Bioconjugate Techniques

1140 Pages·2013·84.157 MB·English

by  Greg T. Hermanson (Auth.)

BIOCONJUGATE
TECHNIQUES
BIOCONJUGATE
TECHNIQUES
Greg T. Hermanson
Pierce Biotechnology, Thermo Fisher Scientific, Rockford, IL
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Third edition 2013
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ISBN: 978-0-12-382239-0
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Back cover review quote reprinted by permission from Macmillan Publishers Ltd: Nature Chemical Biology; Book Review;
Francis, M.B.; Updating the bioconjugation catalog: Vol. 4, No. 12, pp. 717, copyright 2008
Printed and bound in China
13 14 15 16 10 9 8 7 6 5 4 3 2 1
On the cover: Molecular model of R-phycoerythrin, a light-harvesting protein from red algae having bright fluorescent
properties. The crystal structure was published in Contreras-Martel, C., Martinez-Oyanedel, J., Bunster, M., Legrand,
P. Piras, C., Vernede, X., and Fontecilla-Camps, J.C. (2001) Crystallization and 2.2 A resolution structure of
R-phycoerythrin from Gracilaria chilensis: a case of perfect hemihedral twinning. Acta Crystallogr. Sect.D 57, 52–60. The
coordinates are available on the RCSB Protein Data Bank as structure 1eyx. Molecular graphics and analyses were
performed with the UCSF Chimera software package. Chimera is developed by the Resource for Biocomputing,
Visualization, and Informatics at the University of California, San Francisco (Sanner, M.F., Olson, A.J., Spehner, J.C. (1996)
Reduced surface: an efficient way to compute molecular surfaces. Biopolymers 38(3), 305–320).
Dedication
For Amy and Meghan, who, since the second edition was published, have now graduated
from college and are pursuing careers in biology and medicine. Also for baby Violet, who hopefully
someday will be able to read and appreciate a future edition of this book.
Preface to the Third Edition
In the years since the publication of the second edi- basic principles of bioconjugation along with presenting
tion, the field of bioconjugation has continued to important strategies for designing optimal conjugates for
advance at an incredible pace. Since 2008, over 54,000 a wide range of applications. Chapter 1 also reviews the
additional journal publications have appeared in the major application areas where bioconjugates are being
biological, medical, polymer, material science, and used today and describes the conjugate designs associ-
chemistry journals that at least mention the terms “bio- ated with each of these applications.
conjugate” or “bioconjugation.” In addition, many tens In addition, the new Chapter 15 contains one of the
of thousands of new links to Internet sites with biocon- most extensive overviews of immobilization chemistry
jugation information also have appeared in this time ever presented. It begins by reviewing the basic princi-
frame, including sources from academic, corporate, and ples of affinity chromatography along with the resins and
personal web pages. These journal articles and links other solid-phase options used to couple affinity ligands
describe many new reagents and reactions for forming of every type, including proteins, antibodies, enzymes,
bioconjugates of all types, including the formation of peptides, nucleic acids, other biomolecules, metal che-
unique complexes in solution as well as the coupling of lates, and organic mimetics. It also presents numerous
molecules to solid-phase surfaces or particles. In addi- options for activating supports and the coupling reactions
tion, exciting new methods are appearing for the appli- that can be used for covalently attaching ligands onto
cation of bioconjugates in highly sensitive assays and them. These methods can also be successfully applied
detection schemes, for in vivo imaging and diagnosis, to the immobilization of molecules onto any other par-
for therapeutic drug targeting, in the capture and puri- ticle or surface material desired. The resultant affinity
fication of biomolecules, for catalysis and chemical supports can be used for capturing target molecules, in
modification, and for vaccine development and immune the purification of proteins, for studying protein or oli-
modulation. These recent advances in bioconjugate tech- gonucleotide interactions, for proteolysis and enzyme
niques have resulted in two new chapters and many new catalysis, for removing contaminants from solution, or for
sections and updates throughout the book, as well as the developing biosensors and assays for a host of analytes.
rearrangement and consolidation of chapters to more Another major change that is immediately notice-
logically group topics together having common themes. able with this edition is the use of full-color illustrations.
The third edition also contains two major chap- Literally hundreds of new and updated figures now use
ters that were obvious gaps in the previous editions: color to better illustrate reactions or to show how bio-
Chapter 1 is an extensive introduction to the vast field of conjugates are being used in applications. While the
bioconjugation, while Chapter 15 describes the reagents design of the book may have been radically changed
and techniques used for the immobilization of ligands and updated with this edition, it is my hope that the
onto chromatography supports. The new comprehen- reader will continue to find it useful in the design of new
sive introduction to the book begins by describing the bioconjugates.
xiii
Acknowledgments
I thank the thousands of researchers, many of whose Funmilayo Suleman for literally reading the entire sec-
names appear in the reference section, who have devel- ond edition and providing helpful feedback on how to
oped and optimized hundreds of reagents and appli- make this edition even better. I also thank Peter Bell,
cations related to the modification, conjugation, and Julie Kremer, and Alan Doernberg for providing corpo-
immobilization of biomolecules and affinity ligands. rate approval for this entire endeavor. Although Thermo
Their work made this book possible. I also want to Fisher Scientific did not sponsor the project, the com-
thank Barb Tanaglia, Sally Etheridge, Crystal Gomez, pany provided great motivation for me to undertake the
Heather Flynn, and Brian Weathers for their expert help effort and complete the third edition.
in obtaining journal references. I also greatly appreci- Finally, special thanks to the one who made it all
ate Craig Smith for reviewing the new material and possible.
being supportive of my writing. In addition, I thank
xv
Important Information
HEALTH AND SAFETY INTELLECTUAL PROPERTY
This book describes hundreds of reagents, reactions, Throughout this book I have provided references
and applications for use in bioconjugation. Most of the related to the reagents, reactions, and techniques used in
compounds are highly specialized and we have very lit- bioconjugation. There are many additional references that
tle information regarding their toxicological properties. can be found by performing the appropriate key word
At a minimum, bioconjugation reagents should be con- searches on the Internet. However, such knowledge does
sidered irritants and handled with care. However, the not necessarily provide the liberty to legally use these
overwhelming majority are known to be reactive and reagents and applications for commercial purposes with-
any individual compound or solvent can be corrosive, out consideration for existing intellectual property rights.
hazardous, toxic, volatile, flammable, explosive, or oth- While in some cases pertinent patent references are pro-
erwise dangerous to personal health and safety. For this vided within the book, this is done only to supply addi-
reason, the use of any reagent or protocol described in tional technical details about the topic being discussed
this book should be carried out by taking the appropri- and not to imply anything about freedom to operate.
ate precautions. Before utilizing any of these methods, Today, nearly every important reagent or method
the user agrees to take complete responsibility and per- reported in the literature has a patent or patent applica-
sonal liability for any and all risks associated with the tion associated with it, especially if it has potential com-
reagents and reactions described in this book or within mercial value. A search of the patent databases, such as
the references cited. Before starting an experiment, the United States Patent and Trademark Office (http://
the user agrees to reference the appropriate Material www.uspto.gov/) or the European Patent Office (http://
Safety Data Sheets (MSDS) relative to every compound ep.espacenet.com/) for key words or the names of inven-
or component used in a reaction and to completely tors can provide a list of existing issued patents or pat-
understand the properties of the reactions being con- ent applications related to a bioconjugate technique or
templated. The use of personal protective equipment compound.
(PPE), fume hoods, and proper laboratory techniques It is the responsibility of the reader to become famil-
can ensure safety for both the user and other people iar with the patents and claims that may cover par-
in the immediate vicinity. In addition, the disposal of ticular compounds, compositions, reactions, or their
waste materials should be performed according to the methods of use in bioconjugate applications. If patents
appropriate environmental regulations to prevent toxic or patent applications exist, it is important that the
elements or compounds from entering the water, air, or appropriate permission or a license be obtained from
soil. The inappropriate disposal of excess reagents or the owner of such intellectual property before exploit-
reaction byproducts may be harmful to people and the ing it for commercial use.
environment.
xvii
C H A P T E R
1
Introduction to Bioconjugation
The field of bioconjugation has had a deceptively today. In fact, most activities in biological research
quiet, but at the same time enormous, impact on sci- could not be done easily, if at all, without the use of one
ence and technology. The use of bioconjugates may or more bioconjugate reagents to assay, detect, track,
not be the focus of many popular press science articles image, or capture target molecules, or effectively target
or noticed as important for news stories; nevertheless, and treat diseases.
the ability to produce discrete bioconjugate complexes Throughout this book, the techniques of produc-
having unique properties suitable for a wide variety of ing and using bioconjugates are presented with a view
applications has become the underlying success story toward providing options for designing similar conju-
of many research endeavors. Bioconjugation has made gates for new or existing applications. This chapter is
possible the discovery of new biomolecules, the eluci- meant to provide an introduction to aid in understand-
dation of complex biological processes, and the spawn- ing the breadth and depth of this field, while acting as
ing of entire industries within the medical, diagnostics, a guide to rationally choosing bioconjugate components
life sciences, microelectronics, and material sciences and reaction strategies for designing effective reagents.
fields. The use of bioconjugates in an almost unending
number of applications has also made possible a multi-
billion dollar worldwide economy that functions to 1. WHAT IS BIOCONJUGATION?
cure diseases and discover the very secrets of life. The
process of securing grant money, venture capital invest- In its most fundamental aspect, bioconjugation sim-
ment, or corporate R&D funding often includes the pro- ply involves the attachment of one molecule to another,
posed use of novel bioconjugates as key components usually through a covalent bond, to create a complex
in reaching technical goals. Indeed, many of the largest consisting of both molecules linked together (Figure 1.1).
pharmaceutical and biotech companies now depend In most cases, at least one of the molecules is of biologi-
upon bioconjugation to design their future product cal origin or is a fragment or derivative of a biomole-
pipelines and maintain a vital edge over the competi- cule. In some situations, the conjugate that is formed
tion. The “magic bullet” and “targeted drug” concepts is entirely synthetic, but its use is directed toward bio-
often desired in drug development are heavily contin- logical or life science applications. When forming such
gent on the creation of highly specific bioconjugates bioconjugates, the process can yield a composite having
with therapeutic efficacy toward certain cells, tissues, approximately equal proportions of each component
or disease states. Without the advance of bioconjugate or create a conjugate purposely designed to have more
techniques to enable this process, the world would not molecules of one component than the other. The final
have anywhere near the invention and innovation that form that the bioconjugate takes is dependent on the
have taken place over the last few decades in the life
science and medical fields.
A search for any particular bioconjugate type using +
Conjugation
PubMed or other major science and technology search
Agent
engines typically yields at least hundreds or, more A B A-B Conjugate
likely, thousands of hits. The actual percentage of all
publications that use bioconjugates to perform methods FIGURE 1.1 Forming a basic conjugate often involves the reac-
tion of two molecules and a crosslinking agent that covalently links
critical to scientific discovery may not be determinable
the components together. In some conjugation schemes an activation
precisely, but it is likely to be an overwhelming major-
agent is used that results in the linking of two molecules without an
ity of all life science research being done and published intervening cross-bridge between them.
Bioconjugate Techniques, Third Edition
1
DOI: http://dx.doi.org/10.1016/B978-0-12-382239-0.00001-7 © 22001133 Elsevier Inc. All rights reserved.
2 1. InTRoduCTIon To BIoConjugATIon
desired application and the components and methods can be used to bind specifically to a desired biomole-
used to couple them together. With an understanding of cule through the antigen binding sites on the antibody,
the basic concepts of bioconjugation, this process can be and then, due to the fluorescent properties of the label,
done without difficulty and even become controllable the targeted biomolecule can be detected—a feature
through the appropriate choice of reagents, reactions, which would not be possible using the unlabeled anti-
and conditions. body alone. Thus, the formation of a useful bioconju-
The process of making bioconjugates from individ- gate begins by envisioning the features that are desired
ual molecules thus creates new complexes having the in the final complex and then choosing the components
combined properties of each constituent from which it necessary to create it. Figure 1.2 illustrates some biocon-
is made. The result forms novel constructs having char- jugate types that can be made by linking two or more
acteristics not normally found among naturally occur- molecules together. These designs are by no means
ring substances. For instance, attaching a fluorescent exhaustive; the functionality that can be built into a
label to an antibody creates a targeting complex that bioconjugate by connecting two or more molecules
FIGURE 1.2 Some of the com-
E mon bioconjugate designs often
used for life science applications
include (A) streptavidin–enzyme
E
conjugate, (B) an immobilized affin-
S E
ity ligand on a particle, (C) an oligo
C
B molecular beacon probe contain-
A
ing two fluorescent labels or a fluor
and a quencher at each end, (D)
fluorescently labeled streptavidin,
S F (E) an affinity ligand attached to a
E surface, (F) a biotinylated enzyme,
D (G) an antibody–enzyme conjugate,
E
(H) a fluorescently labeled anti-
body, (I) a biotinylated antibody,
(J) a biotinylated oligo probe, (K)
an antibody–drug conjugate, (L) a
E
gadolinium chelate-modified den-
drimer containing folate molecules
G E for targeting.
H
E
I
J
G
G
G
G
K G
L G
G G
G G
BIOCONJUGATE TECHNIQUES
1. WHAT Is BIoConjugATIon? 3
together is limited only by the imagination and the reaction can also be accomplished using secondary
individual components available to assemble it. activating agents that create an intermediate reactive
The process of creating bioconjugates is typically car- group on one of the components to be conjugated. Most
ried out using reactive crosslinking agents that have of the reactive groups used for this process can then be
been specially designed for this purpose or through made to couple with specific functional groups on one
the use of an appropriate reactive group on one of the or more of the molecules to be conjugated, thus link-
molecules to facilitate the conjugation. The coupling ing them together into the final complex. Figure 1.3
N–
O
N+ O
O O S O
N O
NH N O N
HN O
O
H O O
HN N
S O
Sulfo-SMCC
Sulfo-SBED
O NH
HN O HN
O
H
O O O N
S N O O S
S
O O
O OH O NHS-PEG4-Biotin
O O
HN OH HO O O
N
O O O O
O S O
O HN O OH OH
S
O Immobilized
O Glutathione O
HO
NH FITC
2
N
O C
O S
O O O
N O O
O O O N N S NH Cl
H 2
O
NHS-PEG4-Maleimide O Traut's
Reagent
O O
O O
O O O O
Si O N S
O O Fe-BABE
O
O O N H
3-Glycidoxypropyl- O 3+
SATA Fe O
trimethoxy silane Br
N N H
H O
O O O
O
O
N O O O O
O O O O O CH
3
O
mPEG -NHS Ester
8
N
CH3 N O
N O O
CH
3
H N N
O O H2N OH NH O O
Azlactone-Activated O
TMT Isobaric Mass Tag
Support Diazirine-Amino
Acid (photo-Met)
FIGURE 1.3 A selection of common bioconjugate reagents used to modify, label, or crosslink biomolecules.
BIOCONJUGATE TECHNIQUES