- Email: [email protected]
Detection of protective antigen, an anthrax specific toxin in human serum by using surface plasmon resonance
Diagnostic Microbiology and Infectious Disease 77 (2013) 14–19
Contents lists available at SciVerse ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Detection of protective antigen, an anthrax specific toxin in human serum by using surface plasmon resonance Neha Ghosh, Nidhi Gupta, Garima Gupta, Mannan Boopathi, Vijay Pal, Ajay Kumar Goel ⁎ Biotechnology Division, Defence Research & Development Establishment, Gwalior-474 002, India
a r t i c l e
i n f o
Article history: Received 10 February 2013 Received in revised form 5 May 2013 Accepted 7 May 2013 Available online 15 June 2013 Keywords: Anthrax Bacillus anthracis Monoclonal antibody Protective antigen Surface plasmon resonance
a b s t r a c t In this study, surface plasmon resonance (SPR) technology was used for the sensitive detection of protective antigen (PA), an anthrax specific toxin in spiked human serum samples. A monoclonal antibody raised against Bacillus anthracis PA was immobilized on carboxymethyldextran-modified gold chip, and its interaction with PA was characterized in situ by SPR. By using kinetic evaluation software, KD (equilibrium constant) and Bmax (maximum binding capacity of analyte) were found to be 20 fM and 18.74 m°, respectively. The change in Gibb's free energy (ΔG= −78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 1 pg/mL purified PA. In PA-spiked human serum samples, 10 pg/mL of PA could be detected. Presence of PA in blood samples serves as an important early diagnostic marker for B. anthracis infections. Thus, SPR test can be a sensitive assay for detection of anthrax at early stages of infection. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Bacillus anthracis is the causative agent of anthrax, a zoonotic disease of domestic live stock and wildlife. Naturally occurring anthrax is transmitted to humans through direct contact with contaminated animals or through consumption of infected animal products or anthrax spores (Iacono-Connors et al., 1994; Walsh et al., 2007). The virulence of B. anthracis is attributed to 2 major factors, i.e., a tripartite toxin and the poly-γ-D-glutamic acid capsule (Collier and Young, 2003). Virulent B. anthracis vegetative cells form capsules of poly-D-glutamic acid, which impede the host immune system and inhibit macrophages from engulfing and destroying the bacteria (Ezzell and Welkos, 1999). The anthrax toxins are secreted as 3 distinct proteins, namely, protective antigen (PA), lethal factor (LF), and edema factor (EF), and their activities have been well described (Mock and Mignot, 2003; Turk, 2007). PA combines with EF and LF to form the binary toxins edema toxin (ETx) and lethal toxin (LTx), respectively (Singh et al., 1999). ETx elevates intracellular cyclic-adenosine monophosphate levels, whereas LTx inactivates members of the mitogen-activated protein kinase family resulting in an imbalance in the production or release of a range of cytokines that may contribute to the pathogenesis of anthrax (Duesbery et al., 1998; Moayeri et al., 2003). Anthrax toxin is the main cause of host system failure and death (Turk, 2007). Hence, sensitive and rapid assays for detection of anthrax toxins are required.
⁎ Corresponding author. Tel.: 91-751-2233742; fax: 91-751-2341148. E-mail address: [email protected] (A.K. Goel). 0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.05.006
Incidences of anthrax can be diagnosed by culture isolation of B. anthracis from the patients (Moayeri et al., 2003) or by detection of B. anthracis antigens or nucleic acid (Jernigan et al., 2002; Sacchi et al., 2002), if antibiotic treatment has not been initiated. Various assays like immunodiffusion, indirect micro-hemagglutination, and different enzyme-linked immunosorbent assays (ELISAs) with varying degrees of specificity and sensitivity have been developed for detection of α-PA or α-LF antibodies in anthrax infections (Ghosh and Goel, 2012; Ghosh et al., 2013b; Ghosh et al., 2013c; Iacono-Connors et al., 1994; Sirisanthana et al., 1988). However, due to acute and often fatal nature of untreated inhalation or systemic anthrax, the measurement of an immune response has not been a prominent feature of diagnosis. Moreover, during any outbreak or biological warfare–like situation, several methods like culture isolation, PCR, and biopsy may not be applicable. Under such circumstances, serological testing involving the direct detection of PA toxin in serum sample may be the confirmatory diagnostic tool. Presence of PA in the serum is a reliable marker for the detection, identification, and assessing the severity of the anthrax infection in human as well as animals (Kobiler et al., 2006). Moreover, PA plays a central role in the pathogenesis of anthrax, and there is a direct correlation between bacteremia and the PA concentration at the time of death (Fish and Lincoln, 1968). Therefore, sensitive and rapid assays for the detection of B. anthracis toxin are urgently needed to facilitate an early-stage diagnosis for successful treatment post exposure. The currently available ELISA for the detection of anthrax PA is able to achieve sensitivity levels of only up to 1 ng/mL (Mabry et al., 2006). In another study, europium nanoparticle-based immunoassay (ENIA) could detect anthrax PA in the range of 0.01 to 100 ng/mL (Tang et al., 2009). Hence, a highly sensitive assay is required which could detect anthrax toxin in human
N. Ghosh et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 14–19
sera just post exposure when the concentration of circulating PA in blood is even in pg/mL. Surface plasmon resonance (SPR) biosensors represent an emerging technology for a label-free, real-time, specific, rapid, and cost-effective diagnostic tool for bacteria that cause major public concern for food safety, bioterrorism, and nosocomial infections (Homola, 2008; Tawil et al., 2012). SPR is a dynamic method of detection in biological world, which could detect and characterize the antigen antibody interaction in the absence of chemical labeling and with minimum sample preparation (Bouffartigues et al., 2007; Dudak and Boyaci, 2009). It is based on optical phenomenon which measures refractive index changes produced by binding of molecule in the mobile phase to its biospecific ligand immobilized on the sensor (solid) surface. Analysis in real time provides the affinity of interactions or kinetic information of interactions, which can discriminate between the specific and nonspecific interactions. The presence of an analyte in the solution and specific binding to its ligands immobilized on the gold surface of the sensor chip results in a change in the refractive index of light reflected from the gold chip surface. Association and dissociation of the analyte are measured by detecting changes in the angle of incident light at which SPR occurs and is reported in millidegree in the sensorgram (Dudak and Boyaci, 2009; Homola, 2003; McDonnell, 2001; Rich and Myszka, 2000). Over the last years, extensive research efforts have been made to implement the SPR biosensors for rapid detection of bacterial pathogens (Ghosh et al., 2013a; Gupta et al., 2010; Taylor et al., 2006; Wang et al., 2012). In this study, we report a sensitive and specific method for the detection of PA in the human serum samples using SPR technology. The proposed assay can be very useful for diagnosis of B. anthracis infections in clinical samples during an outbreak or biological warfare–like situation. 2. Materials and methods 2.1. Materials N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), phosphate-buffered saline (PBS), 1M ethanolamine, and hydrochloric acid (HCl) were procured from Fluka. Citric acid, sodium hydroxide, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate were supplied by Sigma-Aldrich. All chemicals and reagents used were of analytical grade, and purification was performed wherever necessary before use. A 0.05 mol/L phosphate buffer (pH 6.0) was used as coupling buffer in the experiments, and dilution of antibody was also carried out using this buffer.
15
2.4. Production of monoclonal antibodies A mouse monoclonal antibody designated 3E5B8 was raised against 83-kDa rPA. Preparations containing 50 μg of rPA were injected subcutaneously into 8-week-old BALB/c mice. The immunization was repeated 3 times at 2-week intervals before boosting with 25-μg rPA per mouse. The spleen cells were removed 3 d later and fused with myeloma cells, according to the standard procedure (Kohler and Milstein, 1975). The hybridomas were cloned by limit dilution, screened using ELISA. The MAbs were purified by protein A column (Montage PROSEP-A Antibody Purification; Millipore, MD Millipore Corporation, Billerica, MA, USA) and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis to determine the purity. 2.5. ELISA For determination of titre of antibodies, maxisorp flat bottom 96well microtiter plates (Nalge Nunc International, Roskilde, Denmark) were coated with 100 μL per well of carbonate–bicarbonate buffer (pH 9.6) containing 2 μg/mL of rPA and incubated overnight at 4 °C. The antigen coated plates were washed 3 times with wash buffer (PBS containing 0.1% Tween 20) using ELx 508MS microplate washer (BioTek Instruments, Inc, Winooski, VT, USA). The wells were blocked with 300 μL of blocking buffer (5% skimmed milk in PBS) for 2 h at 37 °C. After washing, a concentration series of purified MAbs were added in 100-μL aliquots to individual wells and incubated for 60 min at 37 °C, and the plates were then washed 3 times with wash buffer. Horseradish peroxidase–conjugated goat anti-mouse antibodies (Sigma Aldrich, St Louis, MO, USA) diluted in PBS containing 5% skim milk and 0.5% Tween 20 (100 μL/well) were added at a dilution of 1:1000 and incubated for 60 min to detect the bound α-PA IgG. Plates were again washed 3 times with wash buffer and detected colorimetrically by using 100 μL/well of ortho-phenyl diamine/H2O2 substrate (Sigma Aldrich). Color development was stopped after 20 min by adding 50 μL of 2.5 N H2SO4 solution in each well, and the plate was read at 492 nm using an ELISA plate reader (BioTek Instruments, Inc). 2.6. Immobilization of α-PA monoclonal antibody on the carboxymethyldextran-modified gold SPR sensor chip
The studies of antigen-antibody interactions were conducted using a 2-channel cuvette-based electrochemical SPR system (Autolab ESPRIT; Ecochemie B.V., Utrecht, The Netherlands). The outcome of the SPR measurement was automatically monitored using a software and data acquisition using the SPR software version 4.3.1, and all kinetic data were obtained using the SPR kinetic evaluation software version 5.1 (Ecochemie B.V.). Carboxymethyldextran (20 nm thickness)–modified gold chip for SPR measurements was purchased from Xantec Bioanalytics (Duesseldorf, Germany). The pH of the buffers used was measured with a EUTECH instrument pH meter (pH-1500; Eutech Instruments Pvt Ltd, Ayer Rajah Crescent, Singapore). The temperature of cuvette was controlled by a water bath (Julabo HE-4, JULABO Labortechnik GmbH, Seelbach, Germany).
Prior to start of the experiment, the millidegree change (m°) in resonance angle was recorded as the baseline and stabilized by passing 50 μL of 0.05 mol/L phosphate buffer (pH 8.0) over carboxymethyldextran-modified gold chip at an interval of 120 s for 600 s. The carboxymethyldextran-modified gold chip was then activated by injecting a 50 μL of freshly prepared 1:1 mixture of EDC (400 mmol/L) and NHS (100 mmol/L) over the chip surface for 900 s in order to get more amine reactive NHS esters followed by immediate injection of 50 μL of α-PA monoclonal antibodies (0.34 mg/mL in 0.05 mol/L phosphate buffer) in channel 2 and allowed to interact for 1800 s to get an effective immobilization of α-PA monoclonal antibody over the activated dextran-modified surface. The immobilized surface was blocked for 600 s with 1M ethanolamine (pH 8.5) to deactivate nonreacted NHS ester in order to avoid nonspecific binding of antigen over the immobilized surface during sensing process. For negative control measurement, the modified gold surface was activated with EDC/NHS and then blocked with ethanolamine in channel 1 as mentioned above and was used as the blank control surface.
2.3. Preparation of antigen
2.7. Optimization of experimental conditions
The antigen, B. anthracis recombinant PA (rPA, 83 kDa) used for evaluation procedure in this study, was prepared and purified as described earlier (Shrivastva et al., 2008).
In order to find out the optimum temperature for the interaction of antigen and antibody, temperature variation study was carried out in the range from 10 to 38 °C with an increment of 3 °C.
2.2. Instrumentation
16
N. Ghosh et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 14–19
5.1. The thermodynamic parameter was calculated using van't Hoff equation (Glasstone, 1947). 2.10. Detection of PA in spiked clinical samples Sera from apparently healthy human (Group I, n = 45) and non– anthrax-infected persons (Group II, n = 20) were collected. The serum samples from Group I were pooled and artificially spiked with different concentrations of rPA, ranging from 1 pg/mL to 10 ng/mL. The spiked serum sample was diluted in PBS and used for the determination of detection sensitivity of SPR. For negative control, unspiked pooled serum was used. All the serum samples from Group II were tested individually to determine the specificity. 3. Results and discussion Fig. 1. Reactivity of purified monoclonal antibodies (3E5B8) with PA by plate ELISA.
2.8. Antigen-antibody interaction and characterization For affinity measurements and detection limit of PA, varying concentrations of PA were prepared in PBS and injected into both channels from the 384-well microtiter plate, and then association was performed for 500 s, and dissociation, for 400 s. Subsequently, 10 mmol/L HCl was used to achieve regeneration of the sensor surface after each interaction for 120 s, and PA was recovered in order to bring the signal to baseline level so as to start a new cycle. The above procedure was performed with solutions containing 1 pg/ mL, 10 pg/mL, 100 pg/mL, and 1 ng/mL concentrations of PA. A solution of PBS (pH 7.4) was used throughout the experiment as the running buffer.
2.9. Study of kinetic parameters Kinetic parameters of affinity interactions were characterized on the basis of KD values (equilibrium constant) and Bmax (maximum binding capacity of analyte) using kinetic evaluation software version
3.1. Generation of monoclonal antibodies The purified monoclonal antibody (MAb) 3E5B8 raised against rPA in this study exhibited a very good reactivity (a titre of 1: 512000) with rPA in ELISA (Fig. 1). 3.2. Immobilization of α-PA monoclonal antibody on carboxymethyldextran-modified gold chip for SPR sensor Immobilization of antibody on modified gold SPR sensor chip was done in 9 steps as described in Fig. 2. In the first step, the base line was stabilized for 120 s in order to get a stable baseline signal. In the second step, the carboxyl groups on the dextran layer was activated with EDCNHS (1:1 mixture) for 900 s for the generation of highly reactive Oacylisourea intermediates in order to make amide bonds between the carboxylic acid groups of modified sensor chip and the amino groups of α-PA monoclonal antibody. The activation of the sensor chip resulted with 548 m° angle change in SPR. In the third step, washing was done with PBS, which brought the angle change nearly to the baseline value (Gupta et al., 2010). After washing, in the fourth step, 50 μL of α-PA monoclonal antibody (0.34 mg/mL in 0.05 mol/L phosphate buffer) was injected on the sensor surface and allowed to interact for 1800 s,
Fig. 2. Sensogram showing immobilization of α-PA monoclonal antibody on modified gold chip. 1) Baseline, 2) EDC-NHS activation, 3) Washing, 4) Ligand coupling, 5) Washing, 6) Deactivation, 7) Washing, 8) Regeneration, and 9) Back to baseline.
N. Ghosh et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 14–19
17
Fig. 5. Calibration plot of SPR signal with concentrations of PA. Fig. 3. Effect of temperature on SPR angle change due to antigen-antibody interaction.
which resulted in angle shift of 102.67 m°. Subsequently, to remove unbound antibodies, a second washing was performed with PBS in the fifth step, and during this process, the SPR angle decreased by 1.5 m°. To prevent nonspecific binding of antigen on the sensor surface with unreacted NHS group, blocking was performed for 600 s with 1M ethanolamine (pH 8.5) in the sixth step, which resulted in increase in SPR angle by 659.5 m°. In the seventh step, washing was done with PBS for 30 s followed by regeneration for 120 s in the eighth step. Finally, in the ninth step, baseline was brought back to initial value in 60 s. A net angle change of 131.6 m° (Fig. 2) was observed, which ascribed the attachment of 1.10 ng mm −2 [120 m° = 1 ng mm −2] of antibody on the sensor surface (Stenberg et al., 1991). 3.3. Effect of temperature on interaction of antigen with α-PA monoclonal antibody A varying range of temperature was used in this study. An increase in SPR angle was observed upon increasing the temperature from 10 to 25 °C, and it decreased afterwards (Fig. 3). The maximum angle change in SPR was observed at 25 °C. This observation may be due to temperature-dependent structural changes and electrostatic interactions that occurred on the sensor chip during the antigen antibody interaction as reported earlier (Paynter and Russell, 2002). Hence, the
optimum temperature for the interaction of α-PA monoclonal antibody and PA was found to be 25 °C. 3.4. Detection of anthrax toxin with α-PA antibody immobilized sensor chip of SPR The sensogram for the antigen-antibody interaction for various concentrations of PA is shown in Fig. 4. The response increased in proportion with the concentrations of PA. A calibration curve was plotted using SPR signals from Fig. 4 (net angle change, i.e., working channel minus reference channel) to find out the detection limit of sensor (Fig. 5). In purified condition, the sensor could detect PA up to a concentration as low as 1 pg/mL. Because true anthrax-infected clinical samples were not available, therefore, the study was carried out with human sera spiked with PA toxin at concentrations ranging from 1 pg/mL to 10 ng/mL. For determination of specificity of the assay, different serum samples from Group II were tested and found that the response units obtained were less than 10 m°. Thus, the test was highly specific. Next, pooled human serum sample (Group I) was spiked with different concentrations of PA to evaluate the sensitivity of the assay. An increase in response value of SPR during association phase of 500 s followed by decrease in response value during 400 s of dissociation phase indicated antigen-antibody interaction on the sensor surface resulting
Fig. 4. Sensogram for antigen-antibody interaction for various concentrations of PA a) 1 pg/mL, b) 10 pg/mL, c) 100 pg/mL, and d) 1 ng/mL.
18
N. Ghosh et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 14–19
Fig. 6. Sensogram for antigen-antibody interaction for various concentrations of PA in human sera a) un-spikes human serum, b) 1 pg/mL, c) 10 pg/mL, d) 100 pg/mL, e) 1 ng/mL, f) 10 ng/mL.
in detection of anthrax PA in human serum (Fig. 6). As shown from the calibration plot, in spiked sera, the lowest detection sensitivity of SPR for PA toxin was up to 10 pg/mL in spiked blood samples (Fig. 6). The detection sensitivity of PA by sandwich ELISA was 50 ng/mL (data not shown). The detection sensitivity of PA in blood samples by an advanced engineered sandwich ELISA was N1 ng/mL (Mabry et al., 2006), whereas an ENIA could detect PA in the range of 0.01 to 100 ng/ mL (Tang et al., 2009). However, both ELISA and ENIA are based upon labeling techniques. Thus, high level of sensitivity was obtained with the immobilized α-PA monoclonal antibodies on the SPR sensor surface. The other advantage of the method is that it is the immediate method of detection of PA in the serum without any involvement of multiple antibodies including a labeled antibody. Labeling technologies commonly used in immunoassays include radioactivity, enzyme activity, and chemicals (chemiluminescence and fluorescence). The use of radioisotopic labeling is being discontinued in many laboratories for several reasons, including the safety hazards that they pose. One major disadvantage of the enzyme-based colorimetric ELISA is its relatively low detection sensitivity. Moreover, labeling makes protocol very time consuming for detection as in case of ELISA. As the present SPR methodology is totally based on monoclonal antibody and also provides information about affinity of interaction, proper association, and dissociation constants along with kinetic information, it could easily discriminate between specific and nonspecific binding. Thus, SPR technology does not involve any labeling and is found to be a better diagnostic system of B. anthracis infections in human in short time with very high specificity.
3.5. Evaluation of kinetic parameters involved in the antigen antibody interaction used for detection of B. anthracis The kinetics of the affinity binding between PA and α-PA monoclonal was analysed using the kinetic evaluation software as mentioned in materials and methods. The Bmax, KD, and ΔG were found to be 18.74 m°, 20 fM, and −78.04 kJ mol −1, respectively. The low KD value indicates the high binding affinity of antibody with antigen and negative value of Gibb's free energy shows the spontaneity of reaction.
Thus, due to high sensitivity and specificity of SPR-based methodology, this assay could be suitable for the detection of low levels of PA in clinical samples in the acute phase of infections and proposed as a good detection system for anthrax in humans in early stage of infections. Hence, SPR-based detection of B. anthracis is a very useful tool for screening and confirmation of anthrax-suspected clinical samples during bio-warfare–like situations also. Acknowledgments Authors are thankful to Director, Defence Research & Development Establishment (DRDE), Gwalior for providing necessary facilities and funds for the work. NG is thankful to Indian Council of Medical Research (ICMR) for providing Senior Research Fellowship. References Bouffartigues E, Leh H, Anger-Leroy M, Rimsky S, Buckle M. Rapid coupling of Surface Plasmon Resonance (SPR and SPRi) and ProteinChip based mass spectrometry for the identification of proteins in nucleoprotein interactions. Nucleic Acids Res 2007;35:e39, http://dx.doi.org/10.1093/nar/gkm030. Collier RJ, Young JA. Anthrax toxin. Annu Rev Cell Dev Biol 2003;19:45–70, http:// dx.doi.org/10.1146/annurev.cellbio.19.111301.140655. Dudak FC, Boyaci IH. Rapid and label-free bacteria detection by surface plasmon resonance (SPR) biosensors. Biotechnol J 2009;4:1003–11, http://dx.doi.org/ 10.1002/biot.200800316. Duesbery NS, Webb CP, Leppla SH, Gordon VM, Klimpel KR, Copeland TD, et al. Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science 1998;280:734–7. Ezzell JW, Welkos SL. The capsule of Bacillus anthracis, a review. J Appl Microbiol 1999;87:250. Fish DC, Lincoln RE. In vivo-produced anthrax toxin. J Bacteriol 1968;95:919–24. Ghosh N, Goel AK. Anti-protective antigen IgG enzyme-linked immunosorbent assay for diagnosis of cutaneous anthrax in India. Clin Vaccine Immunol 2012;19:1238–42, http://dx.doi.org/10.1128/CVI.00154-12. Ghosh N, Gupta G, Boopathi M, Pal V, Singh AK, Gopalan N, Goel AK. Surface plasmon resonance biosensor for detection of Bacillus anthracis, the causative agent of anthrax from soil samples targeting protective antigen. Indian J Microbiol 2013a;53:48–55, http://dx.doi.org/10.1007/s12088-012-0334-3. Ghosh N, Tomar I, Goel AK. A field usable qualitative anti-protective antigen enzymelinked immunosorbent assay for serodiagnosis of human anthrax. Microbiol Immunol 2013b;57:145–9, http://dx.doi.org/10.1111/1348-0421.12014. Ghosh N, Tomar I, Lukka H, Goel AK. Serodiagnosis of human cutaneous anthrax in India using an indirect anti-lethal factor IgG enzyme-linked immunosorbent assay. Clin Vaccine Immunol 2013c;20:282–6, http://dx.doi.org/10.1128/CVI.00598-12.
N. Ghosh et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 14–19 Glasstone SD. Thermodynamics for chemists. New York: Van Nostrand Company; 1947. Gupta G, Singh PK, Boopathi M, Kamboj DV, Singh B, Vijayaraghavan R. Surface plasmon resonance detection of biological warfare agent Staphylococcal enterotoxin B using high affinity monoclonal antibody. Thin Solid Films 2010;519:1171–7, http://dx.doi.org/10.1016/j.tsf.2010.08.064. Homola J. Present and future of surface plasmon resonance biosensors. Analytical Bioanalytical Chem 2003;377:528–39, http://dx.doi.org/10.1007/s00216-0032101-0. Homola J. Surface plasmon resonance sensors for detection of chemical and biological species. Chem Rev 2008;108:462–93, http://dx.doi.org/10.1021/cr068107d. Iacono-Connors LC, Novak J, Rossi C, Mangiafico J, Ksiazek T. Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies. Clin Diagn Lab Immunol 1994;1:78–82. Jernigan DB, Raghunathan PL, Bell BP, Brechner R, Bresnitz EA, Butler JC, et al. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg Infect Dis 2002;8:1019–28. Kobiler D, Weiss S, Levy H, Fisher M, Mechaly A, Pass A, Altboum Z. Protective antigen as a correlative marker for anthrax in animal models. Infect Immun 2006;74:5871–6, http://dx.doi.org/10.1128/IAI.00792-06. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495–7. Mabry R, Brasky K, Geiger R, Carrion Jr R, Hubbard GB, Leppla S, et al. Detection of anthrax toxin in the serum of animals infected with Bacillus anthracis by using engineered immunoassays. Clin Vaccine Immunol 2006;13:671–7, http://dx.doi.org/ 10.1128/CVI.00023-06. McDonnell JM. Surface plasmon resonance: towards an understanding of the mechanisms of biological molecular recognition. Curr Opin Chem Biol 2001;5:572–7. Moayeri M, Haines D, Young HA, Leppla SH. Bacillus anthracis lethal toxin induces TNFalpha-independent hypoxia-mediated toxicity in mice. J Clin Invest 2003;112: 670–82, http://dx.doi.org/10.1172/JCI17991. Mock M, Mignot T. Anthrax toxins and the host: a story of intimacy. Cell Microbiol 2003;5:15–23. Paynter S, Russell DA. Surface plasmon resonance measurement of pH-induced responses of immobilized biomolecules: conformational change or electrostatic interaction effects? Anal Biochem 2002;309:85–95.
19
Rich RL, Myszka DG. Advances in surface plasmon resonance biosensor analysis. Curr Opin Biotechnol 2000;11:54–61. Sacchi CT, Whitney AM, Mayer LW, Morey R, Steigerwalt A, Boras A, et al. Sequencing of 16S rRNA gene: a rapid tool for identification of Bacillus anthracis. Emerg Infect Dis 2002;8:1117–23. Shrivastva A, Tripathi NK, Sathyaseelan K, Jana AM, Rao PVL. Production of Bacillus anthracis recombinant protective antigen in Escherichia coli. Adv Biotech 2008;26: 26–9. Singh Y, Klimpel KR, Goel S, Swain PK, Leppla SH. Oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells. Infect Immun 1999;67:1853–9. Sirisanthana T, Nelson KE, Ezzell JW, Abshire TG. Serological studies of patients with cutaneous and oral-oropharyngeal anthrax from northern Thailand. Am J Trop Med Hyg 1988;39:575–81. Stenberg E, Persson B, Roos H, Urbaniczky C. Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins. J Colloid Interface Sci 1991;143:513–26, http://dx.doi.org/10.1016/00219797(91)90284-f. Tang S, Moayeri M, Chen Z, Harma H, Zhao J, Hu H, et al. Detection of anthrax toxin by an ultrasensitive immunoassay using europium nanoparticles. Clin Vaccine Immunol 2009;16:408–13, http://dx.doi.org/10.1128/CVI.00412-08. Tawil N, Sacher E, Mandeville R, Meunier M. Surface plasmon resonance detection of E. coli and methicillin-resistant S. aureus using bacteriophages. Biosens Bioelectron 2012;37:24–9, http://dx.doi.org/10.1016/j.bios.2012.04.048. Taylor AD, Ladd J, Yu Q, Chen S, Homola J, Jiang S. Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor. Biosens Bioelectron 2006;22:752–8, http://dx.doi.org/10.1016/j.bios.2006.03.012. Turk BE. Manipulation of host signalling pathways by anthrax toxins. Biochem J 2007;402:405–17, http://dx.doi.org/10.1042/BJ20061891. Walsh JJ, Pesik N, Quinn CP, Urdaneta V, Dykewicz CA, Boyer AE, et al. A case of naturally acquired inhalation anthrax: clinical care and analyses of anti-protective antigen immunoglobulin G and lethal factor. Clin Infect Dis 2007;44:968–71, http:// dx.doi.org/10.1086/512372. Wang Y, Knoll W, Dostalek J. Bacterial pathogen surface plasmon resonance biosensor advanced by long range surface plasmons and magnetic nanoparticle assays. Anal Chem 2012;84:8345–50, http://dx.doi.org/10.1021/ac301904x.
Contents lists available at SciVerse ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Detection of protective antigen, an anthrax specific toxin in human serum by using surface plasmon resonance Neha Ghosh, Nidhi Gupta, Garima Gupta, Mannan Boopathi, Vijay Pal, Ajay Kumar Goel ⁎ Biotechnology Division, Defence Research & Development Establishment, Gwalior-474 002, India
a r t i c l e
i n f o
Article history: Received 10 February 2013 Received in revised form 5 May 2013 Accepted 7 May 2013 Available online 15 June 2013 Keywords: Anthrax Bacillus anthracis Monoclonal antibody Protective antigen Surface plasmon resonance
a b s t r a c t In this study, surface plasmon resonance (SPR) technology was used for the sensitive detection of protective antigen (PA), an anthrax specific toxin in spiked human serum samples. A monoclonal antibody raised against Bacillus anthracis PA was immobilized on carboxymethyldextran-modified gold chip, and its interaction with PA was characterized in situ by SPR. By using kinetic evaluation software, KD (equilibrium constant) and Bmax (maximum binding capacity of analyte) were found to be 20 fM and 18.74 m°, respectively. The change in Gibb's free energy (ΔG= −78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 1 pg/mL purified PA. In PA-spiked human serum samples, 10 pg/mL of PA could be detected. Presence of PA in blood samples serves as an important early diagnostic marker for B. anthracis infections. Thus, SPR test can be a sensitive assay for detection of anthrax at early stages of infection. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Bacillus anthracis is the causative agent of anthrax, a zoonotic disease of domestic live stock and wildlife. Naturally occurring anthrax is transmitted to humans through direct contact with contaminated animals or through consumption of infected animal products or anthrax spores (Iacono-Connors et al., 1994; Walsh et al., 2007). The virulence of B. anthracis is attributed to 2 major factors, i.e., a tripartite toxin and the poly-γ-D-glutamic acid capsule (Collier and Young, 2003). Virulent B. anthracis vegetative cells form capsules of poly-D-glutamic acid, which impede the host immune system and inhibit macrophages from engulfing and destroying the bacteria (Ezzell and Welkos, 1999). The anthrax toxins are secreted as 3 distinct proteins, namely, protective antigen (PA), lethal factor (LF), and edema factor (EF), and their activities have been well described (Mock and Mignot, 2003; Turk, 2007). PA combines with EF and LF to form the binary toxins edema toxin (ETx) and lethal toxin (LTx), respectively (Singh et al., 1999). ETx elevates intracellular cyclic-adenosine monophosphate levels, whereas LTx inactivates members of the mitogen-activated protein kinase family resulting in an imbalance in the production or release of a range of cytokines that may contribute to the pathogenesis of anthrax (Duesbery et al., 1998; Moayeri et al., 2003). Anthrax toxin is the main cause of host system failure and death (Turk, 2007). Hence, sensitive and rapid assays for detection of anthrax toxins are required.
⁎ Corresponding author. Tel.: 91-751-2233742; fax: 91-751-2341148. E-mail address: [email protected] (A.K. Goel). 0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.05.006
Incidences of anthrax can be diagnosed by culture isolation of B. anthracis from the patients (Moayeri et al., 2003) or by detection of B. anthracis antigens or nucleic acid (Jernigan et al., 2002; Sacchi et al., 2002), if antibiotic treatment has not been initiated. Various assays like immunodiffusion, indirect micro-hemagglutination, and different enzyme-linked immunosorbent assays (ELISAs) with varying degrees of specificity and sensitivity have been developed for detection of α-PA or α-LF antibodies in anthrax infections (Ghosh and Goel, 2012; Ghosh et al., 2013b; Ghosh et al., 2013c; Iacono-Connors et al., 1994; Sirisanthana et al., 1988). However, due to acute and often fatal nature of untreated inhalation or systemic anthrax, the measurement of an immune response has not been a prominent feature of diagnosis. Moreover, during any outbreak or biological warfare–like situation, several methods like culture isolation, PCR, and biopsy may not be applicable. Under such circumstances, serological testing involving the direct detection of PA toxin in serum sample may be the confirmatory diagnostic tool. Presence of PA in the serum is a reliable marker for the detection, identification, and assessing the severity of the anthrax infection in human as well as animals (Kobiler et al., 2006). Moreover, PA plays a central role in the pathogenesis of anthrax, and there is a direct correlation between bacteremia and the PA concentration at the time of death (Fish and Lincoln, 1968). Therefore, sensitive and rapid assays for the detection of B. anthracis toxin are urgently needed to facilitate an early-stage diagnosis for successful treatment post exposure. The currently available ELISA for the detection of anthrax PA is able to achieve sensitivity levels of only up to 1 ng/mL (Mabry et al., 2006). In another study, europium nanoparticle-based immunoassay (ENIA) could detect anthrax PA in the range of 0.01 to 100 ng/mL (Tang et al., 2009). Hence, a highly sensitive assay is required which could detect anthrax toxin in human
N. Ghosh et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 14–19
sera just post exposure when the concentration of circulating PA in blood is even in pg/mL. Surface plasmon resonance (SPR) biosensors represent an emerging technology for a label-free, real-time, specific, rapid, and cost-effective diagnostic tool for bacteria that cause major public concern for food safety, bioterrorism, and nosocomial infections (Homola, 2008; Tawil et al., 2012). SPR is a dynamic method of detection in biological world, which could detect and characterize the antigen antibody interaction in the absence of chemical labeling and with minimum sample preparation (Bouffartigues et al., 2007; Dudak and Boyaci, 2009). It is based on optical phenomenon which measures refractive index changes produced by binding of molecule in the mobile phase to its biospecific ligand immobilized on the sensor (solid) surface. Analysis in real time provides the affinity of interactions or kinetic information of interactions, which can discriminate between the specific and nonspecific interactions. The presence of an analyte in the solution and specific binding to its ligands immobilized on the gold surface of the sensor chip results in a change in the refractive index of light reflected from the gold chip surface. Association and dissociation of the analyte are measured by detecting changes in the angle of incident light at which SPR occurs and is reported in millidegree in the sensorgram (Dudak and Boyaci, 2009; Homola, 2003; McDonnell, 2001; Rich and Myszka, 2000). Over the last years, extensive research efforts have been made to implement the SPR biosensors for rapid detection of bacterial pathogens (Ghosh et al., 2013a; Gupta et al., 2010; Taylor et al., 2006; Wang et al., 2012). In this study, we report a sensitive and specific method for the detection of PA in the human serum samples using SPR technology. The proposed assay can be very useful for diagnosis of B. anthracis infections in clinical samples during an outbreak or biological warfare–like situation. 2. Materials and methods 2.1. Materials N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), phosphate-buffered saline (PBS), 1M ethanolamine, and hydrochloric acid (HCl) were procured from Fluka. Citric acid, sodium hydroxide, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate were supplied by Sigma-Aldrich. All chemicals and reagents used were of analytical grade, and purification was performed wherever necessary before use. A 0.05 mol/L phosphate buffer (pH 6.0) was used as coupling buffer in the experiments, and dilution of antibody was also carried out using this buffer.
15
2.4. Production of monoclonal antibodies A mouse monoclonal antibody designated 3E5B8 was raised against 83-kDa rPA. Preparations containing 50 μg of rPA were injected subcutaneously into 8-week-old BALB/c mice. The immunization was repeated 3 times at 2-week intervals before boosting with 25-μg rPA per mouse. The spleen cells were removed 3 d later and fused with myeloma cells, according to the standard procedure (Kohler and Milstein, 1975). The hybridomas were cloned by limit dilution, screened using ELISA. The MAbs were purified by protein A column (Montage PROSEP-A Antibody Purification; Millipore, MD Millipore Corporation, Billerica, MA, USA) and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis to determine the purity. 2.5. ELISA For determination of titre of antibodies, maxisorp flat bottom 96well microtiter plates (Nalge Nunc International, Roskilde, Denmark) were coated with 100 μL per well of carbonate–bicarbonate buffer (pH 9.6) containing 2 μg/mL of rPA and incubated overnight at 4 °C. The antigen coated plates were washed 3 times with wash buffer (PBS containing 0.1% Tween 20) using ELx 508MS microplate washer (BioTek Instruments, Inc, Winooski, VT, USA). The wells were blocked with 300 μL of blocking buffer (5% skimmed milk in PBS) for 2 h at 37 °C. After washing, a concentration series of purified MAbs were added in 100-μL aliquots to individual wells and incubated for 60 min at 37 °C, and the plates were then washed 3 times with wash buffer. Horseradish peroxidase–conjugated goat anti-mouse antibodies (Sigma Aldrich, St Louis, MO, USA) diluted in PBS containing 5% skim milk and 0.5% Tween 20 (100 μL/well) were added at a dilution of 1:1000 and incubated for 60 min to detect the bound α-PA IgG. Plates were again washed 3 times with wash buffer and detected colorimetrically by using 100 μL/well of ortho-phenyl diamine/H2O2 substrate (Sigma Aldrich). Color development was stopped after 20 min by adding 50 μL of 2.5 N H2SO4 solution in each well, and the plate was read at 492 nm using an ELISA plate reader (BioTek Instruments, Inc). 2.6. Immobilization of α-PA monoclonal antibody on the carboxymethyldextran-modified gold SPR sensor chip
The studies of antigen-antibody interactions were conducted using a 2-channel cuvette-based electrochemical SPR system (Autolab ESPRIT; Ecochemie B.V., Utrecht, The Netherlands). The outcome of the SPR measurement was automatically monitored using a software and data acquisition using the SPR software version 4.3.1, and all kinetic data were obtained using the SPR kinetic evaluation software version 5.1 (Ecochemie B.V.). Carboxymethyldextran (20 nm thickness)–modified gold chip for SPR measurements was purchased from Xantec Bioanalytics (Duesseldorf, Germany). The pH of the buffers used was measured with a EUTECH instrument pH meter (pH-1500; Eutech Instruments Pvt Ltd, Ayer Rajah Crescent, Singapore). The temperature of cuvette was controlled by a water bath (Julabo HE-4, JULABO Labortechnik GmbH, Seelbach, Germany).
Prior to start of the experiment, the millidegree change (m°) in resonance angle was recorded as the baseline and stabilized by passing 50 μL of 0.05 mol/L phosphate buffer (pH 8.0) over carboxymethyldextran-modified gold chip at an interval of 120 s for 600 s. The carboxymethyldextran-modified gold chip was then activated by injecting a 50 μL of freshly prepared 1:1 mixture of EDC (400 mmol/L) and NHS (100 mmol/L) over the chip surface for 900 s in order to get more amine reactive NHS esters followed by immediate injection of 50 μL of α-PA monoclonal antibodies (0.34 mg/mL in 0.05 mol/L phosphate buffer) in channel 2 and allowed to interact for 1800 s to get an effective immobilization of α-PA monoclonal antibody over the activated dextran-modified surface. The immobilized surface was blocked for 600 s with 1M ethanolamine (pH 8.5) to deactivate nonreacted NHS ester in order to avoid nonspecific binding of antigen over the immobilized surface during sensing process. For negative control measurement, the modified gold surface was activated with EDC/NHS and then blocked with ethanolamine in channel 1 as mentioned above and was used as the blank control surface.
2.3. Preparation of antigen
2.7. Optimization of experimental conditions
The antigen, B. anthracis recombinant PA (rPA, 83 kDa) used for evaluation procedure in this study, was prepared and purified as described earlier (Shrivastva et al., 2008).
In order to find out the optimum temperature for the interaction of antigen and antibody, temperature variation study was carried out in the range from 10 to 38 °C with an increment of 3 °C.
2.2. Instrumentation
16
N. Ghosh et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 14–19
5.1. The thermodynamic parameter was calculated using van't Hoff equation (Glasstone, 1947). 2.10. Detection of PA in spiked clinical samples Sera from apparently healthy human (Group I, n = 45) and non– anthrax-infected persons (Group II, n = 20) were collected. The serum samples from Group I were pooled and artificially spiked with different concentrations of rPA, ranging from 1 pg/mL to 10 ng/mL. The spiked serum sample was diluted in PBS and used for the determination of detection sensitivity of SPR. For negative control, unspiked pooled serum was used. All the serum samples from Group II were tested individually to determine the specificity. 3. Results and discussion Fig. 1. Reactivity of purified monoclonal antibodies (3E5B8) with PA by plate ELISA.
2.8. Antigen-antibody interaction and characterization For affinity measurements and detection limit of PA, varying concentrations of PA were prepared in PBS and injected into both channels from the 384-well microtiter plate, and then association was performed for 500 s, and dissociation, for 400 s. Subsequently, 10 mmol/L HCl was used to achieve regeneration of the sensor surface after each interaction for 120 s, and PA was recovered in order to bring the signal to baseline level so as to start a new cycle. The above procedure was performed with solutions containing 1 pg/ mL, 10 pg/mL, 100 pg/mL, and 1 ng/mL concentrations of PA. A solution of PBS (pH 7.4) was used throughout the experiment as the running buffer.
2.9. Study of kinetic parameters Kinetic parameters of affinity interactions were characterized on the basis of KD values (equilibrium constant) and Bmax (maximum binding capacity of analyte) using kinetic evaluation software version
3.1. Generation of monoclonal antibodies The purified monoclonal antibody (MAb) 3E5B8 raised against rPA in this study exhibited a very good reactivity (a titre of 1: 512000) with rPA in ELISA (Fig. 1). 3.2. Immobilization of α-PA monoclonal antibody on carboxymethyldextran-modified gold chip for SPR sensor Immobilization of antibody on modified gold SPR sensor chip was done in 9 steps as described in Fig. 2. In the first step, the base line was stabilized for 120 s in order to get a stable baseline signal. In the second step, the carboxyl groups on the dextran layer was activated with EDCNHS (1:1 mixture) for 900 s for the generation of highly reactive Oacylisourea intermediates in order to make amide bonds between the carboxylic acid groups of modified sensor chip and the amino groups of α-PA monoclonal antibody. The activation of the sensor chip resulted with 548 m° angle change in SPR. In the third step, washing was done with PBS, which brought the angle change nearly to the baseline value (Gupta et al., 2010). After washing, in the fourth step, 50 μL of α-PA monoclonal antibody (0.34 mg/mL in 0.05 mol/L phosphate buffer) was injected on the sensor surface and allowed to interact for 1800 s,
Fig. 2. Sensogram showing immobilization of α-PA monoclonal antibody on modified gold chip. 1) Baseline, 2) EDC-NHS activation, 3) Washing, 4) Ligand coupling, 5) Washing, 6) Deactivation, 7) Washing, 8) Regeneration, and 9) Back to baseline.
N. Ghosh et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 14–19
17
Fig. 5. Calibration plot of SPR signal with concentrations of PA. Fig. 3. Effect of temperature on SPR angle change due to antigen-antibody interaction.
which resulted in angle shift of 102.67 m°. Subsequently, to remove unbound antibodies, a second washing was performed with PBS in the fifth step, and during this process, the SPR angle decreased by 1.5 m°. To prevent nonspecific binding of antigen on the sensor surface with unreacted NHS group, blocking was performed for 600 s with 1M ethanolamine (pH 8.5) in the sixth step, which resulted in increase in SPR angle by 659.5 m°. In the seventh step, washing was done with PBS for 30 s followed by regeneration for 120 s in the eighth step. Finally, in the ninth step, baseline was brought back to initial value in 60 s. A net angle change of 131.6 m° (Fig. 2) was observed, which ascribed the attachment of 1.10 ng mm −2 [120 m° = 1 ng mm −2] of antibody on the sensor surface (Stenberg et al., 1991). 3.3. Effect of temperature on interaction of antigen with α-PA monoclonal antibody A varying range of temperature was used in this study. An increase in SPR angle was observed upon increasing the temperature from 10 to 25 °C, and it decreased afterwards (Fig. 3). The maximum angle change in SPR was observed at 25 °C. This observation may be due to temperature-dependent structural changes and electrostatic interactions that occurred on the sensor chip during the antigen antibody interaction as reported earlier (Paynter and Russell, 2002). Hence, the
optimum temperature for the interaction of α-PA monoclonal antibody and PA was found to be 25 °C. 3.4. Detection of anthrax toxin with α-PA antibody immobilized sensor chip of SPR The sensogram for the antigen-antibody interaction for various concentrations of PA is shown in Fig. 4. The response increased in proportion with the concentrations of PA. A calibration curve was plotted using SPR signals from Fig. 4 (net angle change, i.e., working channel minus reference channel) to find out the detection limit of sensor (Fig. 5). In purified condition, the sensor could detect PA up to a concentration as low as 1 pg/mL. Because true anthrax-infected clinical samples were not available, therefore, the study was carried out with human sera spiked with PA toxin at concentrations ranging from 1 pg/mL to 10 ng/mL. For determination of specificity of the assay, different serum samples from Group II were tested and found that the response units obtained were less than 10 m°. Thus, the test was highly specific. Next, pooled human serum sample (Group I) was spiked with different concentrations of PA to evaluate the sensitivity of the assay. An increase in response value of SPR during association phase of 500 s followed by decrease in response value during 400 s of dissociation phase indicated antigen-antibody interaction on the sensor surface resulting
Fig. 4. Sensogram for antigen-antibody interaction for various concentrations of PA a) 1 pg/mL, b) 10 pg/mL, c) 100 pg/mL, and d) 1 ng/mL.
18
N. Ghosh et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 14–19
Fig. 6. Sensogram for antigen-antibody interaction for various concentrations of PA in human sera a) un-spikes human serum, b) 1 pg/mL, c) 10 pg/mL, d) 100 pg/mL, e) 1 ng/mL, f) 10 ng/mL.
in detection of anthrax PA in human serum (Fig. 6). As shown from the calibration plot, in spiked sera, the lowest detection sensitivity of SPR for PA toxin was up to 10 pg/mL in spiked blood samples (Fig. 6). The detection sensitivity of PA by sandwich ELISA was 50 ng/mL (data not shown). The detection sensitivity of PA in blood samples by an advanced engineered sandwich ELISA was N1 ng/mL (Mabry et al., 2006), whereas an ENIA could detect PA in the range of 0.01 to 100 ng/ mL (Tang et al., 2009). However, both ELISA and ENIA are based upon labeling techniques. Thus, high level of sensitivity was obtained with the immobilized α-PA monoclonal antibodies on the SPR sensor surface. The other advantage of the method is that it is the immediate method of detection of PA in the serum without any involvement of multiple antibodies including a labeled antibody. Labeling technologies commonly used in immunoassays include radioactivity, enzyme activity, and chemicals (chemiluminescence and fluorescence). The use of radioisotopic labeling is being discontinued in many laboratories for several reasons, including the safety hazards that they pose. One major disadvantage of the enzyme-based colorimetric ELISA is its relatively low detection sensitivity. Moreover, labeling makes protocol very time consuming for detection as in case of ELISA. As the present SPR methodology is totally based on monoclonal antibody and also provides information about affinity of interaction, proper association, and dissociation constants along with kinetic information, it could easily discriminate between specific and nonspecific binding. Thus, SPR technology does not involve any labeling and is found to be a better diagnostic system of B. anthracis infections in human in short time with very high specificity.
3.5. Evaluation of kinetic parameters involved in the antigen antibody interaction used for detection of B. anthracis The kinetics of the affinity binding between PA and α-PA monoclonal was analysed using the kinetic evaluation software as mentioned in materials and methods. The Bmax, KD, and ΔG were found to be 18.74 m°, 20 fM, and −78.04 kJ mol −1, respectively. The low KD value indicates the high binding affinity of antibody with antigen and negative value of Gibb's free energy shows the spontaneity of reaction.
Thus, due to high sensitivity and specificity of SPR-based methodology, this assay could be suitable for the detection of low levels of PA in clinical samples in the acute phase of infections and proposed as a good detection system for anthrax in humans in early stage of infections. Hence, SPR-based detection of B. anthracis is a very useful tool for screening and confirmation of anthrax-suspected clinical samples during bio-warfare–like situations also. Acknowledgments Authors are thankful to Director, Defence Research & Development Establishment (DRDE), Gwalior for providing necessary facilities and funds for the work. NG is thankful to Indian Council of Medical Research (ICMR) for providing Senior Research Fellowship. References Bouffartigues E, Leh H, Anger-Leroy M, Rimsky S, Buckle M. Rapid coupling of Surface Plasmon Resonance (SPR and SPRi) and ProteinChip based mass spectrometry for the identification of proteins in nucleoprotein interactions. Nucleic Acids Res 2007;35:e39, http://dx.doi.org/10.1093/nar/gkm030. Collier RJ, Young JA. Anthrax toxin. Annu Rev Cell Dev Biol 2003;19:45–70, http:// dx.doi.org/10.1146/annurev.cellbio.19.111301.140655. Dudak FC, Boyaci IH. Rapid and label-free bacteria detection by surface plasmon resonance (SPR) biosensors. Biotechnol J 2009;4:1003–11, http://dx.doi.org/ 10.1002/biot.200800316. Duesbery NS, Webb CP, Leppla SH, Gordon VM, Klimpel KR, Copeland TD, et al. Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science 1998;280:734–7. Ezzell JW, Welkos SL. The capsule of Bacillus anthracis, a review. J Appl Microbiol 1999;87:250. Fish DC, Lincoln RE. In vivo-produced anthrax toxin. J Bacteriol 1968;95:919–24. Ghosh N, Goel AK. Anti-protective antigen IgG enzyme-linked immunosorbent assay for diagnosis of cutaneous anthrax in India. Clin Vaccine Immunol 2012;19:1238–42, http://dx.doi.org/10.1128/CVI.00154-12. Ghosh N, Gupta G, Boopathi M, Pal V, Singh AK, Gopalan N, Goel AK. Surface plasmon resonance biosensor for detection of Bacillus anthracis, the causative agent of anthrax from soil samples targeting protective antigen. Indian J Microbiol 2013a;53:48–55, http://dx.doi.org/10.1007/s12088-012-0334-3. Ghosh N, Tomar I, Goel AK. A field usable qualitative anti-protective antigen enzymelinked immunosorbent assay for serodiagnosis of human anthrax. Microbiol Immunol 2013b;57:145–9, http://dx.doi.org/10.1111/1348-0421.12014. Ghosh N, Tomar I, Lukka H, Goel AK. Serodiagnosis of human cutaneous anthrax in India using an indirect anti-lethal factor IgG enzyme-linked immunosorbent assay. Clin Vaccine Immunol 2013c;20:282–6, http://dx.doi.org/10.1128/CVI.00598-12.
N. Ghosh et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 14–19 Glasstone SD. Thermodynamics for chemists. New York: Van Nostrand Company; 1947. Gupta G, Singh PK, Boopathi M, Kamboj DV, Singh B, Vijayaraghavan R. Surface plasmon resonance detection of biological warfare agent Staphylococcal enterotoxin B using high affinity monoclonal antibody. Thin Solid Films 2010;519:1171–7, http://dx.doi.org/10.1016/j.tsf.2010.08.064. Homola J. Present and future of surface plasmon resonance biosensors. Analytical Bioanalytical Chem 2003;377:528–39, http://dx.doi.org/10.1007/s00216-0032101-0. Homola J. Surface plasmon resonance sensors for detection of chemical and biological species. Chem Rev 2008;108:462–93, http://dx.doi.org/10.1021/cr068107d. Iacono-Connors LC, Novak J, Rossi C, Mangiafico J, Ksiazek T. Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies. Clin Diagn Lab Immunol 1994;1:78–82. Jernigan DB, Raghunathan PL, Bell BP, Brechner R, Bresnitz EA, Butler JC, et al. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg Infect Dis 2002;8:1019–28. Kobiler D, Weiss S, Levy H, Fisher M, Mechaly A, Pass A, Altboum Z. Protective antigen as a correlative marker for anthrax in animal models. Infect Immun 2006;74:5871–6, http://dx.doi.org/10.1128/IAI.00792-06. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495–7. Mabry R, Brasky K, Geiger R, Carrion Jr R, Hubbard GB, Leppla S, et al. Detection of anthrax toxin in the serum of animals infected with Bacillus anthracis by using engineered immunoassays. Clin Vaccine Immunol 2006;13:671–7, http://dx.doi.org/ 10.1128/CVI.00023-06. McDonnell JM. Surface plasmon resonance: towards an understanding of the mechanisms of biological molecular recognition. Curr Opin Chem Biol 2001;5:572–7. Moayeri M, Haines D, Young HA, Leppla SH. Bacillus anthracis lethal toxin induces TNFalpha-independent hypoxia-mediated toxicity in mice. J Clin Invest 2003;112: 670–82, http://dx.doi.org/10.1172/JCI17991. Mock M, Mignot T. Anthrax toxins and the host: a story of intimacy. Cell Microbiol 2003;5:15–23. Paynter S, Russell DA. Surface plasmon resonance measurement of pH-induced responses of immobilized biomolecules: conformational change or electrostatic interaction effects? Anal Biochem 2002;309:85–95.
19
Rich RL, Myszka DG. Advances in surface plasmon resonance biosensor analysis. Curr Opin Biotechnol 2000;11:54–61. Sacchi CT, Whitney AM, Mayer LW, Morey R, Steigerwalt A, Boras A, et al. Sequencing of 16S rRNA gene: a rapid tool for identification of Bacillus anthracis. Emerg Infect Dis 2002;8:1117–23. Shrivastva A, Tripathi NK, Sathyaseelan K, Jana AM, Rao PVL. Production of Bacillus anthracis recombinant protective antigen in Escherichia coli. Adv Biotech 2008;26: 26–9. Singh Y, Klimpel KR, Goel S, Swain PK, Leppla SH. Oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells. Infect Immun 1999;67:1853–9. Sirisanthana T, Nelson KE, Ezzell JW, Abshire TG. Serological studies of patients with cutaneous and oral-oropharyngeal anthrax from northern Thailand. Am J Trop Med Hyg 1988;39:575–81. Stenberg E, Persson B, Roos H, Urbaniczky C. Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins. J Colloid Interface Sci 1991;143:513–26, http://dx.doi.org/10.1016/00219797(91)90284-f. Tang S, Moayeri M, Chen Z, Harma H, Zhao J, Hu H, et al. Detection of anthrax toxin by an ultrasensitive immunoassay using europium nanoparticles. Clin Vaccine Immunol 2009;16:408–13, http://dx.doi.org/10.1128/CVI.00412-08. Tawil N, Sacher E, Mandeville R, Meunier M. Surface plasmon resonance detection of E. coli and methicillin-resistant S. aureus using bacteriophages. Biosens Bioelectron 2012;37:24–9, http://dx.doi.org/10.1016/j.bios.2012.04.048. Taylor AD, Ladd J, Yu Q, Chen S, Homola J, Jiang S. Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor. Biosens Bioelectron 2006;22:752–8, http://dx.doi.org/10.1016/j.bios.2006.03.012. Turk BE. Manipulation of host signalling pathways by anthrax toxins. Biochem J 2007;402:405–17, http://dx.doi.org/10.1042/BJ20061891. Walsh JJ, Pesik N, Quinn CP, Urdaneta V, Dykewicz CA, Boyer AE, et al. A case of naturally acquired inhalation anthrax: clinical care and analyses of anti-protective antigen immunoglobulin G and lethal factor. Clin Infect Dis 2007;44:968–71, http:// dx.doi.org/10.1086/512372. Wang Y, Knoll W, Dostalek J. Bacterial pathogen surface plasmon resonance biosensor advanced by long range surface plasmons and magnetic nanoparticle assays. Anal Chem 2012;84:8345–50, http://dx.doi.org/10.1021/ac301904x.