[54]
IMMUNOASSAYS: OVERVIEW
467
tion experiments, modification of free sulfhydryls). In the absence of such information, an empirical approach can be taken. Photobiotin affords an easy means for biotinylation of proteins; however, the sites of modification are not always predictable. NHS-biotin derivatives are readily available and are relatively easy to use. Finally, one may wish to biotinylate the protein while liganded to its normal interacting molecule, in the case of calmodulin, calcium ion is the obvious choice. However, calmodulin could also be biotinylated while bound to a specific binding protein such as calcineurin. 3° The latter method may be of use in determination of changes in reactivities of specific amino acid residues subsequent to binding. Finally, it may prove advantageous to biotinylate genetically engineered calmodulin, yeast calmodulin, or plant calmodulin to further define calmodulin-target protein interactions. Thus, the use of biotinylated calmodulin derivatives may offer insights into a range of structural and functional questions relevant to regulation of specific calmodulin-binding proteins. Acknowledgments This research was supported by a research grant from The InternationalLife Sciences Institute Research Foundation and by U.S. Public Health Service Grant R01-AG06337 to M.L.B. The authors thank Ms. Doris Lineweaverfor manuscriptpreparation. 30 A. S. Manalan and C. B. Klee, Biochemistry 26, 1382 (1987).
[54] A v i d i n - B i o t i n M e d i a t e d I m m u n o a s s a y s : O v e r v i e w
By
M E I R W I L C H E K a n d E D W A R D A . BAYER
Progress in the use of avidin-biotin technology in immunoassays has developed together with major advances of the immunodiagnostics field in general. Thus, whenever a particular improvement in a given step was desired, the applicability of the avidin-biotin complex was rapidly demonstrated. At the beginning, in the late 1960s and early 1970s, when the need for improved diagnostics was established and different approaches (e.g., radioimmunoassay1 and bacteriophage 2 assay systems for the quantification i R. S. Yalow and S. A. Berson, Nature (London) 184, 1648 (1959); D. S. Skelley, L. P. Brown, and P. K. Besch, Clin. Chem. 19, 146 (1973). 20. MS.kel~., Immunology 10, 81 (1966).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
468
APPLICATIONS
[54]
of antigens) were suggested, the mediation of the avidin-biotin system was also examined. 3 Later, when enzyme and solid-phase immunoassay systems were introduced, it was immediately clear that the avidin-biotin system would also be incorporated somehow into such approaches. The basis for further work in this direction was established when it was shown that biotin-modified cells or biotin-modified antibodies and lectins can interact simultaneously with their respective antigens or receptors and with an avidin-containing probe. 4,5 In fact, the binding of biotin or biotinylated molecules to cells is, in essence, a solid-phase assay system, where the cells are representative of an immobilizing matrix. The main application of avidin-biotin technology in immunoassays is enhancement of the signal and/or speed of the assay. The signal is enhanced owing to the fact that many biotin residues can be introduced chemically to the antibody molecule. Thus, multiple avidin-containing probes (including enzymes and radiolabels) can be incorporated into the final step of the detection system. In addition, the four biotin-binding sites on the avidin molecule allow further signal enhancement by using a biotinylated detection probe either applied sequentially or complexed with avidin in a predetermined ratio. An alternative approach would be to use the avidin-biotin complex in the capture system. For this purpose, biotinylated antibody or biotinylated protein A can be immobilized to an avidin-containing matrix. The introduction of the avidin-biotin system to enzyme immunoassay can mainly be attributed to Avrameas and co-workers 6 (see also [55] in this volume). Our own efforts in this area have mainly been directed to the "least attractive" immunodiagnostic approach, namely, radioimmunoassay. The inherent disadvantages of radioiodination are further compounded by the presence of only one tyrosine residue on egg-white avidin and the apparent requirement of this residue for biotin binding. Nonetheless, there are various ways to circumvent this apparent "fatal flaw." One possibility is to use bacterial streptavidin, which has 6 tyrosines per subunit. 7 Another possibility is to increase the number of iodinatable groups on egg-white avidin by chemically incorporating phenols. This can be accomplished by various methods. For example, the Bolton-Hunter reagent will modify amino groups of lysine with phenols; the reagent itself 3 j. M. Becker and M. Wilchek, Biochim. Biophys. Acta 264, 165 (1972). 4 H. Heitzmann and F. M. Richards, Proc. Natl. Acad. Sci. U.S.A. 71, 3537 (1974); E. A. Bayer, E. Skutelsky, D. Wynne, and M. Wilchek, J. Histochem. Cytochern. 24, 933 (1976). 5 E. A. Bayer, M. Wilchek, and E. Skutelsky, FEBS Lett. 68, 240 (1976). 6 j._L. Guesdon, T. Ternynck, and S. Avrameas, J. Histochem. Cytochem. 27, 1131 (1979). 7 L. Chaiet and F. J. Wolf, Arch. Biochem. Biophys. 106, 1 (1964).
[55]
ENZYME IMMUNOASSAYS
469
can be radiolabeled, or the modified protein can be iodinated when desired. In this case (and for most amine-specific modifications), egg-white avidin is preferred over streptavidin because of its large number of lysines per subunit. Extensive modification of the lysines would also serve to reduce the high pI of the native protein, thereby reducing one potential source of nonspecific binding. Nonglycosylated avidin 8 is perhaps the protein of choice for such modifications since its employment precludes a second major contributor (the oligosaccharide component of the native avidin molecule) to nonspecific interactions. Another method of increasing the number of phenolic groups in avidin is to prepare conjugates with either polytyrosine or tyrosine-containing copolymers. 9 Chapters [56]-[64] in this volume illustrate the versatility of the avidin-biotin system in immunoassays. A more extensive literature summary providing details of the application of avidin-biotin technology in immunoassays and diagnostics is presented in [3] in this volume. 8 y. Hiller, E. A. Bayer, and M. Wilchek, this volume [6]. 9 M. Wilchek, J. Solid-Phase Biochem. 5, 193 (1980).
[55] A v i d i n - B i o t i n S y s t e m in E n z y m e I m m u n o a s s a y s B y THI~RI~SE TERNYNCK a n d STRATIS AVRAMEAS
Avidin is a 66,000-Da glycoprotein present in egg white. One of its properties is the ability to bind with a high affinity (dissociation constant 10-15 M) to the small molecule biotin (or vitamin H).1,2 Biotin is easily linked to proteins, which modifies little if any of their biological activities, and, when bound, retains its high affinity for avidin. The avidin-biotin system has been used to develop various procedures) In immunoassays, in order to detect or quantify a constituent, biotinylated antibodies are allowed to react with the constituent, and then avidin (linked to a marker substance) is added. A procedure based on this principle was initially developed for the immunocytochemical localization of erythrocyte surface antigens; in this case, the biotin-labeled antibody was revealed by ferritin-labeled avidin. 3-5 N. M. Green, Biochem J. 89, 585 (1963). 2 N. M. Green, Adv. Protein Chem. 29, 85 (1975). 3 M. Wilchek and E. A. Bayer, lmmunol. Today 5, 39 (1984). 4 H. Heitzmann and F. M. Richards, Proc. Natl. Aead. Sci. U.S.A. 71, 3537 (1974). 5 E. A. Bayer, M. Wilchek, and S. Skutelsky, FEBS Lett. 68, 240 (1976).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
IMMUNOASSAYS: OVERVIEW
467
tion experiments, modification of free sulfhydryls). In the absence of such information, an empirical approach can be taken. Photobiotin affords an easy means for biotinylation of proteins; however, the sites of modification are not always predictable. NHS-biotin derivatives are readily available and are relatively easy to use. Finally, one may wish to biotinylate the protein while liganded to its normal interacting molecule, in the case of calmodulin, calcium ion is the obvious choice. However, calmodulin could also be biotinylated while bound to a specific binding protein such as calcineurin. 3° The latter method may be of use in determination of changes in reactivities of specific amino acid residues subsequent to binding. Finally, it may prove advantageous to biotinylate genetically engineered calmodulin, yeast calmodulin, or plant calmodulin to further define calmodulin-target protein interactions. Thus, the use of biotinylated calmodulin derivatives may offer insights into a range of structural and functional questions relevant to regulation of specific calmodulin-binding proteins. Acknowledgments This research was supported by a research grant from The InternationalLife Sciences Institute Research Foundation and by U.S. Public Health Service Grant R01-AG06337 to M.L.B. The authors thank Ms. Doris Lineweaverfor manuscriptpreparation. 30 A. S. Manalan and C. B. Klee, Biochemistry 26, 1382 (1987).
[54] A v i d i n - B i o t i n M e d i a t e d I m m u n o a s s a y s : O v e r v i e w
By
M E I R W I L C H E K a n d E D W A R D A . BAYER
Progress in the use of avidin-biotin technology in immunoassays has developed together with major advances of the immunodiagnostics field in general. Thus, whenever a particular improvement in a given step was desired, the applicability of the avidin-biotin complex was rapidly demonstrated. At the beginning, in the late 1960s and early 1970s, when the need for improved diagnostics was established and different approaches (e.g., radioimmunoassay1 and bacteriophage 2 assay systems for the quantification i R. S. Yalow and S. A. Berson, Nature (London) 184, 1648 (1959); D. S. Skelley, L. P. Brown, and P. K. Besch, Clin. Chem. 19, 146 (1973). 20. MS.kel~., Immunology 10, 81 (1966).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
468
APPLICATIONS
[54]
of antigens) were suggested, the mediation of the avidin-biotin system was also examined. 3 Later, when enzyme and solid-phase immunoassay systems were introduced, it was immediately clear that the avidin-biotin system would also be incorporated somehow into such approaches. The basis for further work in this direction was established when it was shown that biotin-modified cells or biotin-modified antibodies and lectins can interact simultaneously with their respective antigens or receptors and with an avidin-containing probe. 4,5 In fact, the binding of biotin or biotinylated molecules to cells is, in essence, a solid-phase assay system, where the cells are representative of an immobilizing matrix. The main application of avidin-biotin technology in immunoassays is enhancement of the signal and/or speed of the assay. The signal is enhanced owing to the fact that many biotin residues can be introduced chemically to the antibody molecule. Thus, multiple avidin-containing probes (including enzymes and radiolabels) can be incorporated into the final step of the detection system. In addition, the four biotin-binding sites on the avidin molecule allow further signal enhancement by using a biotinylated detection probe either applied sequentially or complexed with avidin in a predetermined ratio. An alternative approach would be to use the avidin-biotin complex in the capture system. For this purpose, biotinylated antibody or biotinylated protein A can be immobilized to an avidin-containing matrix. The introduction of the avidin-biotin system to enzyme immunoassay can mainly be attributed to Avrameas and co-workers 6 (see also [55] in this volume). Our own efforts in this area have mainly been directed to the "least attractive" immunodiagnostic approach, namely, radioimmunoassay. The inherent disadvantages of radioiodination are further compounded by the presence of only one tyrosine residue on egg-white avidin and the apparent requirement of this residue for biotin binding. Nonetheless, there are various ways to circumvent this apparent "fatal flaw." One possibility is to use bacterial streptavidin, which has 6 tyrosines per subunit. 7 Another possibility is to increase the number of iodinatable groups on egg-white avidin by chemically incorporating phenols. This can be accomplished by various methods. For example, the Bolton-Hunter reagent will modify amino groups of lysine with phenols; the reagent itself 3 j. M. Becker and M. Wilchek, Biochim. Biophys. Acta 264, 165 (1972). 4 H. Heitzmann and F. M. Richards, Proc. Natl. Acad. Sci. U.S.A. 71, 3537 (1974); E. A. Bayer, E. Skutelsky, D. Wynne, and M. Wilchek, J. Histochem. Cytochern. 24, 933 (1976). 5 E. A. Bayer, M. Wilchek, and E. Skutelsky, FEBS Lett. 68, 240 (1976). 6 j._L. Guesdon, T. Ternynck, and S. Avrameas, J. Histochem. Cytochem. 27, 1131 (1979). 7 L. Chaiet and F. J. Wolf, Arch. Biochem. Biophys. 106, 1 (1964).
[55]
ENZYME IMMUNOASSAYS
469
can be radiolabeled, or the modified protein can be iodinated when desired. In this case (and for most amine-specific modifications), egg-white avidin is preferred over streptavidin because of its large number of lysines per subunit. Extensive modification of the lysines would also serve to reduce the high pI of the native protein, thereby reducing one potential source of nonspecific binding. Nonglycosylated avidin 8 is perhaps the protein of choice for such modifications since its employment precludes a second major contributor (the oligosaccharide component of the native avidin molecule) to nonspecific interactions. Another method of increasing the number of phenolic groups in avidin is to prepare conjugates with either polytyrosine or tyrosine-containing copolymers. 9 Chapters [56]-[64] in this volume illustrate the versatility of the avidin-biotin system in immunoassays. A more extensive literature summary providing details of the application of avidin-biotin technology in immunoassays and diagnostics is presented in [3] in this volume. 8 y. Hiller, E. A. Bayer, and M. Wilchek, this volume [6]. 9 M. Wilchek, J. Solid-Phase Biochem. 5, 193 (1980).
[55] A v i d i n - B i o t i n S y s t e m in E n z y m e I m m u n o a s s a y s B y THI~RI~SE TERNYNCK a n d STRATIS AVRAMEAS
Avidin is a 66,000-Da glycoprotein present in egg white. One of its properties is the ability to bind with a high affinity (dissociation constant 10-15 M) to the small molecule biotin (or vitamin H).1,2 Biotin is easily linked to proteins, which modifies little if any of their biological activities, and, when bound, retains its high affinity for avidin. The avidin-biotin system has been used to develop various procedures) In immunoassays, in order to detect or quantify a constituent, biotinylated antibodies are allowed to react with the constituent, and then avidin (linked to a marker substance) is added. A procedure based on this principle was initially developed for the immunocytochemical localization of erythrocyte surface antigens; in this case, the biotin-labeled antibody was revealed by ferritin-labeled avidin. 3-5 N. M. Green, Biochem J. 89, 585 (1963). 2 N. M. Green, Adv. Protein Chem. 29, 85 (1975). 3 M. Wilchek and E. A. Bayer, lmmunol. Today 5, 39 (1984). 4 H. Heitzmann and F. M. Richards, Proc. Natl. Aead. Sci. U.S.A. 71, 3537 (1974). 5 E. A. Bayer, M. Wilchek, and S. Skutelsky, FEBS Lett. 68, 240 (1976).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
