Secondary Immunoreagents - Section 7.2

Figure 1.28 Photobleaching resistance of the red-fluorescent Alexa Fluor 647, Alexa Fluor 633, PBXL-3 and Cy5 dyes and the allophycocyanin fluorescent protein, as determined by laser-scanning cytometry. EL4 cells were labeled with biotin-conjugated anti-CD44 antibody and detected by Alexa Fluor 647 (S21374), Alexa Fluor 633 (S21375), PBXL-3, Cy5 or allophycocyanin (APC, S868) streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6). The cells were then fixed in 1% paraformaldehyde, washed and wet-mounted. After mounting, cells were scanned eight times on a laser-scanning cytometer; laser power levels were 18 mW for the 633 nm spectral line of the He–Ne laser. Scan durations were approximately five minutes apiece, and each repetition was started immediately after completion of the previous scan. Data are expressed as percentages derived from the mean fluorescence intensity (MFI) of each scan divided by the MFI of the first scan. Data contributed by Bill Telford, Experimental Transplantation and Immunology Branch, National Cancer Institute.

Figure 1.9 Photobleaching resistance of the green-fluorescent Alexa Fluor 488, Oregon Green 488 and fluorescein dyes, as determined by laser-scanning cytometry. EL4 cells were labeled with biotin-conjugated anti-CD44 antibody and detected by Alexa Fluor 488 (S11223), Oregon Green 488 (S6368) or fluorescein (S869) streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6). The cells were then fixed in 1% paraformaldehyde, washed and wet-mounted. After mounting, cells were scanned 10 times on a laser-scanning cytometer; laser power levels were 25 mW for the 488 nm spectral line of the argon-ion laser. Scan durations were approximately five minutes apiece, and each repetition was started immediately after completion of the previous scan. Data are expressed as percentages derived from the mean fluorescence intensity (MFI) of each scan divided by the MFI of the first scan. Data contributed by Bill Telford, Experimental Transplantation and Immunology Branch, National Cancer Institute.

Figure 7.56 Labeling scheme utilized in the Zenon Antibody Labeling Kits. An unlabeled IgG antibody is incubated with the Zenon labeling reagent, which contains a fluorophore-labeled, Fc-specific anti-IgG Fab fragment (panel A). This labeled Fab fragment binds to the Fc portion of the IgG antibody (panel B). Excess Fab fragment is then neutralized by the addition of a nonspecific IgG (panel C), 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.

Anti-IgG Antibodies

Molecular Probes offers secondary antibody conjugates directed against IgG from a variety of species, including human, mouse, rabbit, rat, chicken, goat, guinea pig, hamster and sheep (Summary of Molecular Probes' secondary antibody conjugates - Table 7.1). These anti-IgG antibodies are available with a wide selection of fluorophores, including our:

• Blue-fluorescent Alexa Fluor 350 (), Alexa Fluor 405, Marina Blue, Cascade Blue and Pacific Blue dyes
• Green-fluorescent Alexa Fluor 488 (, , , ), Alexa Fluor 500, Oregon Green 488, Oregon Green 514 (), BODIPY FL () and fluorescein dyes
• Yellow-green–fluorescent Alexa Fluor 430 and Alexa Fluor 514 dyes
• Yellow-fluorescent Alexa Fluor 532 dye ()
• Orange-fluorescent Pacific Orange, Alexa Fluor 546 (), Alexa Fluor 555 and tetramethylrhodamine dyes
• Red-orange–fluorescent Alexa Fluor 568 and Rhodamine Red-X dyes
• Red-fluorescent Alexa Fluor 594 (), Texas Red and Texas Red-X fluorophores
• Far-red–fluorescent Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647 (), Alexa Fluor 660 and Alexa Fluor 680 dyes
• Infrared-fluorescent Alexa Fluor 700 and Alexa Fluor 750 dyes
• Alexa Fluor dye–R-phycoerythrin (R-PE) tandem conjugates, which can each be excited with the 488 nm spectral line of the argon-ion laser, but exhibit long-wavelength emission maxima (627 nm for the Alexa Fluor 610–R-PE conjugates, 667 nm for the Alexa Fluor 647–R-PE conjugates and 702 nm for the Alexa Fluor 680–R-PE conjugates)
• Alexa Fluor dye–allophycocyanin (APC) tandem conjugates, which can each be excited by the He–Ne laser at 633 nm or by the krypton-ion laser at 647 nm with emission beyond 700 nm
• Red-orange–fluorescent DyeMer 488/605, red-fluorescent DyeMer 488/615 and far-red–fluorescent DyeMer 488/630 dyes, bifluorophores that can each be excited by the 488 nm spectral line of the argon-ion laser and emit at 605, 615 or 630 nm

Our species-specific anti-IgG antibodies, which are raised against IgG heavy and light chains, are affinity purified and adsorbed against the sera of a number of species to minimize crossreactivity. For multilabeling experiments in which crossreactivity is critical, we offer highly cross-adsorbed goat anti–mouse IgG and goat anti–rabbit IgG antibodies. See Cross-adsorption chart for Molecular Probes' labeled secondary antibody conjugates - Table 7.2 for a complete list of IgG and sera against which our anti-IgG antibodies have been cross-adsorbed.

Molecular Probes also offers chicken anti-mouse, -rabbit, -rat, -goat and -human secondary antibodies (Molecular Probes' affinity-purified chicken anti-mouse, -human, -goat, -rat and -rabbit IgG conjugates - Table 7.3). Chicken secondary antibodies have gained popularity because they demonstrate a lower level of nonspecific binding. Chicken IgY antibodies, which are functionally equivalent to mammalian IgG antibodies, lack a classical "Fc" domain and are not bound by protein A or protein G, nor do they bind to Fc receptors for mammalian IgG.

The CMNB-caged fluorescein conjugates of the goat anti–mouse IgG and goat anti–rabbit IgG antibodies (G21061, G21080) permit some unique experimental protocols, including the light-mediated targeted tagging of single cells or a few cells in tissues (Photoactivatable Reagents, Including Photoreactive Crosslinkers and Caged Probes - Section 5.3) and use as a photoaddressable hapten, in a manner similar to photolithography. These conjugates are essentially colorless and nonfluorescent until illuminated with ultraviolet light, whereupon sites labeled by the caged fluorophore conjugate yield green-fluorescent staining. This photoactivated fluorescence (PAF) can be measured as an increase in signal, even in the presence of a highly autofluorescent background or other green-fluorescent probes. Furthermore, the fluorescein dye that is liberated serves as a hapten that can be specifically detected and the signal amplified by anti-fluorescein/Oregon Green antibody conjugates (Anti-Dye and Anti-Hapten Antibodies - Section 7.4, Figure 7.71).

Figure 7.71 Schematic representation of photoactivated fluorescence combined with sample masking. Initially, no fluorescence is observed from samples stained with a CMNB-caged fluorescein-labeled secondary detection reagent (panel A). The desired mask is then placed over the sample (panel B), after which the sample is exposed to UV light. The mask is then removed; fluorescein molecules present in the unmasked portion of the sample are uncaged by the UV light and fluoresce brightly when viewed with the appropriate filters (panel C). Uncaged fluorescein may now also serve as a hapten for further signal amplification using our anti-fluorescein/Oregon Green antibody (A889). For example, probing with the anti-fluorescein/Oregon Green antibody followed by staining with the Alexa Fluor 594 goat anti–mouse IgG antibody (A11005) can be used to change the color of the uncaged probe to red fluorescent (panel D).

We offer the high-activity horseradish peroxidase and alkaline phosphatase conjugates of goat anti–mouse IgG, rabbit anti–mouse IgG, goat anti–rabbit IgG and rabbit anti–goat IgG antibodies (Alkaline phosphatase and horseradish peroxidase enzyme conjugates and Zenon Labeling Kits - Table 7.4), as well as biotin conjugates of goat anti–mouse IgG antibody and of the F(ab')₂ fragments of goat anti–mouse IgG antibody and goat anti–rabbit IgG antibody (B2763, B11027, B21078). By using an avidin, streptavidin or NeutrAvidin biotin-binding protein bridge, scientists can link our biotinylated or DSB-X biotin–labeled secondary antibodies to a biotinylated enzyme — a method that is often preferred because it tends to reduce nonspecific staining. Links to avidin and streptavidin conjugates made through DSB-X biotin are readily reversible (see Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6 for a description of our unique DSB-X biotin technology). Enzyme and hapten conjugates of secondary antibodies are also commonly used in histochemical amplification schemes such as the tyramide signal amplification (TSA) technology (Tyramide Signal Amplification (TSA) Technology - Section 6.2) and Enzyme-Labeled Fluorescence (ELF) technology (Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3, ). Our Zenon Alkaline Phosphatase and Horseradish Peroxidase Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14) permit the formation of enzyme-labeled antibodies using as little as submicrogram quantities of a primary antibody. Enzyme-conjugated antibodies are also used in a wide variety of ELISA methods, such as in our Amplex Red ELISA Kits (see below).

Anti-IgM Antibodies

In response to requests from researchers wanting to apply Alexa Fluor dye technology to the detection of IgM monoclonal antibodies, we have added goat anti–mouse IgM, goat anti–rat IgM and goat anti–human IgM antibodies to the list of antibodies we offer as conjugates of our Alexa Fluor dyes (Summary of Molecular Probes' secondary antibody conjugates - Table 7.1). The anti-IgM conjugates are prepared from well-characterized antibodies that have been purified by IgM affinity chromatography and react specifically with IgM heavy chains (µ chains) (Antibody Structure and Classification - Note 7.2). To minimize crossreactivity, the goat anti–mouse IgM antibodies have been adsorbed against human IgG₁, IgG_2a, IgG_2b, IgG₃, IgA, human serum and purified human paraproteins. The goat anti–human IgM antibodies have been adsorbed against human IgG and IgA. Due to their large size, IgM antibodies do not diffuse well into tissue, and because the IgM µ chain is more highly conserved across different species than are IgG, IgA, or light chains, anti-IgM antibodies may react with IgM from other species.

Isotype-Specific Antibodies

Molecular Probes offers isotype-specific antibodies to aid in multilabeling experiments (Molecular Probes' goat anti-mouse isotype-specific antibodies - Table 7.5). The Alexa Fluor goat anti–mouse IgG isotype-specific antibodies have been cross-adsorbed against mouse IgM, IgA, pooled human sera, purified human paraproteins and other isotypes to minimize crossreactivity.

F(ab')₂ Fragments

Our range of goat anti–mouse IgG and goat anti–rabbit IgG antibodies has been expanded to include fluorescent dye–, alkaline phosphatase–, horseradish peroxidase– and biotin-labeled F(ab')₂ fragments (Summary of Molecular Probes' secondary antibody conjugates - Table 7.1). These F(ab')₂ fragments are often preferred to whole antibody conjugates because they lack the Fc region (Antibody Structure and Classification - Note 7.2), thereby eliminating nonspecific interactions with Fc receptor–bearing cell membranes and allowing for better penetration into tissue. Please note that the rabbit Fc region may bind nonspecifically to human tissue; consequently, Molecular Probes recommends the F(ab')₂ fragment when using rabbit-derived secondary antibodies on human tissues.

Image-iT FX Kits

The Image-iT FX Kits (Image-iT FX Kits - Table 7.6) provide some of our best secondary detection reagents and the supporting materials needed for optimal imaging of fixed cells and tissue sections:

• Alexa Fluor conjugates of goat anti–mouse IgG antibody, goat anti–rabbit IgG antibody or streptavidin deliver superior photostability and brightness (Fluorescence Characteristics of the Alexa Fluor Dyes in the Image-iT FX Kits - Table 7.7)
• ProLong Gold antifade reagent reduces photobleaching (Figure 23.25, , ; see Fluorescence Microscopy Reference Standards and Antifade Reagents - Section 23.1 for more details)
• Image-iT FX signal enhancer improves the signal-to-noise ratio (, , )
• A sample pack of two CultureWell chambered coverglasses makes sample processing more convenient (Figure 23.35, see Accessories for Fluorescence Microscopy and Magnetic Separation - Section 23.3 for more details)

Each Image-iT FX Kit provides sufficient materials to perform 50–100 assays. Furthermore, the components of each kit are available separately (Alexa Fluor secondary antibodies, Summary of Molecular Probes' secondary antibody conjugates - Table 7.1; Alexa Fluor streptavidins, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23; ProLong Gold antifade reagent, P36930, Fluorescence Microscopy Reference Standards and Antifade Reagents - Section 23.1; Image-iT FX signal enhancer, I36933; CultureWell chambered coverglasses, C37000, C37005; Accessories for Fluorescence Microscopy and Magnetic Separation - Section 23.3) for flexibility in experimental design.

Image-iT FX Signal Enhancer

By efficiently blocking nonspecific interactions of a wide variety of fluorescent dyes with cell and tissue constituents, the Image-iT FX signal enhancer (I36933) dramatically improves the signal-to-noise ratio of immunolabeled cells and tissues, allowing clear visualization of targets that would normally be indistinguishable due to background fluorescence (, , ). Background staining seen with fluorescent conjugates of streptavidin (Fluorescent dyes successfully tested with the Image-iT FX signal enhancer * - Table 7.8), goat anti–mouse IgG antibody or goat anti–rabbit IgG antibody is largely eliminated when Image-iT FX signal enhancer is applied to fixed and permeabilized cells prior to staining. Image-iT FX signal enhancer may also effectively prevent nonspecific staining that is typically blocked with 1–2% BSA or 10% serum treatment, in some cases eliminating the need for another step in the staining protocol.

Molecular Probes' Alexa Fluor Signal Amplification Kits are designed to substantially increase the signals obtained by immunofluorescence techniques, thus permitting detection of low-abundance targets (Figure 7.46, Figure 7.47). Each kit takes advantage of the superior brightness and photostability of Alexa Fluor antibody conjugates. The Alexa Fluor 488 Signal Amplification Kit for Fluorescein-Conjugated Probes (A11053) dramatically enhances the fluorescence and photostability of virtually any fluoresceinated probe. The three kits for mouse antibodies (Molecular Probes' Alexa Fluor Signal Amplification Kits for Mouse Antibodies - Table 7.9) can be used to sensitively detect mouse primary antibodies. All of the Alexa Fluor Signal Amplification Kits contain detailed protocols for staining adherent cells grown on coverslips. The kits also contain protocols for use in flow cytometry.

Alexa Fluor 488 Signal Amplification Kit for Fluorescein- and Oregon Green Dye–Conjugated Probes

The Alexa Fluor 488 Signal Amplification Kit for Fluorescein-Conjugated Probes (A11053, Alexa Fluor(R) 488 Signal-Amplification Kit for Fluorescein and Oregon Green(R) Dye–Conjugated Probes) is designed to simultaneously enhance the fluorescence and the photostability of virtually any fluorescein- or Oregon Green dye–containing probe (Figure 7.46, Figure 7.47). This kit takes advantage of the superior properties of Alexa Fluor 488 conjugates. Alexa Fluor 488 conjugates are considerably brighter and more photostable than fluorescein-labeled probes (Figure 1.9, , ). In addition, the fluorescence of Alexa Fluor 488 conjugates is not sensitive to pH over a wide pH range, unlike the fluorescence of fluorescein conjugates (Figure 1.12).

Figure 7.46 Demonstration of the amplification obtained with the Alexa Fluor 488 Signal Amplification Kit for Fluorescein- and Oregon Green Dye–Conjugated Probes (A11053). Bovine pulmonary artery endothelial cells were labeled with anti–α-tubulin antibody (A11126) in combination with fluorescein goat anti–mouse IgG antibody (F2761) (left panel). The center panel shows the cells after treatment with Alexa Fluor 488 rabbit anti-fluorescein/Oregon Green antibody (A11090), and the right panel show the cells after additional labeling with the Alexa Fluor 488 goat anti–rabbit IgG antibody (A11008). The images were acquired using identical exposure times, and a bandpass filter set appropriate for fluorescein.

Figure 7.47 An example of flow cytometry results obtained using the Alexa Fluor 488 Signal Amplification Kit for Fluorescein- and Oregon Green Dye–Conjugated Probes (A11053). Human T-cell leukemia cells (Jurkat) were stained with fluorescein (FITC) mouse anti-CD4 antibody and, as indicated, with Alexa Fluor 488 rabbit anti-fluorescein/Oregon Green antibody (A11090) and Alexa Fluor 488 goat anti–rabbit IgG antibody (A11008). The fluorescence values of the negative controls, in which the FITC anti-CD4 antibody was omitted, are shown (black) together with the fluorescence values of the experimental samples (green). The fluorescence values represent the average signals from the population of cells analyzed.

Figure 1.12 Comparison of pH-dependent fluorescence of the Oregon Green 488 (), carboxyfluorescein () and Alexa Fluor 488 () fluorophores. Fluorescence intensities were measured for equal concentrations of the three dyes using excitation/emission at 490/520 nm.

The Alexa Fluor 488 Signal Amplification Kit for Fluorescein- and Oregon Green Dye–Conjugated Probes uses Alexa Fluor 488 conjugates of two different antibodies to amplify the signals from fluorescein-labeled probes. Alexa Fluor 488 anti-fluorescein/Oregon Green antibody, which is prepared from a rabbit IgG fraction, is first used to bind to the fluorescein- or Oregon Green dye–labeled target. The fluorescence signal is then dramatically enhanced by addition of the Alexa Fluor 488 goat anti–rabbit IgG antibody. Because the spectra of Alexa Fluor 488 conjugates are remarkably similar to those of fluorescein conjugates (Figure 7.48), the kit can be used with optical filters or instrument settings appropriate for fluorescein (Optical Filters for Fluorescence Microscopy - Section 23.5, Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11). The Alexa Fluor 488 Signal Amplification Kit for Fluorescein-Conjugated Probes can be used for fluorescence microscopy, flow cytometry, blots, microarrays and probably any other application that uses fluorescein-conjugated probes; it contains sufficient reagents for 60–120 assays by microscopy or flow cytometry.

Figure 7.48 Absorption and fluorescence emission spectra of fluorescein goat anti–mouse IgG antibody (F2761, (–)) and Alexa Fluor 488 goat anti–mouse IgG antibody (A11001, (---)). The fluorescence intensity of the Alexa Fluor 488 conjugate was significantly higher than that of the fluorescein conjugate. The data are normalized to show the spectral similarity.

Alexa Fluor Signal Amplification Kits for Mouse Antibodies

Molecular Probes offers three Alexa Fluor Signal Amplification Kits for Mouse Antibodies, permitting enhanced detection of mouse primary antibodies using conjugates of our superior Alexa Fluor 488, Alexa Fluor 568 and Alexa Fluor 594 dyes, which yield green, red-orange and red fluorescence, respectively (Molecular Probes' Alexa Fluor Signal Amplification Kits for Mouse Antibodies - Table 7.9). These kits each use two Alexa Fluor conjugates to detect antibodies derived from mouse. An Alexa Fluor rabbit anti–mouse IgG antibody conjugate is first used to bind to the mouse-derived primary antibody. The fluorescence is then dramatically enhanced by the addition of an Alexa Fluor conjugate of a goat anti–rabbit IgG antibody (Figure 7.49). The Alexa Fluor 488 (Alexa Fluor(R) 488 Signal-Amplification Kit for Mouse Antibodies), Alexa Fluor 568 (Alexa Fluor(R) 568 Signal-Amplification Kit for Mouse Antibodies) and Alexa Fluor 594 (Alexa Fluor(R) 594 Signal-Amplification Kit for Mouse Antibodies) Signal Amplification Kits for Mouse Antibodies can be used for both fluorescence microscopy and flow cytometry and contain sufficient materials for 60–300 assays.

Figure 7.49 Antibody amplification scheme using our superior Alexa Fluor conjugates, permitting enhanced detection of mouse primary antibodies. Molecular Probes offers three Alexa Fluor Signal Amplification Kits for Mouse Antibodies containing antibody conjugates of the Alexa Fluor 488 (A11054), Alexa Fluor 568 (A11066) and Alexa Fluor 594 (A11067) dyes, which yield green, red-orange and red fluorescence, respectively. These kits each use two Alexa Fluor conjugates to detect antibodies derived from mouse. An Alexa Fluor rabbit anti–mouse IgG antibody conjugate is first used to bind to the mouse primary antibody. The fluorescence signal is then dramatically enhanced by the addition of an Alexa Fluor conjugate of goat anti–rabbit IgG antibody.

The SensiFlex ELISA Development Kits (S33853, S33854) offer an extremely sensitive fluorometric detection method for β-lactamase–amplified enzyme-linked immunosorbent assays (ELISAs). The Fluorocillin Green 495/525 reagent provided in these kits is a novel substrate and, following cleavage by β-lactamase, emits a green-fluorescent signal (emission maxima ~525 nm). Fluorocillin Green reagent has a broad dynamic range of fluorescence signal, is more sensitive than common colorimetric substrates and displays only modest hydrolysis when incubated for extended periods from pH 5.5 to 8.0. Additionally, Fluorocillin Green reagent effectively reports β-lactamase activity in the presence of EDTA, many detergents, salts and sodium azide. Developing ELISA protocols that maximize both dynamic range and limit of detection involves optimizing the concentrations of both the enzyme and substrate. The robust enzyme kinetics of the reaction between β-lactamase and Fluorocillin Green reagent are consistent, even in the face of changes in enzyme and substrate concentration. This feature makes assay development using β-lactamase and Fluorocillin Green reagent easier than for assays that employ an enzyme with faster kinetics (e.g., horseradish peroxidase). The SensiFlex ELISA Development Kits contain:

• Fluorocillin Green 495/525 β-lactamase substrate
• Dimethylsulfoxide (DMSO)
• Concentrated phosphate-buffered saline (PBS)
• β-Lactamase TEM-1 conjugate of goat anti–mouse IgG antibody (in Kit S33853) or anti–rabbit IgG antibody (in Kit S33854)
• Fluorocillin stop reagent
• Sodium bicarbonate buffer, pH ~9.3
• Bovine serum albumin (BSA)
• Tween 20
• Nunc-Immuno MaxiSorp U96 plate
• Detailed protocols (SensiFlex ELISA Development Kit with Fluorocillin Green Reagent)

Sufficient reagents are provided in each kit for performing approximately 500 assays using 100 µL reaction volumes. Fluorocillin Green 495/525 β-lactamase substrate (F33952), as well as the β-lactamase TEM-1 conjugates of the goat anti–mouse IgG and anti–rabbit IgG secondary antibodies (G31567, G31568), are also available from Molecular Probes as standalone products.

Amplex Red ELISA Kits

Molecular Probes' Amplex Red ELISA Kits offer an extremely sensitive fluorometric or colorimetric detection method for horseradish peroxidase (HRP)–amplified enzyme-linked immunosorbent assays (ELISAs). The Amplex Red ELISA Kit #1 (A22170) contains an HRP goat anti–mouse IgG antibody conjugate, which can be used for the ELISA detection of any mouse IgG antibody. The Amplex Red ELISA Kit #2 (A22171) contains the versatile protein G conjugate of HRP, which can be used for the ELISA detection of IgGs from most commonly used species, including human, mouse, rabbit, goat, sheep, bovine and horse. The Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine, ) provided in these kits is a highly sensitive and stable probe for the detection of HRP activity. In the presence of HRP, the Amplex Red reagent reacts with hydrogen peroxide with a 1:1 stoichiometry to form the fluorescent product resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1; absorption/emission maxima ~571/585 nm, Figure 10.59). Because resorufin also has strong absorption, the assay can be performed either fluorometrically or spectrophotometrically. The Amplex Red ELISA Kit #1 with the HRP–goat anti–mouse IgG antibody conjugate has detection limits of as little as 10 pg/microplate well of a mouse IgG by fluorometry or 50 pg/microplate well by colorimetry (Figure 7.50). The Amplex Red ELISA Kit #2 with HRP–protein G has detection limits of as little as 1 ng/microplate well of a mouse IgG by fluorometry or 3 ng/microplate well by colorimetry (Figure 7.51) in 96-well plates.

Figure 7.51 Detection of a mouse monoclonal antibody using the Amplex Red ELISA Kit #2 (A22171). The assay can detect as little as 1 ng of a monoclonal antibody in the well of a microplate by fluorometry (panel A) or 3 ng by colorimetry (panel B). Conditions of the assay are essentially as described in the caption of Figure 7.50, except that a horseradish peroxidase conjugate of protein G was used instead of the horseradish peroxidase conjugate of goat anti–mouse IgG antibody.

Each Amplex Red ELISA Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Concentrated reaction buffer
• Hydrogen peroxide (H₂O₂)
• Horseradish peroxidase (HRP) conjugate of goat anti–mouse IgG antibody (in Kit #1, A22170) or of protein G (in Kit #2, A22171)
• Detailed ELISA protocols (Amplex(R) Red ELISA Kit #1, with Goat Anti–Mouse IgG, Horseradish Peroxidase Conjugate, Amplex(R) Red ELISA Kit #2, with Protein G, Horseradish Peroxidase Conjugate)

Each kit provides sufficient reagents for approximately 1000 ELISAs using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay. Our HRP conjugates of the goat anti–mouse IgG antibody (G21040), goat anti–rabbit IgG antibody (G21234) and protein G (P21041) are available separately. HRP conjugates of additional antibodies that can be used with the Amplex Red reagent (A12222, A22177; Substrates for Oxidases, Including Amplex Red Kits - Section 10.5) are listed in Alkaline phosphatase and horseradish peroxidase enzyme conjugates and Zenon Labeling Kits - Table 7.4.

Figure 10.59 Principle of coupled enzymatic assays using our Amplex Red reagent. Oxidation of glucose by glucose oxidase results in generation of H₂O₂, which is coupled to conversion of the Amplex Red reagent to fluorescent resorufin by HRP. The detection scheme shown here is used in our Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189).

Amplex ELISA Development Kits for Rabbit and Mouse IgG

The Amplex ELISA Development Kits for Mouse IgG (A33851) and for Rabbit IgG (A33852) provide a comprehensive set of components for creating a fluorescence-based ELISA using a mouse or rabbit primary antibody, respectively. This assay is based on the Amplex UltraRed reagent, a fluorogenic substrate for horseradish peroxidase (HRP) that reacts with H₂O₂ in a 1:1 stoichiometric ratio to produce a brightly fluorescent and strongly absorbing reaction product (excitation/emission maxima ~568/581 nm) (). Because the Amplex UltraRed peroxidation product has long-wavelength spectra, there is little interference from the blue or green autofluorescence found in most biological samples. With a high extinction coefficient, good quantum efficiency and resistance to autooxidation, the fluorescence-based Amplex UltraRed reagent delivers better sensitivity and a broader assay range than colorimetric reagents. In a sandwich ELISA format using C-reactive protein, Molecular Probes scientists are routinely able to 75 pg of antigen using goat anti–mouse IgG antibody and 1 pg using goat anti–rabbit IgG antibody); these detection limits are 25-fold lower than those obtained from the same sandwich ELISA format using the common colorimetric reagent TMB. Each Amplex ELISA Development Kit contains:

• Amplex UltraRed reagent
• Dimethylsulfoxide (DMSO)
• Concentrated phosphate-buffered saline (PBS), pH 7.2
• Horseradish peroxidase conjugate of goat anti–mouse IgG antibody (in A33851) or goat anti—rabbit IgG antibody (in A33852)
• Amplex stop reagent
• Hydrogen peroxide (H₂O₂)
• 0.1 M sodium bicarbonate buffer, pH ~9.3
• Bovine serum albumin (BSA)
• Tween 20
• Nunc-Immuno MaxiSorp U96 plate
• Detailed protocols (Amplex(R) ELISA Development Kit for Mouse IgG with Amplex(R) UltraRed Reagent, Amplex(R) ELISA Development Kit for Rabbit IgG with Amplex(R) UltraRed Reagent)

Sufficient reagents are provided in each kit for 500 microplate assays in a 96-well fluorescence microplate format (100 µL per assay).

NANOGOLD and Alexa Fluor FluoroNanogold Conjugates

In collaboration with Nanoprobes, Inc. (http://www.nanoprobes.com/), Molecular Probes offers NANOGOLD and Alexa Fluor FluoroNanogold conjugates of antibodies and streptavidin to facilitate immunoblotting, light microscopy and electron microscopy applications (NANOGOLD(R) and FluoroNanogold Conjugates). These reagents include affinity-purified Fab fragments of the goat anti–mouse IgG, goat anti–rabbit IgG and rabbit anti–goat IgG antibodies, as well as of streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, NANOGOLD, Alexa Fluor FluoroNanogold and colloidal gold conjugates - Table 7.10). Also available are NANOGOLD mono(sulfosuccinimidyl ester) (N20130, Long-Wavelength Rhodamines, Texas Red Dyes and QSY Quenchers - Section 1.6, NANOGOLD(R) Mono(sulfosuccinimidyl ester)) and NANOGOLD monomaleimide (N20345, Thiol-Reactive Probes Excited with Visible Light - Section 2.2, NANOGOLD(R) Monomaleimide), which can be conjugated to amines (Figure 1.89) and thiols (Figure 2.22), respectively, in the same way that dyes are conjugated to proteins and nucleic acids. NANOGOLD conjugates are covalently conjugated to the 1.4 nm NANOGOLD gold cluster label, whereas Alexa Fluor FluoroNanogold conjugates are coupled to both a NANOGOLD label and either the Alexa Fluor 488 or Alexa Fluor 594 fluorophore, resulting in gold clusters with green or red fluorescence, respectively. Alexa Fluor FluoroNanogold conjugates have all the advantages of the NANOGOLD cluster, with the additional benefit that they may be used for correlative fluorescence, light and electron microscopy ().

Figure 1.89 Reaction of NANOGOLD mono(sulfosuccinimidyl ester) (N20130) with a primary amine. Image courtesy of Nanoprobes, Inc.

Figure 2.22 Reaction of NANOGOLD monomaleimide (N20345) with a thiol. Image courtesy of Nanoprobes, Inc.

NANOGOLD gold clusters have several advantages over colloidal gold. They develop better with silver than do most gold colloids and as a result, provide higher sensitivity. Additionally, NANOGOLD particles do not have as high affinity for proteins as do gold colloids, thereby reducing any background due to nonspecific binding. Several additional advantages of NANOGOLD and Alexa Fluor FluoroNanogold streptavidin over colloidal gold conjugates include:

• The NANOGOLD gold clusters are an extremely uniform (1.4 nm ± 10% diameter) and stable compound, not a gold colloid.
• NANOGOLD gold clusters are smaller than a complete IgG (H+L) antibody — approximately 1/15 the size of an Fab fragment — and therefore will be able to better penetrate cells and tissues, reaching antigens that are inaccessible to conjugates of larger gold particles.
• NANOGOLD conjugates contain absolutely no aggregates, as they are chromatographically purified through gel filtration columns. This feature is in sharp contrast to colloidal gold conjugates, which are usually prepared by centrifugation to remove the largest aggregates and frequently contain significantly smaller aggregates.
• The ratio of NANOGOLD particle to F(ab) is nearly 1:1, making this product distinct from the 0.2–10 variable stoichiometry of most colloidal gold–antibody preparations.
• NANOGOLD cluster–stained targets develop better with silver than do most gold colloids, resulting in higher sensitivity. Silver enhancement, such as the system provided in the LI Silver Enhancement Kit (L24919), is described below.

NANOGOLD and Alexa Fluor FluoroNanogold products can be used in immunoblotting, light microscopy, and electron microscopy to provide clear visibility (). Standard immunostaining methodologies can be used successfully with NANOGOLD and Alexa Fluor FluoroNanogold immunoreagents. Also, because the concentration of antibody and gold is similar to most commercial preparations of colloidal gold antibodies, similar dilutions and blocking agents are appropriate.

Colloidal Gold Complexes

Molecular Probes offers Alexa Fluor 488 dye–labeled colloidal gold conjugates, including affinity-purified goat anti–mouse IgG and goat anti–rabbit IgG antibodies and streptavidin (NANOGOLD, Alexa Fluor FluoroNanogold and colloidal gold conjugates - Table 7.10). These conjugates, which have been adsorbed to 5 nm or 10 nm gold colloids, may be used as probes in immunoblotting, light microscopy, fluorescence microscopy or electron microscopy. The fluorescence of these conjugates can be easily detected by standard techniques, but visualization of colloidal gold can be greatly improved using silver-enhancement methods, such as those we provide in the LI Silver Enhancement Kit (L24919) described below.

Combining fluorescent secondary detection reagents with colloidal gold to form functional complexes is difficult because the fluorescence of fluorophores such as fluorescein is significantly quenched by proximity to the colloidal gold. Molecular Probes makes fluorescent colloidal gold complexes with our Alexa Fluor 488 dye, a dye that has superior brightness and photostability. Our Alexa Fluor 488 dye–labeled colloidal gold complexes of anti-IgG antibody and of streptavidin may be used to perform correlated immunofluorescence and electron microscopy in a two-step labeling procedure, rather than in the three-step indirect labeling procedure that is required with conventional nonfluorescent colloidal gold complexes of anti-IgG antibodies or streptavidin.

LI Silver Enhancement Kit

The LI Silver Enhancement Kit (L24919) provides a convenient, light-insensitive silver-enhancement system for use with the NANOGOLD, Alexa Fluor FluoroNanogold and colloidal gold reagents that can be used for electron or light microscopy or to visualize gold particles on blots. LI silver is nucleated quickly by NANOGOLD gold clusters or colloidal gold, resulting in the precipitation of metallic silver and the formation of a dark brown to black signal. The system has markedly delayed self-nucleation, resulting in high contrast and very low backgrounds.

Gold particles in the presence of silver (I) ions and a reducing agent such as hydroquinone act as catalysts to reduce silver (I) ions to metallic silver (Figure 7.54). The silver is deposited onto the gold, enlarging the particles to between 30 and 100 nm in diameter. Tissues or blots stained with NANOGOLD gold clusters or colloidal gold are "developed" by this autometallographic procedure to give black staining that can be seen in a light microscope. This method — known as immunogold silver staining (IGSS) — has been widely used with the NANOGOLD cluster probe; it is one of the most sensitive immunodetection systems available and gives highly visible, permanent staining with no fading, with detection limits rivaling that of chemiluminescence and radionuclide labeling. Silver-enhanced NANOGOLD staining is compatible with double-labeling techniques, including enzyme-mediated staining. In blots, as little as 0.1 pg of a target IgG antibody can be detected using a NANOGOLD gold cluster labeled with an Fab' fragment of a secondary antibody. NANOGOLD streptavidin (N24918, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) has proved to be highly sensitive in detecting biotinylated nucleic acid probes in in situ hybridization studies.

Figure 7.54 Mechanism of silver deposition utilized by the LI Silver Enhancement Kit (L24919). Image courtesy of Nanoprobes, Inc.

The LI Silver Enhancement Kit (L24919, LI Silver Enhancement Kit), which is useful for light microscopy, gels and Western blots, is ideal for use with the NANOGOLD and Alexa Fluor FluoroNanogold reagents and for enhancing colloidal gold products. The advantages of LI silver enhancement include:

• High-contrast signal for easy light microscope and immunoblot visibility
• Lower background than other commercial developers
• High sensitivity
• Light-insensitive signal; development can be observed under normal room lighting
• Relatively slow development (10–30 minutes) for precise monitoring of the extent of development
• Compatibility with all immunogold reagents

The use of horseradish peroxidase (HRP) for enzyme-amplified immunodetection — commonly referred to as immunoperoxidase labeling — is a well-established standard histochemical technique. The most widely used HRP substrate for these applications is diaminobenzidine (DAB), which generates a brown-colored polymeric oxidation product localized at HRP-labeled sites. The DAB reaction product can be visualized directly by bright-field light microscopy or, following osmication, by electron microscopy. We offer DAB Histochemistry Kits for detecting mouse IgG primary antibodies (D22185) and biotinylated antibodies and tracers (D22187, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6). Each kit contains:

• Diaminobenzidine (DAB)
• HRP-labeled goat anti–mouse IgG antibody (in Kit D22185) or streptavidin (in Kit D22187) conjugate
• H₂O₂ reaction additive
• Blocking reagent
• Staining buffer
• A detailed staining protocol (Diaminobenzidine Histochemistry Kits)

Each kit provides sufficient materials to stain approximately 200 slides.

Magnetic separation is a quick, simple technique for isolating cells, organelles, proteins and nucleic acids from complex mixtures, based on bioaffinity principles. The sample containing a target ligand to be isolated is incubated with the superparamagnetic particles coupled with the appropriate affinity reagent, e.g., streptavidin, an antibody or a complementary nucleic acid. The bound target ligand is then separated efficiently from the mixture using a high field-strength magnet.

Molecular Probes, in association with Immunicon Corporation, offers Captivate ferrofluid conjugates of goat anti–mouse IgG antibody (C21473), goat anti–rabbit IgG antibody (C21474) and streptavidin (C21476). Ferrofluids are superparamagnetic particles ~200 nm in diameter that respond to a magnetic field but completely demagnetize when the field is removed. The key feature of the Captivate ferrofluid is its small and relatively uniform particle size, which results in efficient diffusion of the ferrofluid conjugate and rapid kinetics of the binding reaction. Once added to the sample, however, no further mixing is required. Furthermore, the ferrofluid conjugates exhibit significantly higher ligand-binding capacities per mass, as compared with larger-diameter superparamagnetic particles from other suppliers. We also offer the Captivate microscope-mounted magnetic yoke assembly (C24700) and disposable sample chambers (C24701), which have been specially designed for use with the Captivate ferrofluid conjugates (Figure 7.55), for optimum capture of Captivate ferrofluid–tagged cells from such diverse liquid samples as culture media, blood and biological buffers. The Captivate microscope-mounted magnetic yoke assembly includes one free set of 10 disposable sample chambers.

Figure 7.55 Flow chart for the magnetic separation and analysis of a cell suspension. Cells are treated with an antibody or a biotinylated or DSB-X biotin–labeled probe that binds to cell-surface markers. The treated cells are incubated with the appropriate Captivate ferrofluid conjugates, which bind to target cells. The mixture is then transferred to a chamber that is inserted into a magnetic yoke. Under the influence of a strong magnetic field, the cells bound to Captivate ferrofluid conjugates are rapidly separated from the unbound cells. The separate cell populations can be analyzed by both fluorometry and fluorescence microscopy.

The unique particle size of the Captivate ferrofluid permits fluorescence microscope–based cell sorting, imaging and analysis — analytical options that rival the capabilities of much more expensive instrumentation. To accomplish this, the cell suspension or blood sample is incubated with the appropriate fluorescent probes and then mixed with the Captivate ferrofluid conjugate. The mixture is loaded into a sample chamber, which is inserted into the magnetic yoke assembly and positioned on a microscope stage. Under influence of the strong magnetic gradient present in the chamber, cells labeled with the Captivate ferrofluid conjugate move quickly into position at the upper surface of the sample chamber. Cells that are not magnetically labeled sediment to the bottom. The captured cells can then be imaged and analyzed, or recovered free from unselected cells (Figure 7.55).

Molecular Probes also has available Captivate magnetic separators for both microplates (C24702, Figure) and microtubes (C24703, Figure) that we find to be particularly useful with the Captivate ferrofluid products. The microplate separator is compatible with most 96-well microplates, whereas the microtube separator can accommodate six 1.5 mL microcentrifuge tubes. Both separators provide excellent separation efficiency and pull magnetic particles to one side, allowing easier removal of supernatants.

Our DSB-X biotin technology permits the fully reversible labeling of DSB-X biotin derivatives by avidin and streptavidin conjugates under extremely mild conditions (Figure 7.100). DSB-X biotin (Figure 4.1) has moderate affinity for avidin and streptavidin; however, its binding is rapidly reversed by addition of excess D-biotin (B1595, B20656) or D-desthiobiotin (D20657). This facile reversibility has several potential applications:

• Live (or fixed) cells can be selectively isolated from a complex suspension of cells with a combination of Captivate ferrofluid streptavidin (C21476, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6), a DSB-X biotin–labeled secondary antibody (Molecular Probes' biotinylated and desthiobiotinylated secondary antibodies - Table 7.11) and a cell-selective primary antibody, then released into fresh medium at neutral pH and physiological temperatures with D-biotin (B1595, B20656; Figure 7.104).
• Cells that have been isolated with the Captivate ferrofluid streptavidin by the above method retain the DSB-X biotin immunolabel; consequently, they can be detected and subsequently analyzed with any of our wide array of avidin and streptavidin conjugates (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23).
• Targets in cells and tissues or on Western or Southern blots labeled with DSB-X biotin conjugates of antibodies or other DSB-X biotin–labeled reagents can initially be stained with fluorescent avidin or streptavidin conjugates, then the fluorescent staining can be reversed with D-biotin and the sample restained with an enzyme-conjugated avidin or streptavidin derivative in combination with a permanent stain, such as diaminobenzidine (DAB; D22185, D22187) or the combination of NBT and BCIP (N6495, B6492, N6547; Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3) or with a chemiluminescent enzyme substrate such as the BOLD APB chemiluminescent substrate for membrane-based alkaline phosphatase detection (B21901, Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4).
• DSB-X biotin conjugates can be reversibly immobilized on Captivate ferrofluid streptavidin or streptavidin agarose (S951, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) and, while on the matrix, the bioconjugate — an antibody, an enzyme, an oligonucleotide or nucleic acid, a drug or some other DSB-X biotin conjugate — can bind to their targets, which may be from cell or tissue extracts or other sources. Gentle elution of the entire complex facilitates subsequent analysis of the affinity-isolated products by electrophoresis or other means. A potentially important application of this technique is the detection of specific protein–protein and protein–nucleic acid interactions through selective isolation and release of their intact complexes. Our DSB-X Biotin Bioconjugate Isolation Kit #1 (D20658, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) contains the reagents and a protocol (DSB-X Bioconjugate Isolation Kit #1, with streptavidin agarose) for isolation and recovery of complexes of DSB-X biotin conjugates with streptavidin agarose.

Figure 4.1 Comparison of the structures of D-biotin (top) and D-desthiobiotin (bottom).

Figure 7.104 Cell separation using Captivate ferrofluid streptavidin and DSB-X biotin conjugates. A mixed population of cells is first mixed with a DSB-X biotin–labeled antibody against an appropriate surface antigen (panel A); subsequent incubation results in the labeling of a specific subpopulation (panel B). The sample is then incubated with Captivate ferrofluid streptavidin (C21476), which binds to the DSB-X biotin hapten, allowing the labeled cells to be isolated via a magnetic field (panel C). After the unlabeled cells have been washed away, the captured cells can be released by reversing the streptavidin linkage to DSB-X biotin with unlabeled biotin (panel D).

DSB-X biotin conjugates of antibodies and other proteins can be efficiently prepared using our DSB-X Biotin Protein Labeling Kit (D20655), which is described in Kits for Labeling Proteins and Nucleic Acids - Section 1.2. Alternatively, our Zenon DSB-X Biotin Mouse IgG₁ Labeling Kit (Z25053, Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3) can be used to rapidly and quantitatively prepare DSB-X biotin–labeled complexes of any intact mouse IgG₁ antibody (Figure 7.56).