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BIOCONJUGATE
TECHNIQUES
BIOCONJUGATE
TECHNIQUES

Greg T. Hermanson
Pierce Biotechnology, Thermo Fisher Scientific, Rockford, IL

AMSTERDAM • BOSTON • HEIDELBERG • LONDON

NEW YORK • OXFORD • PARIS • SAN DIEGO
SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
Academic Press is an imprint of Elsevier
Acquiring Editor: Janice Audet
Development Editor: Mary Preap
Project Managers: Karen East and Kirsty Halterman
Designer: Alan Studholme
Academic Press is an imprint of Elsevier
32 Jamestown Road, London NW1 7BY, UK
225 Wyman Street, Waltham, MA 02451, USA
525 B Street, Suite 1800, San Diego, CA 92101-4495, USA
Third edition 2013
Copyright © 2013, 1996, 2008 Elsevier Inc. All rights reserved.
No other part of this publication may be reproduced, stored in a retrieval system or transmitted in any
form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior
written permission of the publisher.
Permissions may be sought directly from Elsevier’s Science & Technology Rights. Department in Oxford,
UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively,
visit the Science and Technology Books website at www.elsevierdirect.com/rights for further information.
Notice
No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a
matter of products liability, negligence or otherwise, or from any use or operation of any methods,
products, instructions or ideas contained in the material herein.

Because of rapid advances in the medical sciences, in particular, independent verification of

diagnoses and drug dosages should be made.
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the British Library
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the Library of Congress
ISBN: 978-0-12-382239-0

For information on all Academic Press publications

visit our website at elsevierdirect.com

Typeset by MPS Limited, Chennai, India

www.adi-mps.com

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

DOI: http://dx.doi.org/10.1016/B978-0-12-382239-0.00001-7 1 © 2013 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
E conjugate, (B) an immobilized affin-
S
C ity ligand on a particle, (C) an oligo
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)
E an antibody–drug conjugate, (L) a
gadolinium chelate-modified den-
drimer containing folate molecules
E for targeting.
G
H
E
I

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 O
N+
O O S O
N O
NH N O N
HN O
O
H O O
HN N O
S
Sulfo-SMCC
Sulfo-SBED O
HN O HN NH
O H
O O O N
S N O O S
S
O O
O NHS-PEG4-Biotin
O OH

O O HN OH HO O O
O N O O O
O S O
O HN O OH OH
S Immobilized
O
O Glutathione O
HO
NH2 FITC
O N
C
O S
O O O
N O O
O O O N N S NH2 Cl
O H
NHS-PEG4-Maleimide Traut's
O
Reagent
O O
O O O
O O O
Si O N S Fe-BABE
O O
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 CH3
O mPEG8-NHS Ester

N
CH3 N O
N O O
CH3
H N N
O OH N O
O H
H2N 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
4 1. Introduction to Bioconjugation

illustrates some of the major types of reagents used for may also allow more copies of a certain molecule to be
bioconjugation techniques. These are just a few of the coupled and therefore become more actively present
tremendous number of reactive compounds that are in the final conjugate than would be possible without
available today for modification or conjugation pur- its use. Multivalent scaffolds in particular might pro-
poses. Many other specific examples of bioconjugation vide greater numbers of attachment points for cou-
reagents can be found throughout this book. pling detection molecules, thus potentially increasing
The reactions commonly utilized to form bioconju- the detection sensitivity of a bioconjugate in an assay.
gates can be selected from perhaps 1 to 2 dozen princi- Therefore, a given bioconjugate might contain compo-
pal reactive groups that are commonly used for creating nents directly linked together to form a complex or it
covalent bonds with various functional groups on bio- can be made with intermediary molecules which do
molecules. Of these dozens of choices for reactions, not have functional activity in the final reagent, but
probably less than 10 are used routinely to effect the nevertheless contribute to its properties as a whole.
conjugation of the overwhelming majority of biocon- In addition, bioconjugates can be designed to contain
jugates created. The reader will notice that throughout small molecule affinity ligands that specifically inter-
this book time and again the same basic reactions and act with other proteins, and these proteins in turn can
reagents are used to form diverse conjugates that are function as scaffolds for increasing conjugate function-
useful in a broad range of applications. ality through noncovalent interactions. For instance,
The key to forming a successful bioconjugate is to biotin can be attached to one molecule and used as an
select the proper crosslinking reagents that in turn con- affinity handle to interact with (strept)avidin reagents.
tain the appropriate reactive groups, which will couple The interaction between biotin and (strept)avidin is
with the chemical functionalities available on the mol- extremely strong (Chapter 11), and this permits highly
ecules to be linked together. In a sense, it is like choos- specific interactions to occur with the biotinylated con-
ing the right building blocks needed to construct the jugate. For instance, forming a bioconjugate by attach-
final structure of a molecular assembly. Just like a square ing a biotin label to an antibody permits detection of a
peg that will not fit into a round hole, improper choice targeted protein through use of a second bioconjugate,
of crosslinkers or reactive groups will not yield the such as a fluorescently labeled (strept)avidin com-
desired bioconjugate or may result in a suboptimal one. plex. A biotinylated bioconjugate can also be captured
Therefore, a basic understanding of the reactions of bio- and purified from a complex sample solution using an
conjugation is essential to understanding how to build a immobilized (strept)avidin particle or resin.
useful conjugate for a particular application. This is not The intermediary use of a biotin–(strept)avidin com-
to say one has to become an expert at synthetic organic plex in assays provides another type of bioconjugate
chemistry to make a successful bioconjugate; rather, one scaffold for increasing assay or detection sensitivity. A
only needs to gain enough knowledge to make the right biotinylated antibody is often used to target an analyte,
reagent choices. Matching the correct crosslinking agents while a secondary detection conjugate formed using
or reactive groups with the accessible functional groups (strept)avidin linked to another detection molecule pro-
on the components to be conjugated is the first step to vides the signaling agent. The docking of the (strept)
building a successful and active bioconjugate complex. avidin conjugate onto the biotinylated antibody carries
Thus, bioconjugates can be constructed by the judi- with it more detection molecules than could be coupled
cious coupling of two or more individual components to the antibody directly. The use of two conjugates in
to create a multifunctional complex having two or this assay design therefore provides increased sensitiv-
more properties combined into a single macromol- ity beyond that possible using a single antibody-detec-
ecule. The conjugate may contain one or more affin- tion conjugate alone.
ity molecules, which can be used to target, capture, The differences in function and use between a directly
or detect another biomolecule; it may also contain labeled antibody-detection conjugate versus a biotinylated
one or more detection molecules or enzymes, toxins, antibody plus a secondary labeled (strept)avidin conjugate
drugs, or other components having some other specific reflect the fact that the function of a given bioconjugate is
activity or purpose. In addition, some bioconjugates usually determined by the individual constituent prop-
are made using supplementary molecular scaffolds erties making up the whole. In this sense, a fluorescently
that may not be an active component of the biocon- labeled antibody can be used and detected directly, while
jugate, but are nevertheless important in constructing a biotinylated antibody has to be used with another bio-
the final complex. Just like girders in a building, a conjugate, such as a fluorescently labeled (strept)avidin
molecular scaffold can be used to construct the bio- molecule, to provide the same type of detection capability
conjugate by providing the core structure and attach- as the direct bioconjugate (Figure 1.5).
ment points for linking additional molecules into the The combined properties of a given bioconjugate—
overall assemblage of parts (Figure 1.4). The scaffold all its various components linked together—thus

BIOCONJUGATE TECHNIQUES
 1. What is Bioconjugation? 5

HO O
HO O HO O HO
OH
O
O O
H H HO O
N N
N N N Alpha-D-Glucose O
H H H OH
O O polymers in dextran O
O OH
HO O
O OH O OH HO
O OH
OH O
Poly-L-glutamic acid
O OH OH

NH2 NH2
O OH
OH
O O
H H
N N
N N N NH2
H H H H2N
O O

HN
H2N O NH
NH2 NH2 O NH2
NH
HN
Poly-L-lysine
O N
O N O
HN O
NH
N
N
HO HO HO
HN
HN O HN O HN O O NH
O N O
N O
O NH O NH O NH NH
H2N O G1 PAMAM HN
OH OH OH
HN dendrimer O NH2
NH

Poly(N-(2-hydroxypropyl)methacrylamide)
NH2
(HPMA) H2N

FIGURE 1.4 Polymeric molecular scaffolds often used in bioconjugate design to carry targeting molecules, detection components, or bio-
therapeutic agents.

govern the properties, applications, and methods for its

S use. Designing a useful bioconjugate entails envisioning
the preferred characteristics and methods of use for the
S final complex and then choosing the best components
S
and reaction strategies to produce the desired product
or products. On the surface, it may seem that the use of
a fluorescently labeled antibody produces a simpler and
thus a better conjugate than the use of a biotinylated
A B antibody combined with a fluorescently labeled (strept)
avidin complex, because a single conjugate is easier
FIGURE 1.5 Comparison of the use of a fluorescently labeled anti- to make and use. However, that straightforward con-
body versus a biotinylated antibody used with a fluorescently labeled clusion does not tell the whole story. The biotinylated
streptavidin conjugate. The multi-layered effect of the streptavidin–
biotin interaction provides additional detection molecules able to
antibody can have multiple biotins on its surface which
dock at the site of a target molecule, thus providing potentially higher permit the docking of multiple (strept)avidin molecules
sensitivity in assays. on each antibody. If each (strept)avidin molecule also

BIOCONJUGATE TECHNIQUES
6 1. Introduction to Bioconjugation

Incubate
+

Biotinylated Antibody Sample Antigen Captured

Incubate
S & Wash S

Add Immobilized
Isolated Antibody–Antigen Complex
Streptavidin

FIGURE 1.6 Immobilized streptavidin used to isolate a biotinylated antibody–target protein complex by immunoprecipitation (IP). A pri-
mary labeled antibody is allowed to incubate with a sample containing proteins and the antibody binds to its target. The biotin affinity tag facili-
tates isolation of this complex by binding to the immobilized streptavidin on an insoluble particle.

contains multiple fluorescent labels, then the resultant best is a result of careful design and is often meticulously
complex can possess many more fluorescent molecules optimized for its intended use (see Section 3, this chapter).
than a directly labeled antibody can have attached to Each component of the bioconjugate should be considered
it. Therefore, when used in an assay to detect a target as being an active part of the entire reagent. Every element
molecule, the biotinylated antibody combined with a has a role to perform that is essential to the functioning
fluorescently labeled (strept)avidin conjugate can result of the bioconjugate in its intended application. If any one
in much higher fluorescence signals than the fluores- part performs poorly then the activity or specificity of the
cently labeled antibody alone. Multi-layered conjugate entire conjugate is also likely to suffer. For this reason, it is
complexes often result in greater numbers of molecules essential to optimize each bioconjugate with its application
being assembled at the site of a targeted analyte, thus in mind. This section provides some general guidelines to
recruiting greater conjugate activity at that site as well. the design of bioconjugates along with some strategies for
In addition, some bioconjugates like a biotin-mod- choosing the right components and reactions to form the
ified antibody can have multiple uses. Not only can a final complex. Reference will also be made to the appro-
biotinylated antibody be used in an assay or detection priate chapters within the book for further information
process but it can also be used to bind to a target pro- regarding functional groups that can be used as well as
tein in a cell lysate and then isolate the target by use of the conjugation reagents, scaffolds, targeting agents, detec-
an immobilized (strept)avidin affinity support (Figure tion components, polymers, particles, antibodies, and any
1.6). Thus, the preparation of a single bioconjugate con- other molecules that may be incorporated into the final
sisting of a biotin-modified antibody can have multiple bioconjugation reagent. In addition, it is recommended
uses due to the combined properties of its constituent that the Table of Contents and the Index be consulted to
parts. It is clear that envisioning how a bioconjugate cross-reference the appropriate pages that best relate to
ultimately will be used is essential in choosing the opti- the type of bioconjugate being designed and the reactions
mal bioconjugate to make. The next section will explore used to produce it. This section then leads into an exten-
this aspect in greater detail. sive review of bioconjugation, especially highlighting how
it has been used to create reagents that have become essen-
tial to countless fields in science and technology.
2. BIOCONJUGATION STRATEGY
AND DESIGN 2.1. Start with the Application in Mind
The strategies used to develop bioconjugates for par- The ultimate use of a bioconjugate should be care-
ticular applications can be as highly varied as the reactions fully considered in order to properly design its com-
and components that are used to form such complexes. ponents to be appropriate to the envisioned task they
Bioconjugates can be used in a multitude of applications must perform. In addition, the conditions under which
and in each case the particular bioconjugate that performs the bioconjugates are to be used should be taken into

BIOCONJUGATE TECHNIQUES
 2. Bioconjugation Strategy and Design 7
account, because certain components might not per- conjugations using exactly the same fluorescent label
form well in the anticipated medium or environment. that you want to use again, the first variable in the pro-
For this reason, it is important that when beginning to cess is to choose an appropriate reactive dye. The choice
design any bioconjugate its ultimate application must may be governed by the wavelength of excitation and
first be considered before initiating any experimen- emission or it may be determined by the general wave-
tal procedures to actually make the conjugate. If the length region that is required for successful use in the
bioconjugate is to be used in an assay for example, the desired application. For in vivo imaging applications,
most appropriate components might consist of a target- for instance, the antibody should be labeled with a flu-
ing molecule coupled to a detection element, so that the orescent dye that has spectral properties in the far-red
final complex can bind and interact with its target and to near-infrared (NIR) region of the spectrum, whereas
then be detected. Alternatively, if the final bioconjugate for general cell staining applications the dye could have
is to be used in the purification of a target molecule, an emission properties anywhere from the UV to the NIR
appropriate conjugate might consist of an insoluble sup- region, which also covers all of the visible wavelengths.
port or particle to which an affinity ligand is attached. Another set of criteria for dye selection in making this
Therefore, each bioconjugate application may have radi- type of conjugate is limitations that may exist related
cally different conjugate construction requirements. to the instrument used for fluorescence detection. For
The design of a new bioconjugate for a unique appli- instance, if there is access to a fluorescent microscope
cation might best be explored by first considering the with filter sets already available for dyes in the fluo-
bioconjugates that others have used in similar applica- rescein (488), Cy3, and Cy5 wavelength ranges, then it
tions. Section 3 of this chapter should therefore be care- would be appropriate to make the antibody conjugate
fully reviewed as a guide to the bioconjugate types that using dyes that correlate with these wavelengths.
have been used successfully for published applications. In addition, fluorescent dyes may be chosen for anti-
In addition, the entire history of bioconjugation may be body conjugation based on their relative hydrophobicity
consulted through the literature to determine the scope or hydrophilicity. In certain applications, it is far bet-
of bioconjugate designs useful in different types of situ- ter to choose a dye that is more hydrophilic to prevent
ations. Table 1.1 lists some of the major applications antibody aggregation or to reduce the potential for non-
of bioconjugation along with the conjugate styles that specific binding of the conjugate in an assay. Most dyes
have been applied to work with them. This table is not have highly hydrophobic, aromatic core structures that
meant to be exhaustive in its presentation of every con- can interact nonspecifically with many hydrophobic
jugate that has ever been reported, but only to provide regions on biomolecules or interact with surfaces used in
major examples, which can be used as a starting point assay procedures. The use of hydrophilic dyes contain-
for the development of new bioconjugates. Since the ing negatively charged sulfonates or neutral PEG chain
component options in designing a bioconjugate are vast modifications can dramatically reduce nonspecific bind-
and the reactions that can be used in producing it are ing and enhance assay signal-to-noise ratios. Conversely,
many, the options available in creating a new conjugate the use of dyes with somewhat less water solubility may
can be almost daunting. For this reason, it is extremely be appropriate if the antibody conjugate is to be used
useful to gain knowledge of the bioconjugate types and for in vivo imaging or cell-based imaging where efficient
construction details that others have used successfully penetration of membrane structures is required to reach
in similar situations before embarking on a new design. a desired target. In this case, the use of dyes contain-
Even with this knowledge, however, it is likely that ing fewer sulfonates (e.g., perhaps no more than two to
for a new application, the bioconjugation construction three negative charges on a large cyanine-type dye) can
methods will still have to be optimized to work well in perform better in the intended application than dyes
the intended application. containing more hydrophilic groups or a greater num-
For instance, the design of a fluorescently labeled ber of charges. Thus, the performance of the conjugate in
antibody may appear to be quite trivial on the surface, its intended application often dictates the best choice of
especially with so many years of history in the literature fluorescent dye derivative to use in the conjugate design.
involving the fluorescent modification of antibodies. Testing of several potential choices to optimize the per-
To prepare this type of conjugate, it seems to be merely formance of a dye–antibody conjugate should be carried
a matter of coupling a reactive fluorescent molecule out in order to obtain the best possible result.
with the desired purified antibody. Using known reac- Another important consideration when making a con-
tion protocols, such as those described in Chapter 10, jugate is the relative ratio of each component in the final
one can obtain a labeled antibody that could be used to complex. In the dye-labeled antibody example, typi-
detect a target molecule. Upon closer inspection, how- cally it is important to have more than one dye modifi-
ever, the process for creating this conjugate is much cation on each antibody molecule. This has the effect of
more complex. Unless you have experience with similar increasing the fluorescence intensity of each antibody as

BIOCONJUGATE TECHNIQUES
TABLE 1.1 Bioconjugate Components and Designs Used for Major Applications

Application Bioconjugate Components Bioconjugate Reagents Bioconjugate Designs

Enzyme Immunoassays Antibodies; enzymes; scaffolds; biotin; Heterobifunctional aliphatic crosslinkers; Antibody–enzyme; biotinylated antibody plus
streptavidin; particles; microwells; planar heterobifunctional PEG-based crosslinkers; streptavidin–enzyme; biotinylated antibody,
surfaces; antigens; affinity ligands multifunctional scaffolds; zero length crosslinkers; biotinylated enzyme, plus streptavidin; antibody–
homobifunctional crosslinkers; thiolation reagents; particle or antibody–surface for capture; antibody–
biotinylation reagents; activated particles, polymer–enzyme; streptavidin–-polymer–enzyme
microwells, or surfaces plus biotinylated antibody; immobilized antigens or
affinity ligands
Fluorescent Fluorescent dyes; antibodies; scaffolds; biotin; Reactive fluorescent dyes; multifunctional Antibody–dye; biotinylated antibody plus
Immunoassays streptavidin; phycobiliproteins or tandem scaffolds; reactive phycobiliproteins or reactive streptavidin–dye; antibody–phycobiliprotein;
dyes; lanthanide chelates; quantum dots; tandem dyes; bifunctional lanthanide chelators; streptavidin–phycobiliprotein; antibody–lanthanide
particles; microwells; planar surfaces activated particles, microwells, or surfaces; chelate fluor; antibody–quantum dot; antibody–
heterobifunctional crosslinkers; PEG-based dye-doped-particle; antibody–tandem dye;
crosslinkers; zero length crosslinkers streptavidin–quantum dot; streptavidin–tandem dye;
quencher–fluor–oligonucleotide molecular beacon;
quencher–fluor–peptide molecular beacon; inhibitor–
dye; antibody–polymer dye; antibody–particle or
antibody–surface for capture
Chemiluminescent Acridinium esters; antibodies; streptavidin; Reactive acridinium ester compounds; Antibody–acridinium ester; biotinylated antibody
Immunoassays biotin; particles; microwells; planar surfaces biotinylation reagents; activated particles, plus streptavidin–acridinium ester; antibody–particle
microwells, or surfaces or antibody–surface for capture
Hybridization Assays, Fluorescent dyes; synthetic or genomic Reactive fluorescent dyes; modified or activated Oligonucleotide–dye; hapten–labeled oligo;
including FISH oligonucleotide probes; enzymes; biotin; oligo probes; activated particles, microwells, or biotinylated oligo; quencher-dye–labeled oligo for
streptavidin; particles; microwells; planar surfaces; biotinylation reagents; DIG-11-dUTP molecular beacon; oligo–particle or oligo–surface for
surfaces; digoxigenin (DIG); haptens capture; reactive oligo probe
ChIP Assays Oligonucleotide probes; particles; microwells; Functionalized oligo probes; activated particles, Oligonucleotide–biotin; oligo–particle or oligo–
planar surfaces; zero length crosslinkers; microwells, or surfaces; crosslinking agents surface for capture; Antibody–enzyme; antibody–dye;
antibodies streptavidin–enzyme; streptavidin–dye; biotinylated
antibody; oligo–particle or oligo–surface for capture
IP and Co-IP Assays Antibodies; interacting prey protein; particles; Activated particles, microwells, or surfaces; Antibody–particle or antibody–surface for capture;
microwells; planar surfaces crosslinking agents; biotinylation agents biotinylated antibody; streptavidin–particle or
streptavidin–surface for capture
Cellular Imaging and in Antibodies; enzymes; fluorescent dyes; biotin Heterobifunctional aliphatic crosslinkers; Antibody–enzyme; biotinylated antibody; antibody–
vivo Imaging modifications; streptavidin; nanoparticles; heterobifunctional PEG-based crosslinkers; dye; streptavidin–dye; fluorescent active site warhead
oligonucleotides; peptides; fluorescent multifunctional scaffolds; zero length crosslinkers; probes; molecular beacon fluor–peptide–quencher or
proteins; bioluminescent enzymes; split homobifunctional crosslinkers; thiolation reagents; fluor–oligo–quencher probes
enzymes or fluorescent proteins biotinylation reagents; activated particles; reactive
fluorescent dyes
Flow Cytometry Antibodies; fluorescent dyes; Heterobifunctional aliphatic crosslinkers; Antibody–dye; biotin–antibody; antibody–
phycobiliproteins; tandem dyes; streptavidin; heterobifunctional PEG-based crosslinkers; phycobiliprotein; streptavidin–dye; streptavidin
biotin; quantum dots; nanoparticles; magnetic multifunctional scaffolds; zero length crosslinkers; –phycobiliprotein; antibody–tandem dye;
particles homobifunctional crosslinkers; thiolation reagents; streptavidin–tandem dye; antibody–quantum dot;
biotinylation reagents; activated particles; reactive antibody–magnetic particle; antibody–nanoparticle
fluorescent dyes
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