Secondary Reagents

Antibody Forms
Sigma offers antibodies as whole (intact) molecules and as F(ab)₂ fragments of antibodies.

Whole Antiserum Fractionated Antiserum (Ig fraction) IgG Fraction of Antiserum Affinity Isolated Antibody (AIA)

Ascites Fluid Tissue Culture Supernatant Purified Immunoglobulin F(ab)2 Fragment

Whole Antiserum
Description: Whole antiserum contains specific antibody as well as all the other host serum proteins.

Fractionated Antiserum (Ig fraction)
Description: Whole antiserum is fractionated to yield primarily the immunoglobulin fraction of antiserum. This fraction may contain small amounts of the other host serum proteins.

Uses: IgG fractions and fractionated antiserum may be considered useful in situations where very high affinity is required, most commonly when the antigen of interest is rare or present in low abundance. IgG fractions may have the benefit of containing very high affinity antibodies. Affinity isolation may remove some very high affinity antibodies because they bind so tightly to the affinity matrix that they are not eluted.

IgG Fraction of Antiserum
Description: Whole antiserum is fractionated and then further purified by ion exchange chromatography to provide the IgG fraction of antiserum. This fraction is essentially free of other host serum proteins.

Uses: IgG fractions and fractionated antiserum may be considered useful in situations where very high affinity is required, most commonly when the antigen of interest is rare or present in low abundance. IgG fractions may have the benefit of containing very high affinity antibodies. Affinity isolation may remove some very high affinity antibodies because they bind so tightly to the affinity matrix that they are not eluted.

Affinity Isolated Antibody (AIA)
Description: These products are the most purified, therefore, give the lowest amount of non-specific binding. Affinity isolated antigen specific antibody is obtained from antiserum by immunospecific purification using antigen-bound agarose, which removes essentially all the host serum proteins, including immunoglobulins, which do not specifically bind to the antigen.

Uses: Universal

Ascites Fluid
Description: Ascites fluid is intraperitoneal fluid extracted from mice that have had hybridoma cells expressing specific monoclonal antibodies injected into their peritoneal cavity, which serves as a growth chamber for the cells. The hybridoma cells grow to high densities secrete high-titered antibodies. Ascites fluid is clarified by centrifugation to remove the lipid layer and the cell pellet. It contains specific antibody as well as other host serum proteins including immunoglobulins. Specific antibody concentration range is 1-10 mg/ml. Total protein concentration is approximately 20 mg/ml.

Uses: Universal

Tissue Culture Supernatant
Description: Supernatant is the fluid resulting from centrifugation of hybridomas in tissue culture that are secreting specific monoclonal antibodies. Supernatants therefore typically contain culture medium and 5-10% fetal calf serum. Speicifc antibody concentration is in the range of 50 5g/ml.

Uses: Universal

Purified Immunoglobulin
Description: Purified immunoglobulin typically refers to monoclonal antibodies that have been purified using protein A or protein G affinity chromatography.

Uses: Universal

F(ab)₂ Fragment
Description: F(ab)₂ fragments are produced by digestion of IgG with pepsin yielding a divalent molecule (containing two antibody binding sites) but lacking the Fc portion.

Uses: F(ab) ₂ antibody fragments are used in assay systems where the presence of the Fc region may cause problems. Samples such as lymph nodes, spleen, and peripheral blood preparations contain cells with Fc receptors (macrophages, B lymphocytes, and natural killer cells) which could bind the Fc region of intact antibodies, causing high background staining. Use of F(ab)₂ fragments ensures that any antibody binding observed is not due to Fc receptors.¹ These fragments may also be desirable for staining cell preparations in the presence of plasma, because they are not able to bind complement, which could lyse the cells. F(ab)₂ fragments, because of their smaller size, are able to localize antigen more precisely than intact IgG, particularly in staining tissue for electron microscopy. The divalency of F(ab)₂ fragments enables cross-linking of antigens, allowing use of these reagents in precipitation assays, rosetting assays,² or for cellular aggregation via surface antigens.³

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 end user will need to determine which specificity is best suited to his/her 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.

Anti-IgG (whole molecule)
Description: 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.

Uses: 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)
Description: 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.

Uses: 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-Ig, Heavy Chain Specific (alpha, gamma, mu)
Description: 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.

Uses: 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)
Description: 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.

Uses: Same as for Anti-Ig, Heavy-Chain Specific, but for IgG only.

Anti-IgG, Adsorbed with Various Species
Description: 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.

Uses: 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.

References

1. Saito, K. et al., Decreased Fc gamma receptor III (CD16) expression on peripheral blood mononuclear cells in patients with Sjogren's syndrome. J. Rheumatol., 25, 689-696 (1998).
2. de Saint Martin, J., Idiotypic and anti-idiotypic determinants on lymphocytes during anti-Rh immunization. Rev. Fr. Transfus. Immunohematol., 26, 573-583 (1983).
3. De Reys, S., et al., Fc-independent cross-linking of a novel platelet membrane protein by a monoclonal antibody causes platelet activation. Blood, 84, 547-555 (1994).These kits are comprised of universal reagents for use with primary antibodies in immunohistology, ELISA, and immunoblotting.

ExtrAvidin® Staining Kits
ExtrAvidin is a unique form of avidin, available only from Sigma, 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
• Monoclonal Anti-Goat IgG antibodies in EXTRA-1A and EXTRA-1 show no cross-reactivity with human IgG. These antibodies will also recognize sheep IgG.
• Affinity Isolated Antibodies in EXTRA-2A and EXTRA-3A and EXTRA-2, EXTRA-3 and EXTRA-6 have been adsorbed with human IgG and IgM to minimize cross-reactivity.
• Biotinylated antibodies contain a spacer that improves accessibility for the ExtrAvidin conjugates

Kit Components
3 ml ExtrAvidin®-Alkaline Phosphatase or ExtrAvidin®-Peroxidase

3 ml Biotinylated Secondary Antibodies

Complete Instructions and Assay Protocols

Approximate Working Dilutions and Tests per Kit for Various Applications

Optimal dilutions for individual assays should be determined experimentally. Dilutions used will determine number of tests/kit.

References

1. Navarro, A., et al., Pattern of apolipoprotein D immunoreactivity in human brain. Neurosci. Lett., 254, 17-20 (1998).
2. Nitta, H., et al., Localization of 3-beta-hydroxysteroid dehydrogenase in the chicken ovarian follicle shifts from the theca layer to granulosa layer with follicular maturation. Biol. Reprod., 48, 110-116 (1993).
3. Macri, J. and Adeli, K., Development of an amplified enzyme-linked immunosorbent assay for sensitive measurement of apolipoprotein B. Eur. J. Clin. Chem. Clin. Biochem., 31, 441-446 (1993).
4. Rodriguez, E., et al., Detection of cows' milk in ewes' milk and cheese by an indirect enzyme-linked immunosorbent assay (ELISA). J. Dairy Res., 57, 197-205 (1990).
5. Kang, J.H., et al., Reassembly and reconstitution of separate alpha and beta chains of human leukocyte antigen DR4 molecule isolated from Escherichia coli. Mol. Cells, 7, 237-243 (1997).
6. Gharbia, S.E., et al., Characterization of Prevotella intermedia and Prevotella nigrescens isolates from periodontic and endodontic infections. J. Periodontol., 65, 56-61 (1994).

Protein A, G and L

Protein A and Protein G
Description: Protein A is derived from Staphylococcus aureus. Protein G is derived from a Streptococcus species. Both have binding sites for the Fc portion of mammalian IgG. The affinity of these proteins for IgG varies with the animal species. Protein G has a higher affinity for rat, goat, sheep, and bovine IgG, as well as for mouse IgG1 and human IgG3. Protein A has a higher affinity for cat and guinea pig IgG (see Table). In addition to IgG Fc binding sites, native Protein G contains binding sites for albumin, the Fab region of Igs, and membrane binding regions, which can lead to nonspecific staining. These problems have been addressed by creating recombinant forms of the protein. Recombinant Protein G has been engineered to eliminate the albumin binding region, and recombinant Protein G is a truncated protein which lacks the albumin, Fab, and membrane binding sites while retaining the Fc binding site, making it more specific for IgG than the native form.

Uses: Use as a general, non-species-specific reagent for binding primary antibodies or surface IgG in mammalian tissues. Protein G is recommended for most species, including mouse and rat. Protein A is recommended for cat and guinea pig. Neither is recommended for detection of IgA or IgM, for detection of Fab fragments, or for detection of avian IgG. When bound to a resin like agarose, Protein A and Protein G may be used to affinity purify immunoglobulins from serum or ascites fluid.

Note: See PURE1-A Protein A Purification Kit

Protein L
Description: Protein L, from Peptostreptococcus magnus, has an affinity for kappa light chains (see Table) from various species. (see Table) It will detect monoclonal or polyclonal IgG, IgA, and IgM as well as Fab, F(ab)₂, and recombinant single-chain Fv (scFv) fragments that contain kappa light chains. It will also bind chicken IgG. Note: Species such as bovine, goat, sheep, and horse whose Igs contain almost exclusively lambda chains will not bind well, if at all, to Protein L.

Uses: Use as a general reagent for binding primary mammalian or avian antibodies or surface Igs of all classes. Especially useful for detection of Fab, F(ab)₂ fragments, and recombinant scFv fragments, for detection of Igs bound to Fc receptors, or for detection of monoclonal antibodies in the presence of bovine Igs. Use is limited to detection of Igs bearing kappa light chains.

References

1. Kronvall, G., et al., Phylogenetic insight into evolution of mammalian Fc fragment of gamma G globulin using staphylococcal protein A. J. Immunol., 104, 140-147 (1970).
2. Richman, D.D., et al., The binding of staphylococcal protein A by the sera of different animal species. J. Immunol., 128, 2300-2305 (1982).
3. Langone, J.J., [¹²⁵I]Protein A: a tracer for general use in immunoassay. J. Immunol. Meth., 24, 269-285 (1978).
4. Ey, P.L., et al., Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry, 15, 429-436 (1978).
5. Kronvall, G., A surface component in group A, C, and G streptococci with non-immune reactivity for immunoglobulin G. J. Immunol., 111, 1401-1406 (1973).
6. Ekerstrom, B., and Bjorck, L., A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties. J. Biol. Chem., 261, 10240-10247 (1986).
7. Ekerstrom, B., Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J. Immunol. 135, 2589-2592 (1985).
8. Bjorck, L., and Kronvall, G., Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J. Immunol., 133, 969-974 (1984).
9. De Chateau, M., et al., On the interaction between protein L and immunoglobulins of various mammalian species. Scand. J. Immunol., 37(4), 399-405 (1993).
10. Svensson, H.G., et al., Protein LA, a novel hybrid protein with unique single-chain Fv antibody- and Fab-binding properties. Eur. J. Biochem,. 258, 890-896 (1998).

Unlabeled Antibodies
These reagents are offered unconjugated for maximum flexibility. They may be used as secondary reagents for amplification of a primary antibody signal, or as primary reagents for immunoglobulin localization, followed by visualization with conjugated secondary reagents.

Specificity and titer of antibodies to immunoglobulins is determined by ELISA, immunoelectrophoresis and/or Ouchterlony double diffusion. Affinity isolated antibodies are assayed for specific antibody content by quantitative precipitin assay (QPA). In this assay purified antigen is combined with the antibody preparation at various ratios. The amount of antibody specifically precipitated by the antigen is determined at equivalence, the ratio of antibody to antigen at which complete precipitation of both occurs. For product specific information, see the product data sheets under each listing.

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.

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 (Product Code A 3312) using SIGMA FAST™ Fast Red TR/Napthol AS-MX Tablets (Product Code F 4523) as substrate. Click image for larger view

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 (Product Code L 9756) 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. (See Substrates)

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

Antibodies are tested prior to conjugation for specificity and immunoreactivity by ELISA. As indicated in the listing, some antibodies are adsorbed by solid phase techniques with human or animal immunoglobulins or serum proteins to minimize cross-reactivity. Titers obtained by immunohistology, dot blotting or immunoblotting may also be listed.

For product specific information, see the product data sheets under each listing.

References

1. O'Sullivan, M.S., et al., A simple method for the preparation of enzyme-antibody conjugates. FEBS Lett., 95, 311-313 (1978).
2. Lundberg, B.B., et al., Conjugation of an anti-B-cell lymphoma monoclonal antibody, LL2, to long-circulating drug-carrier lipid emulsions. J. Pharm. Pharmacol., 51, 1099-1105 (1999).
3. Pretorius, A., et al., The binding potential of commercial antibody conjugates with sera of various small terrestrial mammals. Onderstepoort J. Vet. Res., 64, 201-203 (1997).
4. Heider, H., and Schroeder, C., Focus luminescence assay: macroscopically visualized foci of human cytomegalovirus and varicella zoster virus infection. J. Virol. Methods, 66, 311-316 (1997).
5. Roe, I.H., et al., Changes in the evolution of the antigenic profiles and morphology during coccoid conversion of Helicobacter pylori. Korean J. Intern. Med., 14, 9-14 (1999).
6. Beesley, J.E., Immunochemistry: A Practical Approach, p. 213 (IRL Press, Oxford, England, 1993).

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.

Photomicrography of a 10 µm cryosection of embryonic mouse optic stalk, labeled with anti-Pax2, Goat Anti-Rabbit IgG Peroxidase Conjugate (Product Code A 9169) and SIGMA FAST™ DAB Table or PDF filets (Product Code D 4393). [From D. Otteson, Department of Anatomy and Cell Biology, University of Michigan, Ann Arbor, MI.] Click image for larger view

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. (See Substrates.)

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

Horseradish peroxidase is used for all conjugates. Antibodies are conjugated using a modification of the two-step glutaraldehyde method of Avrameas,¹ or conjugated using a modification of the periodate conjugation method of Wilson and Nakane² as described in the listing. Antibodies are tested prior to conjugation for specificity and immunoreactivity by ELISA. As indicated in the listing, some antibodies are adsorbed by solid phase techniques with human or animal immunoglobulins or serum proteins to minimize cross-reactivity. Titers obtained by immunohistology, dot immunoblotting or western immunoblotting may also be listed. For product specific information, see the product data sheets under each listing.

References

1. Avrameas, S., et al., Scand. J. Immunol., 8, Suppl. 7, 7 (1978).
2. Wilson, M. and Nakane, P., Immunofluorescence and Related Staining Techniques, p. 215 (Elsevier/North Holland Biomedical Press Amsterdam, 1978) p. 215.
3. O'Sullivan, M.S., et al., A simple method for the preparation of enzyme-antibody conjugates. FEBS Lett., 95, 311-313 (1978).
4. Sanz, C. et al., An enzyme-linked immunosorbent assay applicable to screen blood donors for IgA deficiency. Haematologica, 84, 887-890 (1999).
5. Piza, A.S., et al., 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. Sao Paulo, 41, 39-43 (1999).
6. Hanas, J.S., et al., Expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and p53 tumor suppressor in dysplastic progression and adenocarcinoma in Barrett esophagus. Cancer, 86, 756-763 (1999).
7. Krajewski, S., et al., Detection of multiple antigens on western blots. Anal. Biochem., 236, 221-228 (1996).
8. Beesley, J.E., Immunochemistry: A Practical Approach, p.213 (IRL Press, Oxford, England, 1993).

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 fluorophores.

Cultured rat hepatic stellate cells (Ito cells, passage 4) were incubated for 24 hrs in 1% serum media contianing interferon-gamma. Cells were stained with Monoclonal Anti-gamma-Smooth Muscle Actin (Clone No. 1A4, A 2547) and Sheep Anti-Mouse IgG-C Click image for larger view

Fluorescent dyes are used to detect molecules in a variety of biological applications¹. 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™, 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.

Affinity isolated antibodies and IgG fractions are tested for specificity by immunoelectrophoresis and Ouchterlony double diffusion prior to conjugation with fluorescent dyes. Monoclonal antibodies are tested for specificity by indirect ELISA prior to conjugation.

For product specific information, see the product data sheets under each listing.

PE conjugates are covered under U.S. Patent No. 4,859,582.
Cy is a trademark of Amersham Life Science, Inc. and is distributed under license.

References

1. Lausch, R. et al., Analysis of immunoglobulin G using a capillary electrophoretic affinity assay with protein A and laser-induced fluorescence detection. Electrophoresis, 16, 636-641 (1995).
2. Lloyd-Evans, P., et al., 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, 155-160 (1999).
3. Stewart, C.C., and Stewart, S.J., Four color compensation. Cytometry, 38, 161-175 (1999).

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

Frozen section of mouse spleen stained for B cells using Goat Anti-Mouse IgM-Biotin Conjugate (Product Code B 9265). Visualized using ExtrAvidin-Peroxidase (Product Code E 2886), 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.] Click image for larger view

All biotin conjugates are tested for immunoreactivity by ELISA. Biotin anti-human immunoglobulin conjugates are tested against a panel of human immunoglobulins for reactivity and specificity. Biotin anti-animal immunoglobulin conjugates that are adsorbed with human or animal proteins are tested by ELISA to ensure minimal cross-reactivity.

For product specific information, see the product data sheets under each listing.

References

1. Lee, L. H., et al., Comparison of the labeled avidin-biotin and the conventional enzyme-linked immunosorbent assay for detecting antibody to reovirus in chickens. J. Virol. Meth., 48, 343-347 (1994).
2. Dale, G. L., Rapid production of quasi-stable antibody-phycoerythrin conjugates for use in flow cytometry. Cytometry, 33, 482-486 (1998).
3. Wang, J. J., Immunocytochemical demonstration of the binding of growth-related polypeptide hormones on chick embryonic tissues. Histochem., 93, 133-141 (1989).
4. Erdamar S., et al., Levels of expression of p27KIP1 protein in human prostate and prostate cancer: an immunohistochemical analysis. Mod. Pathol., 12, 751-755 (1999).
5. Soderberg, A., et al., Monoclonal antibodies to human thioredoxin reductase. Biochem. Biophys. Res. Commun., 249, 86-89 (1998).
6. Payette, P.J., et al., Development of an enzyme-linked immunosorbent assay for measurement of serum-associated ALX40-4C. Clin. Diagn. Lab. Immunol., 4, 671-675 (1997).
7. Bayer, E. A., et al., The avidin-biotin complex in affinity cytochemistry. Meth. Enzymol., 62, 308-315 (1979).
8. Beesley, J.E., Immunochemistry: A Practical Approach, p. 213 (IRL Press, Oxford, England, 1993).

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, Sigma offers 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.