[2]
INTRODUCTION
TO AVIDIN--BIOTIN
TECHNOLOGY
5
Completely independently, Tom Broker and Norman Davidson at the California Institute of Technology had the same general ideas for using the avidin-biotin system, and they developed procedures for gene mapping by labeling RNA molecules, especially tRNA, and then using them as hybridization probes to locate genes in double-stranded DNA. 8 After hearing about this work at meetings, we did have some casual discussions with David Ward in Human Genetics at Yale, University. 9 It was clear that the biotin-avidin system as a general cytochemical labeling procedure had arrived. 10 I doubt that any of us at the time imagined the explosion in applications that would occur over the 1980s ~1 and that is documented in this volume. With the basic underpinnings for the technology now in hand, as well as cloned genes for the avidins ~2 and crystal structures of the proteins, 13 the future is bright indeed. 8 j. E. Manning, N. D. Hershey, T. R. Broker, M. Pellegrini, H. K. Mitchell, and N. Davidson, Chromosoma 53, 107 (1975); T. R. Broker, L. M. Angerer, P. Yen, N. D. Hershey, and N. Davidson, Nucleic Acids Res. 5, 363 (1978). 9 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). to E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980); M. Wilchek and E. A. Bayer, lmrnunol. Today 5, 39 (1984). 11 M. Wilchek and E. A. Bayer, Anal. Biochem. 171, 1 (1988). t2 M. L. Gope, R. A. Keinanen, P. A. Kristo, O. M. Conneely, W. G. Beattie, T. ZaruckiSchulz, B. M. O'Malley, and M. S. Kulomaa, Nucleic Acids. Res. 15, 3595 (1987); G. Chandra and J. G. Gray, this volume [7]; C. E. Argarana, I. D. Kuntz, S. Birken, R. Axel, and C. R. Cantor, Nucleic Acids Res. 14, 1871 (1986). ~3 E. Pinn, A. P/ihler, W. Saenger, G. Petsko, and N. M. Green, Eur. J Biochem. 123, 545 (1982); A. P~ihler, W. A. Hendrickson, M. A. G. Kolks, C. E. Argarana, and C. R. Cantor, J. Biol. Chem. 262, 13933 (1987).
[2] I n t r o d u c t i o n to A v i d i n - B i o t i n T e c h n o l o g y
By MEIR WILCHEK and EDWARD A. BAYER The avidin-biotin system has many uses in both research and technology. This general introductory chapter presents an overview of the principles and advantages of this system, and illustrates the numerous applications of avidin-biotin technology. It also explains in general and explicit terms the basic idea behind avidin-biotin technology. METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
6
INTRODUCTION
[2]
Principle The major distinguishing feature of the avidin-biotin system is the extraordinary affinity (Ka = 1015 M -i) that characterizes the complex formed between the vitamin biotin and the egg-white protein avidin (or streptavidin, its bacterial relative from Streptomyces avidinii). ~The interaction is so strong that even biotin coupled to proteins (such as native biotin-requiring carboxylases) is available for binding by avidin. The rationale in using the avidin-biotin system is based on the premise that if one chemically modifies any biologically active compound with biotin through its valeric acid side chain, the biological and physicochemical properties of the biotin-modified molecule will not be changed significantly. Likewise, the biotin moiety of the derivatized molecule would still be available for interaction with avidin. Another distinctive feature of this system is the multiple (four) binding sites of avidin for biotin. This provides us with the possibility of cross-linking between different biotincontaining molecules and adds another dimension to the use of this multifaceted system. If a reporter group of some sort is attached to the avidin molecule, the conjugate can be used for many different purposes (Fig. 1). The avidinconjugated probe can be added as a single chemically conjugated entity, or avidin (by virtue of its four biotin-binding sites) can be applied in native form together with a biotinylated probe. The latter alternative can be instituted either sequentially in a stepwise fashion or in a single step as preformed complexes. Figure 2 (which was first published in an earlier volume in this series 2) presents these two alternatives for labeling. Figure 1 also provides a list of some of the types of binders that can be used to label a given target site, as well as a list of probes that can be either conjugated with avidin (for direct interaction with the biotinylated binder) or derivatized with biotin (for complex formation with underivatized avidin). The various applications of avidin-biotin technology reported to date are also presented in Fig. 1.
Advantages Figure 1 demonstrates why the avidin-biotin system has become so popular: a simplified and unified approach can be taken. Some of the major advantages in using avidin-biotin technology can be summarized as follows: 1. The exceptionally high affinity and stability of the avidin-biotin complex ensures the desired conjunction of binder and probe. N. M. Green, Adv. Protein Chem. 29, 85 (1975). z E. A. Bayer, E. Skutelsky, and M. Wilchek, this series, Vol. 62, p. 308.
[2]
INTRODUCTION
TO AVIDIN--BIOTIN
OTINYLATED
TARGET MOLECULE
~
~
T A R G E T : BINDER Antigens Antibodies Lectins Glycoconjugates Enzymes Receptors Transport proteins Hydrophobic sites Membranes Nucleic acids, genes
7
TECHNOLOGY
PROBES
Antibodies AntigeDs Glycoconjugates Lectins Substrates, cofactors, mhibitors, etc ]tormones, eifeetors, :oxins, etc Vitamins, amino acid:s, sugars, etc Lipids, fatty acids Liposomes DNA/RNA probes
AVIDIN-BIOTIN COMPLEX
Phages, viruses, bacteria, ] subcellular organelles, cells,~AIl of the above tissues, whole organisms J
Enzymes Radiolabe[s Fluorescent agents Chemiluminescent agents Chromophores Heavy metals Colloidal gold Ferrltm Bemocyanul Phages Macromolecular carriers Liposomes Solid supports
APPLICATIONS 1 Affinity chromatography isolation studies 2 Affinity eytochemistry localization studies a light microscopy b fluorescence rnieroscopy c electron microscopy 3 Hlstoehemistry 4 Pathological probe 5 Diagnostics 6 Signal amplification 7 lmmunoassay 8 Hybridoma technology 9 Blotting technology
10 11 12 13 14 15 16 17 18 19 20 21
Bioaffinity sensor Gene probe Crosslinkmg agent Affinity targeting Affinity perturbation Drug delivery Fusogenic agent lmmobdizing agent Selective retrieval Selective elimination Flow cytometry Cytological probe
FIG. I. Generalized scheme illustrating the essentials of avidin-biotin technology. A biologically active target molecule is recognized by a biotinylated binder that is subsequently recognized by avidin conjugated to an appropriate probe. Lists of target-binder pairs, the various probes that have been used, and the broad spectrum of applications of avidin-biotin technology are also presented.
2. Biotin can readily be attached to m o s t binders and p r o b e s , and, f o l l o w i n g biotinylation, the biological activity and p h y s i c a l characteristics are c o m m o n l y retained. In fact, in m a n y c a s e s , a binder can be b i o t i n y l a t e d w i t h o u t e x t e n s i v e purification, since its interaction w i t h the target m o l e c u l e s e r v e s to r e m o v e "irrelevant" biotinylated s u b s t a n c e s .
8
[2]
INTRODUCTION
M~M~IRANE
APPROACH
MEMBRANE:
MEMBI~ANE
£
B a
-
BIOTIN
- LABELED
MEMBRANE SITE
B MEMB'RA,~IE: ;::
APPROACH
2
- AVIDIN
~,~ key
- FERRITIN-AVIDIN CONJUGATE 8
e
I
B
B F ER RITIN- BtC'TrN CONJUGATE
B
FlG. 2. Schematic of two alternative approaches for labeling biotin-modifiedcell membrane sites: (1) one-step method, using ferritin-conjugatedavidin, and (2) two-step method, employing sequential treatment with avidin and biotin-labeledferritin.
3. The multiplicity of biotin groups per binder combined with the tetrameric structure of avidin leads to amplification of the desired signal. 4. The system is amenable to double-labeling and kinetics studies. 5. The system is extremely versatile. A given molecular target can interact with a single type of biotinylated binder that can then be analyzed by various means using different avidin-conjugated probes. Conversely, the status of different target molecules in a given system can be compared using a variety of biotinylated binders and a single avidin-conjugated probe. Of course, the versatility is further extended through the combined use of different biotinylated binders and avidin-associated probes. 6. A wide spectrum of different biotinylating reagents, biotinylated binders, and both biotinylated and avidin-containing probes is available from a variety of commercial sources.
Application The development of avidin-biotin technology was not the outcome of a single event, nor was it the brainchild of a single individual or research group. Rather, it developed through an evolutionary process (the collec-
[2]
9
I N T R O D U C T I OTO N AVIDIN-BIOTIN TECHNOLOGY TABLE I MILESTONES IN DEVELOPMENTOF AV1DIN-BIOT1NTECHNOLOGY
Year 1916 1936 1942 1943 1952 1958 1963 1965 1968 1969 1970 1971
1972 1973 1974 1975
1976 1977 1978 1979 1980 1981
1982 1983 1986 1988 1990
Historical highlights "Toxic substances" in egg white Biotin isolated Structure of biotin determined Synthesis of biotin hydrazide Biotin synthesized Avidin purified from egg white Coenzyme function of biotin described Extensive protein chemical studies on avidin Streptavidin and stravidin isolated Retardation of avidin on biotinyl cellulose Affinity chromatography of avidin on biocytin-Sepharose Structure of stravidin determined Avidin crystallized Biotin-containing peptides isolated on avidin column Avidin sequenced N-Hydroxysuccinimide and p-nitrophenyl esters of biotin synthesized Biotinylated phages used for assay of avidin and biotin Avidin monomer column Localization of biotinylated sites on purified membranes; ferritin-avidin conjugates Biotinylated RNA Spacer-containing biotinylating reagents Comprehensive review on avidin Biotinylated antibodies and lectins (affinity cytochemistry) Biotinylated hormones TIBS review: "Emerging Techniques" ABC approach for enzyme immunoassay Biotinylated lipids First comprehensive review of applications Biotinylated DNA probes Premade complexes Iminobiotin columns and reagents Insulin receptor isolated using biotinylated insulin Protein and DNA blotting using biotinylated binders Cloning of streptavidin gene Nonglycosylated avidin Crystallographic structure of streptavidin Methods in Enzymology, "Avidin-Biotin Technology"
Ref, 3 4 5,6 7 8-10 11,12 13,14 15-18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 32 33-35 36 37 38 39 40 41 42,43 44-46 47-49 50 51 52,53 This volume
Live p r o d u c t o f c u r i o s i t y , n e c e s s i t y , o b s e r v a t i o n , i m a g i n a t i o n , an d c r e a t i v e t h o u g h t ) w h i c h b e g a n w i t h t h e e a r l y d i s c o v e r y o f t o x i c s u b s t a n c e s in e g g w h i t e a n d c u l m i n a t e d w i t h the c o m m e r c i a l a v a i l a b i l i t y o f literally hundreds of "avidin-biotin" products from dozens of companies. Some
10
INTRODUCTION
[2]
of the historical milestones in the development of avidin-biotin technology 3-53 are presented in Table I. 3 W. G. Bateman, J. Biol. Chem. 26, 263 (1916). 4 F. K6gl and B. TOnnis, Z. Physiol. Chem. 242, 43 (1936). V. du Vigneaud, K. Hofmann, and D. B. Melville, J. Am. Chem. Soc. 64, 188 (1942). 6 V. du Vigneaud, D. B. Melville. K. Folkers, D. E. Wolf, R. Mozingo, J. C. Kereszteolf, and S. A. Harris, J. Biol. Chem. 146, 475 (1942). 7 K. Hofmann, D. B. Melville, and V. du Vigneaud, J. Biol. Chem. 144, 513 (1942). s S. A. Harris, D. E. Wolf, R. Mozingo, and K. Folkers, Science 97, 447 (1943). 9 S. A. Harris, D. E. Wolf, R. Mozingo, R. C. Anderson, G. E. Arth, N. R. Easton, D. Heyl, A. N. Wilson, and K. Folkers, J. Am. Chem. Soc. 66, 1756 (1944). ~0S. A. Harris, N. R. Easton, D. Heyl, A. N. Wilson, and K. Folkers, J. Am. Chem. Soc. 66, 1757 (1944). 1~ H. FraenkeI-Conrat, N. S. Snell, and E. D. Ducay, Arch. Biochem. Biophys. 92, 80 (1952). lZ H. Fraenkel-Conrat, N. S. Snell, and E. D. Ducay, Arch. Biochem. Biophys. 92, 97 (1952). z3 S. J. Wakil, E. B. Titchener, and D. M. Gibson, Biochim. Biophys. Acta 29, 225 (1958). t4 F. Lynen, J. Knappe, E. Lorch, G. Jutting, and E. Ringelmann, Angew. Chem. 71, 481 (1959). t5 N. M. Green, Biochem. J. 89, 585 (1963). 16 N. M. Green, Biochem. J. 89, 599 (1963). 17 N. M. Green, Biochem. J. 89, 609 (1963). 18 M. D. Melamed and N. M. Green, Biochem. J. 89, 591 (1963). t9 L. Chaiet, T. W. Miller, F. Tausig, and F. J. Wolf, Antimicrob. Agents Chemother. 3, 28 (1963). 20 D. M. McCormick, Anal. Biochem. 13, 194 (1965). 21 p. Cuatrecasas and M. Wilchek, Biochem. Biophys. Res. Commun. 33, 235 (1968). z2 K. H. Baggaley, B. Blessington, C. P. Falshaw, and W. D. Ollis, Chem. Commun., 101 (1969). 23 N. M. Green and E. J. Toms, Biochem. J. 118, 67 (1970). 24 A. Bodanszky and M. Bodanszky, Experientia 26, 327 (1970). z5 R. J. DeLange and T.-S. Huang, J. Biol. Chem. 246, 698 (1971). 26 j. M. Becket, M. Wilchek, and E. Katcbalski, Proc. Natl. Acad. Sci. U.S.A. 68, 2604 (1971). 27 j. M. Becket and M. Wilchek, Biochim. Biophys. Acta 264, 165 (1972). zs N. M. Green and E. J. Toms, Biochem. J. 133, 687 (1973). 29 H. Heitzmann and F. M. Richards, Proc. Natl. Acad. Sci. U.S.A. 71, 3537 (1974). 30 j. E. Manning, N. D. Hershey, T. R. Broker, M. Pellegrini, H. K. Mitchell, and N. Davidson, Chromosoma 53, 107 (1975). 3~ E. A. Bayer, T. Viswanatha, and M. Wilchek, FEBS Lett. 60, 309 (1975). 32 E. A. Bayer, M. Wilchek, and E. Skutelsky, FEBS Lett. 68, 240 (1976). 33 K. Hofmann and Y. Kiso, Proc. Natl. Acad. Sci. U.S.A. 73, 3516 (1976). 34 K. Hofmann, F. M. Finn, H.-J. Friesen, C. Diaconescu, and H. Zahn, Proc. Natl. Acad. Sci. U.S.A. 74, 2697 (1977). 35 D. Atlas, D. Yaffe, and E. Skutelsky, FEBS Lett. 95, 173 (1978). 36 E. A. Bayer and M. Wilchek, Trends Biochem. Sci. 3, N257 (1978). 37 J.-L. Guesdon, T. Ternynck, and S. Avrameas, J. Histochem. Cytochem. 27, 1131 (1979). 38 E. A. Bayer, B. Rivnay, and E. Skutelsky, Biochem. Biophys. Acta 550, 464 (1979).
[2]
INTRODUCTION TO AVIDIN-BIOTIN TECHNOLOGY
11
We initially intended to include in this volume all the available applications which have thus far appeared in the primary literature, since each contribution has its own special message to convey. We soon discovered that this would be impossible, since the literature is simply too vast. Indeed, many of the methodologies involving avidin and/or biotin have already found their way into this series (Table II), although their contribution to the emerging technology has not generally been emphasized. A more extensive view of the widespread usage of avidin-biotin technology is presented elsewhere in this volume) 4 In assembling this volume, we therefore attempted to include only papers that either set a historical precedence or present a special method or message. Judging from the articles and tables ~4 that appear in this volume, the avidin-biotin system is extremely versatile, the applications are numerous, and the scope of its future use is virtually limitless. We cite the following from our first major review on this subject39: Naturally, we will be unable to cover all possible applications; it seems that the potential of the avidin-biotin complex as a tool in molecular biology is unlimited, and that its successful implementation is directly dependent on the needs and imagination of the user . . . . We cannot, of course, foresee all its possible uses, given our own limited imagination and/or restricted knowledge. We do hope, however, that this review will serve to stimulate the imagination of the readers and convert some of them to users of the method.
39 E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980). 4o p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). 4J S.-M. Hsu, L. Raine, and H. Fanger, J. Histochem. Cytochem. 29, 577 (1981). 4z K. Hofmann, S. W. Wood, C. C. Brinton, J. A. Montibeller, and F. M. Finn, Proc. Natl. Acad. Sci. U.S.A. 77, 4666 (1980). 43 G. A. Orr, J. Biol. Chem. 256, 761 (1981). 44 F. M. Finn, G. Titus, H. Nemoto, T. Noji, and K. Hofmann, Metabolism 31, 691 (1982). 05 F. M. Finn, G. Titus, D. Horstman, and K. Hofmann, Proc. Natl. Acad. Sci. U.S.A. 81, 7328 (1984). 46 R. A. Kohanski and M. D. Lane, J. Biol. Chem. 260, 5014 (1985). 47 B. B. Gordon and S. D. J. Pena, Biochem. J. 208, 351 (1982). 48 K. Ogata, M. Arakawa, T. Kasabara, K. Shioiri-Nakano, and K. Hiraoka, J. Immunol. Methods 65, 75 (1983). 49 j. j. Leary, D. J. Brigati, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 80, 4045 (1983). 50 C. E. Argarana, I. D. Kuntz, S. Birken, R. Axel, and C. R. Cantor, Nucleic Acids Res. 14, 1871 (1986). 5J y . Hiller, J. M. Gershoni, E. A. Bayer, and M. Wilchek, Biochem. J. 248, 167 (1987). 52 A. P~ihler, W. A. Hendrickson, M. A. Gawinowicz Kolks, C. E. Argarana, and C. R. Cantor, J. Biol. Chem. 262, 13933 (1987). s3 p. C. Weber, D. H. Ohlendorf, J. J. Wendoloski, and F. R. Salemme, Science 243, 85 (1989). 54 See M. Wilchek and E. A. Bayer, this volume [3].
TABLE II EARLIER COVERAGE OF AVIDIN--BIOTIN TECHNIQUES IN THIS SERIES
Subject Biotin Assays for biotin Biotin analogs Biotin derivatives
Biotinylated proteins Biotinylated hormones Biotinylated glycoconjugates Biotinyl lipids Biotinylated nucleic acids Immobilized biotin Iminobiotin and derivatives Avidin Assays for avidin Modified avidins Fluorescent avidin Radioactive avidin Avidin conjugates Immobilized avidin Antiavidin antibodies Preparation of biotin-binding proteins Avidin Streptavidin Egg-yolk protein Antibiotin antibodies Holocarboxylase synthetase Applications Affinity cytochemistry Affinity labeling Immunoassay Flow cytometry Affinity chromatography Blotting
Volume [Chapter]"
18A[62], 18A[63], 18A[74], 57137], 62149], 62[50], 621511, 122[12], 1221131 18A[66], 18A[67], 18A[68], 18A[70], 18A[71], 109137], 18A[64], 18A[65], 34120], 46[72], 57[37], 62[54], 62155], 62163], 70[13], 83112], 92137], 109137], 138135], 151139] 62155], 62157], 83112], 92[20], 92[37], 121143], 121168], 133125], 149[11], 150138], 159156] 98122], 109137], 12415] 62155], 83112], 138135] 138135], 149111] 164125], 168154], 17014] 34120], 7118], 122113], 17014] 109137], 122[14], 122[15], 13517] 18A174], 18A[75], 18A[76], 57[37], 62151], 62[52], 62[53], 122112], 122113] 92[20], 109137], 133125] 85148], 92137], 122112], 150138] 62[53], 70113] 62155], 83[12], 92[37], 93120], 149111] 11216], 121168], 122[13], 12415], 125140] 18A[76], 122115] 18A[73], 34120], 122114] 1091371 62[56] 62157] 107[151 62155], 83112], 85[48], 103111], 1291191, 151139], 168154] 62[63], 164125] 73[33], 92120], 103127], 121143], 133125] 103[13], 150138] 34120], 109137], 122[14], 122[15], 12415], 125140], 13517], 17014] 138135], 159156]
Vol. 18A (1970) [62] A. F. Carlucci; [63] D. B. McCormick and J. A. Roth; [64] H. Ruis, D. P. McCormick, and L. D. Wright; [65] J. E. Christner and M. J. Coon; [66] K. Ogata; [67] J. P. Tepper, H.-C. Li, D. B. McCormick, and L. D. Wright; [68] K. Ogata; 1701S. lwahara, D. B. McCormick, and L. D. Wright; [71] H. Ruis, R. N. Brady, D. B. McCormick, and L. D. Wright; [731 N. M. Green; [74] N. M. Green; [75] R.-D. Wei; [76] S. G. Korenman and B. W. O'Malley Vol. 34 (1974) [20] E. A. Bayer and M. Wilchek
" M e t h o d s in E n z y m o l o g y ,
[2]
INTRODUCTION TO AVID1N--BIOTIN TECHNOLOGY
13
References to TABLE I1 (continued) Vol. 46 (1977) [72] E. A. Bayer and M. Wilchek Vol. 57 (1978) [37} H. R. Schroeder, R. C. Boguslaski, R. J. Carrico, and R. T. Buckler Vol. 62 (1979) [491 R. L. Hood; [50] K. Dakshinamurti and L. Allan; [511 H. J. Lin and J. F. Kirsch; [52] H. A. Elo and P. J. Tuohimaa; [53] M. S. Kulomaa, H. A. EIo, and P. J. Tuohimaa; [541 B. K. Sinha and C. F. Chignell; [55] E. A. Bayer, E. Skulelsky, and M, Wilchek; [56] H. W. Meslar and H. B. White 111; [57] M. Berger; [63] E. A. Bayer and M. Wilchek Vot. 70 (1980) [13] J. J. Langone Vol. 71 (1981) [8] M. L. Ernst-Fonberg and J. S. Wolpert Vol. 73 (1981) [33] J.-L. Guesdon and S. Avrameas Vol. 83 (1982) [12] E. A. Bayer, E. Skutelsky, and M. Wilchek Vol. 85 (1982) [48] K. Wang, J. R. Feramisco, and J. F. Ash Vol. 92 (1983) [20] C. Stfihli, V. Miggiano, J. Stocker, Th. Staehelin, P. Hating, and B. Takacs; [37] L. Wofsy Vol. 93 (1983) [20] T. I. Ghose, A. H. Blair, and P. N. Kulkarni Vol. 98 (1983) [22] H. T. Haigler Vol. 103 (1983) [11] R. D. Broadwell and M. W. Brightman; [13] J. C. Cambier and J. G. Monroe; [27] G. E. Trivers, C. C. Harris, C. Rougeot, and F. Dray Vol. 107 (1984) [151 N. H. Goss and H. G. Wood Vol. 109 (1985) [37] F. M. Finn and K. H. Hofmann Vol. 112 (1985) [6] L. Ilium and P. D. E. Jones Vol. 121 (1986) [431J. E. Shively, C. Wagener, and B. R. Clark; [681T. V, Updyke and G. L. Nicolson Vol. 122 (1986) [12] R. D. Nargessi and D. S. Smith; [13] L. Goldstein, S. A. Yankofsky, and G. Cohen; [141 G. A. Orr, G. C. Heney, and R. Zeheb; 115[ R. Zeheb and G. A. Orr Vol. 124 (1986) [5] E. Hazum Vo[. 125 (1986) [401 P. Dimroth Vol. 129 (1986) [19] C.-T. Lin and L. Chan Vol. 133 (1986) [25] G. Barnard, E. A. Bayer, M. Wilchek, Y. Amir-Zaltsman, and F. Kohen Vol. 135 (1987) [7] M. T. W. Hearn Vol. 138 (1987) [35] M. Wilchek and E. A. Bayer Vol. 149 (1987) [11] B. Rivnay, E. A. Bayer, and M. Wilchek Vol. 150 (1987) 1381 D. M. Segal, J. A. Titus, and D. A. Stephany Vol, 151 (1987) [39] G. H. Smith Vol. 159 (1988) [56] R. L. Kincaid, M. L. Billingsley, and M. Vaughan Vol. 164 (1988) [25] J. Ofengand, R. Denman, K. Nurse, A. Liebman, D. Malarek, A. Focella, and G. Zenchoff Vol. 168 (1989) [54] P. C. Emson, H. Arai, S. Agrawal, C. Christodoulou, and M. J. Gait Vol. 170 (1989) [4] T. M. Herman I n d e e d , t h e p r e d i c t i o n o f f u t u r e b r e a k t h r o u g h s in t h e u s e o f a v i d i n - b i o t i n t e c h n o l o g y is b e y o n d o u r i m a g i n a t i v e c a p a c i t y , a n d w e l e a v e d e v e l o p m e n t o f n e w d i r e c t i o n s t o t h e r e a d e r s o f this v o l u m e a n d f u t u r e u s e r s o f this universally applicable technology.
INTRODUCTION
TO AVIDIN--BIOTIN
TECHNOLOGY
5
Completely independently, Tom Broker and Norman Davidson at the California Institute of Technology had the same general ideas for using the avidin-biotin system, and they developed procedures for gene mapping by labeling RNA molecules, especially tRNA, and then using them as hybridization probes to locate genes in double-stranded DNA. 8 After hearing about this work at meetings, we did have some casual discussions with David Ward in Human Genetics at Yale, University. 9 It was clear that the biotin-avidin system as a general cytochemical labeling procedure had arrived. 10 I doubt that any of us at the time imagined the explosion in applications that would occur over the 1980s ~1 and that is documented in this volume. With the basic underpinnings for the technology now in hand, as well as cloned genes for the avidins ~2 and crystal structures of the proteins, 13 the future is bright indeed. 8 j. E. Manning, N. D. Hershey, T. R. Broker, M. Pellegrini, H. K. Mitchell, and N. Davidson, Chromosoma 53, 107 (1975); T. R. Broker, L. M. Angerer, P. Yen, N. D. Hershey, and N. Davidson, Nucleic Acids Res. 5, 363 (1978). 9 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). to E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980); M. Wilchek and E. A. Bayer, lmrnunol. Today 5, 39 (1984). 11 M. Wilchek and E. A. Bayer, Anal. Biochem. 171, 1 (1988). t2 M. L. Gope, R. A. Keinanen, P. A. Kristo, O. M. Conneely, W. G. Beattie, T. ZaruckiSchulz, B. M. O'Malley, and M. S. Kulomaa, Nucleic Acids. Res. 15, 3595 (1987); G. Chandra and J. G. Gray, this volume [7]; C. E. Argarana, I. D. Kuntz, S. Birken, R. Axel, and C. R. Cantor, Nucleic Acids Res. 14, 1871 (1986). ~3 E. Pinn, A. P/ihler, W. Saenger, G. Petsko, and N. M. Green, Eur. J Biochem. 123, 545 (1982); A. P~ihler, W. A. Hendrickson, M. A. G. Kolks, C. E. Argarana, and C. R. Cantor, J. Biol. Chem. 262, 13933 (1987).
[2] I n t r o d u c t i o n to A v i d i n - B i o t i n T e c h n o l o g y
By MEIR WILCHEK and EDWARD A. BAYER The avidin-biotin system has many uses in both research and technology. This general introductory chapter presents an overview of the principles and advantages of this system, and illustrates the numerous applications of avidin-biotin technology. It also explains in general and explicit terms the basic idea behind avidin-biotin technology. METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
6
INTRODUCTION
[2]
Principle The major distinguishing feature of the avidin-biotin system is the extraordinary affinity (Ka = 1015 M -i) that characterizes the complex formed between the vitamin biotin and the egg-white protein avidin (or streptavidin, its bacterial relative from Streptomyces avidinii). ~The interaction is so strong that even biotin coupled to proteins (such as native biotin-requiring carboxylases) is available for binding by avidin. The rationale in using the avidin-biotin system is based on the premise that if one chemically modifies any biologically active compound with biotin through its valeric acid side chain, the biological and physicochemical properties of the biotin-modified molecule will not be changed significantly. Likewise, the biotin moiety of the derivatized molecule would still be available for interaction with avidin. Another distinctive feature of this system is the multiple (four) binding sites of avidin for biotin. This provides us with the possibility of cross-linking between different biotincontaining molecules and adds another dimension to the use of this multifaceted system. If a reporter group of some sort is attached to the avidin molecule, the conjugate can be used for many different purposes (Fig. 1). The avidinconjugated probe can be added as a single chemically conjugated entity, or avidin (by virtue of its four biotin-binding sites) can be applied in native form together with a biotinylated probe. The latter alternative can be instituted either sequentially in a stepwise fashion or in a single step as preformed complexes. Figure 2 (which was first published in an earlier volume in this series 2) presents these two alternatives for labeling. Figure 1 also provides a list of some of the types of binders that can be used to label a given target site, as well as a list of probes that can be either conjugated with avidin (for direct interaction with the biotinylated binder) or derivatized with biotin (for complex formation with underivatized avidin). The various applications of avidin-biotin technology reported to date are also presented in Fig. 1.
Advantages Figure 1 demonstrates why the avidin-biotin system has become so popular: a simplified and unified approach can be taken. Some of the major advantages in using avidin-biotin technology can be summarized as follows: 1. The exceptionally high affinity and stability of the avidin-biotin complex ensures the desired conjunction of binder and probe. N. M. Green, Adv. Protein Chem. 29, 85 (1975). z E. A. Bayer, E. Skutelsky, and M. Wilchek, this series, Vol. 62, p. 308.
[2]
INTRODUCTION
TO AVIDIN--BIOTIN
OTINYLATED
TARGET MOLECULE
~
~
T A R G E T : BINDER Antigens Antibodies Lectins Glycoconjugates Enzymes Receptors Transport proteins Hydrophobic sites Membranes Nucleic acids, genes
7
TECHNOLOGY
PROBES
Antibodies AntigeDs Glycoconjugates Lectins Substrates, cofactors, mhibitors, etc ]tormones, eifeetors, :oxins, etc Vitamins, amino acid:s, sugars, etc Lipids, fatty acids Liposomes DNA/RNA probes
AVIDIN-BIOTIN COMPLEX
Phages, viruses, bacteria, ] subcellular organelles, cells,~AIl of the above tissues, whole organisms J
Enzymes Radiolabe[s Fluorescent agents Chemiluminescent agents Chromophores Heavy metals Colloidal gold Ferrltm Bemocyanul Phages Macromolecular carriers Liposomes Solid supports
APPLICATIONS 1 Affinity chromatography isolation studies 2 Affinity eytochemistry localization studies a light microscopy b fluorescence rnieroscopy c electron microscopy 3 Hlstoehemistry 4 Pathological probe 5 Diagnostics 6 Signal amplification 7 lmmunoassay 8 Hybridoma technology 9 Blotting technology
10 11 12 13 14 15 16 17 18 19 20 21
Bioaffinity sensor Gene probe Crosslinkmg agent Affinity targeting Affinity perturbation Drug delivery Fusogenic agent lmmobdizing agent Selective retrieval Selective elimination Flow cytometry Cytological probe
FIG. I. Generalized scheme illustrating the essentials of avidin-biotin technology. A biologically active target molecule is recognized by a biotinylated binder that is subsequently recognized by avidin conjugated to an appropriate probe. Lists of target-binder pairs, the various probes that have been used, and the broad spectrum of applications of avidin-biotin technology are also presented.
2. Biotin can readily be attached to m o s t binders and p r o b e s , and, f o l l o w i n g biotinylation, the biological activity and p h y s i c a l characteristics are c o m m o n l y retained. In fact, in m a n y c a s e s , a binder can be b i o t i n y l a t e d w i t h o u t e x t e n s i v e purification, since its interaction w i t h the target m o l e c u l e s e r v e s to r e m o v e "irrelevant" biotinylated s u b s t a n c e s .
8
[2]
INTRODUCTION
M~M~IRANE
APPROACH
MEMBRANE:
MEMBI~ANE
£
B a
-
BIOTIN
- LABELED
MEMBRANE SITE
B MEMB'RA,~IE: ;::
APPROACH
2
- AVIDIN
~,~ key
- FERRITIN-AVIDIN CONJUGATE 8
e
I
B
B F ER RITIN- BtC'TrN CONJUGATE
B
FlG. 2. Schematic of two alternative approaches for labeling biotin-modifiedcell membrane sites: (1) one-step method, using ferritin-conjugatedavidin, and (2) two-step method, employing sequential treatment with avidin and biotin-labeledferritin.
3. The multiplicity of biotin groups per binder combined with the tetrameric structure of avidin leads to amplification of the desired signal. 4. The system is amenable to double-labeling and kinetics studies. 5. The system is extremely versatile. A given molecular target can interact with a single type of biotinylated binder that can then be analyzed by various means using different avidin-conjugated probes. Conversely, the status of different target molecules in a given system can be compared using a variety of biotinylated binders and a single avidin-conjugated probe. Of course, the versatility is further extended through the combined use of different biotinylated binders and avidin-associated probes. 6. A wide spectrum of different biotinylating reagents, biotinylated binders, and both biotinylated and avidin-containing probes is available from a variety of commercial sources.
Application The development of avidin-biotin technology was not the outcome of a single event, nor was it the brainchild of a single individual or research group. Rather, it developed through an evolutionary process (the collec-
[2]
9
I N T R O D U C T I OTO N AVIDIN-BIOTIN TECHNOLOGY TABLE I MILESTONES IN DEVELOPMENTOF AV1DIN-BIOT1NTECHNOLOGY
Year 1916 1936 1942 1943 1952 1958 1963 1965 1968 1969 1970 1971
1972 1973 1974 1975
1976 1977 1978 1979 1980 1981
1982 1983 1986 1988 1990
Historical highlights "Toxic substances" in egg white Biotin isolated Structure of biotin determined Synthesis of biotin hydrazide Biotin synthesized Avidin purified from egg white Coenzyme function of biotin described Extensive protein chemical studies on avidin Streptavidin and stravidin isolated Retardation of avidin on biotinyl cellulose Affinity chromatography of avidin on biocytin-Sepharose Structure of stravidin determined Avidin crystallized Biotin-containing peptides isolated on avidin column Avidin sequenced N-Hydroxysuccinimide and p-nitrophenyl esters of biotin synthesized Biotinylated phages used for assay of avidin and biotin Avidin monomer column Localization of biotinylated sites on purified membranes; ferritin-avidin conjugates Biotinylated RNA Spacer-containing biotinylating reagents Comprehensive review on avidin Biotinylated antibodies and lectins (affinity cytochemistry) Biotinylated hormones TIBS review: "Emerging Techniques" ABC approach for enzyme immunoassay Biotinylated lipids First comprehensive review of applications Biotinylated DNA probes Premade complexes Iminobiotin columns and reagents Insulin receptor isolated using biotinylated insulin Protein and DNA blotting using biotinylated binders Cloning of streptavidin gene Nonglycosylated avidin Crystallographic structure of streptavidin Methods in Enzymology, "Avidin-Biotin Technology"
Ref, 3 4 5,6 7 8-10 11,12 13,14 15-18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 32 33-35 36 37 38 39 40 41 42,43 44-46 47-49 50 51 52,53 This volume
Live p r o d u c t o f c u r i o s i t y , n e c e s s i t y , o b s e r v a t i o n , i m a g i n a t i o n , an d c r e a t i v e t h o u g h t ) w h i c h b e g a n w i t h t h e e a r l y d i s c o v e r y o f t o x i c s u b s t a n c e s in e g g w h i t e a n d c u l m i n a t e d w i t h the c o m m e r c i a l a v a i l a b i l i t y o f literally hundreds of "avidin-biotin" products from dozens of companies. Some
10
INTRODUCTION
[2]
of the historical milestones in the development of avidin-biotin technology 3-53 are presented in Table I. 3 W. G. Bateman, J. Biol. Chem. 26, 263 (1916). 4 F. K6gl and B. TOnnis, Z. Physiol. Chem. 242, 43 (1936). V. du Vigneaud, K. Hofmann, and D. B. Melville, J. Am. Chem. Soc. 64, 188 (1942). 6 V. du Vigneaud, D. B. Melville. K. Folkers, D. E. Wolf, R. Mozingo, J. C. Kereszteolf, and S. A. Harris, J. Biol. Chem. 146, 475 (1942). 7 K. Hofmann, D. B. Melville, and V. du Vigneaud, J. Biol. Chem. 144, 513 (1942). s S. A. Harris, D. E. Wolf, R. Mozingo, and K. Folkers, Science 97, 447 (1943). 9 S. A. Harris, D. E. Wolf, R. Mozingo, R. C. Anderson, G. E. Arth, N. R. Easton, D. Heyl, A. N. Wilson, and K. Folkers, J. Am. Chem. Soc. 66, 1756 (1944). ~0S. A. Harris, N. R. Easton, D. Heyl, A. N. Wilson, and K. Folkers, J. Am. Chem. Soc. 66, 1757 (1944). 1~ H. FraenkeI-Conrat, N. S. Snell, and E. D. Ducay, Arch. Biochem. Biophys. 92, 80 (1952). lZ H. Fraenkel-Conrat, N. S. Snell, and E. D. Ducay, Arch. Biochem. Biophys. 92, 97 (1952). z3 S. J. Wakil, E. B. Titchener, and D. M. Gibson, Biochim. Biophys. Acta 29, 225 (1958). t4 F. Lynen, J. Knappe, E. Lorch, G. Jutting, and E. Ringelmann, Angew. Chem. 71, 481 (1959). t5 N. M. Green, Biochem. J. 89, 585 (1963). 16 N. M. Green, Biochem. J. 89, 599 (1963). 17 N. M. Green, Biochem. J. 89, 609 (1963). 18 M. D. Melamed and N. M. Green, Biochem. J. 89, 591 (1963). t9 L. Chaiet, T. W. Miller, F. Tausig, and F. J. Wolf, Antimicrob. Agents Chemother. 3, 28 (1963). 20 D. M. McCormick, Anal. Biochem. 13, 194 (1965). 21 p. Cuatrecasas and M. Wilchek, Biochem. Biophys. Res. Commun. 33, 235 (1968). z2 K. H. Baggaley, B. Blessington, C. P. Falshaw, and W. D. Ollis, Chem. Commun., 101 (1969). 23 N. M. Green and E. J. Toms, Biochem. J. 118, 67 (1970). 24 A. Bodanszky and M. Bodanszky, Experientia 26, 327 (1970). z5 R. J. DeLange and T.-S. Huang, J. Biol. Chem. 246, 698 (1971). 26 j. M. Becket, M. Wilchek, and E. Katcbalski, Proc. Natl. Acad. Sci. U.S.A. 68, 2604 (1971). 27 j. M. Becket and M. Wilchek, Biochim. Biophys. Acta 264, 165 (1972). zs N. M. Green and E. J. Toms, Biochem. J. 133, 687 (1973). 29 H. Heitzmann and F. M. Richards, Proc. Natl. Acad. Sci. U.S.A. 71, 3537 (1974). 30 j. E. Manning, N. D. Hershey, T. R. Broker, M. Pellegrini, H. K. Mitchell, and N. Davidson, Chromosoma 53, 107 (1975). 3~ E. A. Bayer, T. Viswanatha, and M. Wilchek, FEBS Lett. 60, 309 (1975). 32 E. A. Bayer, M. Wilchek, and E. Skutelsky, FEBS Lett. 68, 240 (1976). 33 K. Hofmann and Y. Kiso, Proc. Natl. Acad. Sci. U.S.A. 73, 3516 (1976). 34 K. Hofmann, F. M. Finn, H.-J. Friesen, C. Diaconescu, and H. Zahn, Proc. Natl. Acad. Sci. U.S.A. 74, 2697 (1977). 35 D. Atlas, D. Yaffe, and E. Skutelsky, FEBS Lett. 95, 173 (1978). 36 E. A. Bayer and M. Wilchek, Trends Biochem. Sci. 3, N257 (1978). 37 J.-L. Guesdon, T. Ternynck, and S. Avrameas, J. Histochem. Cytochem. 27, 1131 (1979). 38 E. A. Bayer, B. Rivnay, and E. Skutelsky, Biochem. Biophys. Acta 550, 464 (1979).
[2]
INTRODUCTION TO AVIDIN-BIOTIN TECHNOLOGY
11
We initially intended to include in this volume all the available applications which have thus far appeared in the primary literature, since each contribution has its own special message to convey. We soon discovered that this would be impossible, since the literature is simply too vast. Indeed, many of the methodologies involving avidin and/or biotin have already found their way into this series (Table II), although their contribution to the emerging technology has not generally been emphasized. A more extensive view of the widespread usage of avidin-biotin technology is presented elsewhere in this volume) 4 In assembling this volume, we therefore attempted to include only papers that either set a historical precedence or present a special method or message. Judging from the articles and tables ~4 that appear in this volume, the avidin-biotin system is extremely versatile, the applications are numerous, and the scope of its future use is virtually limitless. We cite the following from our first major review on this subject39: Naturally, we will be unable to cover all possible applications; it seems that the potential of the avidin-biotin complex as a tool in molecular biology is unlimited, and that its successful implementation is directly dependent on the needs and imagination of the user . . . . We cannot, of course, foresee all its possible uses, given our own limited imagination and/or restricted knowledge. We do hope, however, that this review will serve to stimulate the imagination of the readers and convert some of them to users of the method.
39 E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980). 4o p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). 4J S.-M. Hsu, L. Raine, and H. Fanger, J. Histochem. Cytochem. 29, 577 (1981). 4z K. Hofmann, S. W. Wood, C. C. Brinton, J. A. Montibeller, and F. M. Finn, Proc. Natl. Acad. Sci. U.S.A. 77, 4666 (1980). 43 G. A. Orr, J. Biol. Chem. 256, 761 (1981). 44 F. M. Finn, G. Titus, H. Nemoto, T. Noji, and K. Hofmann, Metabolism 31, 691 (1982). 05 F. M. Finn, G. Titus, D. Horstman, and K. Hofmann, Proc. Natl. Acad. Sci. U.S.A. 81, 7328 (1984). 46 R. A. Kohanski and M. D. Lane, J. Biol. Chem. 260, 5014 (1985). 47 B. B. Gordon and S. D. J. Pena, Biochem. J. 208, 351 (1982). 48 K. Ogata, M. Arakawa, T. Kasabara, K. Shioiri-Nakano, and K. Hiraoka, J. Immunol. Methods 65, 75 (1983). 49 j. j. Leary, D. J. Brigati, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 80, 4045 (1983). 50 C. E. Argarana, I. D. Kuntz, S. Birken, R. Axel, and C. R. Cantor, Nucleic Acids Res. 14, 1871 (1986). 5J y . Hiller, J. M. Gershoni, E. A. Bayer, and M. Wilchek, Biochem. J. 248, 167 (1987). 52 A. P~ihler, W. A. Hendrickson, M. A. Gawinowicz Kolks, C. E. Argarana, and C. R. Cantor, J. Biol. Chem. 262, 13933 (1987). s3 p. C. Weber, D. H. Ohlendorf, J. J. Wendoloski, and F. R. Salemme, Science 243, 85 (1989). 54 See M. Wilchek and E. A. Bayer, this volume [3].
TABLE II EARLIER COVERAGE OF AVIDIN--BIOTIN TECHNIQUES IN THIS SERIES
Subject Biotin Assays for biotin Biotin analogs Biotin derivatives
Biotinylated proteins Biotinylated hormones Biotinylated glycoconjugates Biotinyl lipids Biotinylated nucleic acids Immobilized biotin Iminobiotin and derivatives Avidin Assays for avidin Modified avidins Fluorescent avidin Radioactive avidin Avidin conjugates Immobilized avidin Antiavidin antibodies Preparation of biotin-binding proteins Avidin Streptavidin Egg-yolk protein Antibiotin antibodies Holocarboxylase synthetase Applications Affinity cytochemistry Affinity labeling Immunoassay Flow cytometry Affinity chromatography Blotting
Volume [Chapter]"
18A[62], 18A[63], 18A[74], 57137], 62149], 62[50], 621511, 122[12], 1221131 18A[66], 18A[67], 18A[68], 18A[70], 18A[71], 109137], 18A[64], 18A[65], 34120], 46[72], 57[37], 62[54], 62155], 62163], 70[13], 83112], 92137], 109137], 138135], 151139] 62155], 62157], 83112], 92[20], 92[37], 121143], 121168], 133125], 149[11], 150138], 159156] 98122], 109137], 12415] 62155], 83112], 138135] 138135], 149111] 164125], 168154], 17014] 34120], 7118], 122113], 17014] 109137], 122[14], 122[15], 13517] 18A174], 18A[75], 18A[76], 57[37], 62151], 62[52], 62[53], 122112], 122113] 92[20], 109137], 133125] 85148], 92137], 122112], 150138] 62[53], 70113] 62155], 83[12], 92[37], 93120], 149111] 11216], 121168], 122[13], 12415], 125140] 18A[76], 122115] 18A[73], 34120], 122114] 1091371 62[56] 62157] 107[151 62155], 83112], 85[48], 103111], 1291191, 151139], 168154] 62[63], 164125] 73[33], 92120], 103127], 121143], 133125] 103[13], 150138] 34120], 109137], 122[14], 122[15], 12415], 125140], 13517], 17014] 138135], 159156]
Vol. 18A (1970) [62] A. F. Carlucci; [63] D. B. McCormick and J. A. Roth; [64] H. Ruis, D. P. McCormick, and L. D. Wright; [65] J. E. Christner and M. J. Coon; [66] K. Ogata; [67] J. P. Tepper, H.-C. Li, D. B. McCormick, and L. D. Wright; [68] K. Ogata; 1701S. lwahara, D. B. McCormick, and L. D. Wright; [71] H. Ruis, R. N. Brady, D. B. McCormick, and L. D. Wright; [731 N. M. Green; [74] N. M. Green; [75] R.-D. Wei; [76] S. G. Korenman and B. W. O'Malley Vol. 34 (1974) [20] E. A. Bayer and M. Wilchek
" M e t h o d s in E n z y m o l o g y ,
[2]
INTRODUCTION TO AVID1N--BIOTIN TECHNOLOGY
13
References to TABLE I1 (continued) Vol. 46 (1977) [72] E. A. Bayer and M. Wilchek Vol. 57 (1978) [37} H. R. Schroeder, R. C. Boguslaski, R. J. Carrico, and R. T. Buckler Vol. 62 (1979) [491 R. L. Hood; [50] K. Dakshinamurti and L. Allan; [511 H. J. Lin and J. F. Kirsch; [52] H. A. Elo and P. J. Tuohimaa; [53] M. S. Kulomaa, H. A. EIo, and P. J. Tuohimaa; [541 B. K. Sinha and C. F. Chignell; [55] E. A. Bayer, E. Skulelsky, and M, Wilchek; [56] H. W. Meslar and H. B. White 111; [57] M. Berger; [63] E. A. Bayer and M. Wilchek Vot. 70 (1980) [13] J. J. Langone Vol. 71 (1981) [8] M. L. Ernst-Fonberg and J. S. Wolpert Vol. 73 (1981) [33] J.-L. Guesdon and S. Avrameas Vol. 83 (1982) [12] E. A. Bayer, E. Skutelsky, and M. Wilchek Vol. 85 (1982) [48] K. Wang, J. R. Feramisco, and J. F. Ash Vol. 92 (1983) [20] C. Stfihli, V. Miggiano, J. Stocker, Th. Staehelin, P. Hating, and B. Takacs; [37] L. Wofsy Vol. 93 (1983) [20] T. I. Ghose, A. H. Blair, and P. N. Kulkarni Vol. 98 (1983) [22] H. T. Haigler Vol. 103 (1983) [11] R. D. Broadwell and M. W. Brightman; [13] J. C. Cambier and J. G. Monroe; [27] G. E. Trivers, C. C. Harris, C. Rougeot, and F. Dray Vol. 107 (1984) [151 N. H. Goss and H. G. Wood Vol. 109 (1985) [37] F. M. Finn and K. H. Hofmann Vol. 112 (1985) [6] L. Ilium and P. D. E. Jones Vol. 121 (1986) [431J. E. Shively, C. Wagener, and B. R. Clark; [681T. V, Updyke and G. L. Nicolson Vol. 122 (1986) [12] R. D. Nargessi and D. S. Smith; [13] L. Goldstein, S. A. Yankofsky, and G. Cohen; [141 G. A. Orr, G. C. Heney, and R. Zeheb; 115[ R. Zeheb and G. A. Orr Vol. 124 (1986) [5] E. Hazum Vo[. 125 (1986) [401 P. Dimroth Vol. 129 (1986) [19] C.-T. Lin and L. Chan Vol. 133 (1986) [25] G. Barnard, E. A. Bayer, M. Wilchek, Y. Amir-Zaltsman, and F. Kohen Vol. 135 (1987) [7] M. T. W. Hearn Vol. 138 (1987) [35] M. Wilchek and E. A. Bayer Vol. 149 (1987) [11] B. Rivnay, E. A. Bayer, and M. Wilchek Vol. 150 (1987) 1381 D. M. Segal, J. A. Titus, and D. A. Stephany Vol, 151 (1987) [39] G. H. Smith Vol. 159 (1988) [56] R. L. Kincaid, M. L. Billingsley, and M. Vaughan Vol. 164 (1988) [25] J. Ofengand, R. Denman, K. Nurse, A. Liebman, D. Malarek, A. Focella, and G. Zenchoff Vol. 168 (1989) [54] P. C. Emson, H. Arai, S. Agrawal, C. Christodoulou, and M. J. Gait Vol. 170 (1989) [4] T. M. Herman I n d e e d , t h e p r e d i c t i o n o f f u t u r e b r e a k t h r o u g h s in t h e u s e o f a v i d i n - b i o t i n t e c h n o l o g y is b e y o n d o u r i m a g i n a t i v e c a p a c i t y , a n d w e l e a v e d e v e l o p m e n t o f n e w d i r e c t i o n s t o t h e r e a d e r s o f this v o l u m e a n d f u t u r e u s e r s o f this universally applicable technology.
