Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

Several strategies that target anthrax toxin are being developedas therapies for infection by Bacillus anthracis. Although theaction of the tripartite anthrax toxin has been extensivelystudied in vitro, relatively little is known about the presenceof toxins during an infection in vivo. We developed a seriesof sensitive sandwich enzyme-linked immunosorbent assays (ELISAs)for detection of both the protective antigen (PA) and lethalfactor (LF) components of the anthrax exotoxin in serum. Theassays utilize as capture agents an engineered high-affinityantibody to PA, a soluble form of the extracellular domain ofthe anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwichimmunoassays were used to detect and quantify PA and LF in animalsinfected with the Ames or Vollum strains of anthrax spores.PA and LF were detected before and after signs of toxemia wereobserved, with increasing levels reported in the late stagesof the infection. These results represent the detection of freePA and LF by ELISA in the systemic circulation of two animalmodels exposed to either of the two fully virulent strains ofanthrax. Simple anthrax toxin detection ELISAs could prove usefulin the evaluation of potential therapies and possibly as a clinicaldiagnostic to complement other strategies for the rapid identificationof B. anthracis infection.

INTRODUCTION

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Introduction

The gram-positive Bacillus anthracis bacterium is the causativeagent of anthrax. During inhalational anthrax, spores are inhaledinto the lungs, leading to germination followed by the emergenceof vegetative anthrax bacteria in circulation. Key anthrax virulencefactors include the three components of the anthrax exotoxin,protective antigen (PA), lethal factor (LF), and edema factor(EF), which are secreted into the host's system (11). The PAand LF toxins combined are often referred to as anthrax lethaltoxin (LeTx) and operate analogous to other A-B toxin systems(18).

The PA component is secreted as an 83-kDa protein that is cleavedby a host furin-like protease to yield a 63-kDa fragment (5,24) that readily heptamerizes. The PA₆₃ heptamer is bound tothe host cell surface receptor ANTXR1 (ATR/TEM8) (45) or ANTXR2(CMG2) (7) with relatively high affinity (170 pM) (47). TheLF component binds to the PA heptamer and gains entry into thecell through clathrin-dependent endocytosis (1). A drop in endosomalpH leads to a conformational change in PA and transport of LFinto the host cell cytosol (17, 26). In the cytosol, LF toxinacts as a zinc metalloprotease, cleaving the N termini of mitogen-activatedprotein kinase kinases (9, 13), leading to a number of signsassociated with toxemia and ultimately death (2, 32).

In the rodent model, signs of anthrax infection are rapidlyfollowed by death within a few hours, resulting in a very small,if existent, window for postexposure therapy after observedtoxemia. However, in the nonhuman primate model and in humandisease, the infection is characterized by delayed death upto 10 days following initial symptom onset (8, 10). It is thereforebelieved that a window of opportunity for treating anthrax existsduring the second phase of human infection.

The 2001 U.S. anthrax attack led to 11 cases of inhalation anthraxresulting in five deaths (23). The mean incubation period ±standard deviation from the time of exposure (when known) toonset of symptoms was 5 ± 1 days. Of the five patientswho died, B. anthracis infection was confirmed before deathin only one patient (4, 23), even though initial visits by threeof the patients involved typical cell blood count (CBC) or cerebrospinalfluid analysis which yielded results consistent with bacterialinfection (23). However, there was no available test for anthraxtoxemia to provide more-specific information about the natureof the infections.

Both small molecule- and protein-based therapeutic approachesare being developed that rely on antitoxin agents intended toprovide preexposure prophylaxis and/or postexposure treatmentfor infection (16, 22, 27, 30, 34-36, 38, 43, 44). The PA toxinis considered a particularly promising therapeutic target becauseprevention of PA activity would eliminate subsequent LF andEF toxin action as well (29). Additionally, previous studieshave shown that antibody-based anti-PA toxin neutralizationcan result in passive immunity without antibiotic treatment(28, 30, 34), whereas anti-LF antibody approaches have thusfar failed to provide protective immunity in spore challenges(28).

Recently, engineered anti-PA monoclonal antibodies (MAbs) haveproven to be effective in both prophylactic and postexposurelive anthrax spore challenges in the rabbit model (6, 34). Wehave reported a high-affinity single-chain antibody fragment(scAb) called M18, which was modified by conjugation to polyethyleneglycol (30). Like the parent 14B7 murine monoclonal antibody,the engineered M18 scAb targets PA domain IV, the same domainknown to bind the cellular receptors ANTXR1 and ANTXR2. TheM18 antibody fragment-polyethylene glycol conjugate providedpassive immunity against a 250x to 625x 50% lethal dose (LD₅₀)of anthrax spore challenge (Vollum 1b) in the guinea pig modelin the absence of Fc immunity-mediated functions (30).

Surprisingly, despite these successes with anthrax antitoxintherapeutics in animal models, the stage of infection in whichPA/LF is secreted in vivo after exposure remains relativelyunexplored. PA has been previously detected in the serum ofinfected guinea pigs and rabbits after signs of toxemia areobserved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Westernmethods (15, 37), yet without any detection limits reported.More recently, a microarray was engineered to detect unlabelednative toxin (40). Although the reported limit of detectionfor PA was 1.3 µg/ml, there exists potential in expandingupon immunoassays for toxin detection for infected hosts.

Here, we have engineered sandwich capture enzyme-linked immunosorbentassays (ELISAs) for the detection of both PA and LF. The M18scAb (19, 30) was utilized as the capture agent in a sensitiveELISA capable of detecting PA ( $≥$ 1 ng/ml PA) in serum from infectedguinea pigs and rabbits. In addition, an LF-specific sandwichELISA was developed that utilizes the PA heptamer complex asthe capture ligand ( $≥$ 20 ng/ml LF in serum). Preliminary resultsshow that PA is detectable during anthrax infection in the guineapig model, before and after onset of signs of toxemia. PA anda mutant PA (PA Y688A) were also detected by an ANTXR2/CMG2assay showing equivalent levels of detection, as expected. Inaddition, both PA and LF were detected in blood in systemiccirculation in the rabbit model at time of death. The applicationof straightforward, robust immunoassays for diagnosis of anthraxtoxemia could prove useful in both research and clinical settings.

MATERIALS AND METHODS

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Materials and Methods

Sandwich ELISA for PA detection. The M18 scFv construct had previously been cloned into pMoPac16(20), expressed as a scAb, and purified by immobilized metalaffinity chromatography (IMAC) via a C-terminal polyhistidinetag and size-exclusion chromatography (30). ANTXR2 was a giftfrom Robert Liddington's group (The Burnham Institute, La Jolla,Calif.) and was prepared as previously mentioned (41). Rabbitanti-PA polyclonal, native PA, and Y688A PA were produced aspreviously described (39). Human serum was obtained from a donorat the University of Texas Health Center, Austin. For overnightcoating, 50 µl of M18 scAb (4 µg/ml) or ANTXR2 (4to 30 µg/ml) was incubated overnight in a 96-well ELISAplate (no. 3590; Corning Inc., Corning, NY) at 4°C. Theplate was then brought to room temperature and incubated with2% dried milk (Carnation, Nestle)-phosphate-buffered saline(PBS) or 2% bovine serum albumin (BSA; Sigma)/PBS for 2 h. Theplate was washed three times with 1x PBS-0.5% Tween 20 and thenonce with 1x PBS. For initial assays, native or Y688A PA wasdiluted into 2% milk-PBS or human serum at 1 µg/ml andserially diluted onto the ELISA plate. For assays detectingPA in blood of infected animals, serum was added to the plateinitially diluted 1:1 in 2% milk-PBS and then serially dilutedacross the plate. Preexposed serum spiked with quantified PAserved as the positive control and was serially diluted as well.After a 1-h incubation, the plate was washed as described above.Rabbit anti-PA polyclonal serum was diluted 1:1,000 in 2% milk-PBSand added to the plate for a 1-h incubation. The plate was thenwashed again, followed by the addition of rabbit anti-goat immunoglobulinG (IgG)-horseradish peroxidase (HRP) conjugate (Bio-Rad) dilutedin 2% milk-PBS for 1 h. ELISA reactions were developed witho-phenylenediamine (OPD) tablets (Sigma) and quenched by theaddition of 50 µl of 4.5 M H₂SO₄. All ELISAs were runin duplicate, averaging each data point with standard deviation.Experimental assay data determining the detection of PA wereapplied to Prizm 3.03 software (GraphPad Software Inc., SanDiego, CA) to calculate the statistical significance for differencesbetween detection methods of each model. In addition, data werecurve fitted to a nonlinear regression model to determine "goodnessof fit."

Sandwich ELISAs for LF detection. Recombinant PA₈₃ and PA₆₃ were purchased from List Laboratories(New Jersey). PA₆₃ is a cleavage product that is capable ofbinding LF (45). For the sandwich ELISA, 50 µl of PA at63 kDa and 83 kDa (6 µg/ml) was applied to a 96-well plateand blocked with 2% milk-PBS as described above. For initialassays, LF was diluted in PBS or human serum at 5 µg/ml.For assays detecting LF in infected animals, serum was addedto the plate initially diluted 1:1 in 2% milk-PBS and then seriallydiluted across the plate in duplicate. After a 1-h incubation,the plate was washed as described above. Goat anti-LF polyclonalserum (List Labs) was diluted 1:1,000 in 2% milk-PBS and addedto the plate for a 1-h incubation in duplicate. The plate wasthen washed, followed by the addition of goat anti-rabbit IgG-HRPconjugate (Bio-Rad) diluted in 2% milk-PBS for 1 h. ELISA reactionswere developed with OPD tablets (Sigma) and quenched by theaddition of 50 µl of 4.5 M H₂SO₄. Observed concentrationswere calculated from positive control. Data were applied toPrizm 3.03 (GraphPad Software Inc., San Diego, CA) softwarefor statistical analysis.

Animal spore exposure and histology. Female Hartley guinea pigs (225 to 305 g) (Charles River Laboratories,Massachusetts) were housed individually in a One Cage 2100 AllerZoneInterchangeable Micro-Isolator high-density housing system (LabProducts Inc., Seaford, DE), and New Zealand rabbits (2.0 to2.5 kg) (Charles River Laboratories, Wilmington, MA) were housedindividually in ventilated rabbit cage racks (model no. RBV243116US6-2X;Allentown Caging Equipment Company, Inc., Allentown, NJ). Animalswere housed in a biosafety level 4 facility at The SouthwestFoundation for Biomedical Research (San Antonio, TX) for sporechallenge. Anthrax spore inocula, both the Ames and Vollum strains,were prepared on the day of challenge and diluted to the desiredconcentration in PBS. Both guinea pigs and rabbits were sedatedwith ketamine (80 mg/kg of body weight) and xylazine (10 mg/kg)during all bleeds, injections, and inhalation instillations,according to the approved IACUC protocol. Guinea pigs were exposedto B. anthracis Vollum 1B (3) spore inoculum of 500x LD₅₀ (2.0x 10⁷) (100-µl total volume), and rabbits were exposedto a 20x LD₅₀ (2.1 x 10⁶) (48) of B. anthracis Ames spores viaintranasal inoculation in both cases. Blood samples were takenfrom the femoral artery at various times. The guinea pigs weremonitored for signs of toxemia and euthanized by cardiac injectionof sodium pentobarbital when considered moribund. Symptoms oftoxemia include labored breathing, curling of digits, edema,abnormal color in nose/feet/tail, and immobility. For the rabbitstudies, euthanasia was not used and blood samples were takenonly at the time of death. Serum was obtained by centrifugationand separation using StatSampler columns (StatSpin, Norwood,MA).

For histology, lungs and spleens were removed from the guineapigs postmortem. A 100-mg section was excised and homogenizedwith 0.4 ml of sterile PBS in a sterile tissue grinder. Thehomogenates were serially diluted in sterile PBS and then platedin duplicate on sheep blood agar (SBA) plates. After 24 h ofincubation at 37°C, the colonies were enumerated to determinethe number of CFU/100 mg of tissue.

RESULTS

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Results

PA detection ELISAs. A highly sensitive sandwich ELISA for PA capture was developedbased on the engineered M18 antibody. Briefly, the M18 murinescFv (19) was isolated from an error-prone library derived fromthe anti-PA 14B7 murine monoclonal antibody (29) using a newbacterial display technology referred to as anchored periplasmicexpression (APEx) (19). The M18 scFv was cloned into the pMoPac16 vector (20) to produce a fusion between a human kappa chainand the heavy-chain variable domain of the scFv. The resultingconstruct is referred to as a scAb (21). The purified M18 scAbfragment binds PA with an equilibrium dissociation constant(K_D) of 35 pM (19).

Figure 1A shows a schematic of the anti-PA detection ELISA.The well bottoms of a 96-well plate were coated with the M18scAb to serve as the capture agent. PA was serially dilutedin 2% milk-PBS and human serum and then incubated for 1 h onthe plate. After washing, rabbit anti-PA polyclonal serum wasdiluted in 2% milk-PBS and added to the plate. After washing,goat anti-rabbit polyclonal IgG-HRP conjugate was added fordetection of the bound rabbit IgG. Note that having two polyclonaldetection antibodies adds potential signal amplification steps.The results from these serial dilution assays revealed thatPA can be detected in 2% milk-PBS and human serum with an observedlower limit of detection of ${approx}$ 1 ng/ml (Fig. 1B) based upon theconcentration of PA at which a threefold-higher signal overbackground or negative control was seen. The significance betweenthe curves was calculated as a two-way analysis of variance(ANOVA) model using Prism 3.03 software (GraphPad Software,San Diego, CA), resulting in an overall P value of <0.0001."Goodness of fit" for each curve was determined via a nonlinearregression model using the same software to reveal R² valuesof 0.990 (serum) and 0.994 (milk) for PA detection.

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FIG. 1. PA detection immunoassay. (A) Schematic for PA detection assay with M18 scAb. (B) For experimental detection, PA was detected at 1 µg/ml in milk-PBS (purple squares) or human serum (blue diamonds) and serially diluted in the plate assay. After probing with the primary and secondary antibodies as described, results showed that PA is detectable in milk-PBS and serum at very similar levels, with an overall lower detection limit of ${approx}$ 1 ng/ml.

Engineered ELISA for PA variant. The 14B7 antibody was originally identified as a neutralizingantibody and shown to act by blocking binding of PA to cells(29). It has been shown to bind to the PA domain 4 small loopregion involved in receptor binding (39, 41). Domain IV is responsiblefor the majority of binding to the ANTXR2 (CMG2) (42) and ANTXR1(ATR/TEM8) (7) receptors, although a contribution from PA domainII was recently identified by X-ray crystallographic analysisof the complex (41).

The ability to genetically modify a potential biowarfare agentto evade current and future therapeutics must be taken intoconsideration. Rosovitz and colleagues showed that a PA mutanthaving the Y688A substitution in domain IV retains full toxicity(39), even though binding by some anti-PA antibodies was disrupted.We sought to develop an assay capable of detecting anthrax strainscontaining a genetically modified PA component that retainsreceptor binding, and thus virulence, but might evade any singlecapture ELISA antibody. The ANTXR2 receptor was chosen as acapture agent due its reported high affinity to PA (7). Thesoluble component of the ANTXR2 receptor was substituted intothe capture ELISA described above in place of the M18 scAb construct.The ELISA was then performed corresponding to the schematicshown in Fig. 2A. Plates containing either the ANTXR2 solublereceptor or the M18 scAb were analyzed for detection of seriallydiluted native PA and PA Y688A. Results from the assays areshown in Fig. 2B. Assays using ANTXR2 as the capture agent revealedvery similar curves for both native PA (R² = 0.9935) and PAY688A (R² = 0.9979), with a reported P value of <0.0001 usingthe two-way ANOVA model. On the other hand, the M18-based assayshowed a marked difference in sensitivity between the two PAsamples, with the assay for PA Y688A (R² = 0.9851) being closeto an order of magnitude less sensitive than M18 binding nativePA (R² = 0.9986). Interestingly, despite a relatively high affinityreported for this receptor binding to PA, the ANTXR2-based assaywas considerably less sensitive than the ELISA based on M18,even in the case of PA Y688A detection. The discrepancy couldinvolve the way in which the ANTXR2 receptor binds to the plate,possibly leading to lower overall assay sensitivity by precludingunobstructed access to the PA toxin binding site.

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FIG. 2. Sandwich detection ELISA for native PA and mutant PA Y688A. (A) The soluble anthrax receptor, ANTXR2, and M18 scAb were capture ligands for detection of native PA and the mutant PA Y688A in domain IV. (B) Experimental results show that ANTXR2 bound to both native and mutant PAs with similar affinities. There was an order-of-magnitude decrease in sensitivity for M18 to the mutant PA compared to native toxin; however, overall the M18 assay was still more sensitive in both cases.

LF detection ELISA. The PA 63-kDa fragment was purchased from List Labs (New Jersey)and applied as the capture ligand for LF detection in a sandwichELISA (Fig. 3A). Full-length PA₈₃ was used as a negative controlsince this fragment cannot bind LF, whereas PA₆₃ was presumedto be in heptameric form and possess binding sites for LF (33)due to solution conditions from the supplier. As for our PAELISA, detection of captured LF was accomplished by primarypolyclonal anti-LF followed by HRP-conjugated secondary antibodies.As expected, using PA₆₃, but not PA₈₃, as the capture agentproduced an ELISA signal. "Goodness of fit" resulted in R² valuesof 0.9904 for serum and 0.9899 for milk PA detection. Two-wayANOVA calculated a P value of <0.0001 for comparison betweenthe two detection methods. A lower detection limit of ${approx}$ 20 ng/mlLF (Fig. 3B) was determined for the PA₆₃-based assay in serialdilution experiments, which may correspond to the binding affinity(low nM) of LF to PA (14).

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FIG. 3. Sandwich detection ELISA for LF. (A) Schematic shows the PA 63-kDa fragment as the capture ligand in the ELISA for LF. (B) For experimental detection, the PA 63-kDa or 83-kDa (control) fragment was applied as the capture ligand for LF detection. LF was detected at 4 µg/ml in milk-PBS (blue diamonds) or human serum (yellow triangles). The results showed that LF is detectable in milk-PBS and serum at very similar levels, with an overall lower detection limit of ${approx}$ 20 ng/ml when the PA 63-kDa fragment was used as the capture ligand. There was no detection of LF when the PA 83-kDa fragment was the capture ligand. The lower detection limit of the assay was calculated as a signal threefold higher than the PA 83-kDa negative control (purple squares and turquoise circles).

Diagnostic detection of PA from exposed guinea pigs. To examine the ability of the assay to detect PA produced duringinfection, female Hartley guinea pigs (225 to 300 g) were exposedto a 500x LD₅₀ (2 x 10⁷) of Vollum anthrax spores (3) and thenbled twice daily until euthanasia or death. PA was detectedin blood in systemic circulation using the M18 capture ELISAin 4 of the 5 animals before death (Table 1). As mandated bythe approved IACUC protocol, animals displaying any observedsigns of toxemia were immediately euthanized. However, two animalsexpired from anthrax in spite of constant monitoring for signs,and their serum samples were taken postmortem. For the threeanimals euthanized, two showed detectable levels of circulatingPA at the time of euthanasia. Both of the animals that diedfrom anthrax possessed detectable serum levels of PA prior toonset of toxemic signs as well as after death. One animal (25539)was found to have a relatively high serum PA level (24 ±5 µg/ml) at the time of death, compared to the level of0.4 ± 0.06 µg/ml detected in the same animal 9h earlier.

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TABLE 1. Detection of PA in sera from animals exposed to B. anthracis spores^a

Taken together, our results demonstrate that PA can be detectedin the late stages of guinea pig infection, especially within12 h of death. Tissue samples from all five animals were takenfor histology tests and revealed that animals considered moribund,and thus euthanized, possessed equivalent CFU ( ${approx}$ 10⁷ to 10⁹) ofbacteria compared to those that expired due to infection (datanot shown).

Toxin detection from rabbits exposed to Ames spores. As a preliminary investigation into the generality of the approachto PA detection and to evaluate the effectiveness of the LFassay, another animal model, New Zealand White rabbits, wasinvestigated using the other fully virulent laboratory strainof anthrax, the Ames strain. Two rabbits were exposed to a 20xLD₅₀ of Ames spores via intranasal inoculation. Upon death (mean, ${approx}$ 48 h), serum samples were taken for detection of PA and LF componentsof the toxin. Table 2 summarizes the ELISA results. The twoanimals expired with a mean time to death (MTTD) of ${approx}$ 48 h, anMTTD very similar to those of previous reports of exposure toa 150x LD₅₀ (34, 48). Upon death, both animals possessed detectablelevels of PA and LF in the systemic circulation. PA was foundto be present at concentrations of 80 ± 7 µg/mland 100 ± 13 µg/ml in the two animals, using theM18 antibody capture ELISA. The elevated PA levels comparedto those in the guinea pigs might indicate that toxin secretionsdiffer among animal models and/or between different strains(Ames versus Vollum 1b). The two animals expired with an MTTDof ${approx}$ 48 h, an MTTD very similar to those of previous reports ofexposure to a 150x LD₅₀ (34, 48). At time of death, blood sampleswere taken and PA values of 80 to 100 µg/ml were detected.The elevated PA levels compared to those of the guinea pigsmight indicate that toxin secretions differ among animal modelsand/or between different strains (Ames versus Vollum 1b).

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TABLE 2. Detection of B. anthracis in samples from rabbits exposed to Ames spores

DISCUSSION

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Discussion

We have engineered a PA detection ELISA based on single-epitopecapture ligands and secondary polyclonal detection antibodies.The assay using the engineered M18 high-affinity anti-PA antibodyfragment was able to detect PA with a lower detection limitof ${approx}$ 1 ng/ml in serum. PA was detected in the sera of four outof five guinea pigs exposed to spores of the Vollum 1b strainof B. anthracis. Of these, PA was detected in two of the threeanimals that were euthanized. In addition, both of the animalsthat expired due to anthrax displayed levels of circulatingPA before initial signs of toxemia. It thus appears that theM18-based ELISA can detect serum PA toxin secretion in animalsboth before and after external onset of toxemic signs. It mustbe noted that this assay strategy does not distinguish betweenPA₈₃ and PA₆₃ in the serum of the animal (15). However, theseassays are capable of detecting PA via the mammalian cell bindingdomain (PA domain IV), with M18 scAb serving as a detectioncapture ligand as well as a potential therapeutic for passiveimmunity (30).

An unambiguous time course of PA levels is difficult to extrapolatefrom these data. For example, although the administered sporedosages were expected to be uniform among all five guinea pigs,the amounts that reached the lungs may have differed tremendouslyamong the different animals. Such variation could affect incubation/germinationperiods, growth of nascent vegetative bacteria, toxin secretion,and eventually toxemia onset. Therefore, experiments for time-dependenttoxin secretion might be best executed by injection of sporesor bacteria to avoid respiratory incubation period differences.Nevertheless, our guinea pig data represent successful in vivoserum PA detection in animals exposed to anthrax spores by utilizingplate assays. Unfortunately, the LF detection assay was notyet available for investigation of LF levels in these guineapig samples.

Previously, capsular antigen and bacteremia in infected animalswere detected well before time of death (25). Therefore, itis somewhat surprising that PA detection occurred only in thelast stages of infection in four of the five guinea pigs weexamined. However, PA is secreted in an environment surroundedby tissue in which PA receptors, specifically ANTXR2, are highlyexpressed. With such a high affinity to ANTXR2 (K_D ${approx}$ 170 pM)(47), detection might occur only at or near saturation of thevascular system with PA. LF detection, as well, might vary accordingto the quantity remaining unbound and free in the blood.

We also used the soluble form of the ANTXR2 anthrax receptorfor the detection of a Y688A PA variant that was reported tobe capable of evading murine monoclonal 14B7 binding yet retainingcell toxicity (39). As expected, the ANTXR2 ELISA was able todetect both native PA and the PA Y688A variant with similarsensitivities, while M18 showed an order-of-magnitude decreasein sensitivity with samples of PA Y688A. Nevertheless, the overallsensitivity of the M18 scAb-based ELISA was far superior tothat of the ANTXR2 assay in both cases: 2 orders of magnitudemore sensitive in the case of native PA and an order of magnitudemore sensitive with the PA Y688A variant. While demonstratingthe value of the engineered M18 antibody, these results alsoindicate that a sandwich ELISA format may be useful as a wayto screen potential therapeutic molecules for relative bindingto native PA and PA variant molecules.

As a diagnostic, the new sandwich detection ELISAs could provideevidence of toxin secretion in a clinical setting. The incorporationof a toxin or bacterial diagnostic specific to anthrax couldprove useful during this time frame, as some patients did notreceive the ciprofloxacin or other antibiotic treatment untilother antimicrobials failed to improve patient status. Recentanthrax diagnostic tools focus on PCR (31) and bacterial surfaceantigens (12) for B. anthracis confirmation. Our ELISAs canbe run in under 2 h, which is comparable to PCR and other antigenassays. In addition, an immunoassay could readily be incorporatedinto an automated, multiplexed assay platform such as the Luminexbead assay (46).

ACKNOWLEDGMENTS

We would like to thank Monica A. Gonzales, Anysha E. Ticer,Michelle Reynolds, Laurie Condell, Elaine Windhorst, Marie Silva,Antonio Perez, and Marie Tehas for technical assistance withspore preparation, animal studies, and tissue samples. We wouldalso like to thank Barrett Harvey for M18 scAb, Andrew Hayhurstfor pMoPac16 and mentorship of R.M., and Lanling Zou from NIAIDfor many helpful discussions.

This work was supported by the NIH (U01 AI56431) and the Departmentof Defense Army (DAAD17-01-D0001).

FOOTNOTES

* Corresponding author. Mailing address for B. L. Iverson: University of Texas at Austin, 1 University Station, Austin, TX 78712. Phone: (512) 471-5053. Fax: (512) 471-7963. E-mail: biverson{at}mail.utexas.edu. Mailing address for G. Georgiou: Deparment of Chemical Engineering and Biomedical Engineering, University of Texas at Austin, 1 University Station, Austin, TX 78712. Phone: (512) 471-6975. Fax: (512) 471-7963. E-mail: gg@che.utexas.edu.

REFERENCES

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