The number of target molecules per unit volume of sample is a key variable in all biological detection applications. Although it is possible to control target abundance through strategies such as recombinant protein overexpression and siRNA knockdowns, there is an associated risk of fundamentally perturbing the finely balanced and intertwined molecular interaction networks that underlie cellular function. There is therefore always some degree of need to detect molecules at their native abundance levels, which can vary by many orders of magnitude. For example, proteins in mammalian cells have abundances varying by at least seven orders of magnitude (~101–108 copies per cell). Furthermore, the distribution of target molecules within the cell is neither spatially uniform nor temporally static—indeed, these spatial and temporal variations are often the subject for experimental investigation. Many functionally important proteins such as transcription factors and cell-surface cytokine receptors have native expression levels below the detection threshold of labeled primary and secondary antibodies and other affinity reagents. In this chapter, we describe a collection of signal amplification strategies that can be used facilitate the detection of low-abundance molecular targets either in situ or ex situ (e.g., on microarrays). The signal amplification strategies described below are essentially of two types—enzyme labeling and macrofluorophore labeling. These two approaches are not necessarily singular and may be used in combination for additive effect.
Enzyme labeling utilizes an enzyme linked to a target-specific affinity reagent, either by direct conjugation or through a secondary complex (Figure 6.1.1). The enzyme turns over multiple copies of a fluorogenic or chromogenic substrate (Enzyme Substrates and Assays—Chapter 10), resulting in much higher target-associated signal levels than are obtainable by dye-labeled affinity reagents. The two most widely used enzymes for this purpose are horseradish peroxidase (HRP, TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2) and alkaline phosphatase (Phosphatase-Based Signal Amplification Techniques—Section 6.3). Major applications of enzyme labeling include immunocytochemical and immunohistochemical detection and enzyme-linked immunosorbent assays (ELISAs). In immunocytochemical and immunohistochemical detection applications, it is essential that the product of the enzyme reaction is localized in the vicinity of the enzyme conjugate in order to convey information on the spatial distribution of the target. Tyramide substrates for HRP (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2, ) and our ELF substrates for alkaline phosphatase (Phosphatase-Based Signal Amplification Techniques—Section 6.3, ) fulfill this requirement. In ELISAs, the objective is macroscopic quantitation rather than microscopic localization of the target, so substrates that yield diffusible products such as our fluorogenic Amplex UltraRed substrate for HRP (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2) and our CSPD and CDP-Star chemiluminescent substrates for alkaline phosphatase (Phosphatase-Based Signal Amplification Techniques—Section 6.3) are typically used. Detection signals that are amplified using enzyme reactions are necessarily time dependent. Therefore, in both immunocytochemical and immunoassay applications of enzyme labeling, careful control of timing is an essential prerequisite for obtaining quantitative and reproducible results.
Macrofluorophores are collections of fluorophores numbering in the tens (phycobiliproteins, Phycobiliproteins—Section 6.4) to millions (fluorescent microspheres, Microspheres—Section 6.5) attached to or incorporated in a common scaffold. The scaffold is coupled to a target-specific affinity reagent such as an antibody or streptavidin, and the incorporated fluorophores are thereby collectively associated with the target upon binding. From a physical perspective, quantum dot nanocrystals (Qdot nanocrystals, Qdot Nanocrystals—Section 6.6) are single fluorophores, albeit ones with extraordinary photon output capacity. From a utilization standpoint however, they resemble macrofluorophores and are similar in size to our smallest fluorescent microspheres. Macrofluorophores are not subject to the time-dependent signal development considerations introduced by enzyme labeling but are more susceptible to nonspecific binding. Even phycobiliproteins, the smallest and most biocompatible of these macrofluorophores, are not immune to these effects.
There are several other noteworthy approaches to the detection of low-abundance targets that may be applied either in combination with or as alternatives to the labeling technologies described in this chapter. In the case of nucleic acids, the capacity for self-replication allows the amount of target to be amplified through application of the polymerase chain reaction (PCR). Attempts to increase target-associated signals should generally be pursued in parallel with attempts to decrease off-target background signals. Blocking reagents such as our BlockAid blocking solution for use with fluorescent microspheres (B10710, Microspheres—Section 6.5) and our Image-iT FX signal enhancer (I3922, Fluorescence Microscopy Accessories and Reference Standards—Section 23.1) for use with dye-labeled antibodies can be employed in pursuit of this objective. For fluorescence detection in general, single-molecule detection techniques represent perhaps the ultimate in background reduction strategies.
Figure 6.1.1 Schematic diagram of primary and secondary detection reagents. A) In primary detection methods, the target-specific molecule includes one or more detectable moieties, shown here as radiant orbs. B) In secondary detection methods, the target-specific molecule contains binding sites or haptens that can be selectively recognized by secondary detection reagents. For example, these sites might be antigenic epitopes that bind antibodies. Alternatively, the target-specific molecule might be conjugated to either biotin or fluorescent dyes, thereby creating a molecule that can be detected with any of our avidin and streptavidin conjugates or our anti–fluorescent dye antibodies (Antibodies, Avidins, Lectins and Related Products—Chapter 7). As shown here, the target-specific molecule may contain multiple sites for binding the secondary detection reagent, thereby providing a simple system for amplifying the signal.
Both enzyme and macrofluorophore labels can be coupled directly to target-specific affinity reagents (primary detection) or to more generic affinity reagents that form stable complexes with unlabeled primary reagents, usually on the basis of immunorecognition (secondary detection). As indicated schematically in Figure 6.1.1, secondary detection inherently provides some degree of signal amplification, although sometimes at the expense of additional background due to nonspecific binding. These basic concepts of primary and secondary detection apply not only to the signal amplification techniques addressed in the current chapter but also to the dye-labeled affinity reagents described in Antibodies, Avidins and Lectins—Chapter 7.
Primary Detection Reagents
Any easily detectable molecule that binds directly to a specific target is a primary detection reagent. Such reagents are detected by fluorescence, chemiluminescence, absorption or electron diffraction conferred by stably attached labels. The conjugation and crosslinking chemistries used to create these stable attachments are discussed in detail in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1, Thiol-Reactive Probes—Chapter 2 and Crosslinking and Photoactivatable Reagents—Chapter 5. In addition to our fluorophore-labeled anti-dye antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4) and monoclonal antibodies (www.invitrogen.com/handbook/antibodies), many of the Molecular Probes site-selective products can be considered primary detection reagents. These include our fluorescent lectins (Lectins and Other Carbohydrate-Binding Proteins—Section 7.7), nucleic acid stains (Nucleic Acid Detection and Analysis—Chapter 8), protein and glycoprotein stains (Protein Detection on Gels, Blots and Arrays—Section 9.3, Detecting Protein Modifications—Section 9.4), phallotoxins (Probes for Actin—Section 11.1), membrane probes (Probes for Lipids and Membranes—Chapter 13), annexin V conjugates for detecting apoptotic cells (Assays for Apoptosis—Section 15.5) and various drug and toxin analogs (Probes for Neurotransmitter Receptors—Section 16.2, Probes for Ion Channels and Carriers—Section 16.3). These primary detection reagents can typically be detected by fluorescence microscopy, fluorometry or flow cytometry methods.
Secondary Detection Reagents
Although many biomolecules, such as antibodies and lectins, bind selectively to a biological target, they usually need to be chemically modified before they can be detected. Often the biomolecule is conjugated to a fluorescent or chromophoric dye or to a heavy atom complex such as colloidal gold. However, the researcher may wish to avoid the time and expense required for these conjugations, choosing instead to use a more generic secondary detection reagent. Typically, secondary detection reagents recognize a particular class of molecules. For example, labeled goat anti–mouse IgG antibodies can be used to localize a tremendous variety of target-specific mouse monoclonal antibodies. Our extensive secondary antibody offering (Secondary Immunoreagents—Section 7.2) provides a wide selection of labels including our superior Alexa Fluor dye series, phycobiliproteins, Alexa Fluor dye–phycobiliprotein tandem fluorophores, Qdot nanocrystals, biotin and enzyme labels (HRP and alkaline phosphatase). We also offer many options in terms of immunoreactivity, an essential consideration in avoiding confounding cross-reactivity when performing simultaneous secondary immunodetection of two or more targets. Our labeled secondary antibody portfolio contains antibodies against IgG and IgM from several mammalian species, including various isotypes of mouse IgG, as well as antibodies against avian (chicken) IgY. Our Zenon antibody labeling technology (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3) uses conjugates of an Fc-specific anti-IgG Fab fragment for the rapid and quantitative labeling of the corresponding mouse, rabbit, goat or human antibody.
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