DOI 10.1007/s10517-015-2873-1 Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015
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METHODS Immunochromatographic System for Diagnostics of Pseudotuberculosis M. B. Raev, M. S. Bochkova, P. V. Khramtsov, and V. P. Timganova Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 12, pp. 804-807, December, 2014 Original article submitted October 3, 2013 We developed a model immunochromatographic test-system for serological express-diagnostics of pseudotuberculosis. Nitrocellulose membrane sensitized with species-specific antigen, Yersinia pseudotuberculosis thermostable toxin, was used as the immunosorbent. Detection of antibodies to the pathogen was performed using functionalized carbon nanoparticles. System performance was verified in testing of the reference pseudotuberculosis serum. Tests with salmonella, escherichia, and enteral yersinia diagnostic sera and blood serum from a healthy man demonstrated high specificity of the system. Key Words: pseudotuberculosis diagnostics; immunochromatographic test-system; carbon nanoparticles; Yersinia pseudotuberculosis
Pseudotuberculosis (PT) is an infectious disease caused by Yersinia pseudotuberculosis widely spread in the Russian Federation, in particular, Siberia and FarEastern regions [2,3]. Difficulties in recognition and quantitative recording of the disease and high incidence of misdiagnosis can be explained by polymorphism of clinical manifestations and the absence of typical symptoms [8]. The final clinical diagnosis of PT can be made only by the results of serological and bacteriological tests. There is no universal method of PT diagnostics complying with clinical diagnostic tasks. Bacteriological analysis, the basic method of direct Y. рseudotuberculosis detection, requires timeand labor-consuming procedure of bacterial cell culturing. Accuracy of this method remains insufficient (80%) [3]. PCR is a highly efficient method of PT detection in coprofiltrate from the patients [3], but this methInstitute of Ecology and Genetics of Microorganisms, Ural Division of Russian Academy of Sciences, Perm, Russia. Address for correspondence: [email protected]. M. B. Raev
od requires expensive equipment and qualified staff. These factors complicate timely examination because of equipment and logistic problems: samples transportation from distant institutions (children camps, health resorts, military units). The same is true for ELISA. The only commercial preparation for serological diagnostic of PT is typical erythrocyte diagnosticum of 01 serovar for passive hemagglutination test. Despite obvious advantages such as its simplicity and universal application, this method has some drawbacks: the necessity of preliminary preparation of samples (serum depletion in relation to nonspecific agglutination inhibitors) and insufficient specificity of the analysis due to the nature of the used antigens. Antigens of only one of 9 pathological Y. рseudotuberculosis serovars are used for erythrocyte sensitization in the commercial systems of serological analysis [7,9]. Passive hemagglutination test performed using commercial kits can be false-positive with antisera to the related species of Enterobacteriaceae family [7]. Impossibility of recording and storing analysis results can also be classified as a disadvantage of the method.
0007-4888/15/15860833 © 2015 Springer Science+Business Media New York
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Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 METHODS
In light of this, the development of non-instrumental method of serological diagnostics of PT allowing simultaneous evaluation of great number of samples directly in the epidemic focus is a pressing problem. This method should be highly sensitive, specific, reliable, simple, and unambiguous in result interpretation. At present, methods of instrumental ELISA and agglutination tests based on species-specific Y. рseudotuberculosis proteins: thermostable lethal toxin, thermolabile exotoxin and pore-forming protein of outer membrane [1,7]. The aim of this study was to create a model system of simplified immunochromatography-based serological diagnostics of PT. Species-specific thermostable Y. рseudotuberculosis toxin (TSTYp) was used as a sensitizing agent and nanosized carbon particles functionalized by streptococcus G-protein as a diagnostic reagent. Diagnostic carbon conjugates were previously successfully used in non-instrumental solid-phase systems for HIV antibodies, group A Streptococcus detection, in dot-immunoanalytical systems of semiquantitative PT serological analysis, etc. [6].
MATERIALS AND METHODS Carbon diagnosticum was synthesized by the original method [4,5]. Streptococcal G-protein specifically interacting with Fc-domains of IgG of most of the higher animals, including all human IgG subclasses was used as affinity compound for functionalization of carbon particles [10]. Carbon particles in the conjugate were used as a chromophore that allows visual detection of the target antibodies in the sample by presence/ absence of staining of the sensitized substrate. We constructed the test strips for immunochromatographic analysis (Fig. 1). Nitrocellulose HiFlow Plus 135 Membrane Cards (Millipore) mounted on a polystyrene plate served as the solid phase. Absorbing components made of cellulose fibers (Cellulose fiber Sample Pad; Millipore) were placed at both ends of the strip; a Glass fiber
Fig. 1. Test strip for immunochromatographic analysis.
Conjugate Pad (Millipore) previously soaked with carbon diagnosticum solution and dried (conjugate substrate) was placed between the nitrocellulose membrane and one of the absorbing pads (the adjacent end of the strip was considered the lower). Nitrocellulose membrane was sensitized with TSTYp, a complex of species-specific immunogenic proteins isolated from 512 Y. pseudotuberculosis strain presented by the Collection of Research Institute of Epidemiology and Microbiology, Siberian Branch of the Russian Academy of Medical Science. The toxin was absorbed from a concentration of 0.01 mg/ ml in phosphate buffered physiological saline (BPS; pH=7.25). The site of toxin application on the membrate was designated as “analytic zone”. Human IgG (Sigma) absorbed on the membrane from a solution with a concentration of 0.05 mg/ml in BPS served as the positive control. The corresponding area on the membrane was designated as “control zone”. Model chromatographic system was tested using reference PT serum for passive hemagglutination test (Saint-Petersburg Research Institute of Vaccines and Sera). System specificity was evaluated using standard enteral yersinia, escherichia, and salmonella sera for passive hemagglutination test (Saint-Petersburg Research Institute of Vaccines and Sera). Serum of a healthy man (without history of PT) was used as the negative control. All sera were diluted 1:250 in BPS with 0.05% Tween. The lower end of the strip was put in the solution of the test serum. The solution with conjugate particles moved towards the upper side of the test strip under the effect of capillary forces. When the leading edge of the fluid had reached the upper absorbing pad, the lower end of the strip was placed into BPS with 0.05 Tween. The total duration of the procedure was 15-20 min. The results were evaluated by the appearance of black staining in the analytic zone, which attested to the presence of antibodies to TSTYp in the studied sample. Staining of the control zone verified the results.
M. B. Raev, M. S. Bochkova, et al.
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Fig. 3. Evaluation of specificity of the model immunochromatographic system for detection of antibodies to TSTYp. The strips were put into control reference sera diluted 1:250. 1) Salmonella serum, 2) enteric Yersinia serum, 3) Escherichia serum. Fig. 2. Detection of antibodies to TSTYp by immunochromatography. Incubation of the strip with reference PT-serum (1) and serum of a healthy man,dilution 1:250 (2).
RESULTS Testing of the reference PT-serum showed staining in the analytic and control zone of the strips. Analysis of the serum obtained from a healthy man showed staining only in the control zone (Fig. 2). No staining in analytic zone of the strips was found during evaluation of test specificity with enteral yersinia, salmonella, and escherichia sera; the presence of staining in the control zones of all three strips confirmed proper functioning of the test-system (Fig. 3). Thus, we constructed and tested the model immunochromatographic test-system for simplified and prompt serological diagnostics of PT. Using reference serum we showed principal possibility of detection of antibodies to TSTYp. The specificity of detection of antibodies to TSTYp was proven by the absence of staining in the analytic zone of the strips in testing of antisera to related pathogenic bacteria and serum from a healthy man. The constructed test-system is prospective for further improving of the screening diagnostic system because it has some advantages over the existing analogues: the analysis takes no more than 30 min, the preparation of samples and detection procedure are fast and simple, the system can be used under any
conditions and dose not require sofistivated laboratory equipment and high personel qualification, the results can be clearly presented and saved. The study was supported by Program of the Presidium of Russian Academy of Sciences “Fundamental Sciences to Medicine” (project 12-P-4-1004).
REFERENCES 1. B. G. Andryukov, E. P. Nedashkovskaya, and N. F. Timchenko, Klin. Lab. Diagnost., No. 9, 30 (1998). 2. S. V. Devyatilova, G. A. Zakharova, and T. G. Nesterova, Zdorov’e. Meditsinskaya Ekologiya. Nauka, Nos. 1-2, 124-125 (2010). 3. Yersinia and Yersinioses [in Russian], Ed. G. Ya. Tseneva, St. Petersburg (2006). 4. M. B. Raev, Patent RF No. 2314827, A Method of Preparation of Conjugation for Stereospecific Analysis. Byull. No. 2, January 20, 2008. 5. D. Plaksin, M. Raev, and E. Gromanovskaya, Patent RF No. 2089212, A Method of Stereospecific Analysis and Method of Preparation of Conjugation for Spereospecific Analysis, Byull. Izobreteniya. Poleznye Modeli, No. 25, September 10, 1997. 6. M. B. Raev, Nanobiotechnology in Noninstrumental Immunoanalytics [in Russian], Ekaterinburg (2012). 7. N. F. Timchenko, Yersinia pseudotuberculosis Toxins [in Russian], Vladivostok (2004). 8. E. P. Shuvalova, Infectious Diseases [in Russian], Moscow (1990). 9. H. Fukushima, Y. Matsuda, R. Seki, et al., J. Clin. Microbiol., 39, No. 10, 3541-3547 (2001). 10. J. Gosling, Immunoassays, New York (2006).
833
METHODS Immunochromatographic System for Diagnostics of Pseudotuberculosis M. B. Raev, M. S. Bochkova, P. V. Khramtsov, and V. P. Timganova Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 12, pp. 804-807, December, 2014 Original article submitted October 3, 2013 We developed a model immunochromatographic test-system for serological express-diagnostics of pseudotuberculosis. Nitrocellulose membrane sensitized with species-specific antigen, Yersinia pseudotuberculosis thermostable toxin, was used as the immunosorbent. Detection of antibodies to the pathogen was performed using functionalized carbon nanoparticles. System performance was verified in testing of the reference pseudotuberculosis serum. Tests with salmonella, escherichia, and enteral yersinia diagnostic sera and blood serum from a healthy man demonstrated high specificity of the system. Key Words: pseudotuberculosis diagnostics; immunochromatographic test-system; carbon nanoparticles; Yersinia pseudotuberculosis
Pseudotuberculosis (PT) is an infectious disease caused by Yersinia pseudotuberculosis widely spread in the Russian Federation, in particular, Siberia and FarEastern regions [2,3]. Difficulties in recognition and quantitative recording of the disease and high incidence of misdiagnosis can be explained by polymorphism of clinical manifestations and the absence of typical symptoms [8]. The final clinical diagnosis of PT can be made only by the results of serological and bacteriological tests. There is no universal method of PT diagnostics complying with clinical diagnostic tasks. Bacteriological analysis, the basic method of direct Y. рseudotuberculosis detection, requires timeand labor-consuming procedure of bacterial cell culturing. Accuracy of this method remains insufficient (80%) [3]. PCR is a highly efficient method of PT detection in coprofiltrate from the patients [3], but this methInstitute of Ecology and Genetics of Microorganisms, Ural Division of Russian Academy of Sciences, Perm, Russia. Address for correspondence: [email protected]. M. B. Raev
od requires expensive equipment and qualified staff. These factors complicate timely examination because of equipment and logistic problems: samples transportation from distant institutions (children camps, health resorts, military units). The same is true for ELISA. The only commercial preparation for serological diagnostic of PT is typical erythrocyte diagnosticum of 01 serovar for passive hemagglutination test. Despite obvious advantages such as its simplicity and universal application, this method has some drawbacks: the necessity of preliminary preparation of samples (serum depletion in relation to nonspecific agglutination inhibitors) and insufficient specificity of the analysis due to the nature of the used antigens. Antigens of only one of 9 pathological Y. рseudotuberculosis serovars are used for erythrocyte sensitization in the commercial systems of serological analysis [7,9]. Passive hemagglutination test performed using commercial kits can be false-positive with antisera to the related species of Enterobacteriaceae family [7]. Impossibility of recording and storing analysis results can also be classified as a disadvantage of the method.
0007-4888/15/15860833 © 2015 Springer Science+Business Media New York
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Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 METHODS
In light of this, the development of non-instrumental method of serological diagnostics of PT allowing simultaneous evaluation of great number of samples directly in the epidemic focus is a pressing problem. This method should be highly sensitive, specific, reliable, simple, and unambiguous in result interpretation. At present, methods of instrumental ELISA and agglutination tests based on species-specific Y. рseudotuberculosis proteins: thermostable lethal toxin, thermolabile exotoxin and pore-forming protein of outer membrane [1,7]. The aim of this study was to create a model system of simplified immunochromatography-based serological diagnostics of PT. Species-specific thermostable Y. рseudotuberculosis toxin (TSTYp) was used as a sensitizing agent and nanosized carbon particles functionalized by streptococcus G-protein as a diagnostic reagent. Diagnostic carbon conjugates were previously successfully used in non-instrumental solid-phase systems for HIV antibodies, group A Streptococcus detection, in dot-immunoanalytical systems of semiquantitative PT serological analysis, etc. [6].
MATERIALS AND METHODS Carbon diagnosticum was synthesized by the original method [4,5]. Streptococcal G-protein specifically interacting with Fc-domains of IgG of most of the higher animals, including all human IgG subclasses was used as affinity compound for functionalization of carbon particles [10]. Carbon particles in the conjugate were used as a chromophore that allows visual detection of the target antibodies in the sample by presence/ absence of staining of the sensitized substrate. We constructed the test strips for immunochromatographic analysis (Fig. 1). Nitrocellulose HiFlow Plus 135 Membrane Cards (Millipore) mounted on a polystyrene plate served as the solid phase. Absorbing components made of cellulose fibers (Cellulose fiber Sample Pad; Millipore) were placed at both ends of the strip; a Glass fiber
Fig. 1. Test strip for immunochromatographic analysis.
Conjugate Pad (Millipore) previously soaked with carbon diagnosticum solution and dried (conjugate substrate) was placed between the nitrocellulose membrane and one of the absorbing pads (the adjacent end of the strip was considered the lower). Nitrocellulose membrane was sensitized with TSTYp, a complex of species-specific immunogenic proteins isolated from 512 Y. pseudotuberculosis strain presented by the Collection of Research Institute of Epidemiology and Microbiology, Siberian Branch of the Russian Academy of Medical Science. The toxin was absorbed from a concentration of 0.01 mg/ ml in phosphate buffered physiological saline (BPS; pH=7.25). The site of toxin application on the membrate was designated as “analytic zone”. Human IgG (Sigma) absorbed on the membrane from a solution with a concentration of 0.05 mg/ml in BPS served as the positive control. The corresponding area on the membrane was designated as “control zone”. Model chromatographic system was tested using reference PT serum for passive hemagglutination test (Saint-Petersburg Research Institute of Vaccines and Sera). System specificity was evaluated using standard enteral yersinia, escherichia, and salmonella sera for passive hemagglutination test (Saint-Petersburg Research Institute of Vaccines and Sera). Serum of a healthy man (without history of PT) was used as the negative control. All sera were diluted 1:250 in BPS with 0.05% Tween. The lower end of the strip was put in the solution of the test serum. The solution with conjugate particles moved towards the upper side of the test strip under the effect of capillary forces. When the leading edge of the fluid had reached the upper absorbing pad, the lower end of the strip was placed into BPS with 0.05 Tween. The total duration of the procedure was 15-20 min. The results were evaluated by the appearance of black staining in the analytic zone, which attested to the presence of antibodies to TSTYp in the studied sample. Staining of the control zone verified the results.
M. B. Raev, M. S. Bochkova, et al.
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Fig. 3. Evaluation of specificity of the model immunochromatographic system for detection of antibodies to TSTYp. The strips were put into control reference sera diluted 1:250. 1) Salmonella serum, 2) enteric Yersinia serum, 3) Escherichia serum. Fig. 2. Detection of antibodies to TSTYp by immunochromatography. Incubation of the strip with reference PT-serum (1) and serum of a healthy man,dilution 1:250 (2).
RESULTS Testing of the reference PT-serum showed staining in the analytic and control zone of the strips. Analysis of the serum obtained from a healthy man showed staining only in the control zone (Fig. 2). No staining in analytic zone of the strips was found during evaluation of test specificity with enteral yersinia, salmonella, and escherichia sera; the presence of staining in the control zones of all three strips confirmed proper functioning of the test-system (Fig. 3). Thus, we constructed and tested the model immunochromatographic test-system for simplified and prompt serological diagnostics of PT. Using reference serum we showed principal possibility of detection of antibodies to TSTYp. The specificity of detection of antibodies to TSTYp was proven by the absence of staining in the analytic zone of the strips in testing of antisera to related pathogenic bacteria and serum from a healthy man. The constructed test-system is prospective for further improving of the screening diagnostic system because it has some advantages over the existing analogues: the analysis takes no more than 30 min, the preparation of samples and detection procedure are fast and simple, the system can be used under any
conditions and dose not require sofistivated laboratory equipment and high personel qualification, the results can be clearly presented and saved. The study was supported by Program of the Presidium of Russian Academy of Sciences “Fundamental Sciences to Medicine” (project 12-P-4-1004).
REFERENCES 1. B. G. Andryukov, E. P. Nedashkovskaya, and N. F. Timchenko, Klin. Lab. Diagnost., No. 9, 30 (1998). 2. S. V. Devyatilova, G. A. Zakharova, and T. G. Nesterova, Zdorov’e. Meditsinskaya Ekologiya. Nauka, Nos. 1-2, 124-125 (2010). 3. Yersinia and Yersinioses [in Russian], Ed. G. Ya. Tseneva, St. Petersburg (2006). 4. M. B. Raev, Patent RF No. 2314827, A Method of Preparation of Conjugation for Stereospecific Analysis. Byull. No. 2, January 20, 2008. 5. D. Plaksin, M. Raev, and E. Gromanovskaya, Patent RF No. 2089212, A Method of Stereospecific Analysis and Method of Preparation of Conjugation for Spereospecific Analysis, Byull. Izobreteniya. Poleznye Modeli, No. 25, September 10, 1997. 6. M. B. Raev, Nanobiotechnology in Noninstrumental Immunoanalytics [in Russian], Ekaterinburg (2012). 7. N. F. Timchenko, Yersinia pseudotuberculosis Toxins [in Russian], Vladivostok (2004). 8. E. P. Shuvalova, Infectious Diseases [in Russian], Moscow (1990). 9. H. Fukushima, Y. Matsuda, R. Seki, et al., J. Clin. Microbiol., 39, No. 10, 3541-3547 (2001). 10. J. Gosling, Immunoassays, New York (2006).
