Secondary Antibodies, Conjugates, and Kits

Secondary Antibodies

Secondary antibodies are polyclonal or monoclonal antibodies that bind to primary antibodies, or antibody fragments, such as the Fc or Fab regions. They are typically labeled with probes that make them useful for detection, purification, or sorting applications. Our polyclonal secondary antibodies are produced from the serum of host animals such as rabbit, goat, and sheep, whereas monoclonal secondary antibodies are produced from primarily mouse hybridoma clones. Secondary antibodies are used in many applications including ELISA, immunopurification, Western Blotting, IHC, IF, flow cytometry, and more.

The specific utility of a secondary antibody depends upon its conjugated probe(s). Probes are molecules that support various detection technologies. The most common detection systems for conjugated secondary antibodies are colorimetric or fluorescent. Colorimetric assays are typically based on the use of alkaline phosphatase (AP) or horseradish peroxidase (HRP) or its derivatives. The biotin avidin (streptavidin) conjugate binding system is often used to amplify the colorimetric signal for AP or HRP. The most common fluorescent assays utilize Fluorescein (FITC), Rhodamine or its derivative, TRITC, Cyanine (Cy3), or Phycoerythrin (R-PE), CF™ or Atto dyes.

We offer a wide variety of secondary antibodies.

Antibody Specificity

Secondary antibodies have been raised to immunoglobulins of various species to provide reagents for visualizing unconjugated primary antibodies bound to antigens in immunoassays. Antibodies to IgG that are whole molecule specific or Fab specific will usually react with all Ig classes, whereas heavy chain specific and Fc specific antibodies will react only with the indicated Ig class. The researcher will need to determine which specificity is best suited to their work. The assay system and the presence of extraneous protein targets that could bind the antibody and give rise to false positive results or high background will affect the choice of reagents. Table A summarizes the various types of secondary antibodies and their uses.

Table A Antibody Specificity

Antibody Specificity	Description	Uses
Anti-IgG (whole molecule)	Anti-IgG (whole molecule) antibodies have been generated using the intact IgG molecule as immunogen. Antibodies are developed to all portions of the IgG molecule that are different from the host's own IgG. Some of the epitopes recognized may also be found on Igs of other species. This means that antibodies to IgG, whole molecule have a very broad specificity and may cross-react not only with other Ig classes such as IgA and IgM, but also with IgG from other species. For instance, anti-goat IgG, whole molecule, may react with human IgG.	Use when the presence of multiple Ig classes is not a concern, when it is desirable to detect all Ig present, regardless of subclass, or to give a strong signal. They are also a good choice when the antibody Ig class is not known or when detecting total Ig in cell or tissue samples.
Anti-IgG (Fab specific)	Fab specific antibodies are raised by immunizing the host animal with Fab fragments generated by papain digestion of IgG. Since Fab fragments contain epitopes common to all Ig classes, these antibodies can recognize all Ig classes. They do not bind to the class-specific Fc region. As with anti-IgG, whole molecule, these antibodies may react with Igs from other species via epitopes shared among species.	Use when binding to the Fc region is either not possible or undesirable, such as when the target Ig Fc is bound by Protein A, anti-Fc specific antibodies, or Fc receptors on cell surfaces.
Anti-IgG heavy chain specific (alpha, gamma, mu)	Heavy chain specific antibodies are prepared from anti-whole molecule antibodies that have been adsorbed to remove essentially all cross-reactivity to epitopes on the Ig molecule that are not unique to the Ig class of the immunogen. Adsorption is performed as described below in Anti-IgG, Adsorbed with Various Species, using adsorbants containing immunoglobulins of the same species but different Ig class than the immunogen. Antibodies that recognize epitopes on the adsorbant Ig bind to the adsorbant. The heavy-chain specific antibodies are collected in the unbound portion. The resultant antibodies will only recognize a single Ig class. The specificity is indicated by a Greek letter corresponding to the class-specific determinants in the Fc region of the heavy chain: alpha-chain - IgA specific, gamma-chain - IgG specific, mu-chain - IgM specific.	Use when the target contains multiple Ig classes, but only one class is of interest. A common application is detection of individual Igs on tissue sections or in cell preparations for flow cytometry using anti- IgG, IgA, or IgM, heavy chain specific, conjugated to a fluorochrome to determine the level of an individual Ig on a particular cell type. They are also useful for double labeling, such as the localization of two different antigens in a single tissue section using an IgG monoclonal antibody and an IgM monoclonal antibody and detecting bound antibody with anti-mouse IgG, gamma-chain specific, and anti-mouse IgM, mu-chain specific, labeled with different fluorochromes.
Anti-IgG (Fc specific)	Fc specific antibodies are raised by immunizing the host animal with Fc fragments generated by papain digestion of IgG (see the Antibody Fragmentation section). These antibodies are highly specific for IgG, and do not recognize IgA or IgM. Note: Fc specific and gamma-chain specific antibodies have essentially the same specificity.	Same as for Anti-Ig, Heavy-Chain Specific, but for IgG only.
Anti-IgG, Adsorbed	These antibodies are prepared by adsorption with IgG from species other than the species of the target IgG to remove cross-reactivity to those species. This cross-reactivity arises because immunoglobulins contain epitopes that are common to several species. Adsorption is accomplished by incubating the antibodies with IgG from the other species attached to solid-phase supports (adsorbants). Antibodies that recognize epitopes on IgG from those species bind to the adsorbant. The species-specific antibodies are collected in the unbound portion. The resultant antibodies retain good reactivity with IgG from the target species, but reactivity with the adsorbant species is minimized. Adsorbed secondary antibodies are useful for reducing background in immunoassays caused by the secondary antibody binding to the protein or tissue being assayed in addition to the primary antibody.	Use when the target IgG is attached to a matrix that contains potential targets from other species. For example, when detecting primary antibodies on human cells or tissues in immunohistochemistry, choose a secondary antibody that has been adsorbed with human serum proteins.

Conjugates

Alkaline Phosphatase Conjugates

Alkaline phosphatase (AP) is an intestinal enzyme that dephosphorylates alcohols, phenols and amines at alkaline pH. It is a 140 kDa homodimer. The optimal pH range for activity is 9.5-10.5.

Alkaline phosphatase conjugates are widely used in immunoassays such as ELISA,^2,3 immunohistochemistry and immunocytochemistry,⁴ and immunoblotting.⁵ AP conjugates are useful in tissues where endogenous peroxidase activity may generate high background staining with peroxidase conjugates. They are usually more sensitive than peroxidase conjugates, allowing use of higher dilutions, or detection of lower signals, in ELISA or blotting assays. Endogenous alkaline phosphatase activity in tissue sections may be blocked by adding levamisole (L9756) to the substrate buffer.⁶ Levamisole inhibits all alkaline phosphatase isoenzymes with the exception of intestinal alkaline phosphatase.

Alkaline phosphatase substrates are available to form either soluble or insoluble products.

Alkaline phosphatase conjugates are not recommended for use with intestinal tissue sections or extracts because of endogenous intestinal alkaline phosphatase activity.

Figure 1. Formalin-fixed, paraffin-embedded section of human tonsil stained with Anti-Human IgG (gamma -chain specific)-Alkaline Phosphatase Conjugate, F(ab)2 fragment of goat antibody (Cat. No. A3312) using SIGMA FAST™ Fast Red TR/Naphthol AS-MX Tablets (Cat. No. F4523) as substrate.

References

1. O'Sullivan M, Gnemmi E, Morris D, Chieregatti G, Simmons M, Simmonds A, Bridges J, Marks V. 1978. A simple method for the preparation of enzyme-antibody conjugates. 95(2):311-313. https://doi.org/10.1016/0014-5793(78)81018-7

2. LUNDBERG BB, GRIFFITHS G, HANSEN HJ. 1999. Conjugation of an Anti-B-cell Lymphoma Monoclonal Antibody, LL2, to Long-circulating Drug-carrier Lipid Emulsions. 51(10):1099-1105. https://doi.org/10.1211/0022357991776787

3. Pretorius A. 1997. The binding potential of commercial antibody conjugates with sera of various small terrestrial mammals. Onderstepoort J. Vet. Res..(64):201-203.

4. Heider H, Schroeder C. 1997. Focus luminescence assay: macroscopically visualized foci of human cytomegalovirus and varicella zoster virus infection. Journal of Virological Methods. 66(2):311-316. https://doi.org/10.1016/s0166-0934(97)00063-3

5. Roe IH, Son SH, Oh HT, Choi J, Shin JH, Lee JH, Hah YC. Changes in the Evolution of the Antigenic Profiles and Morphology during Coccoid Conversion of Helicobacter pylori. Korean J Intern Med. 14(1):9-14. https://doi.org/10.3904/kjim.1999.14.1.9

6. Beesley J. 1993. Immunochemistry: A Practical Approach. Oxford, England: IRL Press.

Peroxidase Conjugates

Horseradish peroxidase (HRP) is an enzyme that specifically reduces hydrogen peroxide in the presence of a proton-donor. HRP is a 40 kDa glycoprotein. The optimal pH range for activity is 6.0-6.5. HRP exhibits good thermal stability (up to 60 ⁰C) and pH stability (4-10). It is inhibited by azide.

Peroxidase conjugates are used in a variety of immunoassays such as ELISA,^4,5 immunohistochemistry and immunocytochemistry,⁶ and immunoblotting.⁷ HRP conjugates are useful in tissues such as intestine where endogenous alkaline phosphatase activity may generate high background with alkaline phosphatase conjugates. The selection of available peroxidase substrates is wider than that for alkaline phosphatase, and the colors generated are frequently more intense. Endogenous peroxidase activity may be blocked by treating the tissue with an excess of hydrogen peroxide before addition of the conjugate, or by use of other blocking agents such as phenylhydrazine, azide plus nascent hydrogen peroxide, or periodic acid.⁸ A wide variety of substrates are available to form either soluble or insoluble products.

HRP conjugates are not recommended for use with samples such as blood cells and kidney tubules that contain high levels of endogenous peroxidase activity.

References

1. Avrameas S. 1978. Scand. J. Immunol., 8, Suppl. 7, 7.

2. Wilson M, Nakane P. 1978. Immunofluorescence and Related Staining Techniques. Elsevier/North Holland Biomedical Press.215.

3. O'Sullivan M, Gnemmi E, Morris D, Chieregatti G, Simmons M, Simmonds A, Bridges J, Marks V. 1978. A simple method for the preparation of enzyme-antibody conjugates. 95(2):311-313. https://doi.org/10.1016/0014-5793(78)81018-7

4. Sanz, et. al. C. 1999. An enzyme-linked immunosorbent assay applicable to screen blood donors for IgA deficiency. Haematologica. 84887-890.

5. Piza ASdT, Santos JLF, CHAVES LB, Zanettai CR. 1999. An elisa suitable for the detection of rabies virus antibodies in serum samples from human vaccinated with either cell-culture vaccine or suckling-mouse-brain vaccine. Rev. Inst. Med. trop. S. Paulo. 41(1):39-43. https://doi.org/10.1590/s0036-46651999000100008

6. Hanas, et. al., J. 1999. Expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and p53 tumor suppressor in dysplastic progression and adenocarcinoma in Barrett esophagus. Cancer. 86756-763.

7. Krajewski S, Zapata JM, Reed JC. 1996. Detection of Multiple Antigens on Western Blots. Analytical Biochemistry. 236(2):221-228. https://doi.org/10.1006/abio.1996.0160

8. Beesley J. 1993. Immunochemistry: A Practical Approach. Oxford, England: IRL Press.

Fluorochrome Conjugates

Fluorophores absorb light at one wavelength, the absorption or excitation wavelength, inducing an excited electronic state. This state is unstable, and the molecule quickly returns to the unexcited, or ground, state by emitting light. Due to energy loss, this light is emitted at a longer wavelength (lower energy), which is termed the emission wavelength. The difference between the excitation and emission wavelengths is unique to each fluorophore, and the intensity of excitation and emission drops quickly as the wavelength varies from the maximum. The Fluorescent Dye Properties Table gives the excitation and emission wavelengths and the fluorescent color for several common fluorophores.

Table 2 Fluorescent Dye Properties

Fluorochrome	Excitation (nM)	Emission (nM)	Color	Application
Acridine Orange	500	526 (DNA) 650 (RNA)	Green (DNA) Red (RNA)	Nucleic acid stain, Flow cytometry, fluorescence microscopy.
Cy3	552	568-574	Red-orange	Protein conjugation. Flow cytometry.
Cy5	649	670	Red	Protein conjugation. Fluorescence microscopy.
DAPI	360	450	Blue	AT, minor groove dsDNA stain. Flow Cytometry, fluorescence microscopy.
Dil-C18-(3)	550	565	Red-orange	Membrane stain (ER,CM) - cell fusion/permeabilization. Flow cytometry
Ethidium Bromide	493	620	Red	Nucleic acid stain. Gel electrophoresis.
Fluorescein (FITC)	495	525	Green	Protein conjugation. Flow cytometry, fluorescence microscopy.
Green Fluorescent Protein (GFP)	395 (470)	509	Green	Fusion protein. Recombinant protein expression. Fluorescence microscopy.
Hoeschst 33258	360	470	Blue	AT, minor groove dsDNA stain. Flow cytometry, fluorescence microscopy.
R-Phycoerythrin (PE)	488	578	Red-orange	Protein conjugation. Flow cytometry.
PKH2	490	504	Green	Cell membrane stain-cell tracking. Flow cytometry, fluorescence microscopy.
PKH26	551	567	Red-orange	Cell membrane stain-cell tracking. Flow cytometry, fluorescence microscopy.
PKH67	490	502	Green	Cell membrane stain-cell tracking. Flow cytometry, fluorescence microscopy.
CellVue® Claret	655	675	Far-Red	Cell membrane stain-cell tracking. Flow cytometry, fluorescence microscopy.
Propidium Iodide	493	632	Red	Nucleic acid stain, Flow cytometry, fluorescence microscopy.
Quantum Red™	488	670	Red	Protein conjugation. Tandem dye. Flow cytometry.
Rhodamine (TRITC)	552	570	Red-orange	Protein conjugation. Flow cytometry, fluorescence microscopy.
Texas Red	596	620	Red	Protein conjugation. Flow cytometry, fluorescence microscopy.

Fluorescent dyes are used to detect molecules in a variety of biological applications.¹ Unlike many visible dyes, fluorescent dyes generally may be used under physiological conditions and allow staining of living cells and tissues.² There is comparatively little overlap between emission wavelengths of many fluorophores, allowing staining with two or more fluorescent probes on one sample.^2,3 However, this would require that the excitation wavelengths be very similar and the emission wavelengths be considerably different. Tandem dyes such as Quantum Red™ dye, which is a conjugate of two dyes in which the emission wavelength of one dye matches the excitation wavelength of another, can supply an additional color using the same lamp. Some fluorescent dyes stain cell structures directly, such as acridine orange, DAPI, and Hoechst 33258, which stain nucleic acids. Others, such as fluorescein, rhodamine, and phycoerythrin, are conjugated to antibodies, lectins, nucleotides or other biological probes for localization of specific cell or tissue targets.

Fluorochrome conjugates should be protected from light during storage and use.

References

1. Lausch R, Reif O, Riechel P, Scheper T. 1995. Analysis of immunoglobulin G using a capillary electrophoretic affinity assay with protein A and laser-induced fluorescence detection. Electrophoresis. 16(1):636-641. https://doi.org/10.1002/elps.11501601102

2. Lloyd-Evans, Guest, Austin, Scott. 1999. Use of a phycoerythrin-conjugated anti-glycophorin A monoclonal antibody as a double label to improve the accuracy of FMH quantification by flow cytometry. Transfus Med. 9(2):155-160. https://doi.org/10.1046/j.1365-3148.1999.00187.x

3. Stewart C, Stewart S. 1999. Four color compensation. Cytometry. 38161-175.

CF™ Dyes

CF dyes are a series of highly water-soluble fluorescent dyes spanning the visible and near-infrared (near-IR) spectrum for labeling antibodies, proteins, nucleic acids, and other biomolecules. Developed by scientists using new breakthrough chemistries, the brightness, photostability, and color selection of CF dyes rival or exceed the quality of other commercial dyes as a result of rational dye design.

The CF dye product line includes reactive CF dyes, labeling kits, CF-labeled secondary antibodies, and other bioconjugates. This collection further expands our broad range of carefully selected secondary antibodies and conjugates, allowing scientists to achieve greater sensitivity, brighter results, and better photostability in immunoassays.

For more information on CF Dyes or to view the fluorescent dye comparison chart, visit sigma-aldrich.com/cfdyes.

Biotin Conjugates

Biotin (Vitamin H) is a cofactor that binds with high affinity (K_a = 10¹⁵) to avidin at a ratio of 4:1. The strength of the binding results in an essentially irreversible interaction. This interaction has been exploited for immunolabeling of antigens in histochemical, blotting, and multiwell assays. Biotinylated antibody probes bind to targets on tissue samples, microtiter plates, or membranes. Avidin, conjugated to enzyme,¹ fluorochrome,² or colloidal metal³ binds to multiple sites on the biotinylated probes. Thus the avidin amplifies the signal, resulting in greater sensitivity than that achieved with an antibody-enzyme or antibody-fluorochrome conjugate alone. Visualization may be accomplished by detection of fluorescence, by the colorimetric or chemiluminescent end product of substrate conversion by the attached enzyme, or by microscopic examination. The avidin-biotin system has been used in immunohistology and immunocytology,⁴ immunoblotting,⁵ and ELISA.⁶ Some tissues, such as liver and kidney, contain endogenous biotin which can lead to high background staining when using the biotin-avidin system.⁸

Figure 2. Frozen section of mouse spleen stained for B cells using Goat Anti-Mouse IgM-Biotin Conjugate (Cat. No. B9265). Visualized using ExtrAvidin® -Peroxidase (Cat. No. E2886), DAB and nickel chloride, then counterstained with methyl green. 100X shows germinal center formation. [From W. Lee, Finch University of Health Science, Chicago Medical School, Department of Microbiology and Immunology, N. Chicago, IL.]

References

1. Long Huw Le, Ya Hwei Wang, Jui Huang Shien. 1994. Comparison of the labeled avidin-biotin and the coventional enzyme-linked immunosorbent assay for detecting antibody to reovirus in chickens. Journal of Virological Methods. 48(2-3):343-347. https://doi.org/10.1016/0166-0934(94)90133-3

2. Dale G. 1998. Rapid production of quasi-stable antibody-phycoerythrin conjugates for use in flow cytometry. . Cytometry. 33482-486.

3. Wang JJ. 1989. Immunocytochemical demonstration of the binding of growth-related polypeptide hormones on chick embryonic tissues. Histochemistry. 93(2):133-141. https://doi.org/10.1007/bf00315966

4. Erdamar, et. al. S. 1999. Levels of expression of p27KIP1 protein in human prostate and prostate cancer: an immunohistochemical analysis. Mod. Pathol.. 12751-755.

5. Söderberg A, Sahaf B, Holmgren A, Rosén A. 1998. Monoclonal Antibodies to Human Thioredoxin Reductase. Biochemical and Biophysical Research Communications. 249(1):86-89. https://doi.org/10.1006/bbrc.1998.9053

6. Payette PJ, Cormier M, Dabek B, Yungblut P, Presseault S, Climie S, Sahai J, Cameron WD, Filion LG. 1997. Development of an enzyme-linked immunosorbent assay for measurement of serum-associated ALX40-4C.. 4(6):671-675. https://doi.org/10.1128/cdli.4.6.671-675.1997

7. Bayer EA, Skutelsky E, Wilchek M. 1979. [55] The avidin-biotin complex in affinity cytochemistry.308-315. https://doi.org/10.1016/0076-6879(79)62235-8

8. Beesley J. 1993. Immunochemistry: A Practical Approach. Oxford, England: IRL Press.

Avidin, ExtrAvidin^® and Streptavidin Reagents

Avidin has been reported to exhibit non-specific binding to membranes and tissues. For applications where nonspecific binding of avidin is a problem, we offer Streptavidin and ExtrAvidin^®.

Avidin, ExtrAvidin^® and Streptavidin are available unconjugated or conjugated to enzymes, fluorochromes and colloidal gold for use in immunoassays. Avidin is also available immobilized on agarose for immunoprecipitation or affinity purification procedures.

Avidin
Avidin is a 65 kDa protein found in egg whites. It consists of 4 identical subunits, each with a high-affinity binding site for biotin (Vitamin H). The strength of the binding (K_a = 10¹⁵) results in an essentially irreversible interaction. This interaction has been exploited for immunolabeling of antigens in histochemical, blotting, and multiwell assays. Biotinylated probes, which may be secondary antibodies, lectins, or other bioactive compounds, bind to targets on tissue samples, microtiter plates, or membranes. Avidin conjugated to an enzyme, fluorochrome, or other label binds to the biotinylated probes for visualization, either by detection of fluorescence or enzymatic conversion of substrate to produce a visible end product. In this system avidin serves as a secondary probe, attaching to several sites on the primary biotinylated probe and amplifying the signal. Use of an avidin-enzyme conjugate provides further amplification by conversion of substrate by the enzyme, which will continue to produce a visible product until the substrate is exhausted or the reaction is stopped.

ExtrAvidin^®
ExtrAvidin^® is a modified form of egg white avidin that retains the high affinity and specificity of avidin for biotin, but does not exhibit the nonspecific binding at physiological pH that has been reported for avidin.

Streptavidin
Streptavidin is a form of avidin produced by Streptomyces avidinii that exhibits somewhat less non-specific binding than egg white avidin, although background staining may still sometimes be a problem. Streptavidin is a homotetrameric protein of approximately 60 kDa composed of four identical subunits of approximately 15 kDa each. One molecule of streptavidin binds four molecules of biotin by a non-covalent interaction that is essentially irreversible.

ExtrAvidin^® Staining Kits

Available exclusively in our catalog, ExtrAvidin^® is a unique form of avidin that combines the high specificity and affinity of avidin for biotin with low non-specific binding at physiological pH. ExtrAvidin^® alkaline phosphatase and peroxidase conjugates thus exhibit high sensitivity with low background.

Features and Benefits

• Use in immunohistology,^1,2 ELISA,^3,4 and immunoblotting assays.^5,6
• Affinity Isolated Antibodies in EXTRA-2 and EXTRA-3 have been adsorbed with human IgG and IgM to minimize cross-reactivity.
• Biotinylated antibodies contain a spacer that improves accessibility for the ExtrAvidin^® conjugates

References

1. Navarro A, Tolivia J, Astudillo A, del Valle E. 1998. Pattern of apolipoprotein D immunoreactivity in human brain. Neuroscience Letters. 254(1):17-20. https://doi.org/10.1016/s0304-3940(98)00639-9

2. Nitta H, Ian Mason J, Bahr JM. 1993. Localization of 3?-Hydroxysteroid Dehydrogenase in the Chicken Ovarian Follicle Shifts from the Theca Layer to Granulosa Layer with Follicular Maturation1. 48(1):110-116. https://doi.org/10.1095/biolreprod48.1.110

3. Macri J, Adeli K. 1993. Development of an Amplified Enzyme-Linked Immunosorbent Assay for Sensitive Measurement of Apolipoprotein B in Plasma and Tissue Culture Media. 31(7): https://doi.org/10.1515/cclm.1993.31.7.441

4. Rodríguez E, Martín R, García T, Hernández PE, Sanz B. 1990. Detection of cows' milk in ewes' milk and cheese by an indirect enzyme-linked immunosorbent assay (ELISA). Journal of Dairy Research. 57(2):197-205. https://doi.org/10.1017/s0022029900026807

5. Kang, et. al. J. 1997. Reassembly and reconstitution of separate alpha and beta chains of human leukocyte antigen DR4 molecule isolated from Escherichia coli.. Mol. Cells. 7237-243.

6. Gharbia SE, Haapasalo M, Shah HN, Kotiranta A, Lounatmaa K, Pearce MA, Devine DA. 1994. Characterization ofPrevotella intermediaandPrevotella nigrescensIsolates From Periodontic and Endodontic Infections. Journal of Periodontology. 65(1):56-61. https://doi.org/10.1902/jop.1994.65.1.56