Pushing Antibody-Based Labeling Systems to Higher Sensitivity by

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Pushing Antibody-Based Labeling Systems to Higher Sensitivity by Linker-Assisted Affinity Enhancement Hans H. Gorris,†,‡ Steffen Bade,† Niels R€ockendorf,† Milan Franek,§ and Andreas Frey*,† †

Division of Mucosal Immunology and Diagnostics, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 93040 Regensburg, Germany § Department of Analytical Biotechnology, Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic ‡

bS Supporting Information ABSTRACT: The sensitivity of antibody/hapten-based labeling systems is limited by the natural affinity ceiling of immunoglobulins. Breaking this limit by antibody engineering is difficult. We thus attempted a different approach and investigated if the so-called bridge effect, a corecognition of the linker present between hapten and carrier protein during antibody generation, can be utilized to improve the affinity of such labeling systems. The well-known haptens 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D) were equipped with various linkers, and the resulting affinity change of their cognate antibodies was analyzed by ELISA. Anti-2,4-DNP antibodies exhibited the best affinity to their hapten when it was combined with aminobutanoic acid or aminohexanoic acid. The affinity of anti-2,4-D antibodies could be enhanced even further with longer aliphatic spacers connected to the hapten. The affinity toward aminoundecanoic acid-2,4-D derivatives, for instance, was improved about 100-fold compared to 2,4-D alone and yielded detection limits as low as 100 amoles of analyte. As the effect occurred for all antibodies and haptens tested, it may be sensible to implement the bridge effect in future antibody/hapten-labeling systems in order to achieve the highest sensitivity possible.

’ INTRODUCTION As most biomolecules lack an intrinsic reporter unit for sensitive detection, bioanalytical set-ups usually require analyte labeling, either with direct labels, such as fluorophores,1 or with indirect labels for binding secondary detection reagents.2 Although direct labels are read out faster, indirect labels can be more sensitive when used in combination with signal amplification reagents. For indirect labeling, a small molecule is attached covalently to a biomolecule, e.g. by using click chemistry.3 This ligand is subsequently detected by a specific receptor/probe molecule. One of the most widely used indirect labeling strategies exploits the exceptionally high affinity of (strept)avidin to biotin,4 which lies in the pico- to femtomolar range. Here, biotin usually serves as a ligand and (strept)avidin, equipped with a reporter molecule (enzyme, dye, radio nuclide), acts as the probe. A drawback of labeling with biotin, however, is its endogenous occurrence in biological samples which may interfere with analyte detection.5 Most other indirect labels, such as the digoxigenin (DIG) system,6 are based on the binding of antibodies to ligands of low molecular weight, so-called haptens. Albeit similar in design, such antibody-based labeling systems are inferior to (strep)avidinbiotin as they have a natural upper affinity ceiling7 of approximately 1010 M1. In practice, their affinity may even be lower, as it cannot be ruled out that the antibody requires the context of the carrier protein onto which the hapten was coupled for immunization in order to optimally r 2011 American Chemical Society

recognize the hapten structure, a phenomenon known as the bridge effect.8 The affinity reconstitution or improvement caused by a hapten-carrier bridge has usually been considered an adverse effect in particular for competitive ELISAs that rely on similar antibody affinities to the analyte, i.e. the free hapten, and the labeled hapten used for detection, the so-called tracer.913 For example, the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) can only be detected accurately in environmental samples if the bridge effect is avoided.14,15 As most studies are focused on avoiding the bridge effect of 2,4-D and other haptens, its potential benefits for high affinity labeling have not been investigated in detail before. We have recently introduced 2,4-D as a nonbiogenic label that avoids cross-reactivity with biological materials. This labeling strategy was applied to the detection of large (ovalbumin) and small (insulin) proteins, glycoproteins (mucin), DNA, and peptides.16 Moreover, the efficient immobilization and detection of 2,4-D labeled peptides on microtiter plates allowed the design of a robust proteolysis assay17 that operates even with crude intestinal juice preparations.18 In the course of these studies, we observed that the affinity between the 2,4-D label and its cognate antibody was strongly enhanced when an aliphatic linker was attached to Received: April 7, 2011 Revised: July 11, 2011 Published: July 12, 2011 1619

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the carboxyl group of 2,4-D. Here, we explore this phenomenon in depth by investigating the binding of 2,4-D equipped with various linkers to three anti-2,4-D antibody clones and comparing this setup to the classical and extensively described 2,4dinitrophenyl (2,4-DNP) haptenantibody pair.19,20

’ EXPERIMENTAL SECTION Reagents and Buffers. Synthesis reagents were purchased from Sigma-Aldrich (Taufkirchen, Germany), Merck (Darmstadt, Germany), Merck Biosciences (Schwalbach, Germany), Advanced Chemtech (Louisville, KY, USA), Peptech (Burlington, MA, USA), MP Biomedicals (Eschwege, Germany), Molecular Biosciences (Boulder, CO, USA), and Polypure (Oslo, Norway). Monoclonal anti-2,4-D IgG antibodies (clone E2/G2, E4/C2, and F6/C10) were generated in mice against the 2,4-D hapten coupled to thyroglobulin according to standard procedures.21 Monoclonal rat anti-DNP IgG antibodies (clone LODNP-1 and LODNP-61, affinity purified) were purchased from Interchim (Montlucon Cedex, France), casein (Hammarsten grade) from BDH (Poole, England), and horseradish-peroxidase-labeled streptavidin (SA-HRP) from Vector (Burlingame, CA, USA). L(ite)-PBS: 10 mM sodium phosphate, pH 7.0, 10 mM NaCl. D(ulbecco’s)-PBS: 2.7 mM KCl, 1.5 mM KH2PO4, 136 mM NaCl, 8.1 mM Na2HPO4, pH 7.3. P(hysiological)PBS:22 8 mM phosphate, pH 7.2, 4.6 mM K+, 111.3 mM Na+, 101.5 mM Cl). ELISA substrate solution: 3 mM H2O2, 1 mM 3,30 ,5,50 -tetramethylbenzidine in 200 mM potassium citrate buffer, pH 4.0.23 Conjugate Synthesis. Conjugate libraries were SPOT-synthesized on a cellulose membrane24 using standard fluorenylmethoxycarbonyl (Fmoc) amino acid protection chemistry as described earlier.17 To allow the release of the synthesis products from the cellulose support, each individual peptide was anchored C-terminally on the cellulose via a proline-ε-lysine sequence, which can be cleaved off the support under diketopiperazine formation.25 The cellulose sheets used as synthesis support were first uniformly derivatized with proline. In the next step, synthesis areas (SPOTs) of the individual conjugates were defined by applying an ε-Fmoc-protected lysine. Onto this anchor, the conjugate chain was assembled by stepwise reactions with active esters of Fmoc-protected “amino acid” building blocks: Biocytin was coupled first on the SPOTs, introducing the biocytin label. Next followed O-(3-aminopropyl)-O0 -(N-diglycolyl-3-aminopropyl)nonaethyleneglycol, establishing a poly(ethylene glycol) (PEG) spacer, then a single alanine (or a series of amino acids to form a short peptide sequence) was added. Then, one of various linker moieties was added, either an aminoalkanoic acid (H 2 N(CH2)n-COOH, n = 111) or a PEG (O-(3-aminopropyl)-O0 -(Ndiglycolyl-3-aminopropyl)-diethyleneglycol). As the last building block, 2,4-DNP or 2,4-D was coupled. After the last coupling round, all protection groups were removed, the conjugates were cleaved off the individual membrane SPOTs, dried in vacuo, dissolved in 1.5 mL of L-PBS 0.005% (w/v) Tween 20, and stored at 80 °C. Conjugates were used without further purification as the antibody capture step on the microtiter plate (see below) already constitutes a purification step (for a detailed protocol on conjugate synthesis see Supporting Information). Conjugate and Antibody Concentrations. SPOT synthesis yields of conjugates were quantitated with a competitive ELISA by determining the half maximal effective concentration (EC50), where the signal intensity is reduced to 50% of the maximal

Figure 1. Analyte detection setup. (A) 2,4-Dinitrophenyl (2,4-DNP) and 2,4-dichlorophenoxyacetyl (2,4-D) residues. (B) Analyte constructs consist of alanine flanked C-terminally by a short PEG stretch and a biocytin handle and N-terminally by a linker moiety and 2,4-DNP- or 2,4-D-hapten. Adherence of the analyte onto an antibody-coated microtiter plate occurs via the 2,4-D- or 2,4-DNP-label. For analyte detection, a streptavidinhorseradish peroxidase (SA-HRP) conjugate binds to the biocytin handle and oxidizes chromogenic TMBH2 to the chromophore TMB.

signal, indicating that equal amounts of competitor and conjugate are present.17 2,4-DNP-antibody concentrations were provided by the manufacturer. Purity of 2,4-D-antibodies was confirmed by gel electrophoresis, and their concentrations were determined by Lowry- and BCA-assay. ELISA. High-bind polystyrene 96-well microtiter plates (Corning, Wiesbaden, Germany) were coated overnight at 4 °C with 75 μL/well of 50 ng/mL antibody in L-PBS. The microtiter plates were then washed three times with 300 μL/well of D-PBS 0.05% (w/v) Tween 20 (D-PBST), blocked with 250 μL/well of 1% (w/v) casein/D-PBS for 34 h at RT, and washed again four times with D-PBST. Conjugates were serially diluted in 75 μL/well of P-PBS 0.005% (w/v) Tween 20 (P-PBST) on the antibodycoated microtiter plate. After 2 h 30 min, microtiter plates were washed four times with 300 μL/well of D-PBST and incubated for 60 min at RT using 75 μL/well of 1 μg/mL SA-HRP in 1% (w/v) casein in D-PBS. After six washes with 300 μL/well of D-PBST, microtiter plates were developed by adding 75 μL/well of ELISA substrate solution. The enzyme reaction was terminated after 30 min by adding 125 μL/well of 1 M H2SO4. Signal intensities in wells were measured at λ = 450 nm with a microtiter plate reader and fitted to a four-parameter logistic function (eq 1):26 ODc ¼

ODmax ODbg þ ODbg c s 1 þ EC50

ð1Þ

where c is the conjugate concentration, ODc the optical density as a function of c. The curve fit yields the maximum signal intensity (ODmax), the background intensity (ODbg), the half maximal effective conjugate concentration (EC50), and the slope at the inflection point (s).

’ RESULTS AND DISCUSSION Detection System. The effects of different linkers on hapten antibody interaction were analyzed using a specially designed 1620

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Figure 2. Influence of different linkers on haptenantibody binding. Microtiter plates were coated with 50 ng/mL of either anti-2,4-DNP- (LODNP-1, LODNP-61) or anti-2,4-D-antibodies (E2/G2, E4/C2, F6/C10). Analytes carrying the corresponding haptens in combination with various aliphatic linkers were added in serial dilutions and detected via their biocytin label (mean ( SEM of three experiments).

ELISA where an immobilized antibody captures an analyte molecule that is equipped with a tag via which the binding is recorded. The analyte was designed as a peptidic conjugate with the hapten label on one side and a detection handle on the other and was synthesized from C-terminus to N-terminus using SPOT-synthesis on a cellulose membrane.24 As 2,4-D and 2,4DNP (Figure 1A) do not carry an amino function that is necessary for chain elongation, they were positioned on the N-terminus. On the C-terminus, we introduced biocytin (biotin attached to the side chain of lysine) as a detection handle because of its exceptionally high binding constant with streptavidin (1015 M1).4 In addition to the terminal labels, the conjugates contained an aliphatic linker adjacent to 2,4-D or 2,4-DNP for analyzing the bridge effect and a hydrophilic PEG-spacer next to the biocytin handle to improve water solubility. The two halves of the construct were connected by a single amino acid (alanine). After the synthesis was completed, the conjugates were released into solution for ELISA analysis. Anti-2,4-D- or anti 2,4DNP-antibodies where coated on a microtiter plate to immobilize the conjugates via the hapten constructs of interest. Bound conjugate was detected and quantified by a streptavidin horseradish peroxidase conjugate mediating a colorogenic reaction (Figure 1B). ELISA signals are fitted with a logistic function (eq 1) that provides ODmax and EC50. It has been discussed whether and how the affinity is reflected in ODmax and EC50.27 Although logistic functions appear to represent the law of mass action,28 care has to be taken as hapten binding to surface immobilized antibodies can be diffusion limited rather than reaction limited.29 Consequently, comparing the affinities of various hapten antibody combinations requires similar surface conditions and analyte sizes in all experiments. The analytes used in our experiments were structurally identical except for the hapten/linker combination, and homogeneity was conferred by the defined synthetic nature of the analyte molecules. Moreover, we coated the microtiter plate surface with the same antibody concentration (50 ng/mL) in all experiments, which we had found to result in an optimal absorbance span between ODbg and ODmax. Finally, it

should be noted that the antibodyanalyte binding is not affected by bivalent interactions30 as each conjugate carries a single hapten label only. Label and Antibody Specificity. Franek et al.21 demonstrated that the anti-2,4-D antibodies used in our experiments are highly specific for 2,4-D and display very limited cross reactivities to structural analogues. Accordingly, replacement of the oxy acetic acid moiety of 2,4-D by acetic acid or oxybutanoic acid lead to a complete loss of binding, whereas extensions at the carboxyl group of the 2,4-D yielded similar results as native 2,4-D. Thus, only derivatizations that result from coupling of amino-functionalized linkers to the carboxyl group of 2,4-D and that leave the original hapten structure intact may increase sensitivity. This is consistent with the bridge effect. Nevertheless, two possible scenarios independent of the haptenantibody interaction but also causative for a signal increase with hapten/linker constructs are conceivable. First, the high sensitivity could be a mere spacer effect that facilitates the accessibility of the captured analyte for the detection reagent on the microtiter plate surface. When we substituted the linker aminoundecanoic acid adjacent to 2,4-D by a hydrophilic polyethylene glycol (PEG) of comparable length, however, no increase in the detection sensitivity was observable, ruling out control of the signal intensity by mere sterical constraints. Second, the aliphatic linker could mediate nonspecific binding of the conjugate to the solid support by interactions with the hydrophobic polystyrene body of the microtiter plate. In fact, aminohexanoic acid-conjugation has already been used to immobilize peptides directly on microtiter plates.31 However, when we applied peptide conjugates carrying an aminoundecanoic acid linker and the structural analogue phenoxyacetic acid instead of 2,4-D onto a microtiter plate coated with anti-2,4-D-antibodies, no signals were detectable. Consequently, the high sensitivity observed with our hapten/linker conjugates cannot result from hydrophobic binding of long aliphatic linkers to the plate material. Influence of Antibody Clones and Different Linkers. The bridge effect has been attributed to a corecognition of the chemical linkage between the hapten and the carrier protein 1621



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Table 1. ODmax and EC50 (amole) for Anti-DNP- and Anti2,4-D Antibodies Obtained with Different Hapten/Linker Combinationsa hapten

moAb

no linker (CH2)1 (CH2)3 (CH2)5 (CH2)10

DNP LODNP-1 ODmax nd

0.28

0.25

0.30

nd

nd

2434

2820

2367

nd

LODNP-61 ODmax nd

0.19

0.39

0.41

0.16

nd

2997

2448

5532

38983

0.19

0.33

EC50 EC50 2,4-D E2/G2

ODmax 0.26 EC50

E4/C2

ODmax 2.19 EC50

F6/C10

1.26

3.30

33989 69230

82714

943

2.20

2.44

3.02

2.24

167535 182901 105113 89140

ODmax 0.56 EC50

a

32172

32988

0.62

1.37

59016 89924

4462

2.22

3.56

76128

1019

nd; not determinable.

used for antibody generation.13 Usually, a hapten is coupled to the carrier protein via an amino function, either on a lysine side chain or on the N-terminus. In the latter case, antibodies should recognize the hapten in context with the N-terminal amino acid sequence of the carrier protein. We tested this for three anti-2,4-D-antibody clones which had been generated with thyroglobulin as a carrier.21 When we investigated 2,4-D-Asn-Ile-Phe-Glu-Tyr-Gln-labeled conjugates, however, ODmax and EC50 were similar to those obtained with a 2,4-D-label not bearing this amino acid stretch, which shows that the N-terminal amino acid sequence of the carrier protein did not contribute to the bridge effect for any of our 2,4-D-antibodies. We hence concentrated on the effect of aliphatic linkers which may mimic the configuration of a lysine side chain. Dose response curves with serially diluted analytes reflect the influence of such bridges on assay performance (Figure 2). None of the bridges had a marked adverse effect on assay performance, but not all linkers improved the ELISA curves. Whether or not a gain in slope and ODmax and a reduction of EC50 occurred was dependent on the specific haptenlinkerantibody combination. The ODmax and EC50 values of each combination are summarized in Table 1. For the 2,4-DNP-system, the results were in good agreement with the bridge theory: both anti-2,4-DNP-antibody clones recognize 2,4-DNP most efficiently when an aliphatic linker similar in length (aminobutanoic acid or aminohexanoic acid) to a lysine side chain was used. All anti-2,4-D-antibody clones, by contrast, show a remarkably different relationship between affinity and linker length. While shorter linkers such as glycine lead to a similar ODmax as obtained with the respective construct in the 2,4-DNP label system, the 2,4-D system meets no maximum with aminobutanoic acid or aminohexanoic acid. Instead, ODmax is further increasing up to aminoundecanoic acid. This tendency is followed by all anti-2,4-D antibody clones, albeit with individual variations in the strength of the bridge effect, e.g. with clone E2/G2, we observe a 13-fold increase in ODmax with the aminoundecanoic acid linker compared to the hapten alone, with clone E4/C2 only a 1.4-fold increase was detected. An increased ODmax can be attributed in part to lower release rates (koff) of the analyte with long aliphatic linkers, effectuating an equilibrium shift toward more bound analyte and hence a higher ODmax: the stronger the capture antibody binds the analyte the less the analyte will be released from the complex during subsequent washing and incubation steps.16 Additionally, conformational

Figure 3. Influence of increasing linker length and composition on haptenantibody affinity. Analytes carried linear aliphatic linkers ((CH2)n), varying from glycine (n = 1) to aminododecanoic acid (n = 11), coupled to the carboxyl group of 2,4-D. Microtiter plates were coated with 50 ng/mL of anti-2,4-D antibody clone E2/G2 to capture different analyte constructs, and analyte binding was quantitated via the biotin label. ODmax (0) increases and EC50 ([) decreases with the length of the aliphatic linker. By contrast, a hydrophilic PEG linker of similar length as aminododecanoic acid does not improve analyte binding compared to 2,4-D alone ((CH2)0) (mean ( SEM of three experiments).

effects may play a role. It was shown that haptens can induce conformational changes at the antibody binding sites during the binding process,32,33 and structural evidence was provided for conformational diversity in monoclonal antibodies.34 Thus, a population of monoclonal antibodies can be composed of different preexisting isomers that may be converted to a “highaffinity conformation” by an induced-fit isomerization. If hapten binding is not strong enough, it may not be able to induce a conformational shift in some antibody isomers necessary for accommodating the hapten. Consequently, only a fraction of capture antibodies will be occupied, even if the antibody seems to be “saturated”, eventually resulting in a lower ODmax. When looking at the EC50 values, the 2,4-DNP conjugates performed best when short aliphatic linkers were used. By contrast, 2,4-D binding by all antibody clones improved considerably with linkers above aminobutanoic acid, displaying a 30- to 40-fold decrease in EC50 with the aminoundecanoic acid linker compared to 2,4-D alone. Here, the lowest EC50 of 1 fmole conjugate was obtained with the anti-2,4-D-antibody clones E2/G2 and F6/C10, and an analyte detection limit (the so-called end point titer) of approximately 100 amoles was achieved. Thus, labeling with 2,4-D-aminoundecanoic acid leads to “superoptimal” analyte detection, which seems to be largely independent of the anti2,4-D-antibody clone. The superoptimal bridge effect prompted us to investigate the conjugate detection with 2,4-D in detail. Aliphatic linkers were elongated successively by (CH2)n from glycine (n = 1) to aminododecanoic acid (n = 11) (Figure 3). An increasing aliphatic linker length resulted in a sigmoid-like response profile of ODmax and 1/log10(EC50), with an inflection point between (CH2)7 and (CH2)8. The inflection point indicates where increments in the length of the aliphatic linker enhance the affinity most efficiently. While longer linkers still yield higher affinities, the gain in affinity is less pronounced and levels down to a point where further increases are insignificant.

’ CONCLUSION We could show that aliphatic spacers next to the hapten exert distinct bridge effects, considerably improving the binding of 1622


Bioconjugate Chemistry 2,4-D or 2,4-DNP to their cognate antibodies. In contrast to 2,4DNP conjugates, 2,4-D does not tap the full potential of antibody binding when adjacent to aliphatic linkers which have the length of a lysine side chain, but it clearly outperforms the 2,4DNP system when combined with long aliphatic linkers. Our findings demonstrate the potential of the bridge effect when using haptens in combination with aliphatic linkers for high affinity labeling and provide a guideline for optimizing other antibodyhapten pairs as sensitive labeling/detection systems for biomolecules.

’ ASSOCIATED CONTENT

bS

Supporting Information. Details on conjugate synthesis and analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: + 49-4537-188 5620. Fax: + 49-4537-188 6930. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was funded in part by grants from the German Ministry for Education and Research (BMBF, grants 01KO0113 and 13N8473) and from the German Research Council (DFG, grant Fr 958/4-1). ’ REFERENCES (1) Kele, P., Mezo, G., Achatz, D., and Wolfbeis, O. S. (2009) Dual labeling of biomolecules by using click chemistry: a sequential approach. Angew. Chem. Int. Ed. 48, 344–347. (2) Zhang, Q., and Guo, L. H. (2007) Multiple labeling of antibodies with dye/DNA conjugate for sensitivity improvement in fluorescence immunoassay. Bioconjugate Chem. 18, 1668–1672. (3) Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless, K. B. (2002) A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596–2599. (4) Green, N. M. (1990) Avidin and streptavidin. Methods Enzymol. 184, 51–67. (5) Hamblett, K. J., Kegley, B. B., Hamlin, D. K., Chyan, M. K., Hyre, D. E., Press, O. W., Wilbur, D. S., and Stayton, P. S. (2002) A streptavidin-biotin binding system that minimizes blocking by endogenous biotin. Bioconjugate Chem. 13, 588–598. (6) McCreery, T. (1997) Digoxigenin labeling. Mol. Biotechnol. 7, 121–124. (7) Foote, J., and Eisen, H. N. (1995) Kinetic and affinity limits on antibodies produced during immune-responses. Proc. Natl. Acad. Sci. U.S.A. 92, 1254–1256. (8) Franek, M. (1987) Structural aspects of steroid-antibody specificity. J. Steroid Biochem. 28, 95–108. (9) Luttrell, B. M., and Henniker, A. J. (1991) Reaction coupling of chelation and antigen-binding in the calcium ion-dependent antibodybinding of cyclic-AMP. J. Biol. Chem. 266, 21626–21630. (10) Abuknesha, R. A., and Luk, C. (2005) Paraquat enzymeimmunoassays in biological samples: assessment of the effects of haptenprotein bridge structures on assay sensitivity. Analyst 130, 956–963. (11) Nara, S., Tripathi, V., Chaube, S. K., Rangari, K., Singh, H., Kariya, K. P., and Shrivastav, T. G. (2008) Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay. Anal. Biochem. 373, 18–25.

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Pushing Antibody-Based Labeling Systems to Higher Sensitivity by

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