Hermanson G. Bioconjugate Techniques, Second Edition

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960 25. Modiﬁ cation with Synthetic Polymers

2.4. Sulfhydryl-Reactive Derivatives

Sulfhydryl-reactive dextran derivatives may be prepared through the use of heterobifunctional

crosslinking agents (Chapter 5). In particular, crosslinkers containing pyridyl disulﬁ de, male-

imide, or iodoacetyl groups on one end are quite effective in directing a conjugation reaction

to thiols. Both maleimide and iodoacetyl activation procedures will yield nonreversible bonds

with sulfhydryl-containing molecules. Pyridyl disulﬁ de compounds, by contrast, react with thi-

ols to form cleavable disulﬁ de bonds that can be reversed by reduction.

Noguchi et al. (1992) used an amine-terminal spacer arm derivative of dextran to react

with SPDP (Chapter 5, Section 1.1) in the creation of a pyridyl disulﬁ de-activated polymer

(Figure 25.17 ). Brunswick et al. (1988) used a different amine-terminal spacer arm derivative

of dextran and subsequently coupled iodoacetate to form a sulfhydryl-reactive polymer ( Figure

25.18 ). Heindel et al. (1991) used a unique approach. They modiﬁ ed polyaldehyde dextran

with a heterobifunctional crosslinker containing a hydrazide group on one end and a maleim-

ide group on the other (Chapter 5, Section 2). The hydrazides reacted with the aldehyde groups

to form hydrazone linkages, leaving the maleimide ends free to result in a thiol-reactive dextran

derivative ( Figure 25.19 ).

Enzymes are widely used in bioconjugate chemistry as detection components in assay sys-

tems. The catalytic activity of an enzyme can be used to turn substrate molecules into chro-

mogenic, ﬂ uorescent, or chemiluminescent products, which are easily detectable or quantiﬁ able

by imaging, microscopy, or spectroscopy. If an enzyme is conjugated to a targeting molecule

speciﬁ c for some analyte of interest, then an assay system can be constructed to localize or

measure the analyte. The most common targeting molecule is an antibody having antigen-

binding speciﬁ city for the substance to be measured. An enzyme conjugated to such an anti-

body can be used to visualize the presence of antigen. Due to the advantages of this simple

concept, enzyme linked immunoadsorbent assays (ELISAs) have become the most important

type of immunoassay system available.

The rapid turnover rate of some enzymes allows ELISAs to be designed that surpass the

sensitivity of radiolabeling techniques. In addition, substrates can be chosen to produce soluble

products that can be accurately quantiﬁ ed by their absorbance or ﬂ uorescence. Alternatively,

substrates are available which form insoluble, highly colored precipitates, excellent for local-

izing antigens in blots, cells, or tissue sections. The ﬂ exibility of enzyme-based assay systems

makes the chemistry of enzyme conjugation one of the most important application areas in

bioconjugate techniques.

The following sections brieﬂ y describe the principal enzymes used for conjugation with

other protein molecules, particularly in the design of ELISA and other immunoassay systems.

1. Properties of Common Enzymes

1.1. Horseradish Peroxidase

HRP (donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7), derived from horseradish roots,

is a enzyme of molecular weight 40,000 that can catalyze the reaction of hydrogen peroxide

with certain organic, electron-donating substrates to yield highly colored products. The reac-

tion of HRP with its fundamental substrate, H

, forms a stable intermediate that can disso-

ciate in the presence of a suitable electron donor, oxidizing the donor and potentially creating

a color change. The donor can consist of oxidizable molecules like ascorbate, cytochrome C,

Enzyme Modification and Conjugation

961

962 26. Enzyme Modiﬁ cation and Conjugation

ferrocyanide, or the leuco forms of many dyes. A large variety of electron-donating dye sub-

strates is commercially available for use as HRP detection reagents. Some of them can be used

to form soluble colored products for use in spectrophotometric detection systems, while other

substrates form insoluble products that are especially apropos for staining techniques. In addi-

tion, substrates are available that create ﬂ uorescent or chemiluminescent products upon oxida-

tion with HRP. The chemiluminescent substrates are among the most sensitive of all detection

reagents, facilitating the detection of as little as attogram quantities of many targeted analytes.

The pH optimum for HRP is 7.0.

HRP is a hemoprotein containing photohemin IX as its prosthetic group. The presence

of the heme structure gives the enzyme its characteristic color and maximal absorptivity at

403 nm.The ratio of its absorbance in solution at 403 nm to its absorbance at 275 nm, called

the RZ or Reinheitzahl ratio, can be used to approximate the purity of the enzyme. However,

at least seven isoenzymes exist for HRP (Shannon et al., 1966; Kay et al., 1967; Strickland

et al., 1968), and their RZ values vary from 2.50 to 4.19. Thus, unless the RZ ratio is precisely

known or determined for the particular isoenzyme of HRP utilized in the preparation of an

antibody–enzyme conjugate, subsequent measurement after crosslinking would yield question-

able results in the determination of the amount of HRP present in the conjugate.

HRP is a glycoprotein that contains signiﬁ cant amounts of carbohydrate. Its polysaccharide

chains are often used in crosslinking reactions. Mild oxidation of its associated sugar residues

with sodium periodate generates reactive aldehyde groups that can be used for conjugation to

amine-containing molecules. Reductive amination of oxidized HRP to antibody molecules in

the presence of sodium cyanoborohydride is perhaps the simplest method of preparing highly

active conjugates with this enzyme (Chapter 3, Section 1.4 and Chapter 20, Section 1.3).

Other methods of HRP conjugation include the use of the homobifunctional reagent glu-

taraldehyde (Chapter 4, Section 6.2) and the heterobifunctional crosslinker, SMCC (succin-

imidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (Chapter 5, Section 1.3). Using

glutaraldehyde, a two-step protocol usually is employed to limit the extent of oligomer for-

mation. Nevertheless, this method often causes unacceptable amounts of precipitated conju-

gate. Despite this disadvantage, glutaraldehyde conjugation is still routinely used, especially in

the preparation of some antibody–enzyme reagents that go into established diagnostic assays.

The use of the N-hydroxysuccinimide (NHS) ester–maleimide crosslinker, SMCC, provides bet-

ter control over the conjugation process. SMCC usually is reacted ﬁ rst with HRP to create a

derivative containing sulfhydryl-reactive maleimide groups. The maleimide-activated enzyme

can be puriﬁ ed and freeze-dried, providing a ready source of modiﬁ ed HRP to react with a

sulfhydryl-containing antibody. Several preactivated forms of this enzyme are available from

Thermo Fisher.

The size of HRP is an advantage in preparing antibody–enzyme conjugates, since the overall

complex size also can be designed to be small. Relatively low-molecular-weight conjugates are

able to penetrate cellular structures better than large, polymeric complexes. This is why HRP

conjugates are often the best choice for immunocytochemical staining techniques. Small conju-

gate size means greater accessibility to antigenic structures within tissue sections.

Another distinctive advantage of HRP is its robust nature and stability, especially under the

conditions employed for crosslinking. HRP is stable for years in a freeze-dried state, and the

puriﬁ ed enzyme can be stored in solution at 4°C for many months without signiﬁ cant loss of

activity. The enzyme also retains excellent activity after being modiﬁ ed with a conjugation rea-

gent or periodate-oxidized to form aldehyde groups on its polysaccharide chains. Depending

on the methods used for crosslinking, HRP conjugates can be constructed to have a high ratio

of enzyme-to-antibody or a low ratio—both retaining high speciﬁ c activity.

The disadvantages associated with HRP are several. The enzyme only contains two avail-

able primary -amine groups—extraordinarily low for most proteins—thus limiting its ability

to be activated with amine-reactive heterobifunctionals. HRP is sensitive to the presence of

many antibacterial agents, especially azide. It also is reversibly inhibited by cyanide and sulﬁ de

(Theorell, 1951). Finally, while the enzymatic activity of HRP is extremely high, its useful life-

span or practical substrate development time is somewhat limited. After about an hour of sub-

strate turnover, in some situations its activity can be decreased severely.

Nevertheless, HRP is by far the most popular enzyme used in antibody–enzyme conjugates.

One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody

conjugates, most of them utilized in diagnostic assay systems.

1.2. Alkaline Phosphatase

Alkaline phosphatases [AP, orthophosphoric-monoester phosphorylase (alkaline optimum); EC

3.1.3.1] represent a large family of almost ubiquitous isoenzymes found in organisms from

bacteria to animals. In mammals, there are two forms of AP, one form present in a variety of

tissues and another form found only in the intestines. They share common attributes in that

the phosphatase activity is optimal at pH 8–10, is activated by the presence of divalent cations,

and is inhibited by cysteine, cyanides, arsenate, various metal chelators, and phosphate ions.

Most conjugates created with AP utilize the form isolated from calf intestine.

AP isoenzymes can cleave associated phosphomonoester groups from a wide variety of sub-

strates. The exact biological function of these enzymes is not well understood. They can behave

in vivo in their classic phosphohydrolase role at alkaline pH, but at neutral pH AP isoenzymes

can act as phosphotransferases. In this sense, suitable phosphate acceptor molecules can be

utilized in solution to increase the reaction rates of AP on selected substrates. Typical phos-

phate acceptor additives include diethanolamine, Tris, and 2-amino-2-methyl-1propanol. The

presence of these additives in substrate buffers can dramatically increase the sensitivity of AP

ELISA determinations, even when the substrate reaction is done in alkaline conditions.

Calf intestinal AP has a molecular weight of about 140,000. The active site of AP contains

two zinc atoms and a single magnesium atom, both of which are essential for activity (Kim and

Wyckoff, 1991). Substrate development with AP thus should be done in buffered environments

containing small concentrations of these divalent cations to maintain optimal active site conforma-

tion. Avoid the presence of metal chelators such as EDTA, since they may extract these ions from

the enzyme and inhibit activity. The pH optimum for APs can vary from pH 8–0, depending on the

type of isoenzyme. Calf intestinal AP peaks in activity at the higher pH values of this range, and

substrate reactions are commonly performed in diethanolamine buffer at pH 9.8. The calf intesti-

nal enzyme has the highest catalytic rate constant yet discovered for AP isoenzymes, 3,500 s

 1

Puriﬁ ed preparations of calf intestinal AP maintained in solution are usually stored in the

presence of a stabilizer, which is typically 3 M NaCl. The enzyme also may be lyophilized, but

it may experience activity loss with each freeze-thaw cycle. AP is not stable under acidic con-

ditions. Lowering the pH of an AP solution to 4.5 reversibly inhibits the enzyme. It is rec-

ommended that all handling, storage, and use of AP be done under conditions pH 7.0 to

maintain the highest possible catalytic activity.

1. Properties of Common Enzymes 963

964 26. Enzyme Modiﬁ cation and Conjugation

AP often is a difﬁ cult enzyme to work with when preparing enzyme conjugates. Activity

losses may occur upon modiﬁ cation with a crosslinking agent or after coupling to an antibody

molecule. Simply following established protocols for making antibody–AP conjugates doesn ’t

always assure retention of enzyme activity. Sometimes activity losses can be traced to particular

batches or to certain suppliers of the enzyme. Using a highly puriﬁ ed, high activity AP prepara-

tion helps to maintain good resultant activity in the conjugate.

Ironically, AP is the enzyme of choice for some applications due to its stability. Since it can

withstand the moderately high temperatures associated with hybridization assays better than

HRP, AP often is the enzyme of choice for labeling oligonucleotide probes. AP also is cap able

of maintaining enzymatic activity for extended periods of substrate development. Increased

sensitivity can be realized in ELISA procedures by extending the substrate incubation time to

hours and sometimes even days. These properties make AP the second most popular choice for

antibody–enzyme conjugates (behind HRP), being used in almost 20 percent of all commercial

enzyme-linked assays.

Conjugation methods typically employed with AP include glutaraldehyde-mediated

crosslinking (Chapter 4, Section 6.2) and the use of the heterobifunctional reagents, SMCC

(Chapter 5, Section 1.3) or SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate) (Chapter 5,

Section 1.1). Heterobifunctional crosslinkers provide the best control over the crosslinking

process and typically result in antibody–enzyme conjugates of high activity. Many conjugation

protocols incorporate a sodium phosphate buffer system to block reversibly the AP active site

during chemical modiﬁ cation. This prevents derivatization from occurring in the catalytic site,

thus better retaining activity in the resultant conjugate.

1.3.  -Galactosidase

-Galactosidase (  -Gal;  - D-galactoside galactohydrolase; EC 3.2.1.23; also called lactase) cat-

alyzes the hydrolysis of  - D-galactoside in the presence of water to galactose and alcohol. This

type of enzyme is found widespread in many microorganisms, plants, and animals.  -Gal can

be used to determine lactose in biological ﬂ uids and it is employed in food processing opera-

tions, particularly in immobilized form.  -Gal also has good characteristics when conjugated to

antibody molecules for use in ELISA systems (Wallenfels and Weil, 1972; Byrne and Johnson,

1975; Kato et al ., 1975a, b).

 -Gal has a molecular weight of 540,000 and is composed of four identical subunits of MW

135,000, each with an independent active site (Melchers and Messer, 1973). The enzyme has

divalent metals as cofactors, with chelated Mg

2

ions required to maintain active site confor-

mation. The presence of NaCl or dilute solutions (5 percent) of low-molecular-weight alcohols

(methanol, ethanol, etc.) causes enhanced substrate turnover.  -Gal contains numerous sulfhy-

dryl groups and is glycosylated.

Commercially available  -gal usually is isolated from Escherichia coli and has a pH opti-

mum at 7-7.5. By contrast, mammalian  -galactosidases usually have a pH optimum within the

range of 5.5–6; thus, interference from endogenous  -gal during immunohistochemical staining

can be avoided.

Due to the relatively high-molecular-weight of the enzyme, conjugates formed with antibodies

and  -gal tend to be much bulkier than those associated with AP or horseradish peroxidase. For

this reason, antibody conjugates made with  -gal may have more difﬁ culty penetrating tissue struc-

tures during immunohistochemical staining techniques than those made with the other enzymes.

Although numerous research articles have been written describing the preparation and use

of antibody conjugates with  -gal, the enzyme remains a minor player in ELISA procedures.

Less than 1 percent of all commercial ELISA products utilize this enzyme.

 -Gal may be conjugated to antibody molecules using the heterobifunctional reagent

SMCC. This crosslinker is reacted ﬁ rst with an antibody through its amine-reactive NHS ester

end to form a maleimide-activated derivative. This is in contrast with most antibody–enzyme

conjugation schemes utilizing SMCC, wherein the enzyme is modiﬁ ed ﬁ rst and a sulfhydryl-

containing antibody is coupled secondarily. However, since  -gal already contains abundant

free sulfhydryl residues that can participate in coupling to a maleimide-activated protein, conju-

gations with this enzyme often are done with the antibody being the ﬁ rst component modiﬁ ed.

This route avoids having to create sulfhydryls on the antibody molecule, either by reduction or

modiﬁ cation with a thiolating reagent. Thus, antibody–  -gal conjugates usually are simpler to

make than using other enzymes.

1.4. Glucose Oxidase

Glucose oxidase (  -D-glucose: oxygen 1-oxidoreductase; EC 1.1.3.4; GO) is a ﬂ avoenzyme that

catalyzes the oxidation of  -D-glucose to -D-gluconolactone. The intermediate product of the

catalysis is a reduced enzyme–FADH

complex that in the presence of oxygen gets oxidized back

to enzyme–FAD with release of hydrogen peroxide. The enzyme consists of two identical subu-

nits (MW 80,000 each) bound together by disulﬁ de linkages (O ’Malley and Weaver, 1972). GO

contains two tightly bound ﬂ avin adenine dinucleotide (FAD) cofactors, one per subunit, which

are critical to its oxidoreductase activity. Each subunit also contains one molecule of chelated

iron. The intact protein consists of about 74 percent amino acids, 16 percent neutral carbo-

hydrate, and 2 percent amino-sugars (total molecular weight 160,000). GO operates under a

relatively broad pH range of 4–7, but its pH optimum is 5.5. The commercially available prepa-

ration of GO is typically isolated from Aspergillus niger.

Glucose oxidase is widely used in diagnostic assays for the determination of glucose con-

centration in physiological ﬂ uids. Detectability of the oxidation products is done through an

enzyme-coupled reaction wherein liberated H

is reacted with peroxidase and a suitable

chromogenic substrate. The development of substrate color thus is proportional to the amount

of H

released which is in turn related to the amount of glucose originally present. The

production of hydrogen peroxide also can be quantiﬁ ed using a luminescence procedure with

luminol to produce light in proportion to the glucose concentration (Williams et al ., 1976).

GO often is used in solution phase reactions as well as being immobilized on “ dip-sticks ”

and electrodes. Although its overall clinical usage is widespread, its use as conjugated to anti-

bodies in enzyme-linked assay systems is minor compared to the popularity of other enzymes

like horseradish peroxidase and AP. Of the total number of commercial diagnostic assays uti-

lizing antibody–enzyme conjugates, GO is employed in less than 1 percent of clinical tests. The

enzyme remains, however, an important tool in many assays developed for research use. One

particular advantage to the enzyme is that there is no endogenous GO activity in mammalian

tissues, making it an excellent choice for immunohistochemical staining procedures.

Antibody conjugates with GO can be made using the crosslinking agents glutaraldehyde

(Chapter 4, Section 6.2) or SMCC (Chapter 5, Section 1.3). The heterobifunctional reagent

SMCC provides the best control over the conjugation process and usually results in high-

activity preparations.

1. Properties of Common Enzymes 965

966 26. Enzyme Modiﬁ cation and Conjugation

2. Preparation of Activated Enzymes for Conjugation

Enzymes may be modiﬁ ed to contain reactive groups useful for conjugation with other pro-

teins. This operation may be done using homobifunctional (Chapter 4) or heterobifunctional

(Chapters 5, 17, and 18) reagents that can covalently couple to some chemical target on the

enzyme and result in a terminal reactive group that can crosslink with another molecule.

Enzyme activation also may take advantage of the presence of polysaccharide constituents—

oxidizing them with sodium periodate to form reactive aldehydes.

Whatever the method of conjugate creation, the most important considerations are retention

of activity in the complex and prevention of extensive oligomer generation, which may cause

precipitation. The following methods discuss some of the more common methods for produc-

ing enzyme conjugates. The list, however, is by no means inclusive of every possible procedure

used in the literature.

2.1. Glutaraldehyde-Activated Enzymes

Glutaraldehyde is a homobifunctional crosslinker containing an aldehyde residue at both ends

of a 5-carbon chain. Its primary reactivity is toward amine groups, but the reaction may occur

by more than one mechanism. As discussed in Chapter 4, Section 6.2, glutaraldehyde is able to

form Schiff base linkages with amines that can be reduced with sodium cyanoborohydride to

create stable secondary amine linkages. However, glutaraldehyde also exists as ,  -unsaturated

polymers in aqueous solution. The double bonds of these polymers can undergo an addition

reaction with amines that results in covalent bond formation even without a reductant being

present. Fresh glutaraldehyde may contain little polymer formation. However, the older the

preparation of glutaraldehyde, the more likely it is that it contains appreciable amounts of poly-

mer. Thus, reactions with this crosslinking agent can result in indistinct conjugation products.

The high reactivity of glutaraldehyde also makes it difﬁ cult to control the conjugation pro-

cess. Proteins crosslinked with this reagent often form substantial amounts of precipitated

products due to polymerization. The degree of oligomer formation can be moderated some-

what by using a two-step protocol, but the ﬁ rst protein activated with the molecule still can

form large molecular weight complexes.

Despite the obvious disadvantages of glutaraldehyde-mediated conjugation, the crosslinker

continues to be used to form enzyme–antibody complexes and in other applications. Many

diagnostic tests still utilize antibody–enzyme conjugates prepared through glutaraldehyde

crosslinking procedures.

The one- and two-step procedures for enzyme activation and conjugation using glutaralde-

hyde can be found in Chapter 20, Section 1.2.

2.2. Periodate Oxidation Techniques

Molecules containing polysaccharide chains may be oxidized to possess reactive aldehyde res-

idues by treatment with sodium periodate. Any adjacent carbon atoms containing hydroxyl

groups will be affected, cleaving the carbon–carbon bond and transforming the hydroxyls into

aldehydes. Glycoproteins may be oxidized in this manner to form reactive intermediates useful

for crosslinking procedures involving reductive amination (Chapter 3, Section 4). This conjuga-

tion technique can direct the coupling process away from polypeptide active regions, thus help-

ing to preserve catalytic activity or avoid binding sites.

Enzymes that contain carbohydrate, such as HRP or GO, may be oxidized with periodate to

create reactive derivatives that subsequently can be used to label antibodies or other targeting

molecules at their amine groups. The aldehyde-HRP intermediate may be stored for extended

periods in a frozen or lyophilized state without loss of activity (either enzymatic or coupling

potential). Avoid, however, storage in a liquid state, since polymerization may occur—resulting

in precipitation and loss of activity.

The protocols for periodate oxidation of HRP and its conjugation with other proteins may

be found in Chapter 20, Section 1.3.

2.3. SMCC-Activated Enzymes

The heterobifunctional crosslinker SMCC, or its water-soluble analog sulfo-SMCC, can be

used to activate enzymes through their amines, leaving terminal maleimide groups on the pro-

tein surface (Chapter 5, Section 1.3). The NHS ester end of the crosslinker reacts with  -lysine

or N-terminal amines to form amide bonds. The maleimide end of the reagent is stable enough

in aqueous solution to allow puriﬁ cation of the activated enzyme prior to conjugation with a

second protein. The maleimide group can react with sulfhydryl groups to create thioether link-

ages. A maleimide-activated enzyme may be stored in a lyophilized state for extended periods

without loss of sulfhydryl-coupling capability.

The use of this type of heterobifunctional reagent allows controlled conjugations to take

place, precisely regulating the exact ratio of each protein in the ﬁ nal complex and the size of

the resultant conjugate. In addition, the second-stage conjugation through sulfhydryl groups

provides directed coupling at discrete sites within a protein molecule, thus providing the poten-

tial to better avoid active centers or binding regions. For instance, antibody molecules can be

coupled to enzymes in their hinge region after mild disulﬁ de reduction to effect the crosslink in

an area away from the antigen binding site.

Protocols for the activation of enzyme molecules with SMCC (or sulfo-SMCC) can be found

in Chapter 20, Section 1.1. Conjugates formed using this method usually result in high-activity

complexes giving excellent sensitivity for use in immunoassays or other applications.

2.4. Hydrazide-Activated Enzymes

Hydrazide groups can react with carbonyl groups to form stable hydrazone linkages. Derivatives

of proteins formed from the reaction of their carboxylate side chains with adipic acid dihydrazide

(Chapter 4, Section 8.1) and the water-soluble carbodiimide EDC (Chapter 3, Section 1.1) create

activated proteins that can covalently bind to formyl residues. Hydrazide-modiﬁ ed enzymes pre-

pared in this manner can bind speciﬁ cally to aldehyde groups formed by mild periodate oxidation

of carbohydrates (Chapter 1, Section 4.4). These reagents can be used in assay systems to detect

or measure glycoproteins in cells, tissue sections, or blots (Gershoni et al., 1985).

2. Preparation of Activated Enzymes for Conjugation 967

968 26. Enzyme Modiﬁ cation and Conjugation

Other molecules can be used in this type of assay approach. Hydrazide-modiﬁ ed

(strept)avidin, lectins, biocytin, ﬂ uorescent probes and other detectable molecules can be used

to detect speciﬁ cally glycoconjugates in biological samples (Wilchek and Bayer, 1987).

The activation of enzymes using adipic acid dihydrazide and EDC is identical to the pro-

cedure outlined for the modiﬁ cation of (strept)avidin (Chapter 23, Section 5). Alternatively,

hydrazide groups may be created on enzymes using the heterobifunctional chemoselective rea-

gents described in Chapter 17, Section 2.

2.5. SPDP-Activated Enzymes

SPDP is a heterobifunctional crosslinker containing an NHS ester on one end and a pyridyl disulﬁ de

group on the other end (Chapter 5, Section 1.1). The NHS ester end can be used to modify amine

groups on enzymes, forming amide bonds. The result of this procedure is to create sulfhydryl-

reactive pyridyl disulﬁ de groups on the surface of each enzyme molecule that are able to complex

with thiol-containing proteins and other molecules. SPDP-activated enzymes may be puriﬁ ed and

stored for extended periods without breakdown of the coupling capacity. The reaction with a sulf-

hydryl group forms a reversible disulﬁ de linkage that can be cleaved with reducing agents.

The two-step nature of SPDP crosslinking provides control over the conjugation process.

Complexes of deﬁ ned composition can be constructed by adjusting the ratio of enzyme to sec-

ondary molecule in the reaction as well as the amount of SPDP used in the initial activation.

The use of SPDP in conjugation applications is extensively cited in the literature, perhaps mak-

ing it one of the more popular crosslinkers available. It is commonly used to form immunoto-

xins, antibody–enzyme conjugates, and enzyme-labeled DNA probes. A standard activation

and coupling procedure can be found in Chapter 5, Section 1.1.

3. Preparation of Biotinylated Enzymes

Biotinylated enzymes can be used as detection reagents in (strept)avidin–biotin assay proce-

dures. Particularly, in the bridged avidin–biotin (BRAB) approach or the ABC technique

(Chapter 23, Section 2), a biotin-labeled enzyme is used as the signaling agent after the binding

to an antigen of a biotinylated antibody and an (strept)avidin bridging molecule. The biotins

on the surface of the enzyme can bind with extraordinary afﬁ nity to (strept)avidin–antibody

complexes, providing near-covalent interaction potential with high speciﬁ city.

Adding a biotin label to an enzyme molecule is simple, given the wide variety of options

available. A biotinylation reagent is chosen that has a reactive group that will couple to func-

tional groups on the enzyme (Chapter 11; Chapter 18, Section 3). For instance, NHS-LC-biotin

can be used to modify amine groups—a good choice for most biotinylation procedures involving

proteins (Chapter 11, Section 1). When free sulfhydryls are present, as in  -gal, a thiol-reactive

biotin label may be more appropriate, such as biotin–BMCC (Chapter 11, Section 2). However,

a better choice may be to use hydrophilic biotinylation reagents containing a PEG spacer arm,

which results in better solubility of the biotinylated enzyme (Chapter 18, Section 3). The proto-

cols for labeling proteins with these reagents can be found in Chapters 11 and 18.

969

Molecular biology techniques incorporating highly sensitive detection methods involving

ﬂ uorescence, chemiluminescence, or chromogenic enzyme substrates are used widely for assay-

ing oligonucleotide interactions. A major factor in the development of assay and detection

systems for RNA and DNA measurement is the ability to modify a nucleic acid with a detect-

able component while not affecting base pairing. The attachment of a small label, such as a

ﬂ uorescent molecule, or a large catalytic enzyme to an oligonucleotide probe forms the basis

for constructing sensitive hybridization reagents that can be used to detect genomic sequences.

Unfortunately, the methods developed to crosslink or label proteins do not always apply to the

modiﬁ cation of nucleic acids. The major reactive sites on proteins involve primary amines, sulf-

hydryls, carboxylates, or phenolates—groups that are familiar and relatively easy to derivatize.

RNA and DNA contain none of these functionalities. They also are unreactive with many of

the common electrophilic bioconjugate reagents discussed in Part II.

To modify the unique chemical groups on nucleic acids, novel methods have been developed

that allow derivatization through discrete sites on the available bases, sugars, or phosphate

groups (see Chapter 1, Section 3 for a discussion of RNA and DNA structure). These chemical

methods can be used to add a functional group or a label to an individual nucleotide or to one

or more sites in oligonucleotide probes or full-sized DNA or RNA polymers.

If an individual nucleotide is modiﬁ ed in the appropriate way, various enzymatic tech-

niques can be used to polymerize the derivative into an existing oligonucleotide molecule.

Alternatively, nucleotide polymers can be treated with chemical activators that can facilitate the

attachment of a label at particular reactive sites. Thus, there are two main approaches to modi-

fying DNA or RNA molecules: enzymatic or chemical. Both procedures can produce highly

active conjugates for sensitive assays to quantify or localize the binding of an oligo probe to its

complementary strand in a complex mixture.

The following sections describe the major enzymatic and chemical modiﬁ cation procedures

used to label nucleic acids and oligonucleotides.

Nucleic Acid and Oligonucleotide Modification and

Conjugation