61 © 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/B978-0-08-097037-0.00005-1

IntroductionThe history of immunoassay has been one of a trail of spec-tacular successes. High-performance, fast, and robust meth-ods have been developed for a remarkable array of substances. It is clear, however, that outstanding performance has classi-cally only been seen within the realm of heterogeneous assays for analytes of large enough molecular size such that they can be bound by both capture and detector antibodies simultaneously. These assays benefit from being “reagent excess” systems and offer precise and fast assays equally applicable to complex automated immunoassay machines through to dipsticks. Two-site immunometric assays for large molecules have found large international markets for qualitative home, clinic, or bedside diagnostic testing sys-tems (May, 1994; Bonnar et al., 1999).

In spite of the provision of high-performance immuno-assays for large analytes, simple physical constraints have precluded the use of such sandwich approaches for small molecule analytes not large enough to bind two antibodies simultaneously. Instead, competitive-format assays have been widely used. In these approaches, it is classically the fraction of capture antibody that has not bound analyte that is measured. In effect these assays measure how much ana-lyte is not present. This inverse approach brings a great deal of complexity to any assay systems thus based (Ekins, 1987; Self, 1993a).

Not only are such negative readout competitive immu-noassays counterintuitive (a real problem with visually read dipsticks where in these negative systems development of a positive line means absence of analyte) but the greatest problem of competitive-format systems is simply that the response at low analyte concentration is difficult, if not impossible, to distinguish from that at zero, as both condi-tions give rise to large signals. Also, by definition, competi-tive assays are not “reagent excess” in nature. Limited concentrations of both capture antibody and competing partner must be employed, with the resulting limitations for assay speed. In addition, not only must reagent concen-trations be limited, but they must also be precisely main-tained across an assay series to achieve acceptable results. These assay requirements demand high technical precision

in the addition of reagents, besides posing clear constraints on shelf-life considerations. Even relatively modest varia-tions of concentration of the competitive reagents can have a profound effect on the precision of the competitive assay. All of these factors taken together can lead to variable, rela-tively slow assays of limited sensitivity and range. Consid-ering the very wide range of small molecule analytes where fast, high-performance assay systems are required in for example, human and veterinary medical, forensic and defense, and environmental and quality assurance applica-tions within the water, food, and beverage industries, these are critical problems that required a solution.

It is for these reasons, the positive readout (more analyte—more signal) anti-complex and selective antibody assay systems were developed. These systems provide direct measurement of the actual fraction of capture anti-body bound by analyte and consequently share operational advantages seen with large analyte sandwich methods.

The Anti-complex AssayThe anti-complex immunoassay system represents an extremely simple to use direct readout system for small molecules (Self, 1985). In this system an antibody recog-nizes the changed characteristics of another antibody whose binding site is occupied by target analyte (Fig. 1).

Whether these characteristics are a result of a new epit-ope composed of analyte and antibody, or an altered con-formational state of the occupied antibody, is irrelevant to the utility of the assay. The main point is that the analyte-bound fraction of antibody is determined and provides a signal commensurate with the amount of analyte occupy-ing the binding sites on the capture antibody.

Application of the system to the determination of digoxin (Self et al., 1994) has demonstrated the clear advan-tages of this anti-complex approach in terms of the critical parameters of sensitivity, specificity, precision, and speed. For example, it has been shown that, if required, assays can be conducted with immuno-incubation times of at least as short as 1 min. The assays exhibit high precision with a flat “U-shaped” precision profile characteristic of two-site

Non-competitive Immunoassays for Small Molecules—the Anti-complex and Selective Antibody SystemsColin H. Self (colinself@selectiveantibodies.com)

Stephen ThompsonTheresa StreetKelly J. LambGordon DuffinJohn L. DessiMaggie Turnbull

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62 The Immunoassay Handbook

sandwich immunoassays rather than the more “V-shaped” form typically found with competitive assays.

An example of the high performance of the system is given in Fig. 2, which shows a standard curve for digoxin produced in our laboratory by an anti-complex antibody in conjunc-tion with an anti-digoxin primary antibody. In this case, the primary antibody is labeled with surface-bound anti-complex antibody, but as we have demonstrated, the assay can be used in the converse orientation with surface-bound primary anti-body and labeled anti-complex antibody (Self et al., 1994).

Other secondary antibody systems have been developed. Voss and colleagues (e.g., Carrero et al., 1996) have devel-oped antibodies binding anti-FITC antibody in the pres-ence of its ligand. Ullman et al. (1993) described a secondary antibody that binds an anti-cannabinoid antibody in the presence of THC and results in a system of increased spec-ificity. Similarly, the work by Towbin et al. (1995) for the small peptide angiotensin II has resulted in an assay system of higher performance than obtainable by the conven-tional competitive assay format.

In addition to the other advantages of the anti-complex system, our work has demonstrated the particular benefits of increased specificity resulting from the anti-complex anti-body approach (Self et al., 1994; Winger et al., 1996). We have shown, for example, that the anti-complex assay for digoxin is less intrinsically susceptible to digoxin-like immu-noreactive factors (DLIF) artifacts than competitive formats. Such increase in specificity is not surprising given the require-ment of a hapten to participate as an active component of the assay (whether by contributing to a new determinant in the binding site or mediating a conformational change in the

capture antibody) rather than passively competing with labeled competitor. Such enhanced specificity is also seen in the sys-tems of Ullman (Ullman et al., 1993), Towbin et al. (1995), and Carrero et al. (1996), indicating that systems employing such secondary antibodies can elicit a better specificity pro-file from the primary antibody than is achievable by the use of the same primary antibodies used in comparative competi-tive systems. Even higher specificities were obtained by means of format changes (Self et al., 1996) and the multiple binding assay system (Self, 1993b, Winger et al., 1996).

FIGURE 2 Anti-complex assay. © 2013 Selective Antibodies Ltd.

FIGURE 1 Anti-immune complex assay for small molecules. © 2013 Selective Antibodies Ltd.

63CHAPTER 2.2 Non-competitive Immunoassays for Small Molecules—the Anti-complex and Selective Antibody Systems

Selective Antibody Immunometric AssayThe anti-complex approach convinced us that measuring the bound rather than unbound sites in a positive readout approach was indeed clearly advantageous. We were, however, looking for a simpler means of doing this, not requiring the complexity of anti-complex antibodies. We found that this could indeed be achieved by means of the system that we have named the Selective Antibody System (Self, 1989). The principle of this approach is that (i) a primary antibody against the small molecular weight analyte and (ii) a secondary antibody against this primary antibody are selected such that the secondary antibody can still bind to the primary antibody when it has bound to the small molecule analyte but not when, in the absence of analyte, the primary antibody has bound to a specific blocking substance. However, when an analyte is present and has bound to the primary antibody, binding between primary and secondary antibodies takes place. The system can consequently be employed for the quan-titative determination of the analyte in a positive readout assay system where more analyte results in more signal as shown in Fig. 3.

This masking of binding sites unoccupied by analyte in the test sample can be accomplished, for example, by the use of a hapten conjugate, such that when it is present, its size prevents the binding of selective antibody. Alterna-tively, an anti-idiotypic antibody, which specifically recog-nizes the discrete binding site of the analyte, can be used as a masking reagent. In either situation, binding of selective antibody can then be used to quantitatively determine the presence of analyte.

We have produced a range of selective antibody systems that function not only with blocking anti-idiotypic anti-bodies but also with hapten conjugates working as useful blocking reagents. In addition, Barnard and Kohen (1990) and coworkers have used our approach, with anti-idiotypic blocking reagents in the system they subsequently termed the idiometric assay, for estradiol, and have followed up this demonstration with very interesting assays for proges-terone (Barnard et al., 1995a) and estrone-3-glucuronide (Barnard et al., 1995b). Similarly, Kobayashi demonstrated selective antibody noncompetitive anti-idiotypic assay sys-tems for ursodeoxycholic acid 7-N-acetylglucosamine (Kobayasi et al., 2003a) and 11-deoxycortisol (Kobayasi et al., 2003b).

The Universal Selective Antibody SystemBoth the anti-complex and selective antibody systems were found to be capable of providing rapid analytical methods such as lateral flow dipstick methods. However, speed of analysis needs to be partnered by speed of technical devel-opment of actual devices for each new analyte as required. The limiting factor to both the previous selective systems was that different specific custom-made secondary antibod-ies had to be generated against each new primary antibody used for each new analyte needing very time-consuming development and optimization.

This problem of development time was solved by the invention of the “universal” selective antibody system in which “generic” anti-immunoglobulin secondary antibod-ies function as selective antibodies. These may therefore be held ready for use and optimization with new primary antibodies. Our focus has been very much toward the use of these antibodies on rapid testing lateral flow diagnostic systems in which we have demonstrated that they function exceedingly well, employed, for example, as in Fig. 4.

The components of the system are shown in Fig. 4a with the generic anti-immunoglobulin antibody employed on the dipstick as the trap (visualization) line. The particles to be visualized (such as gold particles) are prepared coated with the primary antibody and are pro-vided with the specific blocking substance. When sample is added, analyte in the sample binds the primary anti-body on the gold, the remaining unbound primary anti-body sites being bound by blocker. The mixture travels up the stick to the visualization capture zone comprising the “generic” secondary antibody against primary anti-body. Blocker-bound gold is prevented from binding in the visualization zone and continues to flow past it as shown in Fig. 4b. Analyte-bound gold can, however, bind and is visualized providing a positive line as shown in Fig. 4c. Within the set limits of the system, the more analyte present the more intense the line over the zero background. A video representation of the system in oper-ation can be viewed on the internet at www.selectiveanti-bodies.com. The results are easily read by eye, being not only easy to see but quite intuitive (a positive signal means a positive presence) or, alternatively, by means of a simple handheld reader.

FIGURE 3 The selective antibody immunometric assay for small molecules. (The color version of this figure may be viewed at www.immunoassayhandbook.com). © 2013 Selective Antibodies Ltd.

64 The Immunoassay Handbook

(a) (b)(c)

FIGURE 4 (a) Components of the universal positive readout lateral flow system. (b) The universal system – no analyte - no line. (c) The universal system – positive analyte – positive line. (The color version of this figure may be viewed at www.immunoassayhandbook.com). © 2013 Selective Antibodies Ltd.

FIGURE 5 Highly sensitive detection of the organophosphate insecticide chlorpyrifos. (The color version of this figure may be viewed at www.immunoassayhandbook.com). © 2013 Selective Antibodies Ltd.

FIGURE 6 Rapid clear detection of trinitrotoluene. (The color version of this figure may be viewed at www.immunoassayhandbook.com). © 2013 Selective Antibodies Ltd.

65CHAPTER 2.2 Non-competitive Immunoassays for Small Molecules—the Anti-complex and Selective Antibody Systems

The system has been found to demonstrate distinct and important advantages over classical competitive- format systems. For example, a clear advantage of this system is that a positive detection can be established very early on—as soon as a positive line develops over the blank white background of the stick—unlike in a classical competitive-format system in which the absence of a developing line needs to be established in comparison to what a zero stan-dard would give. Such a positive detection can be achieved with the system within seconds of addition of sample.

Numerous examples of the system have now been developed for drugs, synthetic toxins (such as chlorpyrifos), natural toxins (such as the mycotoxins), CBRN (chemical, biological, radiological, nuclear) agents and explosives, hor-mones, and the like. These have been developed for rapid simple analysis in the food, beverage, and water industries, human and animal health, the forensic and security sectors as well as environmental fields. The wide general applicability of the system for small molecule detection and quantification is indicated by the diversity of examples shown in Figs 5–7.

Figure 5 demonstrates the system for the organophos-phate insecticide chlorpyrifos in which rapid analysis below 1 ppb is achieved with simple dipsticks that can be machine read or readily read by eye. Figure 6 demonstrates the system in a cassetted form for rapid detection of trini-trotoluene, and Fig. 7 shows results of a system developed for the highly toxic mycotoxin aflatoxin.

ConclusionsOur work has shown that positive readout systems have dis-tinct advantages over the classical competitive-format (neg-ative) system. The strengths of these positive systems are particularly evident in lateral flow dipstick formats where a positive signal developing from a clear zero background is very much easier to see and interpret than the indirect signal obtained in the negative systems, where a positive sample produces a reduction of a high zero signal such as a color. The simplification of the positive read-out systems that we

have now achieved should be appreciated by both users and manufacturers in the generation of high-performance diag-nostic systems in areas such as therapeutic drugs, drugs of abuse, natural and synthetic toxins, and pesticides. Direct applications include the food, beverage and water industries, forensics, security, and human and veterinary medicine.

AcknowledgementsThe authors would like to thank the European Union for the Framework VII Award number 242,377 under which some of the above work was conducted.

References and Further ReadingBarnard, G. and Kohen, F. Idiometric assay: a noncompetitive immunoassay for

small molecules typified by the measurement of estradiol in serum. Clin. Chem. 6, 1945–1950 (1990).

Barnard, G., Osher, J., Lichter, S., Gayer, B., De Boever, J., Limor, R., Ayalon, D. and Kohen, F. The measurement of progesterone in serum by a non- competitive idiometric assay. Steroids 60, 824–829 (1995a).

Barnard, G., Amir-Zaltsman, Y., Lichter, S., Gayer, B. and Kohen, F. The mea-surement of oestrone-3-glucuronide in urine by non-competitive idiometric assay. J. Steroid Biochem. Mol. Biol. 5, 107–114 (1995b).

Bonnar, J., Flynn, A., Freundl, G., Kirkman, R., Royston, R. and Snowden, R. Personal hormone monitoring for contraception. Br. J. Fam. Plann. 24, 128–134 (1999).

Carrero, J., Mallender, W.D. and Voss, Jr., E.W. Anti-metatype antibody stabiliza-tion of Fv 4-4-20 variable domain dynamics. J. Biol. Chem. 271, 11247–11252 (1996).

Ekins, R.P. An overview of present and future ultrasensitive non-isotopic immuno-assay development. Clin. Biochem. Rev. 8, 12–23 (1987).

Self, C.H. The impact of new immunodiagnostic technologies. In: Rapid Methods and Automation in Microbiology and Immunology, (ed Spencer, R.C. et al.) (Pub intercept, England, 1993).

Kobayasi, N., Kubota, K., Oiwa, H., Goto, J., Niwa, T. and Kobayashi, K. Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity. J. Immunol. Methods 272, 1–10 (2003a).

Kobayasi, N., Shibusawa, K., Kubota, K., Hsegawa, N., Sun, P., Niwa, T. and Goto, J. Monoclonal anti-idiotype antibodies recognizing the variable region of high-affinity antibody against 11-deoxycortisol. Production, characterization and application to a sensitive noncompetitive immunoassay. J. Immunol. Methods 274, 63–75 (2003b).

May, K. Unipath Clearblue One Step™, Clearplan One Step™ and Clearview™. In: The Immunoassay Handbook, 1st edn. (ed Wild, D.), (Stockton Press, New York, 1994).

Self, C.H. Antibodies manufacture and use. World Intellectual Patent Cooperation Treaty Publication No. 85/04422 (1985).

Self, C.H. Determination method, use and components. Patent Corporation Treaty Publication No. WO89/05453 (1989).

Self, C.H. The impact of new immunodiagnostic technologies. In: Rapid Methods and Automation in Microbiology and Immunology, (ed Spencer, R.C. et al.) (Intercept, Andover, UK, 1993a).

Self, C.H. Multiple binding patent. World Intellectual Patent Cooperation Treaty Publication No. WO 93/14404 (1993b).

Self, C.H., Dessi, J.L. and Winger, L.A. Wash steps and multiple binding formats in ultra-specific immunoassays for small molecules. Clin. Chem. 42, 1527–1531 (1996).

Self, C.H., Dessi, J.L. and Winger, L.A. High-performance assays of small mole-cules: enhanced sensitivity, rapidity and convenience demonstrated with a noncompetitive immunometric anti-immune complex assay system for digoxin. Clin. Chem. 40, 2035–2041 (1994).

Self, C.H. and Chard, M. Assay devices and methods and components for use therein. World Intellectual Patent Cooperation Treaty Publication WO/2008/129302 (2008).

Towbin, H., Motz, J., Oroszlan, O. and Zingel, O. Sandwich immunoassay for the hapten angiotensin II. A novel assay principle based on antibodies against immune complexes. J. Immunol. Methods 181, 167–176 (1995).

Ullman, E.F., Milburn, G., Jelesko, J., Radika, K., Pirio, M., Kempe, T., et al. Anti-immune complex antibodies enhance affinity and specificity of primary anti-bodies. Proc. Natl Acad. Sci. U. S. A. 90, 1184–1189 (1993).

Winger, L.A., Dessi, J.L. and Self, C.H. Enhanced specificity for small molecules in a convenient format which removes limitations imposed by competitive immunoassay. J. Immunol. Methods 199, 185–191 (1996).

FIGURE 7 Highly sensitive quantitative detection of the mycotoxin aflatoxin. (The color version of this figure may be viewed at www.immunoassayhandbook.com). © 2013 Selective Antibodies Ltd.