As discussed in earlier chapters, immunoassay designs rely on the great specificity of antibodies, whose binding reac-tions can be related quantitatively to the concentration of an analyte of interest. Unfortunately antibody molecules do not possess any intrinsic properties that allow them to be mea-sured directly at low concentrations, and they must therefore be used in combination with a signal-generating technique such as colorimetry, fluorescence, or luminescence (see SIGNAL GENERATION AND DETECTION).
This requirement is typically achieved through the use of conjugates, in which two or more different molecules of interest are coupled to yield a single reagent, and which therefore combine some of the properties of the individual constituents. Thus, many immunoassay conjugates consist of an antibody derivatized with a suitable label, which facilitates the generation of the signal. The antibody with the required specificity for the assay may be labeled directly, and this is the approach taken in the majority of commercial immunoassays. However a second antibody approach can be useful in some circumstances, in which the conjugate incorporates a suitable “anti-species” antibody that binds to the primary antibody; thus if a mouse monoclonal IgG is the primary antibody, a labeled conjugate of a sheep anti-(mouse IgG) antibody allows it to be detected without the need for direct conjuga-tion. A wide range of anti-species conjugates are readily avail-able from commercial sources, therefore this approach can sidestep the need for any actual conjugation work. However this may be at a cost, as a second antibody approach can result in additional reagents and more complex assay protocols, as well as a potential reduction in sensitivity.
In some immunoassay formats it is a protein other than an antibody that must be labeled—the antigen, for exam-ple, or a binding protein fulfilling the same functional role as an antibody. While some of the antibody-specific com-ments above do not apply here, this second category of immunoassay conjugates has similar general requirements.
Thirdly, immunoassays also frequently require the immo-bilization of one of the binding molecules. For proteins this operation is sometimes carried out directly via chemisorp-tion or physisorption to the surface of interest (see Butler, 1992, and Chapter 8 of Aslam and Dent, 1998), but it is often preferable to employ indirect techniques based for example on the biotin–avidin or biotin–streptavidin interac-tion. Frequently these too require conjugate reagents to be prepared—a biotinylated antibody for example.
Regardless of which of these three categories an immu-noassay conjugate belongs to, its properties are often criti-cal to the performance of the assay, and therefore the optimization of the conjugate is typically a key factor in achieving the desired performance goals.
Until relatively recently, the main driver for advances in conjugation technology has been from “within the field”: the need for increasingly sensitive immunoassays to meet market demands. There has always been a parallel field of conjugate development for pharmaceutical applications—most notably
with respect to the use of antibody–drug conjugates (ADCs) or “armed antibodies” to deliver drugs to specific in vivo targets—but this has had little impact on diagnostic uses. In the last few years however, the more demanding requirements of pharmaceutical conjugates have led to improvements in the understanding of topics such as the sta-bility of thioether bonds (Alley et al., 2008) and the behavior of antibody thiol groups on reduction (Liu et al., 2010). Such findings have potential relevance to immunoassay applica-tions, and although it is still hard to discern much practical impact on commercial conjugation practices, it is quite possible that this situation will change with time.
Similarly, the regulatory requirements with respect to biopharmaceutical conjugates will increasingly drive the technology to yield more homogeneous conjugates with better-defined points of chemical linkage. This is one of the drivers for better control of antibody disulfide reduc-tion, and has also led to interest in coupling chemistries where both reactive moieties are completely alien to native proteins, and there is therefore no interference from the native functional groups. The click chemistry approaches based on the reaction of azide–alkyne cycloaddition reac-tions exemplify this philosophy, and have been used in the preparation of protein conjugates (Lallana et al., 2012; Hudak et al., 2012). As evidence of the increasing use of this approach in the life sciences, commercial suppliers of labeling reagents are now offering azide and alkyne deriva-tives designed for click chemistry alongside the “tradi-tional” succinimide esters of biotin and fluorescent dyes.
While the rate of change of diagnostic conjugation tech-nology has typically been very slow, it seems unlikely that better-defined conjugates will not at some point result in better-defined assay performance, and this may ultimately drive techniques such as click conjugation into the immunoassay mainstream.
Categories of Conjugates Employed in Immunoassay
The components that make up the conjugates for these three applications vary with the exact application, but in broad terms they can be categorized very simply (Fig. 1). Sometimes (Fig. 1a) both components of the conjugate are protein molecules, for example where an antibody or antigen is labeled with an enzyme. Because proteins contain only a limited range of functional groups to act as coupling sites, there is a great deal in common between the methods used in the preparation of these protein–protein conjugates. Pro-teins are sensitive to many of the conditions typically employed in chemical synthesis; methods requiring non-aqueous solvent systems or extremes of temperature, pres-sure, or pH are of little use in this field, so only a very small subset of the techniques of organic synthesis are applicable.
Sometimes an antibody may instead be labeled with a small molecule such as biotin or a fluorophore, while in some immunoassay designs a low molecular weight antigen may be labeled with an enzyme. These are examples of protein–small molecule conjugates (Fig. 1b). A great deal more variety may be encountered in the functional chemistry of these small molecules, often referred to as haptens—an immunological term implying broadly that they are too small to elicit an immune response in their own right. This prop-erty itself gives rise to another application of conjugation methods in the development of immunoassays—the need for protein–hapten conjugates as immunogens in the produc-tion of hapten-specific antibodies. Whether the product is a labeled conjugate or an immunogen, however, the small molecule must still be coupled to a protein target. The avail-able reaction routes, therefore, are still constrained by most of the factors that impact protein–protein conjugation.
In contrast, where two small molecules are to be com-bined (for example in the preparation of a biotinylated ste-roid), there are really very few constraints on the chemistry that can be employed. A wide range of reactive functional-ities can be introduced if so desired, and the much greater stability of these small molecules to temperature, pH, etc. allows a broader choice of reaction conditions. Therefore the preparation of these small molecule–small molecule conjugates (Fig. 1c) really constitutes a branch of organic synthesis, and will not be discussed further here.
Finally there are many other components that may be encountered in specific immunoassay applications. These include large, soluble carbohydrates such as dextrans, syn-thetic polymers with useful properties such as the polyeth-ylene glycols (PEGs), the nucleic acids DNA and RNA, and macroscopic structures such as blood cells and their synthetic analogs, liposomes. These will not be discussed further here; neither will the coupling of antibodies or other soluble species to solid-phase components such as latex particles, although these reagents too are sometimes referred to as conjugates. See SEPARATION SYSTEMS for more information or see Aslam and Dent (1998), Chapters 7 and 8, for a comprehensive review of these miscellaneous applications of protein coupling methods.
The vast majority of immunoassay conjugations are pro-tein–protein or protein–small molecule reactions, and the remainder of this chapter therefore concentrates on these two categories.
Protein–Protein CouplingFUNCTIONAL CHEMISTRY OF PROTEINSIn nature, proteins carry out an extraordinary range of functions, many of exquisite subtlety and specificity. It is a testament to the power of the evolutionary process that these properties have been achieved using a very small number of simple starting materials: the twenty common amino acids, some simple carbohydrates and metal ions, and the occasional organic moiety. As a consequence it is relatively simple to list the useful reactive groups that are regularly encountered in protein molecules, and therefore provide the chemical basis for conjugation.
AminesEach peptide chain within a protein molecule provides a terminal amine group, although in vivo derivatization reac-tions can sometimes render this unreactive. More impor-tantly though, lysine is one of the commoner amino acids, and each lysine side-chain provides an amine group. The presence of these in large numbers makes them the com-monest target for conjugation reactions. They are reactive toward a wide range of functionalities, including activated carboxyls (e.g., succinimide esters, acid chlorides, and acid anhydrides), aldehydes, and isothiocyanates. These reac-tions require at least a fraction of the amine group to be present in the unprotonated (NH2) rather than the pro-tonated ( ) form, and are therefore typically carried out at a pH of 7 or above. Although the pKa value for these groups (the pH value above which the unprotonated form predominates) may be as high as 9.5, there are enough unprotonated amines present two or even three pH units lower to allow the reaction to proceed. Some useful reactions of protein amines are shown in Fig. 2.
FIGURE 1 Major categories of conjugates employed in immunoassays: (a) protein–protein; (b) protein–small molecule; (c) small molecule–small molecule.
FIGURE 2 Some useful reactions of protein amines: (a) with an activated carboxyl derivative to form an amide; (b) with an aldehyde to form an imine, and (c) with an isothiocyanate to form a thiourea.
303CHAPTER 3.4 Conjugation Methods
ThiolsThe amino acid cysteine provides a side-chain thiol moi-ety, which has very useful reactivity. However, many of these are incorporated into intramolecular disulfide (cys-tine) links, and are therefore unavailable for derivatization. In fact, unless oxidizing conditions can be avoided, thiols of typical reactivity have an inherent tendency to dimerize via disulfide links, and are thus rarely found in accessible positions in native proteins. Where free thiols are available—or more often where these groups have been artificially introduced—they provide reactivity toward maleimide, disulfide, and haloacetyl groups; again high pH promotes the more reactive thiolate (S−) form (pKa typi-cally 8.5 in proteins, though variable), and these conjuga-tions are also generally carried out at pH 7 or above. Some useful reactions of protein thiols are shown in Fig. 3.
CarbohydratesMany proteins contain carbohydrate moieties, typically linked to the peptide chain via asparagine, serine, or threo-nine residues. The hydroxyl groups of these glycoproteins
are not particularly useful per se, but where two such groups are present on adjacent carbon atoms, a useful cleavage reaction employing sodium periodate generates two amine-reactive aldehyde functions.
Other Native GroupsPerhaps surprisingly, there are very few other groups natu-rally present in proteins that are exploited in common con-jugation reactions. There are generally many carboxylic acid functions present from aspartic and glutamic acid groups and from the C-terminals of the peptide chains; however these are unreactive without activation, which tends to be inefficient in the aqueous protein environment. Their use is therefore more-or-less limited to rare enzyme-catalyzed approaches. Where the acid group is amidated, as in asparagine and glutamine residues, the resulting carbox-amide is even less reactive. Tyrosine and histidine contain ring structures that are susceptible to attack by electrophilic reagents, and this property can form the basis of conjuga-tion via diazonium compounds; these routes were classi-cally popular but are now mainly of historical interest. One remaining use of these residues is in radioiodination for the preparation of radioimmunoassay (RIA) conjugates, where many of the commonest techniques employ electrophilic iodine reagents, but even this use is in steep decline as non-radioactive assay formats increasingly dominate the field.
The hydroxyl functions of serine and threonine can occasionally possess anomalously high reactivity, and there are some very specific reactions of arginine, methionine, and tryptophan. In practical terms, however, the chemis-try of proteins with respect to conjugation can be charac-terized adequately in terms of amines, thiols, and carbohydrates.
Table 1 summarizes the amino acid and carbohydrate content of a number of proteins commonly encountered in immunoassay work, namely labeling enzymes and antibodies.
CATEGORIES OF PROTEIN–PROTEIN COUPLING REACTIONThere are a variety of general approaches to protein–protein coupling, and it is useful to consider these briefly
FIGURE 3 Some useful reactions of protein thiols: (a) with a maleimide to form a succinimidyl thioether; (b) with a disulfide to form a second disulfide, and (c) with an acetyl halide to form a thioether.
TABLE 1 Amino Acid and Carbohydrate Content of Labeling Enzymes and Antibodies. The figures quoted for antibodies are necessarily broad estimates. There is a fairly high degree of homology between the immunoglobulin structures of different species. Therefore, the human-derived figures quoted here can give some indication of levels in other species
Protein SourceMolecular weight (kDa)
Number of lysines
Number of free thiols
Number of free carboxylic acids
Carbohydrate content (%)
Peroxidase (HRP)
Horseradish 44 6 (typically only around 2 accessible)
– 28 20
Alkaline phosphatase
Escherichia coli 94 56 – 98 –
Alkaline phosphatase
Bovine 125 42 – 106 10
B-Galactosidase Escherichia coli 465 80 64 (typically only around 16 accessible)
508 –
IgG Human Around 150 Around 90 Variable Around 120 Around 2IgM Human Around 970 Around 350 Variable Around 620 Around 12
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before describing actual chemistries. The two protein molecules of interest can either be linked without the per-manent introduction of any extra atoms (direct coupling), or via a bifunctional coupling agent. The two functional groups of the latter can be chosen to have the same reactiv-ity (homobifunctional coupling), or to react with separate targets (heterobifunctional coupling).
These different approaches can be illustrated in the con-jugation of two typical proteins (Fig. 4). An amine group from one protein can react with a carboxyl group from the other (Fig. 4a); the latter requires activation to the succin-imide ester shown, but the final product contains only atoms present in the component proteins. In a homobi-functional approach (Fig. 4b), a reagent combining two such succinimide esters can be used to couple the proteins via intrinsic amine functions. A heterobifunctional reagent may combine a succinimide ester with a functional group that reacts with thiols, such as a maleimide; this can be employed to create an amine–thiol link between native groups of the two proteins (Fig. 4c). A fourth approach is to use two complementary heterobifunctional reagents, for example one that introduces a thiol, and one that introduces a thiol-reactive maleimide (Fig. 4d).
For protein–protein conjugates, direct coupling has many disadvantages. First, the approach is limited by the few classes of functional groups available in proteins that possess mutual reactivity. Secondly, there are generally many of each functional group present on each molecule—therefore the reaction is unlikely to stop at the tidy 1:1 conjugate shown in Fig. 4a, and may instead continue to form large cross-linked complexes. Finally, there is very little space between the protein molecules in the product, and steric hindrance may therefore interfere with their behavior.
Homobifunctional coupling gets round two of these problems: virtually all proteins contain enough amine groups to allow a bis-succinimide ester or similar amine-reactive homobifunctional linker to be employed, and the presence of the linker group minimizes steric hindrance issues. Cross-linking remains a problem, however, so that large and poorly-defined conjugates are often obtained.
Heterobifunctional reagents offer the most elegant approaches for protein–protein coupling. Free thiols are generally only present in proteins in small numbers, and the approach shown in Fig. 4c can provide well-defined conjugates in such cases. In the ideal case, the degree of derivatization of Protein 1 is controlled such that there is only one maleimide group per molecule, and there is only one free thiol in Protein 2. Under these circumstances the only possible product of the reaction is a 1:1 conjugate.
In the majority of cases there is not a convenient single free thiol in one of the proteins of interest. However this
FIGURE 4 Major categories of conjugation method: (a) direct coupling between the amine group of one protein and the carboxyl group of another, activated as a succinimide ester; (b) homobifunctional coupling of the amine groups of two proteins using a bis-succinimide ester; (c) use of an amine-reactive heterobifunctional reagent to introduce a maleimide group into one protein, allowing coupling to a thiol group in another; (d) use of two amine-reactive heterobifunctional reagents to introduce a maleimide group into one protein, and a thiol group into another, followed by coupling of these groups.
305CHAPTER 3.4 Conjugation Methods
situation can be approximated to by the approach shown in Fig. 4d, where thiol and maleimide functions are both introduced artificially via the protein’s amine groups. Again if the incorporation of each group is controlled to one per molecule, and if there are no competing groups from the native protein structures, a 1:1 conjugate is the only possible product. In practice, incorporation cannot be controlled quite so closely and there are bound to be some multiple-labeled molecules—and a higher mean incorporation may in any case be desirable from the point of view of immunoassay performance. However these approaches can reproducibly yield well-defined conjugates: as an illustration, the size exclusion chro-matograms obtained from a direct and a dual heterobifunctional approach to the preparation of an IgG–horseradish peroxidase (HRP) conjugate are compared in Fig. 5.
Because of this greater degree of control, heterobifunc-tional approaches have achieved great popularity in recent years—especially those based on mild chemistry such as the use of succinimide esters.
COMMON PROTEIN–PROTEIN COUPLING METHODSSome of the commoner conjugation methods used in the preparation of protein–protein conjugates will now be out-lined briefly, including examples from each of the categories above.
Periodate Method (Direct)The only direct coupling method that continues to enjoy widespread application is the periodate method (Nakane & Kawaoi, 1974), applicable if at least one of the proteins to be conjugated is a glycoprotein (Fig. 6). As mentioned ear-lier, sodium periodate can be used to oxidize carbohydrate residues (Fig. 6a), generating aldehyde groups at positions where there is a vicinal diol (hydroxyl residues on two adja-cent carbon atoms). These aldehyde groups (Fig. 6b) can be coupled to the amine groups of a second protein, gener-ating an imine or Schiff ’s base (Fig. 6c). These groups are considered somewhat unstable, and it is common practice to employ a reducing agent to generate a secondary amine link instead (Fig. 6d). Sodium borohydride remains the most commonly employed reducing agent, but sodium cyanoborohydride exhibits greater selectivity.
Periodate is a powerful oxidizing agent and can cause some damage to protein structure, therefore low concen-trations and short reaction times are typically used. Never-theless multiple aldehyde groups are bound to be generated from the glycoprotein, and as there are normally multiple amines on the protein to be conjugated, extensive cross-linking is often observed (see Fig. 5).
In the immunoassay field, there have been two particularly significant applications of the periodate reaction. The first is in the preparation of conjugates of HRP, one of the com-monest labeling enzymes, which is extensively glycosylated. Under mild oxidation conditions there is good retention of
FIGURE 5 Size exclusion chromatograms of the purification of IgG–HRP conjugates manufactured by (a) a direct periodate approach and (b) a dual heterobifunctional thiol–maleimide approach. Reproduced with permission from Aslam and Dent, 1998 p. 90.
FIGURE 6 Periodate coupling: the carbohydrate group of one protein (a), shown in simplified form, contains a vicinal diol group; this is oxidized by periodate to yield a bis-aldehyde (b), which couples with the amine group of a second protein to form an imine link (c). This is frequently stabilized by reduction to a secondary amine (d).
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enzyme activity, and this has provided a convenient means for the preparation of HRP–IgG conjugates, etc. The second notable use is based on the fact that IgG molecules are glyco-sylated predominantly on the Fc region, distant from the binding site. Mild oxidation of these residues therefore allows conjugation to the amines of a labeling protein such as HRP or alkaline phosphatase, with minimal interference to the antigen-binding activity of the antibody.
Bismaleimide Method (Homobifunctional)There is no shortage of homobifunctional reagents that can link the amines of one protein molecule to those of another—a wide range of bis-succinimide ester reagents, for example, or the once-popular glutaraldehyde—but there is no particular advantage to this approach that out-weighs the overwhelming tendency toward cross-linking and aggregation. Although the occasional glutaraldehyde method is still encountered, immunoassay conjugates based on this approach are now rare. Where the proteins to be coupled both possess small numbers of free thiol
groups however, homobifunctional coupling via these moieties is a more attractive option. Obviously a coupling agent bearing two thiol-reactive groups is called for, and N,N′-o-phenylenedimaleimide (PDM) has seen the most widespread use. The coupling reaction is shown in Fig. 7.
In the immunoassay field a particularly elegant applica-tion of this approach has been to couple the labeling enzyme β-galactosidase to antibody fragments such as Fab or Fab′. Both of these proteins possess limited num-bers of free thiols, and PDM coupling yields largely monomeric conjugates with excellent incorporation and little loss of enzyme activity (Ishikawa et al., 1983). Thiol–maleimide coupling takes place readily at neutral to mildly basic pH.
The more widespread use of this approach has been pre-vented by the rarity of cases where the requisite free thiols are present on each of the proteins to be coupled.
Heterobifunctional Thiol–Maleimide Methods, and Related ApproachesAs discussed earlier, heterobifunctional approaches can offer greater control over the product composition than the direct and homobifunctional procedures described so far. The commonest examples exploit the same thiol–maleimide chemistry just described, as shown in Fig. 4d. Two coupling agents are required, one combining an amine-reactive group with a thiol, and one combining an amine-reactive group with a maleimide (Ishikawa et al., 1983). In practice the thiol is often provided in a protected form such as an S-acetyl compound, because free thiols tend to dimerize under the influence of atmospheric oxidation—an unwelcome side-reaction.
Numerous coupling agents of the types described are commercially available, some of which are shown in Tables 2 and 3. All the common amine-reactive maleimides are based on succinimide esters, sometimes derivatized with a sulfonic acid group that confers water-solubility. It is also important whether the maleimide group is attached to an aromatic or an aliphatic function, the latter being gener-ally more stable. The length of the linker group is another
FIGURE 7 Bismaleimide reagents can be used for homobifunctional coupling of protein thiols.
TABLE 2 Amine-Reactive Heterobifunctional Reagents for Introduction of Thiol Groups Into Proteins
S-Acetylmercaptosuccinic anhydride (SAMSA)
O
O
O O
S C CH3
2-lminothiolane hydrochloride (2-IT)
NH2CI–+
S
N-Succinimidyl-S-acetyl thioacetate (SATA)
N
O
OOO
S OCCH2CH3C
307CHAPTER 3.4 Conjugation Methods
parameter of interest, the very long chains of compounds such as SMTCC being claimed to give good performance in immunoassay conjugates by their inventors.
The thiolation reagents shown in Table 2 are interest-ing in terms of their effect on the charge of the protein—like other succinimide esters SATA reduces the charge at typical pH by one through the loss of an amine group. With SAMSA an extra carboxylate function is introduced, reducing the net charge by two. With 2-IT the original charge of the protein is retained, as the amine group is replaced by a similarly-charged amidine function. The S-acetylthiol yielded from SATA or SAMSA can be depro-tected before use with hydroxylamine, while 2-IT yields the free thiol directly.
Subtle changes of conjugate performance can be obtained by using various combinations of the thiol and
maleimide reagents listed above. The overall approach is highly satisfactory for the preparation of immunoassay conjugates, giving well-defined conjugates of controlled size, and generally high retention of protein functionality (e.g., enzyme activity). The thioether link formed in this method is very stable under typical immunoassay condi-tions; recent evidence of some reversibility under in vivo conditions (Alley et al., 2008) seems unlikely to impact the popularity of this approach for diagnostic applications.
There are alternatives to maleimide as the thiol-reactive function in this approach. In particular it is worth men-tioning the reagent N-Succinimidyl-3-(2-pyridyldithio)propionate, SPDP, (Fig. 8) which incorporates a disulfide moiety. This can be exploited in the same way as a maleimide group, yielding instead a conjugate with a disulfide link (see Fig. 3). This may be unstable in the presence of thiols or
TABLE 3 Amine-Reactive Heterobifunctional Reagents for Introduction of Maleimide Groups into Proteins
Succinimidyl 4-[(N-maleimidomethyl) tricaproamido]cyclohexane-1-carboxylate (SMTCC)] O
C NH
CH2 (CH2)6 O NN
OO
OOO
C3
NB: most of these reagents are also available as the water-soluble sulfosuccinimidyl analogs (cf, Figure 15b, sulfo-NHS-biotin), e.g. sulfo-SMCC.
308 The Immunoassay Handbook
other reducing agents—in fact it is sometimes used in appli-cations where the intention is to facilitate later cleavage of the protein–protein bond in this way. This is not generally the case for immunoassay applications, and the maleimide reagents described above predominate in this field.
GENETIC ENGINEERING APPROACHES TO PROTEIN CONJUGATIONThere is a very different approach to the formation of pro-tein–protein conjugates, which has become available over the last two or three decades—although it is still to have a significant impact on commercial immunoassays. This is the use of recombinant DNA techniques to construct a fusion protein, in which the DNA sequences coding for the two components of the conjugate are spliced together and the resulting sequence expressed in a suitable host organism. Well-defined 1:1 conjugates can be produced by this means, which can then be continually expressed in large quantities if required.
This approach has been used to express enzyme conjugates for immunoassay such as insulin coupled to alkaline phosphatase, and a malaria antigen coupled to β-galactosidase. Fusion proteins containing antibodies have also been produced as the relevant gene sequences have become available. Although still not widespread in com-mercial immunoassays, the use of recombinant antibody fragments such as single chain variable fragments (ScFvs) is an increasingly popular technology that can be tailored to diagnostic applications (see for example Backmann et al., 2005). While this approach has obvious potential advan-tages in terms of well-defined conjugates and reproducible supply, there can be problems with expression and process-ing of these artificial constructs (Teillaud, 2005). So far the investment of cost and effort required to develop fusion protein conjugates has prevented them having a significant impact in the immunoassay field.
Protein-Small Molecule CouplingWhere a small molecule is to be conjugated to a protein for immunoassay purposes, the small molecule concerned is almost always the analyte of interest or a close analog. This is true both for the production of conjugates for use in the assay itself, and for the preparation of immunogens used in the generation of antibodies.
The reactive groups available in protein molecules have already been summarized above. In some cases the small molecule of interest may already possess a group such as an amine, thiol, or carboxylic acid, which can form the basis
of a coupling reaction, in which case no synthetic chemis-try is necessary. Often however, there are no such groups present, and it can present a significant challenge to syn-thesize a derivative of the compound that possesses an appropriately reactive “handle” for attachment to the pro-tein, while retaining the ability to be recognized by the appropriate antibodies.
The choice of derivatization approach needs to be made with care. It is important to identify those parts of the mol-ecule likely to be most important in antibody-binding terms, so that they can be avoided as potential derivatiza-tion sites. This choice is often driven primarily by cross-reactivity arguments. An antibody might be required, for example, to provide discrimination between a steroid ana-lyte and a closely-related analog differing only in the nature of single substituent on the A-ring. An immunogen in which the steroid is coupled to the carrier protein via this region of the molecule is unlikely to yield antibody of the necessary specificity. Similarly—once an appropriately specific antibody has been found—a conjugate coupled to a labeling protein through this site is likely to show poor binding. In both cases therefore, a derivatization site remote from the A-ring would probably give the best results.
A wide variety of “handles” can provide appropriate reactivity for coupling to protein molecules, and some common examples reactive toward amines and thiols are shown in Figs. 2 and 3. These offer attractive synthetic targets when the aim is to derivatize a small molecule for coupling to a protein. Routes that yield a carboxylic acid derivative are particularly popular, however, largely due to the well-established and controlled methods that can then be used for coupling to the protein’s amine groups. A step commonly encountered in such a synthetic route, there-fore, is one which exploits an existing functionality to introduce a carboxylic acid. Two common examples are shown in Fig. 9—the use of succinic anhydride to incorpo-rate an acid at the site of an amine or (less efficiently) a hydroxyl group, and the use of carboxymethoxylamine hydrochloride to “convert” an aldehyde or ketone to a carboxyl-containing oxime function.
Note that an amine group in the small molecule can itself be used as a handle. Homobifunctional coupling methods similar to those mentioned for protein–protein applications are sometimes employed to attach such a mol-ecule to a protein, using a bis-succinimide ester for exam-ple. These approaches are often rather inefficient in terms of incorporation, however, and conversion to a carboxylic acid as just described is often preferred as a consequence. Additionally, the resulting succinate spacer group can yield better steric properties (allowing better antibody access) and hence improved assay performance.
COMMON PROTEIN–SMALL MOLECULE COUPLING METHODSBecause carboxyl-based approaches are so common, some of the commonest examples of this category are described in detail below. So too is the Mannich reaction, a useful if rather unpredictable standby for difficult cases where it is not straightforward to prepare a derivative bearing one of the standard handles.
Carbodiimide MethodsIt is possible to activate carboxylic acids toward amines in many ways, but all of these involve intermediates that are unstable to some extent in an aqueous environment. For reaction with proteins it is therefore necessary to choose examples that strike a balance between reactivity toward amines, and resistance toward hydrolysis. One such cate-gory of intermediate is the O-acylisourea, which is derived from the reaction of a carboxylic acid with a carbodiimide (Fig. 10).
These compounds fall into two classes—those that are water-soluble and those that are not. The commonest example of the latter category is dicyclohexylcarbodi-imide (DCC). In a typical reaction scheme there are two steps: first the small molecule is derivatized with DCC in a water-miscible organic solvent, then the product is added to an aqueous protein solution for the coupling stage.
With a water-soluble carbodiimide, however, it is pos-sible to carry out both reactions simultaneously—the isourea can be formed in the protein solution, so only one step is required and the need for organic solvents is also avoided. The commonest examples of water-soluble carbodiimides are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) and 1-cyclo-hexyl-3-(2-(4-methylmorpholin-4-yl)ethyl)carbodi-imide p-toluenesulfonate (CMC). A drawback of a one-step carbodiimide method is the fact that the pH opti-mum for isourea formation (pH ~5–6) is far from that of maximum amine reactivity (pH 9–10), so two-step proce-dures may be preferred even where all the reagents are water-soluble.
Carbodiimide/N-hydroxysuccinimide MethodsA popular variation on the straightforward carbodiimide methods of the previous section is to convert the isourea in situ into a succinimide ester, which goes on to react with the protein amines (Fig. 11); this can give improvements in yield. Thus in a two-step approach it is common to activate a carboxyl-containing compound in an organic solvent with a mixture of DCC and N-hydroxysuccinimide (NHS) before addition to the aqueous protein solution. Similarly the water-soluble N-hydroxysulfosuccinimide (NHSS), can be used in one-step procedures with EDAC or CMC.
In terms of efficiency of incorporation, a two-step DCC/NHS protocol offers one of the best means of conjugating carboxylic compounds to proteins: yields are very high, and it is generally possible to expose the protein to the small quantity of organic solvent required without causing it much harm. Fully aqueous approaches avoid this potential issue, but there is invariably a loss of yield due to hydrolysis effects.
Mixed Anhydride MethodAcid anhydrides, formed by the loss of water between two carboxyl groups, can survive long enough in aqueous solu-tions to react with protein amines. The conditions required to create a symmetric anhydride by dehydration of the acid of interest are very harsh, so it is more common to form a mixed anhydride intermediate by reaction with a chloro-formate. The commonest example of this class of reagents is isobutyl chloroformate (IBCF). The conversion of the car-boxylic compound to its mixed anhydride must be carried out in the absence of moisture, so a two-step approach is required (Fig. 12), the product from the anhydrous activation reaction being added to the aqueous protein solution for coupling.
Mannich ReactionIn the Mannich reaction an amine (e.g., from a protein) is coupled in the presence of an aldehyde (usually formalde-hyde, despite its toxic properties) to a suitable carbon atom
FIGURE 9 Approaches for the introduction of carboxyl groups into small molecules through succinic anhydride activation of amine or hydroxyl function (a), or derivatization of an aldehyde to form a carboxyl-bearing oxime (b).
FIGURE 10 Carbodiimide activation of a carboxylic acid yields an O-acylisourea, which is reactive towards amines.
310 The Immunoassay Handbook
in a wide range of organic compounds (Fig. 13). The car-bon atom must possess an “active” hydrogen atom, loosely speaking, one with a degree of acidic character. Examples of compounds that can take part in the Mannich reaction are shown in Fig. 14. Reaction times can be slow and yields poor, but this approach can be useful where commoner approaches have failed.
Ready-made Reagents for BiotinylationThe introduction of a biotin moiety into a protein is a com-mon requirement of immunoassay systems based on the biotin–avidin or biotin–streptavidin interaction. This is generally achieved by rather simple means, as this is such a widespread need that one-step biotinylation reagents are
readily available from commercial sources. The biotin mol-ecule presents few challenges as a target for coupling to proteins: it possesses a side-chain ending in a carboxyl func-tion, which only requires suitable activation for coupling to protein amines. The succinimide ester of biotin, biotin–NHS, is commercially available for this purpose. So too are longer-chain analogs that incorporate extra spacer regions; some common biotinylation reagents are shown in Fig. 15. Using these reagents, biotinylation is a simple one-step reaction at pH 7 or above. Yields are usually very high, and there is generally good retention of protein functionality.
Many other biotinylation reagents are commercially available, aimed at other functional groups: an example is N-(6-biotinamidohexyl)-3′-(2′-pyridyldithio)propion-amide, Biotin–HPDP (Fig. 16), which can be coupled to protein thiols.
Other Ready-made Reagents for Protein DerivatizationWherever there is sufficient interest in derivatization of proteins with a particular species, commercial suppliers market suitable reagents for this purpose. Thus all the commonest fluorophores and luminophores can be pur-chased as amine-reactive derivatives, typically succinimide esters or isothiocyanates. Some examples are shown in Fig. 17. Needless to say, these generally provide the sim-plest route for preparation of the relevant protein conju-gate—a fluorescein-labeled IgG for example.
Purification of ConjugatesConjugates are sometimes used in their crude form in immunoassays, but it is generally possible to achieve
FIGURE 11 Reaction of an O-acylisourea with an N-hydroxysuccin-imide yields an amine-reactive succinimide ester.
FIGURE 12 Reaction of a carboxylic acid with a chloroformate yields an amine-reactive mixed anhydride.
FIGURE 13 The Mannich reaction allows a wide variety of compounds bearing a suitably ‘active’ hydrogen atom to be coupled to protein amines.
FIGURE 14 Examples of moieties that can provide an active hydrogen for Mannich reaction. Reproduced with permission from Aslam and Dent, 1998 p. 449.
311CHAPTER 3.4 Conjugation Methods
greater control by carrying out a purification step. Often this is essential to obtain the desired assay performance. By far the commonest approach to conjugate purification is chromatography.
CHROMATOGRAPHIC APPROACHES TO CONJUGATE PURIFICATIONAll branches of chromatography are based on the different interactions of the components of a mixture in one phase with a second phase of some kind. In practical terms in this application, this nearly always means the interaction of solutes in an aqueous solution with a solid-phase column.
In size exclusion chromatography (SEC) or gel fil-tration, the solid phase contains pores that are more acces-sible to smaller molecules than to large ones. The result is that large molecules pass through the column more
quickly. This provides an excellent means for separating protein–small molecule conjugates from unreacted hap-ten, for example, or for separating a thiolated protein from excess thiolating reagent in a heterobifunctional protein–protein conjugation. In these “de-salting” examples there is a huge difference in the molecular weights of the protein components (50–500 kDa) and the impurities (50–500 Da). However high-performance size exclusion chromatogra-phy can also resolve separate protein components, allow-ing unreacted enzyme (44 kDa) to be removed from an IgG–HRP conjugate (200–300 kDa), for example.
In ion exchange chromatography (IEC), the solid phase bears charged groups that interact electrostatically with the solutes, providing an alternative to the size-based separa-tions of gel filtration. So long as the ionic strength of the solvent system is kept reasonably low, species of different charge can be eluted sequentially from a solid phase bearing positive (anion exchange) or negative (cation exchange) groups. Sometimes the pH of the eluent system is altered during the purification to effect elution of the more strongly-bound components, but it is generally simpler to modulate ionic strength instead. As this is increased, the interactions of charged groups with the solid phase are outweighed by those with the solvent, and elution is again the result.
Hydrophobic interaction chromatography (HIC) and reverse phase chromatography (RPC) rely on the interactions of the hydrophobic regions of proteins with a hydrophobic solid phase. In HIC modulation of eluent, ionic strength is again typically employed to effect elution of bound solutes, but in this case it is a low ionic strength that is required; high salt content strengthens hydrophobic interactions. In RPC, which was adapted to protein appli-cations from organic synthesis, the interactions tend to be stronger, and it is often necessary to employ organic sol-vents to disrupt the interactions of the bound solutes with the surface of the solid phase.
Affinity chromatography uses solid phases bearing reagents that have a specific ability to bind one component or class of components, thus separating them selectively from a mixture. This can provide highly specific purifica-tion regimes, often unachievable by the more generic tech-niques described above. There are countless examples of this approach in use, but some of the commoner applica-tions in the immunoassay field use solid phases bearing Protein A, Protein G and their recombinant variants, bacterial proteins with a high affinity for most classes of immunoglobulin from most species. Lectins—naturally-occurring proteins that bind carbohydrates—are also widely employed for the purification of glycoprotein con-jugates. Concanavalin A is the commonest example of this category. Various means are used to elute bound proteins from affinity matrices: low pH is probably the commonest,
FIGURE 15 Some common biotinylation reagents: (a) succinimidyl biotin (biotin-NHS); (b) sulfosuccinimidyl biotin, sodium salt (sulfo-NHS-biotin); (c) succinimidyl 6-(biotinamido)hexanoate (NHS-LC-Biotin II™ (Pierce). Biotin-X-NHS™ (Calbiochem)), (d) succinimidyl 6-[6-biotinamido(hexanamido)]hexanoate (Biotin-XX-NHS™ (Calbiochem)).
but organic cosolvents or the structure-disrupting agents known as chaotropes are also employed.
OTHER APPROACHES TO CONJUGATE PURIFICATIONThere are a number of other common approaches for sep-arating components that have a wide difference in size—alternatives to the de-salting applications of size exclusion chromatography. Dialysis is a time-consuming but well-established technique, where a semi-permeable membrane is employed to retain high molecular weight components while allowing smaller molecules to diffuse out. Good sep-arations can be obtained by repeated buffer exchange. Ultrafiltration techniques use the same principle but employ pressure or centrifugation to drive the protein solution through the membrane—obviously a much quicker technique. Membranes with different molecular weight cut-offs are available, allowing some influence over which components are retained; it should be borne in mind however that these cut-offs are often rather approximate.
Electrophoresis techniques, where the solutes are driven through a suitable medium under the influence of an electric field, have excellent power to resolve protein
components on the basis of their charge. The medium is typically a gelatinous matrix coated onto a flat support of some kind. However, the heat generated in the process puts serious constraints on the dimensions of the appara-tus, and while powerful as an analytical tool this approach is only rarely encountered in preparative applications.
Characterization of ConjugatesAn area that has received surprisingly little attention over the years is the study of the actual molecular composition of conjugates. Taking a typical reaction where a number of a protein’s numerous amine groups are derivatized by a suitably reactive reagent—the succinimide ester of a small molecule for example—it is commonplace to calculate a mean incorporation ratio such as “1.7 hapten groups per protein molecule”. This is generally established by tech-niques such as UV-visible spectrophotometry, which relies on the components of the conjugate bearing different absorbance characteristics at two or more wavelengths. Other techniques may be available in specific circum-stances, for example the use of radiometry to quantify a radiolabeled component, or the quantitation of biotin incorporation using a colorimetric assay based on the dye 4-hydroxyazobenzene-2′-carboxylic acid, HABA. How-ever the result is nearly always a mean incorporation.
Simple statistical calculations using Poisson or binomial distributions can be used to show the expected spread around these means: some examples are shown in Fig. 18. These highlight the danger of taking mean incorporations too literally in understanding the behavior of protein conjugates.
To confirm these distributions of conjugate populations of differing individual stoichiometries, it is necessary to use techniques that can elucidate the composition of conjugate product mixtures at the molecular level. Three technologies have dominated such attempts. The first of these is isoelec-tric focusing, a branch of electrophoresis where fractions of the product are separated into tight bands based on their charge. As many small molecule conjugation procedures result in the replacement of positively-charged amine groups, it is possible to track the extent of derivatization on this basis, and this approach has been demonstrated for example by Barbarakis and Bachas (1991) and Pham et al. (1995). A sec-ond branch of electrophoresis, SDS-PAGE, allows resolu-tion on the basis of size, and can therefore be used in a parallel fashion for the characterization of protein–protein conju-gates; see for example Åkerblom et al. (1993). Capillary electrophoresis is now challenging the traditional gel-based formats for these methods, but to date has seen its widest application in the high-throughput screening arena.
Thirdly, the ability of mass spectrometry (MS) to give accurate mass determinations on large molecules is now well-established, and techniques such as electrospray and matrix-assisted laser desorption ionization (MALDI) MS are increasingly employed for protein conjugate charac-terization (see for example Chiu et al., 2011), or even simply as a tool to track the progress of the conjugation reaction (Safavy et al., 2003). Indeed, the increasing ability of MS to
FIGURE 17 Some useful amine-reactive derivatives of common fluorophores and luminophores: (a) 5-carboxyfluorescein succinimidyl ester; (b) 6-carboxytetramethylrhodamine succinimidyl ester; (c) 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methylacridinium-9-carboxylate fluorosulfonate; (d) fluorescein-5-isothiocyanate (FITC Isomer l); (e) 4-isoluminol isothiocyanate.
313CHAPTER 3.4 Conjugation Methods
identify macromolecules is leading to widespread diagnostic use in clinical applications (Vékey, 2007) and immunoassay-like techniques such as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies) now exist based on MS detection of protein fragments ( Jain, 2010).
Given the central role of conjugates in immunoassay, it is certain that their composition can have a significant effect on assay behavior. More work is needed to increase the understanding of conjugate stoichiometry and its effect on immunoassay performance—and ideally to extend the capability of purification methods such that the individual populations can be resolved adequately at a preparative scale. As mentioned in the introduction, it can be hoped that the drive for better-defined antibody conjugates in the biopharmaceutical field will help to improve understanding of immunoassay conjugate behavior too.
ConclusionIn conclusion, while new technologies such as click conju-gates are gaining wider general acceptance in the life sci-ences, the chemistries employed in the preparation of immunoassay conjugates have remained relatively stable over the last two or three decades. Heterobifunctional methods predominate in the production of protein–pro-tein conjugates—those based on thiol–maleimide chemis-try being particularly widespread—while the popularity of carbodiimide and mixed anhydride methods continues unabated for small molecule coupling.
Advances in chromatography continue to be driven primarily by commercial suppliers, and their regular
introduction of better instrumentation and more efficient matrices is facilitating more effective purification of con-jugates. Software for the electronic manipulation of chro-matographic data has become much more powerful in recent years, providing valuable tools for monitoring sep-aration efficiencies.
The last decade has seen big advances in the availability of techniques for the characterization of immunoassay conjugates—as observed earlier, this remains a rather neglected field. Mass spectrometry is likely to have a major part to play in increasing understanding in this area, and again commercial suppliers are playing a key role through the development of ever-better instrumentation.
Finally, the production of a conjugate for use in a com-mercial immunoassay is a manufacturing process, and as such should receive appropriate attention to ensure its robustness and reproducibility. See Chapter 13 of Aslam and Dent (1998) for a discussion of relevant approaches.
References and Further ReadingÅkerblom, E. et al. Preparation and characterization of conjugates of monoclonal
antibodies and staphylococcal enterotoxin A using a new hydrophilic cross-linker. Bioconjugate Chem. 4, 455–466 (1993).
Alley, S.C. et al. Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjugate Chem. 19, 759–765 (2008).
*Aslam, M. and Dent, A.H. (eds), Bioconjugation: protein coupling methods for the bio-medical sciences (Macmillan, London, 1998)
Backmann, N. et al. A label-free immunosensor array using single-chain antibody fragments. Proc. Nat. Acad. Sci. 102, 14587–14592 (2005).
Barbarakis, M.S. and Bachas, L.G. Isoelectric focusing electrophoresis of protein–ligand conjugates: effect of the degree of substitution. Clin. Chem. 37, 87–90 (1991).
Butler, J.E. The behaviour of antigens and antibodies immobilized on a solid phase. In: Structure of Antigens (ed Van Regenmortel, M.H.V.) vol. I 209–259 (CRC Press, Boca Raton, FL, 1992)
Chiu, M.L. et al. Characterization of morphine-glucose-6-phosphate dehydroge-nase conjugates by mass spectrometry. Bioconjugate Chem. 22, 1595–1604 (2011).
*Hermanson, G.T. Bioconjugate Techniques, 2nd edn (Academic Press, New York, 2008)
Hudak, J.E. et al. Synthesis of heterobifunctional protein fusions using copper-free click chemistry and the aldehyde tag. Angew. Chem. Int. Ed. 51, 4161–4165 (2012).
Ishikawa, E. et al. Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. J. Immunoassay 4, 209–325 (1983).
Jain, K.K. The Handbook of Biomarkers, 49 (Springer, New York, 2010).Lallana, E. et al. Zuverlässige und effiziente Konjugation von Biomolekülen über
Huisgen-Azid-Alkin-Cycloaddition. Angew. Chem. 123, 8956–8966 (2012).Liu, H. et al. Ranking the susceptibility of disulfide bonds in human IgG1 antibod-
ies by reduction, differential alkylation and LC-MS analysis. Anal. Chem. 82, 5219–5226 (2010).
Nakane, P.K. and Kawaoi, A. Peroxidase-labeled antibody: a new method of conju-gation. J. Histochem. Cytochem. 22, 1084–1091 (1974).
Pham, D.T. et al. Electrophoretic method for the quantitative determination of a benzyl-DTPA ligand in DTPA monoclonal antibody conjugates. Bioconjugate Chem. 6, 313–315 (1995).
Safavy, A. et al. Synthesis and biological evaluation of Paclitaxel-C225 conjugate as a model for targeted drug delivery. Bioconjugate Chem. 14, 302–310 (2003).
Singh, K.V. et al. Synthesis and characterization of hapten–protein conjugates for antibody production against small molecules. Bioconjugate Chem. 15, 168–173 (2004).
Teillaud, J.-L. Engineering of monoclonal antibodies and antibody-based fusion proteins: Successes and challenges. Exp. Opin. Biol. Therapy 5, S15–S27 (2005).
Vékey, K. Medical Applications of Mass Spectrometry (Elsevier, Amsterdam, 2007).
* Major publications providing detailed information on conjugation methods.
FIGURE 18 (a) Binomial distribution of thiol incorporation (n) in a protein with four derivatization sites, where the mean incorporation is 1; (b) Poisson distribution of thiol incorporation (n) in a protein with a large, undefined number of derivatization sites, where the mean incorporation is 1. Reproduced with permission from Aslam and Dent, 1998 p. 92–93.
