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Development of colloidal gold-based immunochromatographic assay for rapid detection of Streptococcus suis serotype 2
Veterinary Immunology and Immunopathology 133 (2010) 207–211
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
Development of colloidal gold-based immunochromatographic assay for rapid detection of Streptococcus suis serotype 2 Ying Ju a,d,1, Huai-Jie Hao a,1, Guo-Hua Xiong a, Hong-Ran Geng a, Yu-Ling Zheng a, Jing Wang c, Yuanyin Cao d, Yin-Hui Yang a,*, Xue-Hui Cai b,*, Yong-Qiang Jiang a,** a
State Key Laboratory of Pathogen and Biosecurity, The Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Fengtai District, Beijing 100071, People’s Republic of China National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Science, Harbin 150001, People’s Republic of China c Institute of Health Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100025, People’s Republic of China d College of Plant Protection, Shenyang Agricultural University, Shenyang 110161, People’s Republic of China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 April 2009 Received in revised form 30 July 2009 Accepted 7 August 2009
To develop colloidal gold immunochromatographic strips for the direct detection of the Streptococcus suis serotype 2 antigen, colloidal gold was prepared by reduction of a gold salt with sodium citrate and coupled with polyclonal antibody against S. suis serotype 2. The optimal concentrations of the capture antibody and the coating antibody were determined to be 22 mg/mL and 2.0 mg/mL, respectively, and that of the blocking buffer was determined to be 1.5% bovine serum albumin. Different serotypes of S. suis and other related bacteria were used to evaluate the sensitivity, specificity, and stability of the immunochromatographic strips. The detection sensitivity was found to be as high as 106 CFU/mL. There was no cross-reaction of the antibodies with other serotypes of S. suis (except with SS1/2, which shares some common sugar residues or antigenic determinants with serotype 2) and other related bacteria. In conclusion, we developed colloidal gold immunochromatographic strips that had high sensitivity and specificity. This method proved to be feasible, convenient, rapid, and effective for detecting S. suis serotype 2. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Streptococcus suis serotype 2 Colloidal gold Immunochromatography
1. Introduction Streptococcus suis is a gram-positive bacterium that causes severe pathological conditions such as meningitis, septicemia, sudden death, and pneumonia, mainly in pigs (Clifton-Hadley, 1983; Touil et al., 1988; Luque et al., 1998). It can also infect humans through damaged skin or mucous membrane (Robertson and Blackmore, 1989; Huang et al.,
* Corresponding authors. ** Corresponding author. Tel.: +86 10 66948487. E-mail addresses: [email protected] (Y.-H. Yang), [email protected] (X.-H. Cai), [email protected] (Y.-Q. Jiang). 1 These authors contributed equally to this work. 0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.08.010
2005). It has been identified as a causative agent of meningitis, septicemia, endocarditis, and sometimes, permanent hearing loss in humans (Francois et al., 1998; Charland et al., 2000; Halaby et al., 2000; Heidt et al., 2005). The disease caused by S. suis in pigs is categorized as the second class infectious zoonotic diseases in China. Thirty-five serotypes of S. suis have been characterized on the basis of their capsular polysaccharide antigens. Among these, serotype 2 (SS2) is the most common and the most pathogenic to both humans and pigs (Higgins and Gottschalk, 1995). In recent years, SS2 has caused large-scale epidemics and serious infections in both human and pig populations in China and, thus, poses a serious threat to the society. Two large outbreaks of human SS2 infections in 1998 and 2005 resulted in 229 infections and 53 deaths (Tang et al., 2006).
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Y. Ju et al. / Veterinary Immunology and Immunopathology 133 (2010) 207–211
The current methods for diagnosing SS2 infections include routine biochemistry assays, indirect ELISA, agar gel precipitation tests, and polymerase chain reaction (PCR) (Smith et al., 1999; Okwumabua et al., 2003). These methods are time-consuming, and their use is mostly restricted to well-equipped laboratories. It is very important to develop a method for rapidly detecting SS2 in field research and serological surveillance. In 2007, Yang et al. (2007) developed an immunochromatographic strip for the detection of antibodies against the SS2 antigen; this method is rapid and simple and suitable for the serological surveillance of SS2 infection in the field. However, since this immunochromatographic strip was developed for the detection of the antibody generated against the SS2 antigen and not the antigen directly, early detection of SS2 infection is not possible. In this study, we developed an immunochromatographic strip for the rapid detection of the SS2 antigen using colloidal gold coupled with polyclonal antibody. This strip can be used for rapid detection of SS2 in the field and serological surveillance, with high sensitivity and specificity. 2. Materials and methods 2.1. Bacterial strains and materials S. suis serotype 2, Streptococcus pyogenes GAS-M15, GAS-M16, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi A, Clostridium perfringens, and Listeria monocytogenes were isolated in our laboratory. S. suis strains of different serotypes were supplied by the Harbin Veterinary Research Institute (HVRI), and the other strains were supplied by the national institute for the control of pharmaceutical and biological products. The bacterial strains used in this study have been listed in Table 1. Streptococcus strains were maintained on Columbia blood agar containing 6% sheep blood and cultured in Todd– Hewitt broth (THB) at 37 8C. The strains of E. coli, S. paratyphi A, and C. perfringens were grown in Luria–Bertani (LB) medium at 37 8C. Those of S. aureus and L. monocytogenes were cultivated in nutrient broth (NB) at 37 8C. HiTrapTM rProtein A FF was purchased from GE corporation. Nitrocellulose membrane, sample pad, absorbent pad and conjugate pad were purchased from Millipore Corporation. 2.2. Preparation and identification of antibodies against SS2 Antibodies against SS2 were prepared according to a previously described method, with some modifications (Ying et al., 2005). S. suis 149 was isolated from pigs and cultured in THB at 37 8C for 7 h, and the bacteria were then collected and dissolved in sterile water for blood plate counting. It was inactivated at 37 8C for 7 days in 1% formaldehyde and then used as the immunogen after washing three times with PBS to remove the formaldehyde. SS2-negative New Zealand rabbits weighing about 2 kg were immunized to prepare polyclonal antibodies against SS2. The entire immunization procedure comprised 6 injections. The doses of the immunogen
were 5 108 CFU, 1 109 CFU, 2 109 CFU, 4 109 CFU, 8 109 CFU, and 1.6 109 CFU, respectively, in the first through the sixth injections. The first 2 injections were intravenous and carried out at an interval of 2 weeks, and the subsequent 4 injections were carried out intravenous at intervals of 1 week. Indirect ELISA was performed to measure the serum antibody titers. Polyclonal antibodies against SS2 were purified by affinity chromatography on a protein A-sepharose column according to the manufacturer’s instructions. The concentration of antibodies was determined by the Bradford protein assay. 2.3. Determination of optimal concentration of capture antibody We prepared 25-nm colloidal gold by the citrate– natrium deoxidize method (Chaudhuri and Raychaudhuri, 2001). The pH of the colloidal gold was adjusted to 8.5. The polyclonal antibodies prepared above were further dialyzed for desalination and centrifuged at 10,000 g for removing macromolecular aggregations. The antibodies were then diluted to 0.2 mg/mL with purified H2O. The minimum amount of antibody needed for stabilization of the colloidal gold was determined as that required to change the color of the solution from reddish to blue. Different solutions were added into 10 cuvettes and left standing for 2 h after mixing. If a tube contained the minimum amount of the antibody, the color of the colloidal gold did not change from reddish to blue. To stabilize the colloidal gold, we choose 120% of the minimum amount of capture antibody as its working concentration. 2.4. Preparation of colloidal gold probe The colloidal gold probe was prepared according to a previously described method with slight modifications (Chaudhuri and Raychaudhuri, 2001). The pH of the colloidal gold was adjusted to 8.5 by dropwise addition of 0.1 mol/L K2CO3. One milliliter of colloidal gold was mixed with 110 mL of 0.2 mg/mL antibody, with shaking at intervals for 30 min. Then, another 110 mL of 10% BSA was added, and the mixture was centrifuged at 12,000 g for 30 min at 4 8C. The pellet was re-suspended with 1 mL of the suspension buffer (0.01 mol/L Tris–HCl (pH 8.2), 1% BSA, 5% sucrose). The colloidal gold was centrifuged at 12,000 g for another 30 min at 4 8C, and the pellet was re-suspended in 500 mL of buffer. Finally, it was centrifuged at 1000 g for another 4 min at 4 8C, and the supernatant was collected and stored at 4 8C for use as the colloidal gold probe. 2.5. Determination of optimal concentration of coating antibody The antibody against SS2 was diluted to 3.5 mg/mL, 2.5 mg/mL, 2.0 mg/mL, 1.5 mg/mL, 1.0 mg/mL, and 0.5 mg/ mL with 0.01 mol/L phosphate-buffered saline (PBS; pH 7.2). The BSA concentrations of these diluted solutions were 2.0%, 1.5%, 1.0%, 0.75%, and 0.5%, respectively. The diluted antibody (Test line, T line) and goat anti-rabbit antibody (Control line, C line) were transferred onto a nitrocellulose membrane, which was then dried for 2 h at 37 8C (Jones,
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Table 1 Bacterial strains used in this study. Discriptiona
Strains Streptococcu suis serotypes 2(SS2)
SS1 SS1/2 SS3 SS4 SS5 SS6 SS7 SS8 SS9 SS10 SS11 SS12 SS13 SS14 SS15 Streptococcus sp.
Streptococcus pyogenes
Staphylococcus aureus Escherichia coli Salmonella paratyphi A Clostridium perfringens Listeria monocytogenes a b
S. suis 05ZY S. suis 149 S. suis 606 S. suis 607 S. suis 1940 S. suis 200601 S. suis 200602 S. suis 200502 S. suis 200501 S. suis SUN S. suis T15 S. suis 1330 S. suis S735 S. suis s428 S. suis #2651 S. suis #4961 S. suis #640 S. suis 11538 S. suis #2524 S. suis #8074 S. suis #14636 S. suis #DAN22083 S. suis DAN4417 S. suis #DAN12814 S. suis #DAN8830 S. suis #DAN10581 S. suis #13730 S. suis #GrT639 CMCC32115 CMCC32116 CMCC32118 CMCC32119 CMCC32120 CMCC32121 CMCC32122 CMCC32123 CMCC32124 CMCC32125 CMCC32126 CMCC32134 CMCC32144 CMCC32145 CMCC32146 CMCC32171 CMCC32180 GAS-M15 GAS-M16 S. aureus S-6 S. aureus 137 ATCC25922 CMCC50671 CMCC64601 C5
+
+
+
+
MRP EF SLY 89K MRP+EF+SLY+89K+ MRP+EF+SLY+89K MRP+EF+SLY 89K MRP+EF+SLY+89K MRP+EF+SLY+89K+ MRP+EF+SLY+89K+ MRP+EF+SLY+89K+ MRP+EF+SLY+89K+ MRP+EF+SLY+89K+ MRP EF SLY+89K MRP EF SLY 89K MRP+EF*SLY+89K
Originb
Source
Sichuan, China HP, 2005 Jiangshu, China HP, 1998 China DP, 1980s Japan DP China DP, 1980s Guangxi, China HP, 2006 Guangxi, China HP, 2006 Guangxi, China HP, 2005 Guangxi, China HP, 2005 Jiangsu, China HP, 2006 Europe HPL Canada HPL The Netherlands DP
This work This work This work This work This work This work This work This work This work This work Wisselink HJ Dr. Gottschalk M Wisselink HJ
89K, 89K pathogenicity islands (PAI); MRP, muramidase-released protein; EF, extracellular factor; SLY, suilysin. HP, human patients; DP, diseased piglets; HPL, healthy piglets.
1999a,b). The blank control without S. suis was tested simultaneously to determine the optimal concentrations of the coating antibody and the blocking buffer. 2.6. Preparation of immunochromatographic strip The immunochromatographic strip was composed of a sample pad, a conjugate pad, an immobilized nitrocellulose membrane, and an absorbent pad. A schematic diagram of the immunochromatographic strip is shown in Fig. 1. The colloidal gold probe was added to the
conjugate pad and dried at 37 8C for 2 h. The T line was coated with 2.0 mg/mL rabbit anti-pig antibody against SS2 and the C line was coated with 0.5 mg/mL goat antirabbit immune serum. The sample pad, conjugate pad, immobilized nitrocellulose membrane, and absorbent pad were overlapped and stuck to a polyvinyl chloride (PVC) plate and then cut into 4-mm-wide strips. These strips were stored dried at 4 8C until required. When the samples were applied to the sample pads and allowed to flow through the nitrocellulose membrane, after 5–10 min, a positive test showed two red lines for the
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Fig. 1. Schematic diagram of the immunochromatographic strip.
test and control lines, whereas a negative test only produced one red line in the control region. The strip test was incorrect if there was no red line for the control line.
was regarded as the optimal concentration of the blocking buffer, since the blank control did not show false-positive results at concentrations higher than this.
2.7. Specificity, sensitivity, and stability of immunochromatographic strip
3.2. Sensitivity of immunochromatographic strip
S. suis 149 was inoculated onto a Columbia agar plate containing 5% goat blood and cultured at 37 8C in a 5% CO2 atmosphere for 24 h. The colony was selected and cultured in THB at 37 8C in a 5% CO2 atmosphere for 6 h. The cultures were centrifuged at 8000 g for 10 min at 4 8C and diluted serially with PBS. For the sensitivity assay, 100 mL of each solution of a particular dilution was added to an equal volume of buffer. The culture methods used for other strains were the same as the one described before. We prepared 108 CFU/ mL cultures by the turbidity method. For the specificity assay, 100 mL of the cultures of different bacteria was added to an equal volume of buffer. To evaluate the stability of immunochromatographic strips during storage, they were stored at 4 8C and room temperature respectively for 3 months. Then, the strips were stored at 37 8C for 15 d for stability testing. We used 108 CFU/mL and 106 CFU/mL of S. suis 149 and 109 CFU/mL of S. aureus as samples and PBS as the blank control.
To determine the sensitivity of the immunochromatographic strip, 108 CFU/mL S. suis cultures were serially diluted 10-fold with THB, and samples without SS2 were used as the blank control. The results have been shown in Fig. 2. The sensitivity of the immunochromatographic strip was 1.0 106 CFU/mL. The sensitivity assay was repeated more than 5 times, all replicates were positive at 1.0 106 CFU/mL. 3.3. Specificity of immunochromatographic strip S. aureus S-6, S. aureus 137, E. coli ATCC 25922, S. paratyphi A CMCC 50671, C. perfringens CMC 64601, and L. monocytogenes C5 were cultured in THB at 37 8C in a 5% CO2 atmosphere for 6 h. Different suspensions (concentration >108 CFU/mL) were collected to evaluate the specificity of the immunochromatographic strip. We also included 15 serotypes of S. suis. Only S. suis serotype 2 and serotype 1/2 showed positive results, and all the other serotypes showed clearly negative results. The capsules of SS2 and SS1/2 share some common sugar residues or antigenic determinants (Elliott and Tai, 1978; Smith et al., 1999). No
3. Results 3.1. Optimal concentration of capture antibody, coating antibody, and blocking BSA After 6 immunizations for 7 weeks, the antibody titers in the serum reached 107. The optimal capture antibody was determined to be 22 mg/mL according to the protocol in Section 2. Regarding the coating antibody, with the increase in the concentration, the color of the indicating line deepened gradually. When the antibody concentration reached 2.0 mg/mL, the color did not change any more. Therefore, 2.0 mg/mL was determined as the optimal concentration of the coating antibody. The blank control that did not contain SS2 but contained BSA at different concentrations showed false-positive to negative results with increase in the BSA concentration. Therefore, 1.5% BSA
Fig. 2. Sensitivity testing of immunochromatographic strip. 1: Blank control; 2: S. suis 149, 1.0 108 CFU/mL; 3: S. suis 149, 1.0 107 CFU/mL; 4: S. suis 149, 1.0 106 CFU/mL; 5: S. suis 149, 1.0 105 CFU/mL; 6: S. suis 149, 1.0 104 CFU/mL.
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these polyclonal antibodies. Surprisingly, the coupling of polyclonal antibodies with colloidal gold particles in this study resulted in considerably high specificity in the detection of 15 different serotypes of S. suis. This high specificity may be a result of the inactivation of S. suis 149. Inactivation in 1% formaldehyde at 37 8C for 7 days may help retain the sugar residues or antigenic determinants of SS2. In summary, the colloidal gold immunochromatographic strips we developed in this study can be used directly in serological surveillance and detection of the S. suis serotype 2 antigen in the field. Acknowledgements
Fig. 3. Stability testing of immunochromatographic strip by evaluating the sensitivity and specificity. 1: Blank control; 2: S. suis 149 108 CFU/ mL; 3: S. suis 149 106 CFU/mL; 4: S. aureus 108 CFU/mL.
cross-reaction was observed when testing the other pathogenic bacteria. These results confirmed that the immunochromatographic strip had high specificity for detecting SS2.
This work was supported by grants from the National Natural Science Foundation of China (30870091, 30770117 and 30600023), the National Basic Research Program (973) of China (2006CB504400), the High-Tech Research & Development Project 863 (2006AA02Z404, 2007AA02 1504), and the National Key Technologies R&D program (2006BAD06A02), mega-projects of science research for the 11th Five-year plan (2009ZX10004-103). References
3.4. Stability of immunochromatographic strip The immunochromatographic strips were stored at 4 8C and room temperature for 3 months, respectively, and then kept at 37 8C for another 15 days to assess their stability. As shown in Fig. 3, good results were obtained, as expected, for the detection of S. aureus and S. suis after this time period. This demonstrated that the strips were stable for at least 3 months at room temperature. 4. Discussion It has been suggested that SS2 is an opportunistic pathogen infecting pigs. Therefore, serological surveillance will be useful in determining the infection status and can play an important role in the control of S. suis in both pigs and humans. Colloidal gold-based immunochromatographic assay has been confirmed to be a rapid and sensitive method for serological surveillance and detection of bacterial pathogens in the field. However, the immunochromatographic strip that has been developed (Yang et al., 2007) can only detect antibodies generated against the S. suis type 2 antibody, and not the antigen directly. In this study, we developed an immunochromatographic strip for the rapid detection of the SS2 antigen using colloidal gold coupled with polyclonal antibody. This strip can be used for the rapid detection of SS2 in the field and in serological surveillance, with high sensitivity and specificity. Colloidal gold-based immunochromatographic assay has been used increasingly in the rapid diagnosis of bacterial pathogens. However, the use of polyclonal antibodies generated by immunization with whole bacteria as antigens is associated with the problem of low specificity. The antibodies may react with other related bacteria; such cross-reactions hamper the application of
Charland, N., Nizet, V., et al., 2000. Streptococcus suis serotype 2 interactions with human brain microvascular endothelial cells. Infect. Immun. 68 (2), 637–643. Chaudhuri, B., Raychaudhuri, S., 2001. Manufacturing high quality gold sol. IVD Technol. 8, 46–54. Clifton-Hadley, F.A., 1983. Streptococcus suis type 2 infections. Br. Vet. J. 139 (1), 1–5. Elliott, S.D., Tai, J.Y., 1978. The type-specific polysaccharides of Streptococcus suis. J. Exp. Med. 148 (6), 1699–1704. Francois, B., Gissot, V., et al., 1998. Recurrent septic shock due to Streptococcus suis. J. Clin. Microbiol. 36 (8), 2395. Halaby, T., Hoitsma, E., et al., 2000. Streptococcus suis meningitis, a poacher’s risk. Eur. J. Clin. Microbiol. Infect. Dis. 19 (12), 943–945. Heidt, M.C., Mohamed, W., et al., 2005. Human infective endocarditis caused by Streptococcus suis serotype 2. J. Clin. Microbiol. 43 (9), 4898–4901. Higgins, R., Gottschalk, M., 1995. Distribution of Streptococcus suis capsular types in 1994. Can. Vet. J. 36 (5), 320. Huang, Y.T., Teng, L.J., et al., 2005. Streptococcus suis infection. J. Microbiol. Immunol. Infect. 38 (5), 306–313. Jones, K., 1999a. Troubleshooting protein binding in nitrocellulose membranes. Part 1. Principles. IVD Technol. 5 (2), 32–41. Jones, K., 1999b. Troubleshooting protein binding in nitrocellulose membranes. Part 2. Common problems. IVD Technol. 5 (3), 26–35. Luque, I., Tarradas, C., et al., 1998. Streptococcus suis serotypes associated with different disease conditions in pigs. Vet. Rec. 142 (26), 726–727. Okwumabua, O., O’Connor, M., et al., 2003. A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase. FEMS Microbiol. Lett. 218 (1), 79–84. Robertson, I.D., Blackmore, D.K., 1989. Occupational exposure to Streptococcus suis type 2. Epidemiol. Infect. 103 (1), 157–164. Smith, H.E., Veenbergen, V., et al., 1999. The cps genes of Streptococcus suis serotypes 1, 2 and 9: development of rapid serotype-specific PCR assays. J. Clin. Microbiol. 37 (10), 3146–3152. Tang, J., Wang, C., et al., 2006. Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2. PLoS Med. 3 (5), e151. Touil, F., Higgins, R., et al., 1988. Isolation of Streptococcus suis from diseased pigs in Canada. Vet. Microbiol. 17 (2), 171–177. Yang, J., Jin, M., et al., 2007. Development and evaluation of an immunochromatographic strip for detection of Streptococcus suis type 2 antibody. J. Vet. Diagn. Invest. 19 (4), 355–361. Ying, T., Wang, H., et al., 2005. Immunoproteomics of outer membrane proteins and extracellular proteins of Shigella flexneri 2a 2457T. Proteomics 5 (18), 4777–4793.
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
Development of colloidal gold-based immunochromatographic assay for rapid detection of Streptococcus suis serotype 2 Ying Ju a,d,1, Huai-Jie Hao a,1, Guo-Hua Xiong a, Hong-Ran Geng a, Yu-Ling Zheng a, Jing Wang c, Yuanyin Cao d, Yin-Hui Yang a,*, Xue-Hui Cai b,*, Yong-Qiang Jiang a,** a
State Key Laboratory of Pathogen and Biosecurity, The Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Fengtai District, Beijing 100071, People’s Republic of China National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Science, Harbin 150001, People’s Republic of China c Institute of Health Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100025, People’s Republic of China d College of Plant Protection, Shenyang Agricultural University, Shenyang 110161, People’s Republic of China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 April 2009 Received in revised form 30 July 2009 Accepted 7 August 2009
To develop colloidal gold immunochromatographic strips for the direct detection of the Streptococcus suis serotype 2 antigen, colloidal gold was prepared by reduction of a gold salt with sodium citrate and coupled with polyclonal antibody against S. suis serotype 2. The optimal concentrations of the capture antibody and the coating antibody were determined to be 22 mg/mL and 2.0 mg/mL, respectively, and that of the blocking buffer was determined to be 1.5% bovine serum albumin. Different serotypes of S. suis and other related bacteria were used to evaluate the sensitivity, specificity, and stability of the immunochromatographic strips. The detection sensitivity was found to be as high as 106 CFU/mL. There was no cross-reaction of the antibodies with other serotypes of S. suis (except with SS1/2, which shares some common sugar residues or antigenic determinants with serotype 2) and other related bacteria. In conclusion, we developed colloidal gold immunochromatographic strips that had high sensitivity and specificity. This method proved to be feasible, convenient, rapid, and effective for detecting S. suis serotype 2. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Streptococcus suis serotype 2 Colloidal gold Immunochromatography
1. Introduction Streptococcus suis is a gram-positive bacterium that causes severe pathological conditions such as meningitis, septicemia, sudden death, and pneumonia, mainly in pigs (Clifton-Hadley, 1983; Touil et al., 1988; Luque et al., 1998). It can also infect humans through damaged skin or mucous membrane (Robertson and Blackmore, 1989; Huang et al.,
* Corresponding authors. ** Corresponding author. Tel.: +86 10 66948487. E-mail addresses: [email protected] (Y.-H. Yang), [email protected] (X.-H. Cai), [email protected] (Y.-Q. Jiang). 1 These authors contributed equally to this work. 0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.08.010
2005). It has been identified as a causative agent of meningitis, septicemia, endocarditis, and sometimes, permanent hearing loss in humans (Francois et al., 1998; Charland et al., 2000; Halaby et al., 2000; Heidt et al., 2005). The disease caused by S. suis in pigs is categorized as the second class infectious zoonotic diseases in China. Thirty-five serotypes of S. suis have been characterized on the basis of their capsular polysaccharide antigens. Among these, serotype 2 (SS2) is the most common and the most pathogenic to both humans and pigs (Higgins and Gottschalk, 1995). In recent years, SS2 has caused large-scale epidemics and serious infections in both human and pig populations in China and, thus, poses a serious threat to the society. Two large outbreaks of human SS2 infections in 1998 and 2005 resulted in 229 infections and 53 deaths (Tang et al., 2006).
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Y. Ju et al. / Veterinary Immunology and Immunopathology 133 (2010) 207–211
The current methods for diagnosing SS2 infections include routine biochemistry assays, indirect ELISA, agar gel precipitation tests, and polymerase chain reaction (PCR) (Smith et al., 1999; Okwumabua et al., 2003). These methods are time-consuming, and their use is mostly restricted to well-equipped laboratories. It is very important to develop a method for rapidly detecting SS2 in field research and serological surveillance. In 2007, Yang et al. (2007) developed an immunochromatographic strip for the detection of antibodies against the SS2 antigen; this method is rapid and simple and suitable for the serological surveillance of SS2 infection in the field. However, since this immunochromatographic strip was developed for the detection of the antibody generated against the SS2 antigen and not the antigen directly, early detection of SS2 infection is not possible. In this study, we developed an immunochromatographic strip for the rapid detection of the SS2 antigen using colloidal gold coupled with polyclonal antibody. This strip can be used for rapid detection of SS2 in the field and serological surveillance, with high sensitivity and specificity. 2. Materials and methods 2.1. Bacterial strains and materials S. suis serotype 2, Streptococcus pyogenes GAS-M15, GAS-M16, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi A, Clostridium perfringens, and Listeria monocytogenes were isolated in our laboratory. S. suis strains of different serotypes were supplied by the Harbin Veterinary Research Institute (HVRI), and the other strains were supplied by the national institute for the control of pharmaceutical and biological products. The bacterial strains used in this study have been listed in Table 1. Streptococcus strains were maintained on Columbia blood agar containing 6% sheep blood and cultured in Todd– Hewitt broth (THB) at 37 8C. The strains of E. coli, S. paratyphi A, and C. perfringens were grown in Luria–Bertani (LB) medium at 37 8C. Those of S. aureus and L. monocytogenes were cultivated in nutrient broth (NB) at 37 8C. HiTrapTM rProtein A FF was purchased from GE corporation. Nitrocellulose membrane, sample pad, absorbent pad and conjugate pad were purchased from Millipore Corporation. 2.2. Preparation and identification of antibodies against SS2 Antibodies against SS2 were prepared according to a previously described method, with some modifications (Ying et al., 2005). S. suis 149 was isolated from pigs and cultured in THB at 37 8C for 7 h, and the bacteria were then collected and dissolved in sterile water for blood plate counting. It was inactivated at 37 8C for 7 days in 1% formaldehyde and then used as the immunogen after washing three times with PBS to remove the formaldehyde. SS2-negative New Zealand rabbits weighing about 2 kg were immunized to prepare polyclonal antibodies against SS2. The entire immunization procedure comprised 6 injections. The doses of the immunogen
were 5 108 CFU, 1 109 CFU, 2 109 CFU, 4 109 CFU, 8 109 CFU, and 1.6 109 CFU, respectively, in the first through the sixth injections. The first 2 injections were intravenous and carried out at an interval of 2 weeks, and the subsequent 4 injections were carried out intravenous at intervals of 1 week. Indirect ELISA was performed to measure the serum antibody titers. Polyclonal antibodies against SS2 were purified by affinity chromatography on a protein A-sepharose column according to the manufacturer’s instructions. The concentration of antibodies was determined by the Bradford protein assay. 2.3. Determination of optimal concentration of capture antibody We prepared 25-nm colloidal gold by the citrate– natrium deoxidize method (Chaudhuri and Raychaudhuri, 2001). The pH of the colloidal gold was adjusted to 8.5. The polyclonal antibodies prepared above were further dialyzed for desalination and centrifuged at 10,000 g for removing macromolecular aggregations. The antibodies were then diluted to 0.2 mg/mL with purified H2O. The minimum amount of antibody needed for stabilization of the colloidal gold was determined as that required to change the color of the solution from reddish to blue. Different solutions were added into 10 cuvettes and left standing for 2 h after mixing. If a tube contained the minimum amount of the antibody, the color of the colloidal gold did not change from reddish to blue. To stabilize the colloidal gold, we choose 120% of the minimum amount of capture antibody as its working concentration. 2.4. Preparation of colloidal gold probe The colloidal gold probe was prepared according to a previously described method with slight modifications (Chaudhuri and Raychaudhuri, 2001). The pH of the colloidal gold was adjusted to 8.5 by dropwise addition of 0.1 mol/L K2CO3. One milliliter of colloidal gold was mixed with 110 mL of 0.2 mg/mL antibody, with shaking at intervals for 30 min. Then, another 110 mL of 10% BSA was added, and the mixture was centrifuged at 12,000 g for 30 min at 4 8C. The pellet was re-suspended with 1 mL of the suspension buffer (0.01 mol/L Tris–HCl (pH 8.2), 1% BSA, 5% sucrose). The colloidal gold was centrifuged at 12,000 g for another 30 min at 4 8C, and the pellet was re-suspended in 500 mL of buffer. Finally, it was centrifuged at 1000 g for another 4 min at 4 8C, and the supernatant was collected and stored at 4 8C for use as the colloidal gold probe. 2.5. Determination of optimal concentration of coating antibody The antibody against SS2 was diluted to 3.5 mg/mL, 2.5 mg/mL, 2.0 mg/mL, 1.5 mg/mL, 1.0 mg/mL, and 0.5 mg/ mL with 0.01 mol/L phosphate-buffered saline (PBS; pH 7.2). The BSA concentrations of these diluted solutions were 2.0%, 1.5%, 1.0%, 0.75%, and 0.5%, respectively. The diluted antibody (Test line, T line) and goat anti-rabbit antibody (Control line, C line) were transferred onto a nitrocellulose membrane, which was then dried for 2 h at 37 8C (Jones,
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Table 1 Bacterial strains used in this study. Discriptiona
Strains Streptococcu suis serotypes 2(SS2)
SS1 SS1/2 SS3 SS4 SS5 SS6 SS7 SS8 SS9 SS10 SS11 SS12 SS13 SS14 SS15 Streptococcus sp.
Streptococcus pyogenes
Staphylococcus aureus Escherichia coli Salmonella paratyphi A Clostridium perfringens Listeria monocytogenes a b
S. suis 05ZY S. suis 149 S. suis 606 S. suis 607 S. suis 1940 S. suis 200601 S. suis 200602 S. suis 200502 S. suis 200501 S. suis SUN S. suis T15 S. suis 1330 S. suis S735 S. suis s428 S. suis #2651 S. suis #4961 S. suis #640 S. suis 11538 S. suis #2524 S. suis #8074 S. suis #14636 S. suis #DAN22083 S. suis DAN4417 S. suis #DAN12814 S. suis #DAN8830 S. suis #DAN10581 S. suis #13730 S. suis #GrT639 CMCC32115 CMCC32116 CMCC32118 CMCC32119 CMCC32120 CMCC32121 CMCC32122 CMCC32123 CMCC32124 CMCC32125 CMCC32126 CMCC32134 CMCC32144 CMCC32145 CMCC32146 CMCC32171 CMCC32180 GAS-M15 GAS-M16 S. aureus S-6 S. aureus 137 ATCC25922 CMCC50671 CMCC64601 C5
+
+
+
+
MRP EF SLY 89K MRP+EF+SLY+89K+ MRP+EF+SLY+89K MRP+EF+SLY 89K MRP+EF+SLY+89K MRP+EF+SLY+89K+ MRP+EF+SLY+89K+ MRP+EF+SLY+89K+ MRP+EF+SLY+89K+ MRP+EF+SLY+89K+ MRP EF SLY+89K MRP EF SLY 89K MRP+EF*SLY+89K
Originb
Source
Sichuan, China HP, 2005 Jiangshu, China HP, 1998 China DP, 1980s Japan DP China DP, 1980s Guangxi, China HP, 2006 Guangxi, China HP, 2006 Guangxi, China HP, 2005 Guangxi, China HP, 2005 Jiangsu, China HP, 2006 Europe HPL Canada HPL The Netherlands DP
This work This work This work This work This work This work This work This work This work This work Wisselink HJ Dr. Gottschalk M Wisselink HJ
89K, 89K pathogenicity islands (PAI); MRP, muramidase-released protein; EF, extracellular factor; SLY, suilysin. HP, human patients; DP, diseased piglets; HPL, healthy piglets.
1999a,b). The blank control without S. suis was tested simultaneously to determine the optimal concentrations of the coating antibody and the blocking buffer. 2.6. Preparation of immunochromatographic strip The immunochromatographic strip was composed of a sample pad, a conjugate pad, an immobilized nitrocellulose membrane, and an absorbent pad. A schematic diagram of the immunochromatographic strip is shown in Fig. 1. The colloidal gold probe was added to the
conjugate pad and dried at 37 8C for 2 h. The T line was coated with 2.0 mg/mL rabbit anti-pig antibody against SS2 and the C line was coated with 0.5 mg/mL goat antirabbit immune serum. The sample pad, conjugate pad, immobilized nitrocellulose membrane, and absorbent pad were overlapped and stuck to a polyvinyl chloride (PVC) plate and then cut into 4-mm-wide strips. These strips were stored dried at 4 8C until required. When the samples were applied to the sample pads and allowed to flow through the nitrocellulose membrane, after 5–10 min, a positive test showed two red lines for the
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Fig. 1. Schematic diagram of the immunochromatographic strip.
test and control lines, whereas a negative test only produced one red line in the control region. The strip test was incorrect if there was no red line for the control line.
was regarded as the optimal concentration of the blocking buffer, since the blank control did not show false-positive results at concentrations higher than this.
2.7. Specificity, sensitivity, and stability of immunochromatographic strip
3.2. Sensitivity of immunochromatographic strip
S. suis 149 was inoculated onto a Columbia agar plate containing 5% goat blood and cultured at 37 8C in a 5% CO2 atmosphere for 24 h. The colony was selected and cultured in THB at 37 8C in a 5% CO2 atmosphere for 6 h. The cultures were centrifuged at 8000 g for 10 min at 4 8C and diluted serially with PBS. For the sensitivity assay, 100 mL of each solution of a particular dilution was added to an equal volume of buffer. The culture methods used for other strains were the same as the one described before. We prepared 108 CFU/ mL cultures by the turbidity method. For the specificity assay, 100 mL of the cultures of different bacteria was added to an equal volume of buffer. To evaluate the stability of immunochromatographic strips during storage, they were stored at 4 8C and room temperature respectively for 3 months. Then, the strips were stored at 37 8C for 15 d for stability testing. We used 108 CFU/mL and 106 CFU/mL of S. suis 149 and 109 CFU/mL of S. aureus as samples and PBS as the blank control.
To determine the sensitivity of the immunochromatographic strip, 108 CFU/mL S. suis cultures were serially diluted 10-fold with THB, and samples without SS2 were used as the blank control. The results have been shown in Fig. 2. The sensitivity of the immunochromatographic strip was 1.0 106 CFU/mL. The sensitivity assay was repeated more than 5 times, all replicates were positive at 1.0 106 CFU/mL. 3.3. Specificity of immunochromatographic strip S. aureus S-6, S. aureus 137, E. coli ATCC 25922, S. paratyphi A CMCC 50671, C. perfringens CMC 64601, and L. monocytogenes C5 were cultured in THB at 37 8C in a 5% CO2 atmosphere for 6 h. Different suspensions (concentration >108 CFU/mL) were collected to evaluate the specificity of the immunochromatographic strip. We also included 15 serotypes of S. suis. Only S. suis serotype 2 and serotype 1/2 showed positive results, and all the other serotypes showed clearly negative results. The capsules of SS2 and SS1/2 share some common sugar residues or antigenic determinants (Elliott and Tai, 1978; Smith et al., 1999). No
3. Results 3.1. Optimal concentration of capture antibody, coating antibody, and blocking BSA After 6 immunizations for 7 weeks, the antibody titers in the serum reached 107. The optimal capture antibody was determined to be 22 mg/mL according to the protocol in Section 2. Regarding the coating antibody, with the increase in the concentration, the color of the indicating line deepened gradually. When the antibody concentration reached 2.0 mg/mL, the color did not change any more. Therefore, 2.0 mg/mL was determined as the optimal concentration of the coating antibody. The blank control that did not contain SS2 but contained BSA at different concentrations showed false-positive to negative results with increase in the BSA concentration. Therefore, 1.5% BSA
Fig. 2. Sensitivity testing of immunochromatographic strip. 1: Blank control; 2: S. suis 149, 1.0 108 CFU/mL; 3: S. suis 149, 1.0 107 CFU/mL; 4: S. suis 149, 1.0 106 CFU/mL; 5: S. suis 149, 1.0 105 CFU/mL; 6: S. suis 149, 1.0 104 CFU/mL.
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these polyclonal antibodies. Surprisingly, the coupling of polyclonal antibodies with colloidal gold particles in this study resulted in considerably high specificity in the detection of 15 different serotypes of S. suis. This high specificity may be a result of the inactivation of S. suis 149. Inactivation in 1% formaldehyde at 37 8C for 7 days may help retain the sugar residues or antigenic determinants of SS2. In summary, the colloidal gold immunochromatographic strips we developed in this study can be used directly in serological surveillance and detection of the S. suis serotype 2 antigen in the field. Acknowledgements
Fig. 3. Stability testing of immunochromatographic strip by evaluating the sensitivity and specificity. 1: Blank control; 2: S. suis 149 108 CFU/ mL; 3: S. suis 149 106 CFU/mL; 4: S. aureus 108 CFU/mL.
cross-reaction was observed when testing the other pathogenic bacteria. These results confirmed that the immunochromatographic strip had high specificity for detecting SS2.
This work was supported by grants from the National Natural Science Foundation of China (30870091, 30770117 and 30600023), the National Basic Research Program (973) of China (2006CB504400), the High-Tech Research & Development Project 863 (2006AA02Z404, 2007AA02 1504), and the National Key Technologies R&D program (2006BAD06A02), mega-projects of science research for the 11th Five-year plan (2009ZX10004-103). References
3.4. Stability of immunochromatographic strip The immunochromatographic strips were stored at 4 8C and room temperature for 3 months, respectively, and then kept at 37 8C for another 15 days to assess their stability. As shown in Fig. 3, good results were obtained, as expected, for the detection of S. aureus and S. suis after this time period. This demonstrated that the strips were stable for at least 3 months at room temperature. 4. Discussion It has been suggested that SS2 is an opportunistic pathogen infecting pigs. Therefore, serological surveillance will be useful in determining the infection status and can play an important role in the control of S. suis in both pigs and humans. Colloidal gold-based immunochromatographic assay has been confirmed to be a rapid and sensitive method for serological surveillance and detection of bacterial pathogens in the field. However, the immunochromatographic strip that has been developed (Yang et al., 2007) can only detect antibodies generated against the S. suis type 2 antibody, and not the antigen directly. In this study, we developed an immunochromatographic strip for the rapid detection of the SS2 antigen using colloidal gold coupled with polyclonal antibody. This strip can be used for the rapid detection of SS2 in the field and in serological surveillance, with high sensitivity and specificity. Colloidal gold-based immunochromatographic assay has been used increasingly in the rapid diagnosis of bacterial pathogens. However, the use of polyclonal antibodies generated by immunization with whole bacteria as antigens is associated with the problem of low specificity. The antibodies may react with other related bacteria; such cross-reactions hamper the application of
Charland, N., Nizet, V., et al., 2000. Streptococcus suis serotype 2 interactions with human brain microvascular endothelial cells. Infect. Immun. 68 (2), 637–643. Chaudhuri, B., Raychaudhuri, S., 2001. Manufacturing high quality gold sol. IVD Technol. 8, 46–54. Clifton-Hadley, F.A., 1983. Streptococcus suis type 2 infections. Br. Vet. J. 139 (1), 1–5. Elliott, S.D., Tai, J.Y., 1978. The type-specific polysaccharides of Streptococcus suis. J. Exp. Med. 148 (6), 1699–1704. Francois, B., Gissot, V., et al., 1998. Recurrent septic shock due to Streptococcus suis. J. Clin. Microbiol. 36 (8), 2395. Halaby, T., Hoitsma, E., et al., 2000. Streptococcus suis meningitis, a poacher’s risk. Eur. J. Clin. Microbiol. Infect. Dis. 19 (12), 943–945. Heidt, M.C., Mohamed, W., et al., 2005. Human infective endocarditis caused by Streptococcus suis serotype 2. J. Clin. Microbiol. 43 (9), 4898–4901. Higgins, R., Gottschalk, M., 1995. Distribution of Streptococcus suis capsular types in 1994. Can. Vet. J. 36 (5), 320. Huang, Y.T., Teng, L.J., et al., 2005. Streptococcus suis infection. J. Microbiol. Immunol. Infect. 38 (5), 306–313. Jones, K., 1999a. Troubleshooting protein binding in nitrocellulose membranes. Part 1. Principles. IVD Technol. 5 (2), 32–41. Jones, K., 1999b. Troubleshooting protein binding in nitrocellulose membranes. Part 2. Common problems. IVD Technol. 5 (3), 26–35. Luque, I., Tarradas, C., et al., 1998. Streptococcus suis serotypes associated with different disease conditions in pigs. Vet. Rec. 142 (26), 726–727. Okwumabua, O., O’Connor, M., et al., 2003. A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase. FEMS Microbiol. Lett. 218 (1), 79–84. Robertson, I.D., Blackmore, D.K., 1989. Occupational exposure to Streptococcus suis type 2. Epidemiol. Infect. 103 (1), 157–164. Smith, H.E., Veenbergen, V., et al., 1999. The cps genes of Streptococcus suis serotypes 1, 2 and 9: development of rapid serotype-specific PCR assays. J. Clin. Microbiol. 37 (10), 3146–3152. Tang, J., Wang, C., et al., 2006. Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2. PLoS Med. 3 (5), e151. Touil, F., Higgins, R., et al., 1988. Isolation of Streptococcus suis from diseased pigs in Canada. Vet. Microbiol. 17 (2), 171–177. Yang, J., Jin, M., et al., 2007. Development and evaluation of an immunochromatographic strip for detection of Streptococcus suis type 2 antibody. J. Vet. Diagn. Invest. 19 (4), 355–361. Ying, T., Wang, H., et al., 2005. Immunoproteomics of outer membrane proteins and extracellular proteins of Shigella flexneri 2a 2457T. Proteomics 5 (18), 4777–4793.