amplification buffer
• Hydrogen peroxide (H₂O₂)
• Detailed protocols for Zenon complex formation and fluorescent tyramide labeling (Enhanced Zenon Mouse IgG Labeling Kit)

Use and Applications of Zenon Technology

With the exception of our Zenon Tricolor Labeling Kits, our Zenon Antibody Labeling Kits contain sufficient reagents for 50 labelings (with the low molecular weight dye–derived Zenon labeling reagents), 25 labelings (with the R-PE–, APC-, HRP- and alkaline phosphatase–derived reagents) or 10 labelings (with the Zenon labeling reagents containing one of five different Alexa Fluor dye–phycobiliprotein tandem conjugates). The Zenon Tricolor Labeling Kits #1, #2 and #3 contain sufficient reagents for 10 labelings of the same or different antibodies with each of three different Alexa Fluor dyes. "One labeling" is defined as the amount of Zenon labeling reagent required for labeling 1 µg of an intact mouse, rabbit, goat or human IgG antibody; however, smaller (or larger) quantities of a primary antibody can be reliably labeled with any of the Zenon labeling reagents.

For single-color labeling, it is usually not necessary to block any residual Zenon labeling reagent that has not complexed with the primary antibody; however, in applications that involve multiple antibodies of any type (including antibodies from other species or of other isotypes that may react to a small degree with the Zenon labeling reagent), adsorption of residual Zenon labeling reagent is essential to avoid crossreactivity. Adsorption can be done with a solution of soluble nonspecific IgG, which is included in all of the Zenon Antibody Labeling Kits. When the Zenon complexes are in a solution containing the Zenon labeling reagents adsorbed onto soluble nonspecific IgG, they should be used for staining within an hour to avoid possible transfer of the Zenon labeling reagent to the excess nonspecific IgG. Zenon complexes can be used with standard immunolabeling techniques; staining with Zenon complexes can be performed sequentially or combined with each other or with additional dye- or enzyme-labeled primary antibody conjugates in a one-step, multiparameter labeling protocol. Fixation with aldehyde-based fixatives following staining is recommended to prevent transfer of the Zenon label between antibodies.

We have demonstrated the utility of Zenon staining for imaging in an assortment of cells (, , ) and tissues (). As with the use of any antibody conjugate in tissues, staining with Zenon complexes requires good accessibility of the antibody to the target, which can be affected by the specific conditions of fixation and permeabilization. Zenon complexes of intact primary antibodies have somewhat higher molecular weights than direct conjugates of antibodies, potentially reducing the accessibility of the complexes to tissue antigens in some cases.