Toward the Limits of Sandwich Immunoassay of Very Low Molecular

Feb 24, 2010 - To whom correspondence should be addressed. E-mail: [email protected] (F.T.); [email protected] (C.C.). Tel.: +33-1-69-08-...
0 downloads 5 Views 2MB Size
Download PDF
Anal. Chem. 2010, 82, 2536–2540

Toward the Limits of Sandwich Immunoassay of Very Low Molecular Weight Molecules Julia Quinton,† Lise Charruault,† Marie-Claire Nevers,‡ Herve´ Volland,‡ Jean-Pierre Dognon,§ Christophe Cre´minon,*,‡ and Fre´de´ric Taran*,† CEA, iBiTecS, Gif sur Yvette, F-91191, France A model study aiming at exploring the limits of sandwich immunoassays of very small molecules is described. Combinatorial association of antibody couples to detect small molecules constituted by two small epitopes connected via different linear spacers was used to investigate the minimum size of compounds susceptible to be simultaneously bound by two distinct antibodies. The results clearly indicated that despite the fact that below 10 carbon atoms unfavorable interactions between antibodies took place, molecules bearing two epitopes separated by only 5 carbon atoms might be directly detected by sandwich immunoassays. Enzyme immunoassay is one of the most popular analytical tools for the specific detection and quantification of target molecules in complex media. This method is routinely used by industry mainly for clinical diagnosis purposes. Among the immunoassay techniques, sandwich enzyme-linked immunosorbent assay (ELISA) is the most widely used. This assay requires two distinct antibodies that simultaneously bind the desired analyte. The first antibody, immobilized onto the solid support captures the analyte out of solution, while the second, conjugated to a suitable reporter enzyme (e.g., horseradish peroxidase, alkaline phosphate or acetylcholine esterase) allows for secondary signal amplification through substrate turnover. By definition, this technique is thus a noncompetitive analytical method consequently offering several advantages such as higher sensitivity and specificity, wider dynamic range (excess of reagents), and lower background than competitive immunoassays.1 However, sandwich immunoassays suffer from a fundamental limitations that the molecule to be measured must be large enough to have two epitopes to be bounded. The identification of pairs of antibodies that can bind simultaneously very small molecules (haptens) is therefore assumed to be a very difficult task since the large size of the antibody molecule inherently prevents a second antibody from binding due to steric hindrance. This assumption has been reinforced by X-ray structural analyses of hapten-antibody complexes showing that small molecules are buried deep in the * To whom correspondence should be addressed. E-mail: [email protected] (F.T.); [email protected] (C.C.). Tel.: +33-1-69-08-26-85. Fax: -33-1-6908-79-91. † Service de Chimie Bioorganique et de Marquage, Commissariat a` l’Energie Atomique, Saclay. ‡ Service de Pharmacologie et d’Immunoanalyse, Commissariat a` l’Energie Atomique, Saclay. § Service de Chimie Mole´culaire, Commissariat a` l’Energie Atomique, Saclay. (1) Ekins, R. Nature 1980, 284, 14–15.

2536

Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

binding site, with surface areas of 200-400 Å2.2-6 This drawback prompted researchers to develop alternative strategies such as the use of short peptide loops,7 antimetatype8 or anti-idiotype9 antibodies, solid-phase-covalent immobilization of epitope,10-12 or open sandwich immunoassay approaches.13-15 Direct sandwich immunoassays of small molecules are therefore rare, and to date, the minimum size of molecules allowing the binding of two antibodies remains an unanswered question. To the best of our knowledge, the smallest molecule measured by true sandwich immunoassay was the octapeptide Angiotensine II (MW ) 1048 g · mol-1, 1475 Å2).16 During the course of our efforts in using ELISA for the monitoring of chemical reactions, we proved that two antibodies might simultaneously bind molecules smaller than Angiotensine II.17 In this contribution, we propose to look at the limits of sandwich immunoassays and explore, on a model study, the minimum size of molecules that can be bound by two distinct antibodies. For such an investigation, we used two model antibody populations that have been previously produced by our group and (2) Arevalo, J. H.; Taussig, M. J.; Wilson, I. A. Nature 1993, 365, 859–863. (3) Jeffery, P. D.; Schildbach, J. F.; Chang, C.-Y. Y.; Kussie, P. H.; Margolies, M. N.; Sheriff, S. J. Mol. Biol. 1995, 248, 344–360. (4) Tanaka, F.; Kinoshita, K.; Tanimura, R.; Fujii, I. J. Am. Chem. Soc. 1996, 118, 2332–2339. (5) Romesberg, F. E.; Spiller, B.; Schultz, P. G.; Raymond, C. S. Science 1998, 279, 1929–1933. (6) Xu, J.; Deng, Q.; Chen, J.; Houk, K. N.; Bartek, J.; Hilvert, D.; Wilson, I. A. Science 1999, 286, 2345–2348, and references therein. (7) Gonzalez-Techera, A.; Kim, H. J.; Gee, S. J.; Last, J. A.; Hammock, B. D.; Gonzalez-Sapienza, G. Anal. Chem. 2007, 79, 9191–9196. (8) Voss, J. E. W.; Miklasz, S. D.; Petrossian, A.; Dombrink-Kurtzman, M. A. Mol. Immunol. 1988, 25, 751–759. (9) Kobayashi, N.; Iwakami, K.; Kotoshiba, S.; Niwa, T.; Kato, Y.; Mano, N.; Goto, J. Anal. Chem. 2006, 78, 2244–2253. (10) Volland, H.; Pradelles, P.; Ronco, P.; Azizi, M.; Simon, D.; Cre´minon, C.; Grassi, J. J. Immunol. Meth. 1999, 228, 37–47. (11) Volland, H.; Pradelles, P.; Taran, F.; Buscarlet, L.; Cre´minon, C. J. Pharm. Biomed. Anal. 2004, 34, 737–752. (12) Buscarlet, L.; Volland, H.; Dupret-Carruel, J.; Jolivet, M.; Grassi, J.; Cre´minon, C.; Taran, F.; Pradelles, P. Clin. Chem. 2001, 47, 102–109. (13) Ueda, H.; Tsumoto, K.; Kubota, K.; Suzuki, E.; Nagamune, T.; Nishimura, H.; Schueler, P. A.; Winter, G.; Kumagai, I.; Mahoney, W. C. Nat. Biotechnol. 1996, 14, 1714–1718. (14) Suzuki, C.; Ueda, H.; Mahoney, W.; Nagamune, T. Anal. Biochem. 2000, 286, 238–246. (15) Lim, S.-L.; Ichinose, H.; Shinoda, T.; Ueda, H. Anal. Chem. 2007, 79, 6193– 6200. (16) Grassi, J.; Cre´minon, C.; Frobert, Y.; Etienne, E.; Ezan, E.; Volland, H.; Pradelles, P. Clin. Chem. 1996, 42, 1532–1536. (17) Vicennati, P.; Bensel, N.; Wagner, A.; Cre´minon, C.; Taran, F. Angew. Chem., Int. Ed. 2005, 44, 6863–6866. 10.1021/ac100058f  2010 American Chemical Society Published on Web 02/24/2010

Scheme 2. Synthesis of Compounds 1-8

Figure 1. Schematic diagram of direct sandwich immunoassay for HVA-HIS conjugates. Specific anti-HIS monoclonal antibodies (blue) are immobilized on a solid support, and anti-HVA monoclonal antibodies (red) are conjugated with acetylcholine esterase (AchE).

Scheme 1. Structures of HVA and HIS Haptens for Immunogen Preparation and Antibody Production

whose binding properties have been characterized.18,19 The first set of monoclonal antibodies (mAbs) has been raised against histamine derivatives and bound HIS epitope with good affinities (up to nanomolar; Figure 1). The second set of mAbs was raised against homovanillic acid derivatives and are specific to HVA epitope with lower affinity (∼micromolar; Figure 1). Any structural modification on HIS or HVA results in dramatic loss of recognition and further binding by their corresponding antibodies. The exploration of the combinatorial association of these two mAbs populations with a series of HIS-HVA conjugates allowed us to obtain a rapid readout and to investigate the minimum size of the spacer separating both epitopes required to get efficient sandwich immunoassays. EXPERIMENTAL SECTION Chemicals and Apparatus. All reagents were used directly as obtained commercially (Aldrich). Melting points were determined on a Bu¨chi 535 capillary melting point apparatus and are uncorrected. Flash chromatography was carried out on Merck silica gel (40-63 µm). IR spectra were obtained on a Perkin-Elmer system 2000 FT-IR spectrophotometer. 1H NMR (400 MHz), 13C NMR (100 MHz) were measured on a Bruker Avance 400 MHz spectrometer. Electrospray mass spectra were obtained using a ESI/TOF Mariner Mass Spectrometer. Monoclonal Antibodies. Anti-HVA monoclonal antibodies were produced after immunizing Biozzi’s mice with HVA/KLH immunogen prepared through glutaraldehyde coupling of HVA hapten (Scheme 1) to keyhole limpet hemocyanin (KLH) as we previously described.20,21 Anti-HIS monoclonal antibodies were obtained after immunizing Biozzi’s mice with HIS/KLH immunogen prepared by reacting HIS hapten with KLH previously modified by maleimide moieties. (18) Taran, F.; Frobert, Y.; Cre´minon, C.; Grassi, J.; Olichon, D.; Mioskowski, C.; Pradelles, P. Clin. Chem. 1997, 43, 363–368. (19) Volland H. Application du proce´de´ SPIE-IA au dosage du leucotrie`ne C4, de l’Angiotensine II et de l’Histamine. Ph.D. Thesis, University of Paris VI, 1999. (20) Pradelles, P.; Antoine, C.; Lellouche, J. P.; Maclouf, J. Methods Enzymol. 1990, 187, 82–89. (21) Pradelles, P.; Grassi, J.; Maclouf, J. Anal. Chem. 1985, 57, 1170–1173.

Sandwich Immunoassays. A 100 µL portion of HIS-HVA conjugates 1-8 (Scheme 2) were added at a range of concentration from 0.05 to 50 nM for 6-8 and 5 to 500 nM for 1-5 to the wells of a microtiter plate previously coated with mAb HIS-203 (direct adsorption to the polystyrene support). After 5 h of incubation at room temperature, the plates were washed and 100 µL of biotinilated mAb HVA-46 (200 ng/mL) were added. After 12 h of immunological incubation at 4 °C, the plates were washed and 200 µL of AChE-Streptavidine conjugate (2 UE), prepared and stored as previously described,20,21 were added. After 2 h incubation at room temperature, the plates were washed. Ellman’s reagent was then added, and the absorbance related to the solid phase-bound AChE activity was measured at 414 nm. All measurements for standard or samples were made in duplicate. Kd,app Determination Using Competitive Immunoassays. Kd,app determinations were carried out according to standard protocols previously described.18 Briefly, in wells of a microtiter plate previously coated (direct adsorption to the solid support) with polyclonal goat antimouse antibody (Jackson Immuno. Research Laboratories Inc.), 50 µL of compounds 1-8 solutions prepared in EIA buffer at a range of concentration from 0.1 nM to 10 µM were added to a solution containing 50 µL of the enzymatic tracer HIS-AChE or HVA-AChE (prepared and stored as previously described)18 and 50 µL of HIS mAbs or HVA mAbs in EIA buffer. After 12 h of incubation at 4 °C, the plates were washed and Ellman’s reagent was added. The absorbance related to the solid phase bound AChE activity was measured at 414 nm. Results are expressed as B/Bo(%) as a function of the logarithm of the dose. Calibration curves were fitted using a linear log-logit transformation. All measurements were made in duplicate. Kd,app values are defined as the concentration of competing antigen that results in halfmaximal OD 414 nm. RESULTS AND DISCUSSION Synthesis and Surface Area Calculation of HIS-HVA Conjugates 1-8. These compounds were easily prepared in four steps as shown in Scheme 2. Briefly, homovanillic acid and histamine were connected through linear amino acid spacers using standard peptide bound formation. All products were fully charAnalytical Chemistry, Vol. 82, No. 6, March 15, 2010

2537

Figure 2. (A) Energy-minimized structures of HIS-HVA conjugates 1 and 8 (carbon frameworks in gray, oxygen in red, nitrogen in dark blue and hydrogen in blue) and water accessible surface areas (the Connolly surface is shown as dots). (B) Solvent accessible surface areas of compounds 1-8 and of spacers.

acterized (see the Supporting Information for detailed protocols and analytical data). The geometries of compounds 1-8 were optimized with the TINKER 4.2 program22 using the MM2 force field.23 The solvent accessible surface area were calculated with a probe radius of 1.40 Å and visualized using the CCP4 Molecular Graphics Project software package.24 The results indicated a minimum area of 570 Å2 for the smaller HIS-HVA conjugate (1, n ) 1) and increased gradually as a function of the spacer size to reach a maximum of 842 Å2 for compound 8 (n ) 11). All of these surfaces are largely inferior to those of molecules that have been assayed so far by sandwich immunoassays. The length of the spacers separating both HIS and HVA epitopes was calculated to be 2.5 Å for the smallest to 12.7 Å for the largest which corresponds to surfaces areas ranging from 117 to 394 Å2, respectively (Figure 2). Sandwich Immunoassays of HIS-HVA Conjugates by Combinatorial Association of mAbs. In order to get significant data on the probability of obtaining antibody-(1-8)-antibody ternary complexes, a panel of mAbs was used. Sixteen anti-HIS mAbs and 12 anti-HVA mAbs were selected for sandwich immunoassays experiments that were performed using 96-well microtiter plates, further leading to 192 antibody combinations for each compound to be tested. The sandwich immunoassays were performed in a sequential way. Taking into account their higher affinity, all sandwich immunoassays were performed using antiHIS mAbs to capture HIS-HVA conjugates 1-8 in order to ensure a maximum efficiency of the capture. After eliminating all remaining unbound HIS-HVA compounds, biotin-labeled antiHVA antibodies were then added and incubated at 4 °C overnight. After washing, the ternary complexes were revealed via streptavidin-acetylcholine esterase bioconjugate before Ellman’s reagent was finally added for staining. A total of 1536 combinations was therefore carried out, and the results are summarized in Figure 3 using a color code format for clarifying the presentation. The results demonstrate that most of the antibody couples allow efficient sandwich immunoassays with molecules possessing (22) Ponder, J. W. TINKER, Software Tools for Molecular Design, version 4.2; Washington University School of Medicine, 2004; http://dasher.wustl.edu/ tinker/. (23) Allinger, N. L. J. Am. Chem. Soc. 1977, 99, 8127–8134. (24) Potterton, L.; McNicholas, S.; Krissinel, E.; Gruber, J.; Cowtan, K.; Emsley, P.; Murshudov, G. N.; Cohen, S.; Perrakis, A.; Noble, M. Acta Crystallogr. 2004, D60, 2288–2294.

2538

Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

at least 10 methylene groups separating the two epitope moieties required for binding (compounds 7 and 8). Only two mAbs (HIS 76 and HVA 137) prevented sandwich immunoassay formation whatever the second mAb used, suggesting a deep binding of the HIS-HVA conjugates within their combining sites. An efficiency gap in the sandwich assays is clearly observed with shorter linkers since almost all of the antibody combinations provided lower absorbance signal with compound 6 (n ) 8) than with 7 and 8. Figure 4 shows a global view of the influence of the linker size on the sandwich efficiencies. More than 85% of mAb combinations allowed efficient sandwich immunoassays with compounds 7 and 8 (Figure 4A). The efficiency of ternary complex formation (as estimated by absorbance signal reading) of all of the active mAb combinations was similar for both HIS-HVA conjugates 7 and 8 but decreased dramatically when 6 (n ) 8) was used (Figure 4B). The active mAb combinations (55%) obtained with 6 globally provided an absorbance signal 5 times lower than with 7 and 8. This result suggests that below 10 methylene groups (11.5 Å) separating the epitopes, steric hindrance takes place, resulting in unfavorable interactions of mAbs couples. Interestingly, 15% of mAbs combinations still remained functional using compound 4 (n ) 5). This result represents the smallest sandwich immunoassays ever described and proves that the simultaneous binding of two mAbs to an hapten bearing epitopes connected through a spacer arm as short as 5 Å is possible. Not surprisingly, compounds 1, 2, and 3 involving shorter spacers failed to lead to sandwich immunoassays. Among the tested mAbs, two anti-HIS (HIS-71 and 203) and two anti-HVA (HVA-46 and 120) mAbs were selected due to their better capacity to undergo sandwich immunoassays with 4. Binding Properties and Sandwich Curves of Selected mAbs. The binding properties of these mAbs for compounds 1-8 were evaluated using competitive immunoassays (Table 1). The results clearly indicate a strong increase of the binding properties of HIS-71 mAb, also observed but to a lesser extent with HIS-203 and HVA-46 mAbs, when the size of the spacer is increasing suggesting that mAbs recognized part of the spacer. This phenomenon is less pronounced for HVA-120 mAb that recognize equally all tested HIS-HVA conjugates. Since mAbs HIS203 and HVA-46 exhibited the best binding properties, they were selected to perform sandwich immunoassays of compounds 4-8 used at different concentrations in order to analyze standard curves (Figure 5). As expected for a sandwich immunoassay, standard curves providing a dose-dependent signal were observed for compounds 4-8. On the other hand, no signal was detected for compounds 1-3 even for concentrations as important as 10 µM. Compounds 7-8 exhibited close standard curves with minimum detectable concentration (MDC) near 0.2 nM, thus demonstrating that a spacer arm of eight carbon atoms allows very comfortable binding for both capture and tracer antibodies. A clear-cut modification of the sensitivity was observed for compounds 4 and 5, resulting in at least a 2 order of magnitude decrease of the calculated MDC. Moreover the shape of the generated curves was also strongly different with a slope remaining sharp for compound 5 but lowering for compound 4. However, these signals truly corresponded to sandwich immunoassay since coincubation of 4 or 5 with either

Figure 3. Combinatorial association of mAbs for sandwich immunoassays of 1-8 used at 100 nM. The absorbance (A) values were obtained after 1 h of staining and were corrected by subtracting nonspecific binding (NSB) values obtained without compounds 1-8. All experiments were made in duplicate.

Figure 4. Influence of the linker size on the efficiency of 192 sandwich immunoassays. (A) Number and percentages of mAbs pairs allowing sandwich immunoassays as a function of linker size. (B) Percentage of absorbance signal of sandwich immunoassays (taking the value obtained for 8 as reference) after 1 h of revelation as a function of linker size. Each dot corresponds to one mAbs combination. Table 1. Binding Properties of Selected Anti-HIS and Anti-HVA Monoclonal Antibodies Kd,appa (µM) for HIS-HVA conjugates mAbs

1 (n ) 1)

2 (n ) 3)

3 (n ) 4)

4 (n ) 5)

5 (n ) 6)

6 (n ) 8)

7 (n ) 10)

8 (n ) 11)

HIS-71 HIS-203 HVA-46 HVA-120

0.758 0.026 0.422 3.283

0.504 0.023 0.314 1.845

0.700 0.019 0.445 2.800

0.181 0.005 0.517 2.128

0.007 0.003 0.254 1.978

0.005 0.002 0.157 1.602

0.005 0.002 0.095 1.465

0.006 0.003 0.132 2.484

a

Kd,app determined by competitive ELISA (see the Experimental Section).

histamine or HVA derivatives totally abolished the signal demonstrating competition for the specific binding to either the capture or the tracer antibody (see the Supporting Information). It is worth noting that the simultaneous binding of the two antibodies to 4 and 5 becomes very difficult and probably involves minor structural modifications of the antibodies to achieve the formation of the ternary complex. It should be hypothesized that, even if both antibodies

display surface binding, they should be in very tight contact in the ternary complex and that compounds 4 and 5 are totally buried into the antibody binding sites. CONCLUSION The present model study explored the size limit of molecules that can be detected by sandwich immunoassays. According to Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

2539

proved in this case study that this hypothesis is partially wrong and that molecules constituted of two epitopes of only ∼300 Å2 separated by a spacer as small as 5 Å, may be bound by two antibodies. This finding is intriguing since it proves that the formation of ternary complexes constituted by the association of two proteins via a very short linker, further leading to tight contacts between these proteins, is possible. The data obtained in this case study should therefore be of interest for researchers involvedinthefieldofsmallmoleculedetectionusingimmunoassays. ACKNOWLEDGMENT We warmly thank D. Buisson for experimental assistance with MS measurements and HPLC purification. Figure 5. Calibration curves of sandwich immunoassays of 4-8 using mAb HIS-203 for capture and mAb HVA-46 for detection. Revelation times were 1 h for 8-6 and 5 h for 5 and 4.

this study, the probability of obtaining successful sandwich immunoassay is high if the epitopes of the molecule to be detected are separated by at least 11.5 Å. The hypothesis that small haptens cannot be simultaneously bound by two antibodies and thus cannot be assayed by direct sandwich methods generally drives scientists to chose alternative detection techniques. However, we

2540

Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

SUPPORTING INFORMATION AVAILABLE Experimental procedures, analytical and spectral characterization data of compounds 1-8, and control experiments for sandwich immunoassays. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review January 8, 2010. Accepted February 15, 2010. AC100058F