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Femtomolar Detection of the Anthrax Edema Factor in Human and Animal Plasma Elodie Duriez,† Pierre L. Goossens,‡ Franc¸ois Becher,† and Eric Ezan*,† CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, 91191 Gif-sur-Yvette, France, and Institut Pasteur, Toxines et Pathoge´nie Bacte´rienne, 75724 Paris Cedex 15, France Edema factor (EF), a calmodulin-activated adenylyl cyclase, is a toxin which contributes to cutaneous and systemic anthrax. As a novel strategy to detect anthrax toxins in humans or animals infected by Bacillus anthracis, we have developed a sensitive enzymatic assay to be able to monitor functional EF in human and animal plasma. Samples containing EF are incubated in the presence of calmodulin and ATP, which is converted to cAMP. After oxidation and derivatization, cAMP is monitored by competitive enzyme immunoassay. Because of the high turnover of EF and the sensitivity of cAMP detection, EF can be detected at concentrations of 1 pg/ mL (10 fM) in 4 h in plasma from humans or at 10 pg/ mL in the plasma of various animal species using only a blood volume of 5 µL. The assay has good reproducibility with intra- and interday coefficients of variation in the range of 20% and is not subject to significant interindividual matrix effects. In an experimental study performed in mice infected with the Berne strain, we were able to detect EF in serum and ear tissues. This simple and robust combination of enzymatic reaction and enzyme immunoassay for the diagnosis of anthrax toxemia could prove useful in biological threat detection as well in research and clinical practice. Numerous toxins consist of a component that binds to cell surfaces and a catalytic unit that modifies cell homeostasis leading to deleterious effects in cell targets. The exquisite specificity of their enzymatic components and high turnover provide the basis for the development of assays for pharmacological study of these toxins. Since these toxins or their expression vector are also considered as potential terrorist weapons,1 there is considerable interest in using these activities as a means to monitor infection in human plasma as well as the biological threat in various environmental media.2 This was applied to the development of bioassays for ricin3 and botulism toxins4 with the lowest limit of * To whom correspondence should be addressed. Mailing address: CEA, iBiTecS, Service de Pharmacologie et d’Immunoanalyse, Baˆt 136, CEA Saclay, 91191 Gif-sur-Yvette France. Phone: 33-1-69-08-73-50. Fax: 33-1-69-08-59-07. E-mail:
[email protected]. † CEA. ‡ Institut Pasteur. (1) Centers for Disease Control and Prevention and Department of Health and Human Services. Bioterrorism Agents/Diseases. 2008. (2) Demirev, P. A.; Fenselau, C. J. Mass Spectrom. 2008, 43, 1441–1457. (3) Becher, F.; Duriez, E.; Volland, H.; Tabet, J. C.; Ezan, E. Anal. Chem. 2007, 79, 659–665. 10.1021/ac900827s CCC: $40.75 2009 American Chemical Society Published on Web 06/12/2009
detection in the pico- and femtomolar range. Some of the detection systems were based on mass spectrometry, which offers high sensitivity and specific applications to each subtype of toxins.5 Alternatively, and in a context of field applications, other detection systems such as enzyme immunoassay (EIA)6,7 and fluorescence7 are also applied. Among bioterrorist agents, anthrax is considered as one of most potent and dangerous8 and has been classified in the A list of the Centers for Disease Control.1 In previous events, the 2001 anthrax attacks in the United States occurred over the course of several weeks where letters containing anthrax spores were mailed to several news media offices and U.S. Senators, killing five people and infecting 11 others from inhalational anthax.9 Anthrax is caused by infection with Bacillus anthracis, a grampositive, spore-forming bacterium.10 Anthrax exotoxin, which is secreted into the host’s system, consists of three nontoxic proteins that associate to form toxin complexes at the surface of mammalian cells: protective antigen (PA; 83 kDa), lethal factor (LF; 90 kDa), and edema factor (EF; 89 kDa). At the molecular level, PA in its 83 kDa form binds to a cellular receptor, where it is cleaved to PA20 and PA63.11 PA63 self-associates to form the heptameric prepore, which competitively binds three molecules of EF or LF or both. The complex of PA and LF forms lethal toxin (LTx), and PA complexed with EF forms edema toxin (ETx). These complexes are endocytosed and trafficked before EF and LF are translocated to the cytosol where they target their respective substrates. LF is a Zn2+-dependent metalloprotease that cleaves certain mitogen-activated protein kinase kinases (MAPKKs), leading to impaired innate immune cell responses and death of susceptible macrophages through a Nalp1bdependent mechanism that is beginning to be unraveled.12 EF (4) Kalb, S. R.; Goodnough, M. C.; Malizio, C. J.; Pirkle, J. L.; Barr, J. R. Anal. Chem. 2005, 77, 6140–6146. (5) Barr, J. R.; Moura, H.; Boyer, A. E.; Woolfitt, A. R.; Kalb, S. R.; Pavlopoulos, A.; McWilliams, L. G.; Schmidt, J. G.; Martinez, R. A.; Ashley, D. L. Emerg. Infect. Dis. 2005, 11, 1578–1583. (6) Volland, H.; Lamourette, P.; Nevers, M. C.; Mazuet, C.; Ezan, E.; Neuburger, L. M.; Popoff, M.; Creminon, C. J. Immunol. Methods 2008, 330, 120–129. (7) Ler, S. G.; Lee, F. K.; Gopalakrishnakone, P. J. Chromatogr., A 2006, 1133, 1–12. (8) Inglesby, T. V.; Henderson, D. A.; Bartlett, J. G.; Ascher, M. S.; Eitzen, E.; Friedlander, A. M.; Hauer, J.; McDade, J.; Osterholm, M. T.; O’Toole, T.; Parker, G.; Perl, T. M.; Russell, P. K.; Tonat, K. J. Am. Med. Assoc. 1999, 281, 1735–45. (9) Centers for Disease Control and Prevention (CDC). MMWR, Morb. Mortal. Wkly. Rep. 2001, 50, 1008-1010. (10) Fouet, A.; Mock, M. Curr. Opin. Microbiol. 2006, 9, 160–166. (11) Collier, R. J. J. Appl. Microbiol. 1999, 87, 283. (12) Turk, B. E. Biochem. J. 2007, 402, 405–417.
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is a calmodulin- and Ca2+-dependent adenylyl cyclase which catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP), leading to edema.13,14 Because of the potential use of B. anthracis as an agent of bioterrorism and because of its high infection potential and its persistency as spores in the environment, the development of rapid and accurate detection methods is needed. In the literature, numerous detection methods of B. anthracis15 targeting the entire organism, either spores or vegetative cells, have already been described using antibody-based methods.16-20 Among other techniques available for B. anthracis detection are the nucleic acid amplification-based techniques including polymerase chain reaction (PCR)21,22 and real-time PCR.23-25 These techniques all rely on the selection of nucleic acid probes that are specific for B. anthracis and offer a high specificity. In addition to the detection of the entire organism, the anthrax toxins are also a target. There are many diagnostic techniques designed to be used when an infection is suspected. They include the detection of antibodies to PA or EF by an indirect microhemagglutination test,26 to PA and LF by electrophoretic immunotransblot,27,28 to PA,29 EF, and LF,28 and poly-D-glutamic acid capsule28 by ELISA, or to PA by fluorescent covalent microsphere immunoassay.30 Quantification of anthrax toxins in serum can be performed by Western blot31 or more sensitively by ELISA.32,33 However, DNA-based and antibody-based techniques do not indicate whether the pathogens are still viable or whether anthrax (13) Guo, Q.; Shen, Y.; Zhukovskaya, N. L.; Florian, J.; Tang, W. J. J. Biol. Chem. 2004, 279, 29427–29435. (14) Drum, C. L.; Yan, S. Z.; Bard, J.; Shen, Y. Q.; Lu, D.; Soelaiman, S.; Grabarek, Z.; Bohm, A.; Tang, W. J. Nature 2002, 415, 396–402. (15) Edwards, K. A.; Clancy, H. A.; Baeumner, A. J. Anal. Bioanal. Chem. 2006, 384, 73–84. (16) Farrell, S.; Halsall, H. B.; Heineman, W. R. Analyst 2005, 130, 489–497. (17) Gatto-Menking, D. L.; Yu, H.; Bruno, J. G.; Goode, M. T.; Miller, M.; Zulich, A. W. Biosens. Bioelectron. 1995, 10, 501–507. (18) Taitt, C. R.; Anderson, G. P.; Lingerfelt, B. M.; Feldstein, M. J.; Ligler, F. S. Anal. Chem. 2002, 74, 6114–6120. (19) Stopa, P. J. Cytometry 2000, 41, 237–244. (20) Zahavy, E.; Fisher, M.; Bromberg, A.; Olshevsky, U. Appl. Environ. Microbiol. 2003, 69, 2330–2339. (21) Sjo ¨stedt, A.; Eriksson, U.; Ramisse, V.; Garrigue, E. FEMS Microbiol. Ecol. 1997, 23, 159–168. (22) Fasanella, A.; Losito, S.; Trotta, T.; Adone, R.; Massa, S.; Ciuchini, F.; Chiocco, D. Vaccine 2001, 19, 4214–4218. (23) Qi, Y.; Patra, G.; Liang, X.; Williams, L. E.; Rose, S.; Redkar, R. J.; Delvecchio, V. G. Appl. Environ. Microbiol. 2001, 67, 3720–3727. (24) Oggioni, M. R.; Meacci, F.; Carattoli, A.; Ciervo, A.; Orru, G.; Cassone, A.; Pozzi, G. J. Clin. Microbiol. 2002, 40, 3956–3963. (25) Makino, S.; Cheun, H. I. J. Microbiol. Methods 2003, 53, 141–147. (26) Buchanan, T. M.; Feeley, J. C.; Hayes, P. S.; Brachman, P. S. J. Immunol. 1971, 107, 1631–1636. (27) Harrison, L. H.; Ezzell, J. W.; Abshire, T. G.; Kidd, S.; Kaufmann, A. F. J. Infect. Dis. 1989, 160, 706–710. (28) Sirisanthana, T.; Nelson, K. E.; Ezzell, J. W.; Abshire, T. G. Am. J. Trop. Med. Hyg. 1988, 39, 575–581. (29) Quinn, C. P.; Semenova, V. A.; Elie, C. M.; Romero-Steiner, S.; Greene, C.; Li, H.; Stamey, K.; Steward-Clark, E.; Schmidt, D. S.; Mothershed, E.; Pruckler, J.; Schwartz, S.; Benson, R. F.; Helsel, L. O.; Holder, P. F.; Johnson, S. E.; Kellum, M.; Messmer, T.; Thacker, W. L.; Besser, L.; Plikaytis, B. D.; Taylor, T. H., Jr.; Freeman, A. E.; Wallace, K. J.; Dull, P.; Sejvar, J.; Bruce, E.; Moreno, R.; Schuchat, A.; Lingappa, J. R.; Martin, S. K.; Walls, J.; Bronsdon, M.; Carlone, G. M.; Bajani-Ari, M.; Ashford, D. A.; Stephens, D. S.; Perkins, B. A. Emerg. Infect. Dis. 2002, 8, 1103–1110. (30) Biagini, R. E.; Sammons, D. L.; Smith, J. P.; Page, E. H.; Snawder, J. E.; Striley, C. A.; MacKenzie, B. A. Occup. Environ. Med. 2004, 61, 703–708. (31) Molin, F. D.; Fasanella, A.; Simonato, M.; Garofolo, G.; Montecucco, C.; Tonello, F. Toxicon 2008, 52, 824–828. (32) Sastry, K. S.; Tuteja, U.; Santhosh, P. K.; Lalitha, M. K.; Batra, H. V. J. Med. Microbiol. 2003, 52, 47–49.
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toxins are still functional. An indirect approach to detect functional B. anthracis is to monitor the activity of its functional toxin, such as the lethal factor, which displays a metalloprotease-like activity. Combined with immunoextraction and enzymatic reaction, LF activity on a 45-amino-acid substrate was studied using MALDITOF MS,34 which is a powerful tool to unambiguously detect trace levels of an infection in complex samples.35 Applied to infected monkey, the assay was able to detect the toxin in the femtomolar range.34 EF is another alternative to investigate anthrax exposure and has not been explored so far. EF is a calmodulin (CaM)-dependent adenylyl cyclase14 modulated by physiological calcium concentrations and has been widely studied.36,37 The enzyme has a very high turnover36,38 and may offer a potential for measurement of EF in biological media. In this study, we aimed to exploit these unique biological properties to obtain an original assay of EF in plasma from humans or various animal species with a very high sensitivity. Upon reaction with ATP and in the presence of calmodulin, the enzymatic activity is monitored by measuring the production of cAMP using a competitive enzyme immunoassay (EIA). After assay optimization, we demonstrated that the EIA detection of the reaction product allowed the detection of EF in plasma from human or various animal species with a sensitivity of 1 and 10 pg/mL, respectively. Combined with other methods for the assessment of anthrax infection, this method allows full coverage of anthrax toxin detection in blood. MATERIALS AND METHODS Chemicals and Reagents. Recombinant anthrax edema factor (EF) and anthrax activated protective antigen (PA63) from B. anthracis were from Quadratech Diagnostics (Epsom Surrey, U.K.). Poly γ-D-glutamic (γDPGA) was purified as previously described.39 Adenosine 5′-triphosphate (ATP) disodium salt hydrate, adenosine 3′,5′ cyclic monophosphate, sodium periodate, rhamnose, and calmodulin from bovine heart or from bovine testes were from Sigma-Aldrich (St. Louis, MO). Calmodulin human recombinant (rCaM) was from te´bu-bio (Le Perray en Yvelines, France). cAMP antiserum (polyclonal antibodies), cAMP acetylcholinesterase (AChE) enzymatic tracer, cAMP standard, and acetic anhydride used in the EIA studies were from Spi-Bio (Montigny-Le-Bretonneux, France). Adenylyl Cyclase EF Assay. The different steps of the anthrax edema factor assay are presented in Figure 1. Standards were prepared by dilution of the EF stock solution to 1 mg/mL in water. The standard curve of EF was performed with serial dilutions of EF in a blank matrix (either plasma or tissue extracts). (33) Mabry, R.; Brasky, K.; Geiger, R.; Carrion, R., Jr.; Hubbard, G. B.; Leppla, S.; Patterson, J. L.; Georgiou, G.; Iverson, B. L. Clin. Vaccine Immunol. 2006, 13, 671–677. (34) Boyer, A. E.; Quinn, C. P.; Woolfitt, A. R.; Pirkle, J. L.; McWilliams, L. G.; Stamey, K. L.; Bagarozzi, D. A.; Hart, J. C., Jr.; Barr, J. R. Anal. Chem. 2007, 79, 8463–8470. (35) Duriez, E.; Fenaille, F.; Tabet, J. C.; Lamourette, P.; Hilaire, D.; Becher, F.; Ezan, E. J. Proteome Res. 2008, 7, 4154–4163. (36) Shen, Y.; Lee, Y. S.; Soelaiman, S.; Bergson, P.; Lu, D.; Chen, A.; Beckingham, K.; Grabarek, Z.; Mrksich, M.; Tang, W. J. EMBO J. 2002, 21, 6721–6732. (37) Ulmer, T. S.; Soelaiman, S.; Li, S.; Klee, C. B.; Tang, W. J.; Bax, A. J. Biol. Chem. 2003, 278, 29261–29266. (38) Drum, C. L.; Yan, S. Z.; Sarac, R.; Mabuchi, Y.; Beckingham, K.; Bohm, A.; Grabarek, Z.; Tang, W. J. J. Biol. Chem. 2000, 275, 36334–36340. (39) Candela, T.; Fouet, A. Mol. Microbiol. 2005, 57, 717–726.
Figure 1. EF enzymatic assay: EF in the human sample is incubated with ATP and calmodulin after a 20-fold dilution (step 1). To eliminate interference of ATP and to increase the cAMP sensitivity, oxidation and derivatization were performed with sodium periodate and acetic anhydride, respectively (step 2). Modified cAMP is detected by enzyme immunoassay (step 3).
Before assay, each standard or plasma sample was diluted 20fold (and therefore dilution depending on the EF concentration in the sample) in 20 mM Hepes buffer pH 7.2 with 0.5 mg/mL bovine serum albumin, 10 mM MgCl2, 10 µM CaCl2, 1 mM ATP, and 10 µM CaM. The adenylyl cyclase activities were then assayed in a volume of 100 µL (this corresponds to 5 µL of plasma sample diluted 20-fold in Hepes buffer) for 30 min at 30 °C (Figure 1, step 1). Enzyme Immunoassay for cAMP. After incubation of EF with ATP and calmodulin, the production of cAMP was quantified using an EIA. Before EIA, the samples and standards were chemically transformed (Figure 1, step 2). ATP ring-opening was performed using periodate sodium oxidation following a previously described procedure.40 This allowed the elimination of ATP crossreaction with cAMP analysis. A volume of 45 µL of a 0.5 M (40) Hennere, G.; Becher, F.; Pruvost, A.; Goujard, C.; Grassi, J.; Benech, H. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2003, 789, 273–81.
aqueous solution of sodium periodate was added to each standard or biological sample (100 µL). The samples were gently mixed and incubated at 37 °C for 5 min. A volume of 20 µL of a 1 M aqueous solution of rhamnose were then added to quench sodium periodate, and the samples were again gently mixed for 20 min at 37 °C. The samples were then derivatized by acetylation to increase sensitivity. A volume of 33 µL of a 4 M aqueous solution of potassium hydroxide and 8 µL of acetic anhydride were successively and rapidly added and shaken for 15 s, after which 8 µL of 4 N KOH were added. The final volume of samples was 214 µL, from which we used 50 µL (in duplicate, i.e., 100 µL) for the analysis of cAMP by EIA (Figure 1, step 3). The EIA was a competitive method, in which cAMP was in competition with a cAMP enzymatic tracer, cAMP coupled to acetylcholinesterase (AChE), for binding to anti-cAMP polyclonal antibodies. The amount of tracer bound to the anti-cAMP antibody site was then measured. The response is thus inversely proportional to the concentration of cAMP in the sample. The assay was performed using microtiter plates (Nunc, Denmark) coated with mouse monoclonal antibodies specific for rabbit immunoglobulins (Spi-Bio, Montigny-Le-Bretonneux, France). Before use, the plates were extensively washed with 0.01 M phosphate buffer pH 7.4, containing 0.05% Tween 20 (washing buffer) using a microplate washer Wellwash AC (Labsystem) (300 µL/well, five wash cycles). The assay was performed in a total volume of 150 µL, each component being added in a volume of 50 µL. In the routine assay, reagents were dispensed as follows: 50 µL of standard or biological sample, 50 µL of cAMP-AChE tracer, and 50 µL of cAMP antiserum. The plates were then left for 2 h at room temperature and washed again. Then 200 µL of the AChE substrate (Ellman’s reagent, Spi-Bio, Montigny-Le-Bretonneux, France) were automatically dispensed into each well by using an Autodrop apparatus. During the enzymatic reaction catalyzed by AChE, the plates were gently agitated. The absorbance was recorded at 414 nm using a spectrophotometer microplate reader Multiskan Ascent (Labsystem). The limits of cAMP detection were determined as the concentration inhibiting 20% of the total tracer-antibody binding, i.e., in the absence of cAMP. The limits of EF detection were then determined as the EF concentration producing detectable cAMP. Validation of the EF Assay. The matrix effect was assessed by spiking different human plasma samples (n ) 8) or plasma from animal species (n ) 3 for each species, either mouse, dog, or monkey) with 80 and 100 pg/mL of EF, respectively. Intersubject variability was measured by calculating the mean recovery and its variability. Repeatability (intraday variability) was estimated by the coefficient of variation (CV) for the 8 different human plasma samples which were assessed in 10 replicates the same day. Quality control samples (EF at 50, 200, and 500 pg/mL in human plasma) were measured in five independent experiments in order to assess the interassay variability. In order to check if anthrax toxin components interfere with the assay, human plasma spiked with 20 pg/mL EF was assessed in the presence of various concentrations (from 20 pg/mL to 1 µg/mL) of PA63. We also tested human plasma spiked with 60 pg/mL EF in the presence of various concentrations (from 40 pg/mL to 1 µg/mL) of γDPGA. The recoveries were calculated for each component. Analytical Chemistry, Vol. 81, No. 14, July 15, 2009
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Application to Animal Infection. The method was applied to samples recovered from B. anthracis infected mice. Female A/J mice (6- to 10-week-old; Harlan Laboratories, U.K.) were maintained under specific pathogen-free conditions at the Institut Pasteur in compliance with European animal welfare regulations. Infections were performed as previously described41 with a bioluminescent derivative of the B. anthracis strain 9602R, which is a phenotypic equivalent to the Sterne strain and therefore does not produce a capsule but maintains toxin production. Cutaneous infections (104 spores) were performed under light isoflurane anesthesia by injecting 10 µL of spore suspensions in PBS into the ear with a 0.5 mL insulin syringe as previously described (Becton Dickinson, NJ).41,42 Intravenous infections were performed with 106 spores or bacilli. Bioluminescence images were acquired using an IVIS100 system (Xenogen Corp., CA) as previously described.41,42 Blood and ear samples were taken at the septicemic stage of infection when kidneys were bioluminescent, i.e., 8 h after intravenous infection with bacilli, 24 h after intravenous infection with spores, and 72 h after spore cutaneous infection for the ear samples. Plasma was obtained after intraperitoneal injection of 100 IU of calciparin and centrifugation to pellet erythrocytes. The ears were homogenized in 1 mL of 150 mM PBS supplemented with an antiprotease cocktail (Protease Inhibitor set, Calbiochem) in a Precellys 24 (Bertin Technology, Montigny le Bretonneux, France) at 6500 rpm for 60 s with CK14 ceramic beads; the cell debris was pelleted after a 5 min centrifugation at 13 000g, and the supernatants were sterilized by centrifugation on a 0.22 µm GV Durapore filter tube (Millipore). Blood and ear samples were then kept at -20 °C until the quantitative assays. Blood and ear samples from uninfected mice were used as controls and matrix. Blood and ear samples were diluted 20-fold with uninfected mouse matrix (either blood or ear blank extracts) in Hepes BSA before the assay of adenylyl cyclase activity as described above. Calibration curves of EF were established in each uninfected mouse matrix. RESULTS The principle of this EF enzymatic assay is to quantify the production of cAMP by the EF adenylyl cyclase, in the presence of ATP and calmodulin. Before the quantification of cAMP by EIA, chemical modifications (oxidation and derivatization) were performed to eliminate interference by ATP and to increase the cAMP sensitivity. The flowchart of the method including EF enzymatic assay and cAMP determination is presented in Figure 1. We will first describe the optimization of the cAMP measurement and then the optimization, validation, and application of the EF assay in human and animal plasma. cAMP Competitive Enzyme Immunoassay. In the first step, we sought to increase the sensitivity of cAMP detection and adapted it to the human plasma cAMP EIA already developed by our group using a competitive format.43 A derivatization of cAMP with acetic anhydride increases the assay sensitivity 20-fold (parts A vs B of Figure 2). However, although ATP did not cross-react (41) Glomski, I. J.; Corre, J. P.; Mock, M.; Goossens, P. L. Infect. Immun. 2007, 75, 4754–61. (42) Glomski, I. J.; Piris-Gimenez, A.; Huerre, M.; Mock, M.; Goossens, P. L. PLoS Pathog. 2007, 3, e76. (43) Pradelles, P.; Grassi, J.; Chabardes, D.; Guiso, N. Anal. Chem. 1989, 61, 447–453.
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Figure 2. Enzyme immunoassay standard curves of cAMP and ATP in buffer without (A) or with (B) derivatization and (C) with derivatization/oxidation. The limits of detection for cAMP (inhibition of 20% of the total tracer-antibody binding, i.e., in the absence of cAMP) are 650 (a), 30 (b), and 30 pg/mL (c). The cross-reactivity of ATP is
