Development of a PCR Lateral Flow Assay for Rapid Detection of Bacillus anthracis, the Causative Agent of Anthrax.

Bacillus anthracis, the causative agent of anthrax is one of the most potent listed biological warfare agents. The conventional microbiological methods of its detection are labor intensive and time consuming, whereas molecular assays are fast, sensitive and specific. PCR is one of the most reliable diagnostic tools in molecular biology. The combination of PCR with lateral flow strips can reduce the diagnostic/detection time. It gives an alternative to gel electrophoresis and offers easy and clear interpretation of results. In the present study, a PCR Lateral flow (PCR-LF) assay targeting cya gene present on pXO1 plasmid of B. anthracis has been developed. The forward and reverse primers were tagged with 6-carboxyflourescein (6-FAM) and biotin, respectively, at 5' end. The dual labeled PCR products were detected using lateral flow (LF) strips developed in this study. The PCR-LF assay could detect ≥ 5 pg of genomic DNA and ≥ 500 copies of target DNA harboured in a recombinant plasmid. The assay was able to detect as few as 103 and 10 CFU/mL of B. anthracis Sterne cells spiked in human blood after 6 and 24 h of enrichment, respectively.

Clindamycin Protects Nonhuman Primates Against Inhalational Anthrax But Does Not Enhance Reduction of Circulating Toxin Levels When Combined With Ciprofloxacin.

BACKGROUND: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. METHODS: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. RESULTS: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. CONCLUSION: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.

Furin Protease: From SARS CoV-2 to Anthrax, Diabetes, and Hypertension.

Furin is a protease that is ubiquitous in mammalian metabolism. One of the innovations that make sudden acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) more infectious than its ancestor viruses is the addition of a furin cleavage site. Conditions associated with elevated furin levels, including diabetes, obesity, and hypertension, overlap greatly with vulnerability to the severe form of coronavirus disease 2019 (COVID-19). We suggest that diet and lifestyle modifications that reduce the associated comorbidities may prevent the development of severe COVID-19 by, in part, lowering circulating furin levels. Likewise, natural and pharmaceutical inhibitors of furin may be candidate prophylactic interventions or, if used early in the COVID-19, may prevent the development of critical symptoms.

Placeholder Strategy with Upconversion Nanoparticles-Eriochrome Black T Conjugate for a Colorimetric Assay of an Anthrax Biomarker.

The timely warning of the germination of bacterial spores and their prevention are highly important to minimize their potential detrimental effects and for disease control. Thus, a sensitive and selective assay of biomarkers is most desirable. In this work, a nanoprobe is constructed by conjugating lanthanide upconversion nanoparticles (UCNPs) with sodium tripolyphosphate (TPP) and eriochrome black T (EBT). The nanoprobe, UCNPs-TPP/EBT, serves as a platform for the detection of the anthrax biomarker, dipicolinic acid (DPA). In principle, DPA displaces EBT from the UCNPs-TPP/EBT nanoconjugate, resulting in a color change from magenta to blue because of the release of free EBT into the aqueous solution. The binding sites on UCNPs are partly preblocked with TPP as the placeholder molecule, leaving a desired number of binding sites for EBT conjugation. On the basis of this dye displacement reaction, a novel colorimetric assay protocol for DPA is developed, deriving a linear calibration range from 2 to 200 µM with a detection limit of 0.9 µM, which is well below the infectious dose of the spores (60 µM). The assay platform exhibits excellent anti-interference capability when treating a real biological sample matrix. The present method is validated by the analysis of DPA in human serum, and its practical application is further demonstrated by monitoring the DPA release upon spore germination.

Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System.

Francisella tularensis, Bacillus anthracis, and Yersinia pestis are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of F. tularensis, B. anthracis, and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge-based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative-control blood in a blind manner. Specificity was determined by testing against nontarget pathogens and blood samples from clinical patients. The assay LOD was 8.5 CFU/ml for F. tularensis, 10 CFU/ml for B. anthracis, and 4.5 CFU/ml for Y. pestis The sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. The assay specificity was 100% when it was tested against both nontarget pathogens and clinical patient blood samples. The total assay time was approximately 100 min. This automated assay, which is suitable for use at the point of care, identifies three select agents directly in blood without the need for enrichment with a high sensitivity within 100 min. This assay may enable rapid detection and treatment of patients infected with the target organisms in the event of a bioterrorism attack.

Zeptomole per milliliter detection and quantification of edema factor in plasma by LC-MS/MS yields insights into toxemia and the progression of inhalation anthrax.

Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins: lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7-16.6% with 91.2-99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2-4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments.

Design and use of a novel substrate for simple, rapid, and specific early detection of anthrax infection.

Bacillus anthracis is a major biological warfare threat. The inhalation form of infection can kill quickly. While antibiotic treatment is effective, if diagnosis is delayed, the rapidly produced toxin may already be present in lethal amounts. This report describes a fast, sensitive, specific and accurate method for detection of active infection by Bacillus anthracis in plasma. One of the virulence factors, anthrax lethal factor, is an endopeptidase present in blood early in the infection. However, the use of peptidic substrates to detect endopetidases is problematic in plasma due to the presence of other proteases and the likelihood of nonspecific cleavage of the substrate. The fluorescently labeled peptide substrate MAPKKide Plus designed in this study is not cleaved by plasma proteases and thus is specific for lethal factor. Three detection strategies are described. Two include enrichment by capture from plasma using lethal factor antibody-coated microtiter plates or similarly coated immuno-tubes. The captured lethal factor is exposed to the MAPKKide Plus, and the amount of cleavage is determined either by HPLC or microplate reader. Concentration of lethal factor using the antibody-coated plates aplnd HPLC allows for detection of less than 5 pg lethal factor/ml of neat plasma after 2 hours of incubation. Using antibody-coated immuno-tubes, 20 pg lethal factor/ml plasma can be detected in 5 hours by a simple end point read of fluorescence in a microplate reader. For a third strategy, the substrate is added directly to diluted plasma, and cleavage is monitored by the increase in fluorescence as a function of time. The limit of detection by this simple method is 25 ng lethal factor/ml of plasma in 15 minutes, 5 ng/ml after 45 minutes, and <1 ng lethal factor/ml of plasma after 5 hours.

Development and application of a Bacillus anthracis protective antigen domain-1 in-house ELISA for the detection of anti-protective antigen antibodies in cattle in Zambia.

In Zambia, anthrax outbreaks among cattle are reported on nearly an annual basis. Presently, there is a lack of serological assays and information to develop an anthrax management and control strategy. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) based on recombinant protective antigen domain 1 (rPA-D1) of Bacillus anthracis was developed and used to detect anti-PA antibodies in cattle in Zambia. An antigen coating of 10 ng/well and a serum dilution of 1:100 were determined to be the optimal rPA-D1 ELISA titration conditions. The intra- and inter-assay % coefficients of variation were less than 10% and 15%, respectively. The rPA-D1 ELISA could detect seroconversion in the cattle 1 month after anthrax vaccination. In a cross-sectional study conducted in the Western Province, Zambia, 187 serum samples from 8 herds of cattle were screened for anti-PA antibodies using the rPA-D1 ELISA. The seropositive rate of the serum samples was 8%, and the mean anti-PA antibody was 0.358 ELISA units. Additionally, we screened 131 cattle serum samples from Lusaka, which is a nonendemic area, and found no significant association between the antibody levels and sampling area (endemic versus nonendemic area). Conversely, significant differences were observed between the anti-PA antibody levels and herds, anti-PA antibody levels and vaccination status and anti-PA antibody levels and vaccination timing. Collectively, these findings suggest that the rPA-D1 ELISA is a useful tool for the detection of anti-PA antibodies in cattle in Zambia. The low proportion of seropositive sera indicates that there is inadequate cattle vaccination in the Western Province and, in addition to other epidemiological factors, this may precipitate the anthrax outbreak recurrence.

Bioinspired detoxification of blood: The efficient removal of anthrax toxin protective antigen using an extracorporeal macroporous adsorbent device.

Whilst various remedial human monoclonal antibodies have been developed to treat the potentially life-threatening systemic complications associated with anthrax infection, an optimal and universally effective administration route has yet to be established. In the later stages of infection when antibody administration by injection is more likely to fail one possible route to improve outcome is via the use of an antibody-bound, adsorbent haemoperfusion device. We report here the development of an adsorbent macroporous polymer column containing immobilised B. anthracis exotoxin-specific antibodies, PANG (a non-glycosylated, version of a plant-produced human monoclonal antibody) and Valortim (a fully human monoclonal N-linked glycosylated antibody), for removal of anthrax protective antigen (PA) from freshly frozen human plasma and human whole blood. In addition, we have demonstrated that continuous extracorporeal blood recirculation through a Valortim-bound haemoperfusion column significantly reduced the blood plasma concentration of anthrax PA over 2 hours using an in vivo PA rat infusion model. This work provides proof-of-concept evidence to support the development of such alternative detoxification platforms.

Simultaneous Immunodetection of Anthrax, Plague, and Tularemia from Blood Cultures by Use of Multiplexed Suspension Arrays.

Multiplexed detection technologies are becoming increasingly important given the possibility of bioterrorism attacks, for which the range of suspected pathogens can vary considerably. In this work, we describe the use of Luminex MagPlex magnetic microspheres for the construction of two multiplexed diagnostic suspension arrays, enabling antibody-based detection of bacterial pathogens and their related disease biomarkers directly from blood cultures. The first 4-plex diagnostic array enabled the detection of both anthrax and plague infections using soluble disease biomarkers, including protective antigen (PA) and anthrax capsular antigen for anthrax detection and the capsular F1 and LcrV antigens for plague detection. The limits of detection (LODs) ranged between 0.5 and 5 ng/ml for the different antigens. The second 2-plex diagnostic array facilitated the detection of Yersinia pestis (LOD of 1 × 106 CFU/ml) and Francisella tularensis (LOD of 1 × 104 CFU/ml) from blood cultures. Inoculated, propagated blood cultures were processed (15 to 20 min) via 2 possible methodologies (Vacutainer or a simple centrifugation step), allowing the direct detection of bacteria in each sample, and the entire assay could be performed in 90 min. While detection of bacteria and soluble markers from blood cultures using PCR Luminex suspension arrays has been widely described, to our knowledge, this study is the first to demonstrate the utility of the Luminex system for the immunodetection of both bacteria and soluble markers directly from blood cultures. Targeting both the bacterial pathogens as well as two different disease biomarkers for each infection, we demonstrated the benefit of the multiplexed developed assays for enhanced, reliable detection. The presented arrays could easily be expanded to include antibodies for the detection of other pathogens of interest in hospitals or labs, demonstrating the applicability of this technology for the accurate detection and confirmation of a wide range of potential select agents.

Evaluation of clinical and serological findings for diagnosis of cutaneous anthrax infection after an outbreak.

PURPOSE: Anthrax, caused by the bacterium Bacillus anthracis, is one of the oldest documented infectious diseases in both livestock and humans. We aimed to evaluate clinical findings and risk factors of patients with cutaneous anthrax infection and report anti-lethal factor (LF) IgG and anti-protective antigen (PA) IgG titers in the serologic diagnosis of disease. METHODS: In this study, serum samples of 18 cutaneous anthrax patients were collected and anti-LF IgG and anti-PA IgG titers were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Twelve (67%) males and 6 (33%) females, with a mean age of 36.06 ± 16.58 years were included in the study. Risk factors identified in the patient population studied were slaughtering (28%), flaying (56%), chopping meat (67%), burying diseased animal corpses (17%) and milking (6%) livestock. Black eschar formation (94%), pruritus (78%) and painful lymphadenopathy (61%) were first three common clinical signs and symptoms, respectively. Fourteen (78%) patients produced a positive IgG response against PA, 11 (61%) patients produced against LF. Three (17%) patients had no response to either antigen. CONCLUSIONS: A detailed history of contact with sick animals or animal products along with clinical findings should be taken at the first step for the diagnosis of cutaneous anthrax infection. Serologic detection of anti-LF IgG and anti-PA IgG with ELISA may be useful auxillary method for establishing the diagnosis.

Serum paraoxonase activity and oxidative stress levels in patients with cutaneous anthrax.

INTRODUCTION: Anthrax is a bacterial disease caused by the aerobic sporeforming bacterium Bacillus anthracis. It has been suggested that oxidative stress plays an important role in the pathogenesis of B. anthracis. The aim of this study was to investigate serum paraoxonase 1 (PON1) activity, catalase activity, malondialdehyde (MDA) levels, and superoxide dismutase (SOD) levels in patients with cutaneous anthrax. MATERIALS AND METHODS: Fifteen patients with cutaneous anthrax and 15 healthy controls were enrolled in this study. The serum MDA levels, SOD levels, paraoxonase, arylesterase, and catalase activities were measured using a spectrophotometer. RESULTS: The serum SOD levels, paraoxonase, arylesterase, and catalase activities were significantly lower in patients with cutaneous anthrax than in controls (for all, p < 0.001), whereas MDA levels were significantly higher ( p < 0.001). No significant correlation was found between serum paraoxonase activity, arylesterase activity, SOD levels, and MDA levels (all, p > 0.05) in patients with cutaneous anthrax. CONCLUSIONS: The current study was the first to show decreased antioxidant levels and increased oxidant levels in patients with cutaneous anthrax. Therefore, decreased PON1 activity may play a role in the pathogenesis of cutaneous anthrax.

Validation of high throughput screening of human sera for detection of anti-PA IgG by Enzyme-Linked Immunosorbent Assay (ELISA) as an emergency response to an anthrax incident.

To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r2), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from -4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 µg/mL. The regression analysis results for dilutional linearity data were r2 = 0.952, slope = 1.02 and intercept = -0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate.

Rapid and facile ratiometric detection of an anthrax biomarker by regulating energy transfer process in bio-metal-organic framework.

A ratiometric fluorescent sensor based on luminescent bio-metal-organic framework was prepared by exchanging both Tb(3+) and Eu(3+) cations into anionic bio-MOF-1. Due to a highly efficient energy transfer from Tb(3+) to Eu(3+) (>89%), emission color of Tb/Eu@bio-MOF-1 was orange-red even though Tb(3+) was the dominant content in this Tb/Eu co-doping material. More interestingly, this energy transfer process could be modulated by dipicolinic acid (DPA), an unique biomarker for bacillus spores. With DPA addition, corresponding DPA-to-Tb(3+) energy transfer was gradually enhanced while the energy transfer from Tb(3+) to Eu(3+) was significantly weakened. By regulating the energy transfer process in Tb/Eu@bio-MOF-1, visual colorimetric sensing of DPA in porous MOF was realized for the first time. Detection limit of Tb/Eu@bio-MOF-1 for DPA was 34nM, which was much lower than an infectious dosage of Bacillus anthracis spores (60µM) for human being. Besides, Tb/Eu@bio-MOF-1 showed a remarkable selectivity over other aromatic ligands and amino acids. More importantly, this porous ratiometric sensor worked equally well in human serum. These particularly attractive features of Tb/Eu@bio-MOF-1 made the direct, rapid and naked-eye detection of DPA for practical application possible.

In vivo dynamics of active edema and lethal factors during anthrax.

Lethal and edema toxins are critical virulence factors of Bacillus anthracis. However, little is known about their in vivo dynamics of production during anthrax. In this study, we unraveled for the first time the in vivo kinetics of production of the toxin components EF (edema factor) and LF (lethal factor) during cutaneous infection with a wild-type toxinogenic encapsulated strain in immuno-competent mice. We stratified the asynchronous infection process into defined stages through bioluminescence imaging (BLI), while exploiting sensitive quantitative methods by measuring the enzymatic activity of LF and EF. LF was produced in high amounts, while EF amounts steadily increased during the infectious process. This led to high LF/EF ratios throughout the infection, with variations between 50 to a few thousands. In the bloodstream, the early detection of active LF and EF despite the absence of bacteria suggests that they may exert long distance effects. Infection with a strain deficient in the protective antigen toxin component enabled to address its role in the diffusion of LF and EF within the host. Our data provide a picture of the in vivo complexity of the infectious process.

Serologic Evidence of the Geographic Distribution of Bacterial Zoonotic Agents in Kenya, 2007.

Diseases of zoonotic origin contribute to the burden of febrile illnesses in developing countries. We evaluated serologic evidence of exposure to Bacillus anthracis, Brucella spp., spotted fever group rickettsioses (SFGR), and typhus group rickettsioses (TGR) from samples of persons aged 15-64 years collected during a nationwide human immunodeficiency virus (HIV) serosurvey conducted in 2007 in Kenya. The seropositivity observed for pathogens was B. anthracis 11.3%, Brucella spp. 3.0%, SFGR 23.3%, and TGR 0.6%. On univariate analysis, seropositivity for each pathogen was significantly associated with the following risk factors: B. anthracis with province of residence; Brucella spp. with sex, education level, and wealth; SFGR with age, education level, wealth, and province of residence; and TGR with province of residence. On multivariate analysis, seropositivity remained significantly associated with wealth and province for B. anthracis; with sex and age for Brucella spp; and with sex, education level, and province of residence for SFGR whereas TGR had no significance. High IgG seropositivity to these zoonotic pathogens (especially, B. anthracis and SFGR) suggests substantial exposure. These pathogens should be considered in the differential diagnosis of febrile illness in Kenya.

A S-Layer Protein of Bacillus anthracis as a Building Block for Functional Protein Arrays by In Vitro Self-Assembly.

S-layer proteins create a cell-surface layer architecture in both bacteria and archaea. Because S-layer proteins self-assemble into a native-like S-layer crystalline structure in vitro, they are attractive building blocks in nanotechnology. Here, the potential use of the S-layer protein EA1 from Bacillus anthracis in constructing a functional nanostructure is investigated, and apply this nanostructure in a proof-of-principle study for serological diagnosis of anthrax. EA1 is genetically fused with methyl parathion hydrolase (MPH), to degrade methyl parathion and provide a label for signal amplification. EA1 not only serves as a nanocarrier, but also as a specific antigen to capture anthrax-specific antibodies. As results, purified EA1-MPH forms a single layer of crystalline nanostructure through self-assembly. Our chimeric nanocatalyst greatly improves enzymatic stability of MPH. When applied to the detection of anthrax-specific antibodies in serum samples, the detection of our EA1-MPH nanostructure is nearly 300 times more sensitive than that of the unassembled complex. Together, it is shown that it is possible to build a functional and highly sensitive nanosensor based on S-layer protein. In conclusion, our present study should serve as a model for the development of other multifunctional nanomaterials using S-layer proteins.

Development & validation of a quantitative anti-protective antigen IgG enzyme linked immunosorbent assay for serodiagnosis of cutaneous anthrax.

BACKGROUND & OBJECTIVES: Anthrax caused by Bacillus anthracis is primarily a disease of herbivorous animals, although several mammals are vulnerable to it. ELISA is the most widely accepted serodiagnostic assay for large scale surveillance of cutaneous anthrax. The aims of this study were to develop and evaluate a quantitative ELISA for determination of IgG antibodies against B. anthracis protective antigen (PA) in human cutaneous anthrax cases. METHODS: Quantitative ELISA was developed using the recombinant PA for coating and standard reference serum AVR801 for quantification. A total of 116 human test and control serum samples were used in the study. The assay was evaluated for its precision, accuracy and linearity. RESULTS: The minimum detection limit and lower limit of quantification of the assay for anti-PA IgG were 3.2 and 4 µg/ml, respectively. The serum samples collected from the anthrax infected patients were found to have anti-PA IgG concentrations of 5.2 to 166.3 µg/ml. The intra-assay precision per cent CV within an assay and within an operator ranged from 0.99 to 7.4 per cent and 1.7 to 3.9 per cent, respectively. The accuracy of the assay was high with a per cent error of 6.5 - 24.1 per cent. The described assay was found to be linear between the range of 4 to 80 ng/ml (R [2] = 0.9982; slope = 0.9186; intercept = 0.1108). INTERPRETATION & CONCLUSIONS: The results suggested that the developed assay could be a useful tool for quantification of anti-PA IgG response in human after anthrax infection or vaccination.

Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 µg.

Fast and sensitive detection of an anthrax biomarker using SERS-based solenoid microfluidic sensor.

We report the application of a fully automated surface-enhanced Raman scattering (SERS)-based solenoid-embedded microfluidic device to the quantitative and sensitive detection of anthrax biomarker poly-γ-D-glutamic acid (PGA) in solution. Analysis is based on the competitive reaction between PGA and PGA-conjugated gold nanoparticles with anti-PGA-immobilized magnetic beads within a microfluidic environment. Magnetic immunocomplexes are trapped by yoke-type solenoids embedded within the device, and their SERS signals were directly measured and analyzed. To improve the accuracy of measurement process, external standard values for PGA-free serum were also measured through use of a control channel. This additional measurement greatly improves the reliability of the assay by minimizing the influence of extraneous experimental variables. The limit of detection (LOD) of PGA in serum, determined by our SERS-based microfluidic sensor, is estimated to be 100 pg/mL. We believe that the defined method represents a valuable analytical tool for the detection of anthrax-related aqueous samples.