[21]
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[21] Prevention of Nonspecific Binding of A v i d i n By
RAYMOND
C. DUHAMEL
and JAMES S. WHITEHEAD
For the purposes here, nonspecific binding is defined as any unwanted binding of biotin-containing proteins or avidin conjugates to the material under study. The unwanted binding may in some cases involve specific high-affinity binding to extraneous components, such as endogenous biotin-containing proteins or lectins. Unwanted binding of biotin or avidin conjugates m a y b e classified into four categories: (I) unwanted binding of the avidin or biotin conjugates to endogenous biotin-containing proteins or endogenous avidin, respectively, (2) unwanted binding of avidin to endogenous lectins, (3) unwanted binding of avidin to macromolecules through nonspecific ionic or hydrophobic interactions, and (4) nonspecific binding arising from other components in the detection system besides biotin or avidin (such as the antibodies, lectins, or reporter molecules to which they are conjugated). Although the last category may not directly involve biotin or avidin, a few comments are included concerning frequently encountered causes of nonspecific binding to antibodies and nitrocellulose since these are often used with biotin or avidin reagents. For each category of nonspecific binding, the cellular component or tissue type associated with the binding and the most appropriate method for blocking the nonspecific binding are discussed. For simplicity, the focus of the discussion is avidin, but much of what is said can be equally well applied to streptavidin reagents. The advantages and disadvantages of streptavidin relative to avidin are also discussed.
Unwanted Binding to Endogenous Biotin or Avidin Site of Binding. Endogenous avidin-binding activity is due to the presence of biotin-containing proteins in certain mammalian tissues. Free, unconjugated biotin is also present in tissues, but it is easily washed from the material under study and not relevant to this discussion. Biotin-containing cytoplasmic binding sites for avidin seem to be restricted to kidney, liver, pancreas, ~,2 and brain. 3,4 These tissues contain elevated levels
I G. S. W o o d and R. W a r n k e , J. Histochem. Cytochem. 29, 1196 (1981). 2 R. F. Rowley and G. S. Eisenbarth, Diabetes 31, 107 (1982). 3 W. Y. Naritoku a n d C. R. Taylor, J. Histochem. Cytochern. 30, 253 (1982). 4 S. M. L e v i n e and W. B. Macklin, Brain Res. 444, 199 (1988).
METHODS IN ENZYMOLOGY. VOL. 184
Copyright © 1990by AcademicPress. Inc. All rights of reproduction in any form reserved.
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of biotin-dependent carboxylases, 5 presumably in mitochondria, and it has long been recognized that the biotin moiety of these carboxylases is accessible to avidin binding. In fact, inactivation of carboxylase activity by avidin is a criterion for demonstrating biotin dependence. 6 Avidin also binds to biotin-containing components of the cell surface of certain bacteria, including Escherichia coli, which may be present in cell extracts from bacterial cultures. 7 Other bacteria 8 as well as other microorganisms, 9 plants, ~° and insects H may also contain accessible bound biotin residues. Endogenous biotin-binding activity is not of concern in mammalian tissues since avidin has not been found in mammals, but some tissuestaining protocols involve the use of egg white, which is a rich source of avidin, to aid in attaching sections to glass slides. Avidin is isolated from chicken eggs and is present in certain other tissues of oviparous vertebrates. Avidin is produced under hormonal control in the oviducts of birds, amphibians, and reptiles. It was long thought that avidin was found only in the reproductive tissues, but it has been recognized that avidin production can be induced by tissue injury in a variety of other tissues, primarily as an inflammatory response. Avidin is also produced by avian cells in tissue culture and can be induced by Rous sarcoma virus. 4 Blocking Method. The method of choice for blocking endogenous avidin-binding activity is a two-step pretreatment performed prior to incubation with biotinylated antibody and avidin conjugates. 12-21The two-step 5 M. E. Bramwell, J. Biochem. Biophys. Methods 15, 125 (1987). 6 R. G. Duggleby, P. V. Atwood, J. C. Wallace, and D. B. Keech, Biochem. 21, 3364 (1982). 7 H. A. Elo and J. Korpela, Comp. Biochem. Physiol. 78B, 15 (1984). 8 B. R. Jennings, H. Mincer, J. Turner, V. Baselski, and R. T. Kelly, J. Clin. Microbiol. 18, 1250 (1983). 9 M. E. Collins, M. T. Moss, S. Wall, and J. W. Dale, FEMS Microbiol. Lett. 43, 53 (1987). ~0 B. J. Nikolau, E. S. Wurtele, and P. K. Stumpf, Anal. Biochem. 149, 448 (1985). tl T. F. Tsai, R. A. Bolin, M. Montoya, R. E. Bailey, D. B. Francy, M. Jozan, and J. T. Roehrig, J. Clin. Microbiol. 25, 370 (1987). lz T. Letonja and C. Hammerberg, J. Parasitol. 73, 962 (1987). 13 S. Moil, T. Akiyama, Y. Morishita, S.-I. Shimizu, K. Sakai, K. Sudoh, K. Toyoshima, and T. Yamamoto, Virchows Arch. B 54, 8 (1987). 14 K. Sakai, M. Takiguchi, S. Moil, O. Kobori, Y. Morioka, H. Inoko, M. Sekiguchi, and K. Kano, J. Natl. Cancer Inst. 79, 923 (1987). 15 T. V. Tuazon, E. E. Schneeberger, A. K. Bhan, R. T. McCluskey, A. B. Cosimi, R. T. Schooley, R. H. Rubin, and R. B. Colvin, Am. J. Pathol. 129, 119 (1987). t6 R. Jonsson, L. Klareskog, K. Backman, and A. Tarkowski, Clin. Imrnunol. lmmunopathol. 45, 235 (1987). J7 S. Moil, Y. Morishita, K. Sakai, S. Kuilmoto, M. Okamoto, T. Kawamoto, and T. Kuroki, Acta Pathol. Jpn. 37, 1909 (1987). 18 C. J. Verdi, T. M. Grogan, R. Protell, L. Richter, and C. Rangel, Hepatology 6, 6 (1986). 19 V. Glezerov, J. Histotechnol. 9, 15 (1986).
[21]
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avidin-biotin block consists of incubation with excess unlabeled avidin followed by incubation with excess free biotin. Avidin in the first step binds to the endogenous biotin, and the excess biotin in the second step blocks residual biotin-binding sites in the immobilized avidin. For histological sections, Wood and Warnke recommend 1.0-0.1 mg/ml avidin and 0.1-0.01 mg/ml biotin.1 An abbreviated procedure consists of mixing the avidin conjugate with normal blocking serum and diluting the primary antibody in a buffer containing biotin. The rare occurrence of endogenous avidin can be blocked by preincubation with free biotin alone.
Unwanted Binding of Avidin to Endogenous Lectins Site of Occurrence. Many tissues contain membrane-associated lectins. Most of the endogenous lectins encountered in staining recognize terminal o~-linked mannose or terminal/3-1inked galactose residues. The galactose-specific lectins generally do not present problems, because neither avidin nor most of the enzymes used in conjugates contain terminal galactose residues. Avidin consists of 10% carbohydrate, which includes terminal mannose in the side chains. The mannose-specific lectins reactive with avidin seem to be restricted to liver, kidney, pancreas, and, occasionally, brain. Lectin binding is generally seen only in frozen sections since mammalian lectins are destroyed by most fixatives and paraffin-embedding procedures. Blocldng Method. It is possible to block lectin binding specifically by diluting the avidin conjugate in 200 m M a-methyl-D-mannoside. The glycoside is used rather than the free sugar, because the former competes for the binding site on the lectin more effectively. It should be noted, however, that prevention of lectin binding may not completely eliminate nonspecific binding in a particular tissue, since many of the same tissues that exhibit lectin binding are also rich in biotin-containing proteins. The twostep avidin-biotin blocking procedure described above eliminates nonspecific binding whether it is due to endogenous biotin or to lectins. Nonspecific Binding of Avidin Owing to Ionic and Hydrophobic Interactions Site of Binding. Avidin in some circumstances will bind to the nucleus of cells. Avidin binding has been reported to be specific for condensed
20 T. A. Van Dyke, C. Finlay, D. Miller, J. Marks, G. Lozano, and A. J. Levine, J. Virol. 61, 2029 (1987). 21 j. Bresser and M. J. Evinger-Hodges, Gene. Anal. Techn. 4, 89 (1987).
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[2 1]
chromatin rather than uncondensed chromatin, 22 but diffuse staining of interphase nuclei has also been observed.l,23 Nonspecific binding can also occur in the cytoplasm. For example, avidin conjugates can bind to the cytoplasmic granules of some mast cells. 24 These cells contain heparin, a polysacchatide containing many sulfate and carboxyl groups. Since heparin is highly negatively charged, it has the potential to bind to proteins of high isoelectric point such as avidin (pI > 10), antibodies, and some enzymes. Nonspecific avidin staining of mast cells appears to be highly dependent on the ionic strength and pH of the buffers used in staining, the tissue of origin, and the fixative used in preparing sections for histology. Tissues fixed in Methacarn or Carnoy's fixatives seem to have the greatest potential for nonspecific staining of mast cells. Nonspecific avidin staining of the cell surface has also been reported. It has been suggested that avidin binds to acidic components of the cell capsule. Blocking Method. Ionic binding to avidin conjugates can frequently be suppressed by raising the ionic strength of dilution buffers by the addition of 0.3-0.5 M sodium chloride. 25,26Raising the pH to 9.4 has also proved effective in some instances. 24 High pH does not affect the avidin-biotin interaction, since it is effective across a pH range from 3 to 10, but high pH may be incompatible with the primary interaction under study and must therefore be used with caution. Many lectins, for example, become inactive at high p H . 24 Nonspecific binding that cannot be blocked by raising the ionic strength or pH may be due to hydrophobic or other protein-protein interactions. Addition of protein to diluent solution has proved effective at blocking such interactions. Bovine serum albumin is effective at a concentration of 1-3%, but it should be a crystalline grade. Spurious background staining has been associated with the use of noncrystalline grades. With antibodies, it is common practice to include nonimmune serum in the diluent to serve as a cartier and to minimize nonspecific binding. PolyL-lysine, a highly basic polypeptide, has been used to block nonspecific binding of streptavidin-peroxidase in a nitrocellulose system. 27 Gelatin has been used to prevent nonspecific interactions with avidin-stabilized 22 M. H. Heggeness, Stain Technol. 52, 165 (1977). 23 R. C. Duhamel and D. A. Johnson, J. Histochem. Cytochem. 33, 711 0985). 24 G. Bussolati and P. Gugliotta, J. Histochem. Cytochem. 31, 1419 (1983). 25 C. J. P. Jones, S. M. Mosley, I. J. M. Jeffrey, and R. W. Stoddart, Histochem. J. 19, 264 (1987). 26 R. K. Clark, Y. Tani, and I. Damjanov, J. Histochem. Cytochem. 34, 1509 (1986). z7 L. Scopsi, B.-L. Wang, and L.-I. Larsson, J, Histochem. Cytochem. 34, 1469 (1986).
[9.1]
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gold sols. z8 Nonfat dry milk at a concentration of 5-10% has been used to block nuclear and cytoplasmic nonspecific binding of avidin conjugates in histological sections. 23However, it has been reported that, in a nitrocellulose blot procedure, although nonfat dry milk eliminated the vast majority of the background staining, several distinct bands exhibited spurious binding to avidin-peroxidase, but not to biotin-peroxidase. The use of 0.15 or 0.3 M NaCI instead of nonfat dry milk eliminated both general background staining and binding to the spurious bands. 26 Presence o f Biotin in Protein Solutions. The presence of biotin in protein carrier solutions, such as nonimmune serum and nonfat dry milk, may reduce the signal-to-noise ratio of the detection system. Avidinbiotin detection systems are often so sensitive that even small amounts of free biotin in buffers can significantly suppress the signal. This is especially true with ABC complexes of avidin-biotin-peroxidase. The usual response of the experimenter to a reduced signal is to increase the concentration of the primary reagents, but this can have the effect of increasing the nonspecific background significantly. A better solution is to reduce or eliminate the free biotin in the affected reagent. Biotin-containing solutions can be dialyzed to remove free biotin, if necessary. Nonspecific Binding Unrelated to Biotin or Avidin Antibody Procedures. Since antibodies are frequently conjugated with either biotin or avidin, some commonly encountered causes of nonspecific binding to antibodies deserve comment. Since most tissues contain some immunoglobulin, residual cross-reactivity of the secondary antibody for endogenous immunoglobulin in the target tissue can result in spurious binding of secondary antibodies. Unless the cross-reactivity is very strong, however, adding 2% serum from the species of the tissue to the diluent of the secondary antibody will block the cross-reactivity. IgM antibodies in whole serum are a frequent source of nonspecific staining because IgM is notoriously "sticky." Since many IgM antibodies are cryoglobulins which tend to precipitate when stored in the cold for lengthy periods, they can frequently be eliminated by centrifuging the primary antibody prior to use. If necessary, ammonium sulfate fractionation or DEAE chromatography can be employed to eliminate IgM class immunoglobulins. Many animals, especially rabbits, have constitutive antibodies against tissue elements such as cytokeratins and connective tissues. These are 28 O. Behnke, T. Ammitzboll, H. Jessen, M. Klokker, K. Nilausen, J. Tranum-Jensen, and L. Olsson, Eur. J. Cell Biol. 41, 326 (1986).
206
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generally IgM antibodies of low affinity, however, and false staining may be eliminated by using a higher dilution of primary antibody. Ideally, it is sound practice to circumvent this problem by affinity purification of the primary antibody on an antigen column, but diluting the primary antibody as much as possible and longer incubation of the primary antibody at 4 ° will generally eliminate the binding problems. If protein is used as a blocking agent or carrier it should be compatible with the specificity of the secondary antibody. For example, nonfat dry milk should not be used with primary antibodies made in goat or sheep, because secondary antibodies to goat or sheep IgG generally cross-react strongly with immunoglobulin present in bovine milk. Nitrocellulose Procedures. Detection systems that involve the binding of target macromolecules to nitrocellulose or other membrane materials require that a blocking solution be used to inactivate all remaining binding sites on the membrane. In some protocols, protein solutions such as 1-3% albumin or 5% nonfat dry milk are used as irreversible blocking agents. Subsequent incubations with avidin-biotin reagents are subject to the same problems of nonspecific binding owing to ionic or hydrophobic interactions with the immobilized blocking protein as discussed above. Other protocols use Tween 20, a detergent, as a blocking agent. The latter does not block irreversibly and must be included in all subsequent diluent and wash buffers. RIA grade Tween 20 may be preferred. Since Tween 20 binds to proteins, the addition of excess carrier proteins may render the Tween 20 ineffective.
Advantages~Disadvantages of Streptavidin The previous discussion has focused primarily on avidin, but many of the comments apply equally to streptavidin. Streptavidin is only slightly anionic, contains no carbohydrate, and is assumed to bind biotin with the same affinity as avidin. 29 It has been suggested that the lack of carbohydrate and the lower isoelectric point of streptavidin would eliminate many of the sources of nonspecific binding associated with avidin. 3° The presumption of an advantage to streptavidin does not necessarily hold in all circumstances. In many cases, the native isoelectric point of avidin is irrelevant, since it is altered by derivatization with fluorochromes or by conjugation to enzymes of low isoelectric point. There are certain disadvantages to streptavidin. Streptavidin is usually more expensive than avidin. The biotin-binding cleft in streptavidin is different than that of avidin and, in some cases, may require a longer 29 L. Chaiet and F. J. Wolf, Arch. Biochem. Biophys. 106, 1 (1964). 30 T. V. Updyke and G. L. Nicolson, J. lmmunol. Methods 73, 83 (1984).
[21]
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spacer arm for biotinylation to achieve optimum binding with streptavidin. The lack of carbohydrate potentially eliminates the problem of nonspecific binding to endogenous lectins, but if the reagent system includes other macromolecules containing terminal mannose residues, such as horseradish peroxidase, unwanted binding of the reagent to lectins may still occur. Furthermore, as discussed above, the same tissues that contain endogenous avidin-binding lectins may also contain endogenous biotin-containing proteins. General Recommendations
Frequently, background staining that is initially ascribed to endogenous biotin or avidin binding proves to be due to other components of the system. To determine whether nonspecific binding occurs in a particular system, controls should be performed in which each reagent in the system is systematically omitted. An almost universal method for preventing nonspecific binding in avidin-biotin systems is the two-step sequential pretreatment with avidin and biotin. This approach may add unnecessary time and expense to the staining process, however, and it is essential only when the unwanted binding is due to endogenous biotin-containing protein or avidin. In many applications, raising the ionic strength of dilution buffers by the inclusion of 0.3 M NaC1 will be sufficient to eliminate nonspecific binding arising from ionic interaction. If the unwanted binding is due to endogenous lectins, the addition of 200 mM a-methyl-D-mannoside will usually eliminate it. High salt and the glycoside can be combined, if desired. If the appropriate controls indicate that nonspecific binding has not been fully suppressed, the addition of protein blocking agents such as albumin, nonfat dry milk, or nonimmune serum can be considered, subject to the caution that the biotin content should not diminish the intensity of the reaction. Although the focus of this chapter has been nonspecific staining encountered with the avidin-biotin system, it should not be inferred that nonspecific binding will inevitably occur. In fact, if the proper procedures for general immunohistochemical staining are followed, nonspecific staining is rarely seen. When used in an appropriate way and with a full awareness of its principles, the avidin-biotin system offers the potential for maximum staining sensitivity with no significant nonspecific background.
NONSPECIFICBINDING
201
[21] Prevention of Nonspecific Binding of A v i d i n By
RAYMOND
C. DUHAMEL
and JAMES S. WHITEHEAD
For the purposes here, nonspecific binding is defined as any unwanted binding of biotin-containing proteins or avidin conjugates to the material under study. The unwanted binding may in some cases involve specific high-affinity binding to extraneous components, such as endogenous biotin-containing proteins or lectins. Unwanted binding of biotin or avidin conjugates m a y b e classified into four categories: (I) unwanted binding of the avidin or biotin conjugates to endogenous biotin-containing proteins or endogenous avidin, respectively, (2) unwanted binding of avidin to endogenous lectins, (3) unwanted binding of avidin to macromolecules through nonspecific ionic or hydrophobic interactions, and (4) nonspecific binding arising from other components in the detection system besides biotin or avidin (such as the antibodies, lectins, or reporter molecules to which they are conjugated). Although the last category may not directly involve biotin or avidin, a few comments are included concerning frequently encountered causes of nonspecific binding to antibodies and nitrocellulose since these are often used with biotin or avidin reagents. For each category of nonspecific binding, the cellular component or tissue type associated with the binding and the most appropriate method for blocking the nonspecific binding are discussed. For simplicity, the focus of the discussion is avidin, but much of what is said can be equally well applied to streptavidin reagents. The advantages and disadvantages of streptavidin relative to avidin are also discussed.
Unwanted Binding to Endogenous Biotin or Avidin Site of Binding. Endogenous avidin-binding activity is due to the presence of biotin-containing proteins in certain mammalian tissues. Free, unconjugated biotin is also present in tissues, but it is easily washed from the material under study and not relevant to this discussion. Biotin-containing cytoplasmic binding sites for avidin seem to be restricted to kidney, liver, pancreas, ~,2 and brain. 3,4 These tissues contain elevated levels
I G. S. W o o d and R. W a r n k e , J. Histochem. Cytochem. 29, 1196 (1981). 2 R. F. Rowley and G. S. Eisenbarth, Diabetes 31, 107 (1982). 3 W. Y. Naritoku a n d C. R. Taylor, J. Histochem. Cytochern. 30, 253 (1982). 4 S. M. L e v i n e and W. B. Macklin, Brain Res. 444, 199 (1988).
METHODS IN ENZYMOLOGY. VOL. 184
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[21]
of biotin-dependent carboxylases, 5 presumably in mitochondria, and it has long been recognized that the biotin moiety of these carboxylases is accessible to avidin binding. In fact, inactivation of carboxylase activity by avidin is a criterion for demonstrating biotin dependence. 6 Avidin also binds to biotin-containing components of the cell surface of certain bacteria, including Escherichia coli, which may be present in cell extracts from bacterial cultures. 7 Other bacteria 8 as well as other microorganisms, 9 plants, ~° and insects H may also contain accessible bound biotin residues. Endogenous biotin-binding activity is not of concern in mammalian tissues since avidin has not been found in mammals, but some tissuestaining protocols involve the use of egg white, which is a rich source of avidin, to aid in attaching sections to glass slides. Avidin is isolated from chicken eggs and is present in certain other tissues of oviparous vertebrates. Avidin is produced under hormonal control in the oviducts of birds, amphibians, and reptiles. It was long thought that avidin was found only in the reproductive tissues, but it has been recognized that avidin production can be induced by tissue injury in a variety of other tissues, primarily as an inflammatory response. Avidin is also produced by avian cells in tissue culture and can be induced by Rous sarcoma virus. 4 Blocking Method. The method of choice for blocking endogenous avidin-binding activity is a two-step pretreatment performed prior to incubation with biotinylated antibody and avidin conjugates. 12-21The two-step 5 M. E. Bramwell, J. Biochem. Biophys. Methods 15, 125 (1987). 6 R. G. Duggleby, P. V. Atwood, J. C. Wallace, and D. B. Keech, Biochem. 21, 3364 (1982). 7 H. A. Elo and J. Korpela, Comp. Biochem. Physiol. 78B, 15 (1984). 8 B. R. Jennings, H. Mincer, J. Turner, V. Baselski, and R. T. Kelly, J. Clin. Microbiol. 18, 1250 (1983). 9 M. E. Collins, M. T. Moss, S. Wall, and J. W. Dale, FEMS Microbiol. Lett. 43, 53 (1987). ~0 B. J. Nikolau, E. S. Wurtele, and P. K. Stumpf, Anal. Biochem. 149, 448 (1985). tl T. F. Tsai, R. A. Bolin, M. Montoya, R. E. Bailey, D. B. Francy, M. Jozan, and J. T. Roehrig, J. Clin. Microbiol. 25, 370 (1987). lz T. Letonja and C. Hammerberg, J. Parasitol. 73, 962 (1987). 13 S. Moil, T. Akiyama, Y. Morishita, S.-I. Shimizu, K. Sakai, K. Sudoh, K. Toyoshima, and T. Yamamoto, Virchows Arch. B 54, 8 (1987). 14 K. Sakai, M. Takiguchi, S. Moil, O. Kobori, Y. Morioka, H. Inoko, M. Sekiguchi, and K. Kano, J. Natl. Cancer Inst. 79, 923 (1987). 15 T. V. Tuazon, E. E. Schneeberger, A. K. Bhan, R. T. McCluskey, A. B. Cosimi, R. T. Schooley, R. H. Rubin, and R. B. Colvin, Am. J. Pathol. 129, 119 (1987). t6 R. Jonsson, L. Klareskog, K. Backman, and A. Tarkowski, Clin. Imrnunol. lmmunopathol. 45, 235 (1987). J7 S. Moil, Y. Morishita, K. Sakai, S. Kuilmoto, M. Okamoto, T. Kawamoto, and T. Kuroki, Acta Pathol. Jpn. 37, 1909 (1987). 18 C. J. Verdi, T. M. Grogan, R. Protell, L. Richter, and C. Rangel, Hepatology 6, 6 (1986). 19 V. Glezerov, J. Histotechnol. 9, 15 (1986).
[21]
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avidin-biotin block consists of incubation with excess unlabeled avidin followed by incubation with excess free biotin. Avidin in the first step binds to the endogenous biotin, and the excess biotin in the second step blocks residual biotin-binding sites in the immobilized avidin. For histological sections, Wood and Warnke recommend 1.0-0.1 mg/ml avidin and 0.1-0.01 mg/ml biotin.1 An abbreviated procedure consists of mixing the avidin conjugate with normal blocking serum and diluting the primary antibody in a buffer containing biotin. The rare occurrence of endogenous avidin can be blocked by preincubation with free biotin alone.
Unwanted Binding of Avidin to Endogenous Lectins Site of Occurrence. Many tissues contain membrane-associated lectins. Most of the endogenous lectins encountered in staining recognize terminal o~-linked mannose or terminal/3-1inked galactose residues. The galactose-specific lectins generally do not present problems, because neither avidin nor most of the enzymes used in conjugates contain terminal galactose residues. Avidin consists of 10% carbohydrate, which includes terminal mannose in the side chains. The mannose-specific lectins reactive with avidin seem to be restricted to liver, kidney, pancreas, and, occasionally, brain. Lectin binding is generally seen only in frozen sections since mammalian lectins are destroyed by most fixatives and paraffin-embedding procedures. Blocldng Method. It is possible to block lectin binding specifically by diluting the avidin conjugate in 200 m M a-methyl-D-mannoside. The glycoside is used rather than the free sugar, because the former competes for the binding site on the lectin more effectively. It should be noted, however, that prevention of lectin binding may not completely eliminate nonspecific binding in a particular tissue, since many of the same tissues that exhibit lectin binding are also rich in biotin-containing proteins. The twostep avidin-biotin blocking procedure described above eliminates nonspecific binding whether it is due to endogenous biotin or to lectins. Nonspecific Binding of Avidin Owing to Ionic and Hydrophobic Interactions Site of Binding. Avidin in some circumstances will bind to the nucleus of cells. Avidin binding has been reported to be specific for condensed
20 T. A. Van Dyke, C. Finlay, D. Miller, J. Marks, G. Lozano, and A. J. Levine, J. Virol. 61, 2029 (1987). 21 j. Bresser and M. J. Evinger-Hodges, Gene. Anal. Techn. 4, 89 (1987).
204
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[2 1]
chromatin rather than uncondensed chromatin, 22 but diffuse staining of interphase nuclei has also been observed.l,23 Nonspecific binding can also occur in the cytoplasm. For example, avidin conjugates can bind to the cytoplasmic granules of some mast cells. 24 These cells contain heparin, a polysacchatide containing many sulfate and carboxyl groups. Since heparin is highly negatively charged, it has the potential to bind to proteins of high isoelectric point such as avidin (pI > 10), antibodies, and some enzymes. Nonspecific avidin staining of mast cells appears to be highly dependent on the ionic strength and pH of the buffers used in staining, the tissue of origin, and the fixative used in preparing sections for histology. Tissues fixed in Methacarn or Carnoy's fixatives seem to have the greatest potential for nonspecific staining of mast cells. Nonspecific avidin staining of the cell surface has also been reported. It has been suggested that avidin binds to acidic components of the cell capsule. Blocking Method. Ionic binding to avidin conjugates can frequently be suppressed by raising the ionic strength of dilution buffers by the addition of 0.3-0.5 M sodium chloride. 25,26Raising the pH to 9.4 has also proved effective in some instances. 24 High pH does not affect the avidin-biotin interaction, since it is effective across a pH range from 3 to 10, but high pH may be incompatible with the primary interaction under study and must therefore be used with caution. Many lectins, for example, become inactive at high p H . 24 Nonspecific binding that cannot be blocked by raising the ionic strength or pH may be due to hydrophobic or other protein-protein interactions. Addition of protein to diluent solution has proved effective at blocking such interactions. Bovine serum albumin is effective at a concentration of 1-3%, but it should be a crystalline grade. Spurious background staining has been associated with the use of noncrystalline grades. With antibodies, it is common practice to include nonimmune serum in the diluent to serve as a cartier and to minimize nonspecific binding. PolyL-lysine, a highly basic polypeptide, has been used to block nonspecific binding of streptavidin-peroxidase in a nitrocellulose system. 27 Gelatin has been used to prevent nonspecific interactions with avidin-stabilized 22 M. H. Heggeness, Stain Technol. 52, 165 (1977). 23 R. C. Duhamel and D. A. Johnson, J. Histochem. Cytochem. 33, 711 0985). 24 G. Bussolati and P. Gugliotta, J. Histochem. Cytochem. 31, 1419 (1983). 25 C. J. P. Jones, S. M. Mosley, I. J. M. Jeffrey, and R. W. Stoddart, Histochem. J. 19, 264 (1987). 26 R. K. Clark, Y. Tani, and I. Damjanov, J. Histochem. Cytochem. 34, 1509 (1986). z7 L. Scopsi, B.-L. Wang, and L.-I. Larsson, J, Histochem. Cytochem. 34, 1469 (1986).
[9.1]
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gold sols. z8 Nonfat dry milk at a concentration of 5-10% has been used to block nuclear and cytoplasmic nonspecific binding of avidin conjugates in histological sections. 23However, it has been reported that, in a nitrocellulose blot procedure, although nonfat dry milk eliminated the vast majority of the background staining, several distinct bands exhibited spurious binding to avidin-peroxidase, but not to biotin-peroxidase. The use of 0.15 or 0.3 M NaCI instead of nonfat dry milk eliminated both general background staining and binding to the spurious bands. 26 Presence o f Biotin in Protein Solutions. The presence of biotin in protein carrier solutions, such as nonimmune serum and nonfat dry milk, may reduce the signal-to-noise ratio of the detection system. Avidinbiotin detection systems are often so sensitive that even small amounts of free biotin in buffers can significantly suppress the signal. This is especially true with ABC complexes of avidin-biotin-peroxidase. The usual response of the experimenter to a reduced signal is to increase the concentration of the primary reagents, but this can have the effect of increasing the nonspecific background significantly. A better solution is to reduce or eliminate the free biotin in the affected reagent. Biotin-containing solutions can be dialyzed to remove free biotin, if necessary. Nonspecific Binding Unrelated to Biotin or Avidin Antibody Procedures. Since antibodies are frequently conjugated with either biotin or avidin, some commonly encountered causes of nonspecific binding to antibodies deserve comment. Since most tissues contain some immunoglobulin, residual cross-reactivity of the secondary antibody for endogenous immunoglobulin in the target tissue can result in spurious binding of secondary antibodies. Unless the cross-reactivity is very strong, however, adding 2% serum from the species of the tissue to the diluent of the secondary antibody will block the cross-reactivity. IgM antibodies in whole serum are a frequent source of nonspecific staining because IgM is notoriously "sticky." Since many IgM antibodies are cryoglobulins which tend to precipitate when stored in the cold for lengthy periods, they can frequently be eliminated by centrifuging the primary antibody prior to use. If necessary, ammonium sulfate fractionation or DEAE chromatography can be employed to eliminate IgM class immunoglobulins. Many animals, especially rabbits, have constitutive antibodies against tissue elements such as cytokeratins and connective tissues. These are 28 O. Behnke, T. Ammitzboll, H. Jessen, M. Klokker, K. Nilausen, J. Tranum-Jensen, and L. Olsson, Eur. J. Cell Biol. 41, 326 (1986).
206
GENERAL METHODOLOGY
[21]
generally IgM antibodies of low affinity, however, and false staining may be eliminated by using a higher dilution of primary antibody. Ideally, it is sound practice to circumvent this problem by affinity purification of the primary antibody on an antigen column, but diluting the primary antibody as much as possible and longer incubation of the primary antibody at 4 ° will generally eliminate the binding problems. If protein is used as a blocking agent or carrier it should be compatible with the specificity of the secondary antibody. For example, nonfat dry milk should not be used with primary antibodies made in goat or sheep, because secondary antibodies to goat or sheep IgG generally cross-react strongly with immunoglobulin present in bovine milk. Nitrocellulose Procedures. Detection systems that involve the binding of target macromolecules to nitrocellulose or other membrane materials require that a blocking solution be used to inactivate all remaining binding sites on the membrane. In some protocols, protein solutions such as 1-3% albumin or 5% nonfat dry milk are used as irreversible blocking agents. Subsequent incubations with avidin-biotin reagents are subject to the same problems of nonspecific binding owing to ionic or hydrophobic interactions with the immobilized blocking protein as discussed above. Other protocols use Tween 20, a detergent, as a blocking agent. The latter does not block irreversibly and must be included in all subsequent diluent and wash buffers. RIA grade Tween 20 may be preferred. Since Tween 20 binds to proteins, the addition of excess carrier proteins may render the Tween 20 ineffective.
Advantages~Disadvantages of Streptavidin The previous discussion has focused primarily on avidin, but many of the comments apply equally to streptavidin. Streptavidin is only slightly anionic, contains no carbohydrate, and is assumed to bind biotin with the same affinity as avidin. 29 It has been suggested that the lack of carbohydrate and the lower isoelectric point of streptavidin would eliminate many of the sources of nonspecific binding associated with avidin. 3° The presumption of an advantage to streptavidin does not necessarily hold in all circumstances. In many cases, the native isoelectric point of avidin is irrelevant, since it is altered by derivatization with fluorochromes or by conjugation to enzymes of low isoelectric point. There are certain disadvantages to streptavidin. Streptavidin is usually more expensive than avidin. The biotin-binding cleft in streptavidin is different than that of avidin and, in some cases, may require a longer 29 L. Chaiet and F. J. Wolf, Arch. Biochem. Biophys. 106, 1 (1964). 30 T. V. Updyke and G. L. Nicolson, J. lmmunol. Methods 73, 83 (1984).
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NONSPECIFICBINDING
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spacer arm for biotinylation to achieve optimum binding with streptavidin. The lack of carbohydrate potentially eliminates the problem of nonspecific binding to endogenous lectins, but if the reagent system includes other macromolecules containing terminal mannose residues, such as horseradish peroxidase, unwanted binding of the reagent to lectins may still occur. Furthermore, as discussed above, the same tissues that contain endogenous avidin-binding lectins may also contain endogenous biotin-containing proteins. General Recommendations
Frequently, background staining that is initially ascribed to endogenous biotin or avidin binding proves to be due to other components of the system. To determine whether nonspecific binding occurs in a particular system, controls should be performed in which each reagent in the system is systematically omitted. An almost universal method for preventing nonspecific binding in avidin-biotin systems is the two-step sequential pretreatment with avidin and biotin. This approach may add unnecessary time and expense to the staining process, however, and it is essential only when the unwanted binding is due to endogenous biotin-containing protein or avidin. In many applications, raising the ionic strength of dilution buffers by the inclusion of 0.3 M NaC1 will be sufficient to eliminate nonspecific binding arising from ionic interaction. If the unwanted binding is due to endogenous lectins, the addition of 200 mM a-methyl-D-mannoside will usually eliminate it. High salt and the glycoside can be combined, if desired. If the appropriate controls indicate that nonspecific binding has not been fully suppressed, the addition of protein blocking agents such as albumin, nonfat dry milk, or nonimmune serum can be considered, subject to the caution that the biotin content should not diminish the intensity of the reaction. Although the focus of this chapter has been nonspecific staining encountered with the avidin-biotin system, it should not be inferred that nonspecific binding will inevitably occur. In fact, if the proper procedures for general immunohistochemical staining are followed, nonspecific staining is rarely seen. When used in an appropriate way and with a full awareness of its principles, the avidin-biotin system offers the potential for maximum staining sensitivity with no significant nonspecific background.
