Biosensors and Bioelectronics 55 (2014) 396–399
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Development of colloidal gold-based immunochromatographic assay for rapid detection of Mycoplasma suis in porcine plasma Kai Meng 1, Wenjing Sun 1, Peng Zhao, Limei Zhang, Dongjie Cai, Ziqiang Cheng, Huijun Guo, Jianzhu Liu n, Dubao Yang, Shujing Wang, Tongjie Chai College of Veterinary Medicine, Research Center for Animal Disease Control Engineering Shandong Province, Shandong Agricultural University, Tai’an 271018, China
art ic l e i nf o
a b s t r a c t
Article history: Received 29 September 2013 Received in revised form 11 December 2013 Accepted 22 December 2013 Available online 31 December 2013
A one-step immunochromatographic assay using gold nanoparticles coated with polyclonal antibody (pAb) against Mycoplasma suis (M. suis) was developed in this study for the detection of M. suis in porcine plasma. The colloidal gold was prepared by the reduction of gold salt with sodium citrate coupled with pAb against M. suis. The pAb was produced by immunizing the BALB/c mice with recombinant MSG1 (rMSG1) protein from M. suis expressed in Escherichia coli. The optimal concentrations of the capture antibody and the coating antibody were 12 μg/ml and 1.5 mg/ml, respectively, and that of the blocking buffer was 1% bovine serum albumin. The lower detection limit of the immunochromatographic assay test was 100 ng/ml with visual detection under optimal conditions of analysis. Classical swine fever virus, porcine reproductive and respiratory syndrome virus, swine pneumonia mycoplasma, swine toxoplasma, and porcine parvovirus were used to evaluate the specificity of the immunochromatographic strips. No cross-reaction of the antibodies with other related swine pathogens was observed. This qualitative test based on the visual evaluation of the results did not require any equipment. The assay time for M. suis detection was less than 10 min, suitable for rapid detection at the grassroots level. The one-step colloidal gold immunochromatographic strips that we developed had high specificity and sensitivity. Therefore, this method would be feasible, convenient, rapid, and effective for detecting M. suis in porcine plasma. & 2013 Elsevier B.V. All rights reserved.
Keywords: Mycoplasma suis Polyclonal antibody Colloidal gold Immunochromatographic assay Rapid detection
1. Introduction Mycoplasma suis (M. suis) is an uncultivable, hemotropic pathogen in swine that causes severe hemolytic anemia and enormous economic losses in the swine industry mainly due to chronic infections characterized by chronic anemia, poor growth rates, general unthriftness, anoestrus or delayed estrus, early embryonic deaths and abortion, and predisposing the infected swine to secondary infection (Hoelzle, 2008; Messick, 2004; Rikihisa et al., 1997; Zhang et al., 2012). M. suis infections have been consecutively reported worldwide over the past 75 years (Wu et al., 2006). However, valuable data on the prevalence of M. suis infections are limited because of the difficulty in diagnosing this uncultivable pathogen (Hoelzle et al., 2009). M. suis is an extracorpuscular bacterial microorganism that adheres to the surface of porcine erythrocytes. This characteristic is vital to the pathogenicity of M. suis, although little is known about how the adhesion is achieved (Hoelzle et al., 2003; Zhang
n
Corresponding author. Tel.: þ 86 538 8246287; fax: þ 86 538 8241419. E-mail address: [email protected] (J. Liu). 1 These authors contributed equally to the work.
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.12.048
et al., 2012). Only one M. suis adhesion protein, designated as MSG1 protein, has been characterized. This protein, which is situated on the surface of the microorganism and highly conserved, is a major immunogen of M. suis (Hoelzle et al., 2007b). The encoding gene MSG1 has been found in all tested M. suis isolates (Hoelzle et al., 2009). Both naturally and experimentally infected pigs generate high antibody titers against MSG1 protein (K. Hoelzle et al., 2007; Hoelzle et al., 2006), and the recombinant form (rMSG1) expressed in Escherichia coli can also trigger a strong immune response but fails to induce protection in pigs (Hoelzle et al., 2009). The current methods for diagnosing M. suis infections are routine biochemistry assays, polymerase chain reaction (Hoelzle et al., 2003), indirect ELISA based on rHspA1 (Hoelzle et al., 2007a), whole-cell ELISA (Zhang et al., 2008), indirect ELISA based on rMSG1 (Liu et al., 2011), and the novel blocking ELISA based on rMSG1 protein (Zhang et al., 2012). However, these methods are time consuming, require expensive equipment and well-trained personnel, and can only be used in laboratories. Therefore, a rapid and economical method for the detection of M. suis should be developed. The one-step immunochromatographic assay using gold nanoparticles has been widely used in certain fields (Hua et al., 2010; Ju et al., 2010; Nara et al., 2010; Zhou et al., 2009). This method is easy, rapid, and
K. Meng et al. / Biosensors and Bioelectronics 55 (2014) 396–399
convenient to use and requires few equipments. However, there is no report about the detection of M. suis using the one-step immunochromatographic assay. Taking advantage of the polyclonal antibody (pAb) produced in a previous work (Liu et al., 2011), the present study aims to develop a one-step immunochromatographic assay for the rapid detection of M. suis in porcine plasma samples.
2. Experimental 2.1. Materials and reagents Fifty porcine plasma samples containing Mycoplasma suis (M. suis), swine pneumonia mycoplasma, swine toxoplasma, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus were preserved in our laboratory. The polyclonal antibody (pAb) against M. suis were produced in our laboratory. Bovine serum albumin (BSA) and goat anti-mouse IgG antibody were purchased from the Beijing Dingguo Biotechnology Development Center (Beijing, China). Nitrocellulose (NC) membrane, absorbent pad, sample pad, conjugate pad, and PVC sheets were purchased from Millipore Corporation (Shanghai, China). Hydrogen tetra-chloroaurate hydrate (HAuCl4) and trisodium citrate were purchased from Shanghai Chemical Reagents (Shanghai, China). Approximately 0.02 M sodium phosphatebuffered saline (PBS, pH 8.5) was used as incubating and washing buffer in this study. All glassware used in this experiment was siliconized with sigmacote obtained from Sigma Chemical Company. All the solvents, chemicals, and salts used in this study were of analytical grade. 2.2. Preparation of immunoassay reagents The preparation of pAb using recombinant MSG1 (rMSG1) protein as immunogen is described in the reference (Chen et al., 2011; Liu et al., 2011). The pAb was purified by saturated ammonium sulfate [(NH4)2SO4], conjugated with colloidal gold, and used as a capture reagent fixed on NC membrane (test line). 2.3. Preparation of the colloidal gold suspension All glassware used was siliconized with sigmacote, cleaned in aqua regia [HNO3/HCL (v/3v)], washed in ultrapure water, and oven-dried before use. Colloidal gold particles (mean diameter, 20 nm) were prepared according to the procedure in the literature (Liu et al., 2012). Briefly, 100 ml of 0.01% HAuCl4 solution (in ultrapure water) in a 200 ml beaker was boiled thoroughly, and 2.5 ml of 1% trisodium citrate solution was then added under continuous stirring. When the color of the solution changed from blue to dark red in 2 min, the solution was boiled for another 10 min. Subsequently, the heating source was removed and the colloidal gold solution was continuously stirred for another 5 min. The obtained gold colloid supplemented with 0.01% (m/v) sodium azide (NaN3) was stored in a dark bottle at 4 1C. The solution would be used to prepare the colloidal gold–pAb conjugate as quickly as possible. The gold colloids were characterized by transmission electron microscopy.
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solution (pH 8.5) containing 1% BSA and 0.2% sodium azide] and stored in a dark bottle at 4 1C until use. 2.5. Preparation of the optimal concentration of the coating antibody The pAb against M. suis were diluted to 3.0, 2.5, 2.0, 1.5, 1.0, and 0.5 mg/ml with 0.02 M sodium PBS (pH 8.5). The diluted pAb and 1 mg/ml goat anti-mouse IgG were transferred onto the NC membrane with a volume of 1 μl/cm to form the test (T) and the control (C) lines, respectively. The distance between the T and C lines was 8 mm. The test strips were dried at 37 1C for 2 h. In this study, different concentrations of BSA (3.0%, 2.5%, 2.0%, 1.5%, 1.0%, and 0.5%) were used as the blocking buffer. The positive porcine plasma samples containing M. suis were tested to determine the optimal concentrations of the coating antibody, and the blank control without M. suis was used to determine the optimal concentrations of the blocking buffer.
2.6. Preparation of the immunochromatographic strip The composition of the immunochromatographic test strip is described as follows and shown in Fig. 1. The sample pad was saturated with 0.02 M PBS solution (pH 8.5) containing 0.2% Tween-20 and 1.5% (w/v) BSA. The sample pad was dried at 37 1C for 2 h and stored in a desiccator at 4 1C for future use. The colloidal gold probe was diluted (1:1, v/v) with 0.02 M PBS (pH 8.5) containing 5% (w/v) sucrose and 1.5% (w/v) BSA. The glass fiber was dealt with 0.2% Tween-20 for 12 h and dried for 1 h at 37 1C before use. The colloidal gold probe was then added to the conjugate pad, dried at 37 1C for 1 h, and then stored in a desiccator at 4 1C for future use. The diluted pAb and goat anti-mouse IgG (1 mg/ml) were transferred onto the NC membrane with a volume of 1 μl/cm to form the T and C lines, respectively. The NC membrane was dealt with 0.02 M PBS containing 1.0% BSA, dried for 2 h at 37 1C, and then stored in a desiccator at 4 1C for future use. Pure cellulose fiber of 100% was used as the absorbent pad. The PVC plate was used as the bottom of the test strip. The sample pad, conjugate pad, immobilized NC membrane, and absorbent pad were assembled, as described in Fig. 1. These strips were cut into 5 mm width and stored in a desiccator at 4 1C for future use.
2.4. Preparation of the colloidal gold–pAb conjugate Approximately 0.4 ml of purified optimal pAb (3.0 mg/ml) against M. suis was added to 100 ml colloidal gold solution (pH 8.5) with gentle stirring. The admixture was mixed gently for 15 min, blocked by 11.1 ml of 10% BSA solution for 60 min, and then centrifuged at 12,000 rpm for 25 min. After centrifugation, the pellet was suspended in 10 ml dilution buffer [0.002 mol/L sodium carbonate
Fig. 1. (A) The schematic description of the immunochromatographic device. (B) The illustration of immunochromatographic test results.
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K. Meng et al. / Biosensors and Bioelectronics 55 (2014) 396–399
After the samples (100 μl) were added dropwise to the sample pad and allowed to pass through the NC membrane, the positive result was denoted by the appearance of two red lines in the test and control regions after 10 min. The negative result was denoted by the appearance of only one red line in the control region. The appearance of only a single red line in the test region or the absence of a line in test strip was considered an invalid test (Fig. 1). 2.7. Sensitivity, specificity, and stability of the immunochromatographic strip Different concentrations of the porcine plasma samples containing M. suis (1 mg/ml, 100 mg/ml, 10 mg/ml, 1 mg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, and 0.1 ng/ml) were prepared by diluting a stock solution (2 mg/ml) with 0.9% NaCl (pH 7.2) to evaluate the sensitivity of the immunochromatographic strip. For the sensitivity assay, 100 μl of each solution of a particular dilution was used for the immunochromatographic strip test and 0.9% NaCl (pH 7.2) was used as the blank control. Other samples containing different antigens, such as swine toxoplasma, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus were also tested to evaluate the specificity of the immunochromatographic strip. All samples were diluted 1 mg/ml with 0.9% NaCl (pH 7.2), respectively. For the specificity assay, 100 μl of each solution of a particular dilution was added to the sample pad. All immunochromatographic strips were stored at 4 1C for 16 weeks to evaluate the stability of the immunochromatographic strips during storage. 1 mg/ml and 100 ng/ml of the porcine plasma samples containing M. suis and 1 mg/ml of the porcine samples containing pneumonia mycoplasma were used as samples, with 0.9% NaCl (pH 7.2) as the blank control. 2.8. Detection of clinical samples A total of 350 porcine plasma samples collected in field from different pig farms were detected using the developed test strips. The plasma samples were diluted with 0.9% NaCl (pH 7.2) before detection. These plasma samples were also detected by the ELISA kit to evaluate the accuracy of the immunochromatographic strip test.
3. Results and discussion 3.1. Design of the one-step immunochromatographic assay The porcine plasma samples were obtained by centrifuging at 5000 rpm for 10 min and then diluted with 0.9% NaCl (pH 7.2) for detection. 100 μl sample was added to the sample pad and allowed to flow through the nitrocellulose membrane. After 10 min, the result can be obtained. The positive result is judged by the appearance of two red lines in test and control region. The negative result is judged by the appearance of only a single line in the control region. If no line is present in the test strip or only one line appears in the test region, the test is invalid.
with the increase in pAb concentration. However, the color of the test line was maintained when the pAb concentration reached 1.5 mg/ml. Thus, the optimal concentration of the coating antibody was 1.5 mg/ ml. In this study, the blank control that did not contain M. suis but contained BSA at different concentrations showed false-positive results to negative results with the increase in BSA concentration because the blank control did not display false-positive results when the BSA concentrations were higher than 1%. Thus, 1% BSA was determined as the most advantageous concentration of the blocking buffer. 3.3. Sensitivity of the immunochromatographic strip To confirm the sensitivity of the immunochromatographic strip, different concentrations of the porcine plasma samples containing M. suis (1 mg/ml, 100 mg/ml, 10 mg/ml, 1 mg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, and 0.1 ng/ml) were prepared by diluting a stock solution (2 mg/ml) with 0.9% NaCl (pH 7.2). Whereas the porcine plasma samples without M. suis were used as the blank control. The results are presented in Fig. 2. Standard densitometric analysis based on scanning the strips and measuring the color intensity (gray scale) via standard image software was adopted for the sensitivity test (Fernández-Sánchez et al., 2005). The sensitivity test was repeated more than three times and the results showed that the red line on the testing region could be observed clearly when the concentrations of the porcine plasma samples containing M. suis were up to 100 ng/ml. 3.4. Specificity of the immunochromatographic strip Other porcine samples containing different pathogens, such as swine toxoplasma, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus, were used to evaluate the specificity of the immunochromatographic strip. All the porcine samples were diluted with 0.9% NaCl (pH 7.2), and 0.9% NaCl (pH 7.2) was used as the blank control. Results (Fig. 3(A)) showed that only porcine samples containing M. suis presented two red lines in test and control region and others presented only one red line in control region which indicated that the immunochromatographic strip had high specificity for detecting M. suis. In addition, no cross-reaction was observed when other porcine pathogens were tested. 3.5. Stability of the immunochromatographic strip The same batch of immunochromatographic strips was stored at 4 1C for 16 weeks to assess their stability by evaluating the sensitivity and specificity. It was seen from Fig. 3(B) that the visible red color on the testing region can been seen clearly after 16
3.2. Optimal concentration of the capture antibody, coating antibody, and blocking BSA The pAb using rMSG1 protein as immunogen was produced by the method described in reference (Chen et al., 2011; Liu et al., 2011). After 4 immunizations for 6 weeks, the antibody titers in the porcine serum reached 107. Based on the protocol in Section 2, the most advantageous concentration of the capture antibody was 12 μg/ml. For the coating antibody, the color of the test line deepened gradually
Fig. 2. Sensitivity testing of immunochromatographic strip. 1: Blank control; 2: M. suis, 1 mg/ml; 3: M. suis, 100 mg/ml; 4: M. suis, 10 mg/ml; 5: M. suis, 1 mg/ml; 6: M. suis, 100 ng/ml; 7: M. suis, 10 ng/ml; 8: M. suis, 1 ng/ml; 9: M. suis, 0.1 ng/ml.
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suis in the field and in serological surveillance using colloidal gold coupled with polyclonal antibody, and the immunochromatographic strip was evaluated in sensitivity, specificity and stability. Results of the sensitivity test indicated that the darker the color density of the test line is, the higher the concentration of the M. suis tested in the sample. However, the use of polyclonal antibody is associated with low specificity, and polyclonal antibody may react with other related antigens. In fact, the coupling of polyclonal antibody with colloidal gold particles in this study resulted in high specificity in the detection of M. suis. In subsequent research, we will verify whether the monoclonal antibody has higher specificity than polyclonal antibody.
4. Conclusion In this study, an immunochromatographic assay format of colloidal gold-labeled pAb was developed for the detection of M. suis in porcine plasmas. Although the sensitivity of the assays described in this study is lower than that of PCR and ELISA, the method presented is easy, rapid, and convenient to use and requires few equipment compared with PCR, ELISA, and other techniques. The test can also be accomplished within 10 min without complicated handling procedures; this feature is significant for clinical diagnosis. Moreover, the proposed method is superior to other immunoassays in terms of speed and simplicity. Fig. 3. (A) Specificity testing of immunochromatographic strip. 1: Blank control; 2: M. suis, 1 mg/ml; 3: swine toxoplasma, 1 mg/ml; 4: classical swine fever virus (CSFV), 1 mg/ml; 5: porcine reproductive and respiratory syndrome virus (PRRSV), 1 mg/ml; 6: porcine parvovirus (PPV), 1 mg/ml. (B). Stability testing of immunochromatographic strip by evaluating the sensitivity and specificity. 1: Blank control; 2: M. suis, 1 mg/ml; 3: M. suis, 100 ng/ml; 4: pneumonia mycoplasma, 1 mg/ml.
weeks storage at 4 1C. This demonstrated that the strips were stable for at least 16 weeks at 4 1C. 3.6. Clinical application of the developed immunochromatographic strip A total of 350 plasma samples was detected by the developed test strip and by the ELISA kit respectively. The positive rate was 43.84% in the immunochromatographic strip test and 47.25% in the ELISA. The coincidence was only 92.78% which may be caused by the polyclonal antibody (pAb) against M. suis. In future research, the monoclonal antibodies against M. suis will be applied to improve the reliability of the developed immunochromatographic strip. M. suis is an opportunistic organism that infects pigs (Messick, 2004). Therefore, serological surveillance will be useful in determining the infection status, and it has an important role in the control of M. suis in both pigs and humans. The colloidal gold-based immunochromatographic assay was affirmed to be a rapid, simple, and sensitive method for serological surveillance in the field. Colloidal gold-based immunochromatographic assay is widely used for detecting certain antigens, such as Streptococcus suis serotype 2 (Ju et al., 2010), brevetoxins (Zhou et al., 2009), cortisol (Nara et al., 2010) and botulinum neurotoxin (Klewitz et al., 2006). In this study, we developed an immunochromatographic strip for the rapid detection of M.
References Chen, T.T., Liu, J.Z., Cheng, Z.Q., Zhang, L., Zhou, D., Wang, Z.Y., Liu, H.T., Liu, Y.X., Wang, S.J., Chai, T.J., 2011. Chin. J. Zoono 3, 21. Fernández-Sánchez, C., McNeil, C.J., Rawson, K., Nilsson, O., Leung, H.Y., Gnanapragasam, V., 2005. J. Immunol. Methods 307, 1–12. Hoelzle, K., Doser, S., Ritzmann, M., Heinritzi, K., Palzer, A., Elicker, S., Kramer, M., Felder, K.M., Hoelzle, L.E., 2009. Vaccine 27, 5376–5382. Hoelzle, K., Grimm, J., Ritzmann, M., Heinritzi, K., Torgerson, P., Hamburger, A., Wittenbrink, M.M., Hoelzle, L.E., 2007. Clin. Vaccine Immunol. 14, 1616–1622. Hoelzle, L., 2008. Vet. Microbiol. 130, 215–216. Hoelzle, L., Adelt, D., Hoelzle, K., Heinritzi, K., Wittenbrink, M., 2003. Vet. Microbiol. 93, 185–196. Hoelzle, L., Hoelzle, K., Harder, A., Ritzmann, M., Aupperle, H., Schoon, H., Heinritzi, K., Wittenbrink, M., 2007a. FEMS Immunol. Med. Microbiol. 49, 215–223. Hoelzle, L., Hoelzle, K., Helbling, M., Aupperle, H., Schoon, H., Ritzmann, M., Heinritzi, K., Felder, K., Wittenbrink, M., 2007b. Microbes Infect. 9, 466–474. Hoelzle, L., Hoelzle, K., Ritzmann, M., Heinritzi, K., Wittenbrink, M., 2006. Clin. Vaccine Immunol. 13, 116–122. Hua, X., Qian, G., Yang, J., Hu, B., Fan, J., Qin, N., Li, G., Wang, Y., Liu, F., 2010. Biosens. Bioelectron. 26 (1), 189–194. Ju, Y., Hao, H.J., Xiong, G.H., Geng, H.R., Zheng, Y.L., Wang, J., Cao, Y., Yang, Y.H., Cai, X.H., Jiang, Y.Q., 2010. Vet. Immunol. Immunopathol. 133, 207–211. Klewitz, T., Gessler, F., Beer, H., Pflanz, K., Scheper, T., 2006. Sens. Actuators B Chem. 113, 582–589. Liu, X., Xiang, J.J., Tang, Y., Zhang, X.L., Fu, Q.Q., Zou, J.H., Lin, Y.H., 2012. Anal. Chim. Acta 745, 99–105. Liu, Y., Liu, J., Cheng, Z., 2011. Chin. Agri. Sci. 44, 4508–4515. Messick, J., 2004. Vet. Clin. Pathol. 33, 2–13. Nara, S., Tripathi, V., Singh, H., Shrivastav, T.G., 2010. Anal. Chim. Acta 682, 66–71. Rikihisa, Y., Kawahara, M., Wen, B., Kociba, G., Fuerst, P., Kawamori, F., Suto, C., Shibata, S., Futohashi, M., 1997. J. Clin. Microbiol. 35, 823–829. Wu, J., Yu, J., Song, C., Sun, S., Wang, Z., 2006. Ann. N. Y. Acad. Sci. 1081, 280–285. Zhang, C., Li, Y., Jiang, P., Chen, W., 2012. Vet. J. 193, 535–538. Zhang, S., Ju, Y., Jia, L., Kumagai, S., Li, J., Manabe, N., 2008. J. Vet. Med. Sci. 70, 1143–1145. Zhou, Y., Pan, F.G., Li, Y.S., Zhang, Y.Y., Zhang, J.H., Lu, S.Y., Ren, H.L., Liu, Z.S., 2009. Biosens. Bioelectron. 24 (8), 2744–2747.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Development of colloidal gold-based immunochromatographic assay for rapid detection of Mycoplasma suis in porcine plasma Kai Meng 1, Wenjing Sun 1, Peng Zhao, Limei Zhang, Dongjie Cai, Ziqiang Cheng, Huijun Guo, Jianzhu Liu n, Dubao Yang, Shujing Wang, Tongjie Chai College of Veterinary Medicine, Research Center for Animal Disease Control Engineering Shandong Province, Shandong Agricultural University, Tai’an 271018, China
art ic l e i nf o
a b s t r a c t
Article history: Received 29 September 2013 Received in revised form 11 December 2013 Accepted 22 December 2013 Available online 31 December 2013
A one-step immunochromatographic assay using gold nanoparticles coated with polyclonal antibody (pAb) against Mycoplasma suis (M. suis) was developed in this study for the detection of M. suis in porcine plasma. The colloidal gold was prepared by the reduction of gold salt with sodium citrate coupled with pAb against M. suis. The pAb was produced by immunizing the BALB/c mice with recombinant MSG1 (rMSG1) protein from M. suis expressed in Escherichia coli. The optimal concentrations of the capture antibody and the coating antibody were 12 μg/ml and 1.5 mg/ml, respectively, and that of the blocking buffer was 1% bovine serum albumin. The lower detection limit of the immunochromatographic assay test was 100 ng/ml with visual detection under optimal conditions of analysis. Classical swine fever virus, porcine reproductive and respiratory syndrome virus, swine pneumonia mycoplasma, swine toxoplasma, and porcine parvovirus were used to evaluate the specificity of the immunochromatographic strips. No cross-reaction of the antibodies with other related swine pathogens was observed. This qualitative test based on the visual evaluation of the results did not require any equipment. The assay time for M. suis detection was less than 10 min, suitable for rapid detection at the grassroots level. The one-step colloidal gold immunochromatographic strips that we developed had high specificity and sensitivity. Therefore, this method would be feasible, convenient, rapid, and effective for detecting M. suis in porcine plasma. & 2013 Elsevier B.V. All rights reserved.
Keywords: Mycoplasma suis Polyclonal antibody Colloidal gold Immunochromatographic assay Rapid detection
1. Introduction Mycoplasma suis (M. suis) is an uncultivable, hemotropic pathogen in swine that causes severe hemolytic anemia and enormous economic losses in the swine industry mainly due to chronic infections characterized by chronic anemia, poor growth rates, general unthriftness, anoestrus or delayed estrus, early embryonic deaths and abortion, and predisposing the infected swine to secondary infection (Hoelzle, 2008; Messick, 2004; Rikihisa et al., 1997; Zhang et al., 2012). M. suis infections have been consecutively reported worldwide over the past 75 years (Wu et al., 2006). However, valuable data on the prevalence of M. suis infections are limited because of the difficulty in diagnosing this uncultivable pathogen (Hoelzle et al., 2009). M. suis is an extracorpuscular bacterial microorganism that adheres to the surface of porcine erythrocytes. This characteristic is vital to the pathogenicity of M. suis, although little is known about how the adhesion is achieved (Hoelzle et al., 2003; Zhang
n
Corresponding author. Tel.: þ 86 538 8246287; fax: þ 86 538 8241419. E-mail address: [email protected] (J. Liu). 1 These authors contributed equally to the work.
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.12.048
et al., 2012). Only one M. suis adhesion protein, designated as MSG1 protein, has been characterized. This protein, which is situated on the surface of the microorganism and highly conserved, is a major immunogen of M. suis (Hoelzle et al., 2007b). The encoding gene MSG1 has been found in all tested M. suis isolates (Hoelzle et al., 2009). Both naturally and experimentally infected pigs generate high antibody titers against MSG1 protein (K. Hoelzle et al., 2007; Hoelzle et al., 2006), and the recombinant form (rMSG1) expressed in Escherichia coli can also trigger a strong immune response but fails to induce protection in pigs (Hoelzle et al., 2009). The current methods for diagnosing M. suis infections are routine biochemistry assays, polymerase chain reaction (Hoelzle et al., 2003), indirect ELISA based on rHspA1 (Hoelzle et al., 2007a), whole-cell ELISA (Zhang et al., 2008), indirect ELISA based on rMSG1 (Liu et al., 2011), and the novel blocking ELISA based on rMSG1 protein (Zhang et al., 2012). However, these methods are time consuming, require expensive equipment and well-trained personnel, and can only be used in laboratories. Therefore, a rapid and economical method for the detection of M. suis should be developed. The one-step immunochromatographic assay using gold nanoparticles has been widely used in certain fields (Hua et al., 2010; Ju et al., 2010; Nara et al., 2010; Zhou et al., 2009). This method is easy, rapid, and
K. Meng et al. / Biosensors and Bioelectronics 55 (2014) 396–399
convenient to use and requires few equipments. However, there is no report about the detection of M. suis using the one-step immunochromatographic assay. Taking advantage of the polyclonal antibody (pAb) produced in a previous work (Liu et al., 2011), the present study aims to develop a one-step immunochromatographic assay for the rapid detection of M. suis in porcine plasma samples.
2. Experimental 2.1. Materials and reagents Fifty porcine plasma samples containing Mycoplasma suis (M. suis), swine pneumonia mycoplasma, swine toxoplasma, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus were preserved in our laboratory. The polyclonal antibody (pAb) against M. suis were produced in our laboratory. Bovine serum albumin (BSA) and goat anti-mouse IgG antibody were purchased from the Beijing Dingguo Biotechnology Development Center (Beijing, China). Nitrocellulose (NC) membrane, absorbent pad, sample pad, conjugate pad, and PVC sheets were purchased from Millipore Corporation (Shanghai, China). Hydrogen tetra-chloroaurate hydrate (HAuCl4) and trisodium citrate were purchased from Shanghai Chemical Reagents (Shanghai, China). Approximately 0.02 M sodium phosphatebuffered saline (PBS, pH 8.5) was used as incubating and washing buffer in this study. All glassware used in this experiment was siliconized with sigmacote obtained from Sigma Chemical Company. All the solvents, chemicals, and salts used in this study were of analytical grade. 2.2. Preparation of immunoassay reagents The preparation of pAb using recombinant MSG1 (rMSG1) protein as immunogen is described in the reference (Chen et al., 2011; Liu et al., 2011). The pAb was purified by saturated ammonium sulfate [(NH4)2SO4], conjugated with colloidal gold, and used as a capture reagent fixed on NC membrane (test line). 2.3. Preparation of the colloidal gold suspension All glassware used was siliconized with sigmacote, cleaned in aqua regia [HNO3/HCL (v/3v)], washed in ultrapure water, and oven-dried before use. Colloidal gold particles (mean diameter, 20 nm) were prepared according to the procedure in the literature (Liu et al., 2012). Briefly, 100 ml of 0.01% HAuCl4 solution (in ultrapure water) in a 200 ml beaker was boiled thoroughly, and 2.5 ml of 1% trisodium citrate solution was then added under continuous stirring. When the color of the solution changed from blue to dark red in 2 min, the solution was boiled for another 10 min. Subsequently, the heating source was removed and the colloidal gold solution was continuously stirred for another 5 min. The obtained gold colloid supplemented with 0.01% (m/v) sodium azide (NaN3) was stored in a dark bottle at 4 1C. The solution would be used to prepare the colloidal gold–pAb conjugate as quickly as possible. The gold colloids were characterized by transmission electron microscopy.
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solution (pH 8.5) containing 1% BSA and 0.2% sodium azide] and stored in a dark bottle at 4 1C until use. 2.5. Preparation of the optimal concentration of the coating antibody The pAb against M. suis were diluted to 3.0, 2.5, 2.0, 1.5, 1.0, and 0.5 mg/ml with 0.02 M sodium PBS (pH 8.5). The diluted pAb and 1 mg/ml goat anti-mouse IgG were transferred onto the NC membrane with a volume of 1 μl/cm to form the test (T) and the control (C) lines, respectively. The distance between the T and C lines was 8 mm. The test strips were dried at 37 1C for 2 h. In this study, different concentrations of BSA (3.0%, 2.5%, 2.0%, 1.5%, 1.0%, and 0.5%) were used as the blocking buffer. The positive porcine plasma samples containing M. suis were tested to determine the optimal concentrations of the coating antibody, and the blank control without M. suis was used to determine the optimal concentrations of the blocking buffer.
2.6. Preparation of the immunochromatographic strip The composition of the immunochromatographic test strip is described as follows and shown in Fig. 1. The sample pad was saturated with 0.02 M PBS solution (pH 8.5) containing 0.2% Tween-20 and 1.5% (w/v) BSA. The sample pad was dried at 37 1C for 2 h and stored in a desiccator at 4 1C for future use. The colloidal gold probe was diluted (1:1, v/v) with 0.02 M PBS (pH 8.5) containing 5% (w/v) sucrose and 1.5% (w/v) BSA. The glass fiber was dealt with 0.2% Tween-20 for 12 h and dried for 1 h at 37 1C before use. The colloidal gold probe was then added to the conjugate pad, dried at 37 1C for 1 h, and then stored in a desiccator at 4 1C for future use. The diluted pAb and goat anti-mouse IgG (1 mg/ml) were transferred onto the NC membrane with a volume of 1 μl/cm to form the T and C lines, respectively. The NC membrane was dealt with 0.02 M PBS containing 1.0% BSA, dried for 2 h at 37 1C, and then stored in a desiccator at 4 1C for future use. Pure cellulose fiber of 100% was used as the absorbent pad. The PVC plate was used as the bottom of the test strip. The sample pad, conjugate pad, immobilized NC membrane, and absorbent pad were assembled, as described in Fig. 1. These strips were cut into 5 mm width and stored in a desiccator at 4 1C for future use.
2.4. Preparation of the colloidal gold–pAb conjugate Approximately 0.4 ml of purified optimal pAb (3.0 mg/ml) against M. suis was added to 100 ml colloidal gold solution (pH 8.5) with gentle stirring. The admixture was mixed gently for 15 min, blocked by 11.1 ml of 10% BSA solution for 60 min, and then centrifuged at 12,000 rpm for 25 min. After centrifugation, the pellet was suspended in 10 ml dilution buffer [0.002 mol/L sodium carbonate
Fig. 1. (A) The schematic description of the immunochromatographic device. (B) The illustration of immunochromatographic test results.
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After the samples (100 μl) were added dropwise to the sample pad and allowed to pass through the NC membrane, the positive result was denoted by the appearance of two red lines in the test and control regions after 10 min. The negative result was denoted by the appearance of only one red line in the control region. The appearance of only a single red line in the test region or the absence of a line in test strip was considered an invalid test (Fig. 1). 2.7. Sensitivity, specificity, and stability of the immunochromatographic strip Different concentrations of the porcine plasma samples containing M. suis (1 mg/ml, 100 mg/ml, 10 mg/ml, 1 mg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, and 0.1 ng/ml) were prepared by diluting a stock solution (2 mg/ml) with 0.9% NaCl (pH 7.2) to evaluate the sensitivity of the immunochromatographic strip. For the sensitivity assay, 100 μl of each solution of a particular dilution was used for the immunochromatographic strip test and 0.9% NaCl (pH 7.2) was used as the blank control. Other samples containing different antigens, such as swine toxoplasma, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus were also tested to evaluate the specificity of the immunochromatographic strip. All samples were diluted 1 mg/ml with 0.9% NaCl (pH 7.2), respectively. For the specificity assay, 100 μl of each solution of a particular dilution was added to the sample pad. All immunochromatographic strips were stored at 4 1C for 16 weeks to evaluate the stability of the immunochromatographic strips during storage. 1 mg/ml and 100 ng/ml of the porcine plasma samples containing M. suis and 1 mg/ml of the porcine samples containing pneumonia mycoplasma were used as samples, with 0.9% NaCl (pH 7.2) as the blank control. 2.8. Detection of clinical samples A total of 350 porcine plasma samples collected in field from different pig farms were detected using the developed test strips. The plasma samples were diluted with 0.9% NaCl (pH 7.2) before detection. These plasma samples were also detected by the ELISA kit to evaluate the accuracy of the immunochromatographic strip test.
3. Results and discussion 3.1. Design of the one-step immunochromatographic assay The porcine plasma samples were obtained by centrifuging at 5000 rpm for 10 min and then diluted with 0.9% NaCl (pH 7.2) for detection. 100 μl sample was added to the sample pad and allowed to flow through the nitrocellulose membrane. After 10 min, the result can be obtained. The positive result is judged by the appearance of two red lines in test and control region. The negative result is judged by the appearance of only a single line in the control region. If no line is present in the test strip or only one line appears in the test region, the test is invalid.
with the increase in pAb concentration. However, the color of the test line was maintained when the pAb concentration reached 1.5 mg/ml. Thus, the optimal concentration of the coating antibody was 1.5 mg/ ml. In this study, the blank control that did not contain M. suis but contained BSA at different concentrations showed false-positive results to negative results with the increase in BSA concentration because the blank control did not display false-positive results when the BSA concentrations were higher than 1%. Thus, 1% BSA was determined as the most advantageous concentration of the blocking buffer. 3.3. Sensitivity of the immunochromatographic strip To confirm the sensitivity of the immunochromatographic strip, different concentrations of the porcine plasma samples containing M. suis (1 mg/ml, 100 mg/ml, 10 mg/ml, 1 mg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, and 0.1 ng/ml) were prepared by diluting a stock solution (2 mg/ml) with 0.9% NaCl (pH 7.2). Whereas the porcine plasma samples without M. suis were used as the blank control. The results are presented in Fig. 2. Standard densitometric analysis based on scanning the strips and measuring the color intensity (gray scale) via standard image software was adopted for the sensitivity test (Fernández-Sánchez et al., 2005). The sensitivity test was repeated more than three times and the results showed that the red line on the testing region could be observed clearly when the concentrations of the porcine plasma samples containing M. suis were up to 100 ng/ml. 3.4. Specificity of the immunochromatographic strip Other porcine samples containing different pathogens, such as swine toxoplasma, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus, were used to evaluate the specificity of the immunochromatographic strip. All the porcine samples were diluted with 0.9% NaCl (pH 7.2), and 0.9% NaCl (pH 7.2) was used as the blank control. Results (Fig. 3(A)) showed that only porcine samples containing M. suis presented two red lines in test and control region and others presented only one red line in control region which indicated that the immunochromatographic strip had high specificity for detecting M. suis. In addition, no cross-reaction was observed when other porcine pathogens were tested. 3.5. Stability of the immunochromatographic strip The same batch of immunochromatographic strips was stored at 4 1C for 16 weeks to assess their stability by evaluating the sensitivity and specificity. It was seen from Fig. 3(B) that the visible red color on the testing region can been seen clearly after 16
3.2. Optimal concentration of the capture antibody, coating antibody, and blocking BSA The pAb using rMSG1 protein as immunogen was produced by the method described in reference (Chen et al., 2011; Liu et al., 2011). After 4 immunizations for 6 weeks, the antibody titers in the porcine serum reached 107. Based on the protocol in Section 2, the most advantageous concentration of the capture antibody was 12 μg/ml. For the coating antibody, the color of the test line deepened gradually
Fig. 2. Sensitivity testing of immunochromatographic strip. 1: Blank control; 2: M. suis, 1 mg/ml; 3: M. suis, 100 mg/ml; 4: M. suis, 10 mg/ml; 5: M. suis, 1 mg/ml; 6: M. suis, 100 ng/ml; 7: M. suis, 10 ng/ml; 8: M. suis, 1 ng/ml; 9: M. suis, 0.1 ng/ml.
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suis in the field and in serological surveillance using colloidal gold coupled with polyclonal antibody, and the immunochromatographic strip was evaluated in sensitivity, specificity and stability. Results of the sensitivity test indicated that the darker the color density of the test line is, the higher the concentration of the M. suis tested in the sample. However, the use of polyclonal antibody is associated with low specificity, and polyclonal antibody may react with other related antigens. In fact, the coupling of polyclonal antibody with colloidal gold particles in this study resulted in high specificity in the detection of M. suis. In subsequent research, we will verify whether the monoclonal antibody has higher specificity than polyclonal antibody.
4. Conclusion In this study, an immunochromatographic assay format of colloidal gold-labeled pAb was developed for the detection of M. suis in porcine plasmas. Although the sensitivity of the assays described in this study is lower than that of PCR and ELISA, the method presented is easy, rapid, and convenient to use and requires few equipment compared with PCR, ELISA, and other techniques. The test can also be accomplished within 10 min without complicated handling procedures; this feature is significant for clinical diagnosis. Moreover, the proposed method is superior to other immunoassays in terms of speed and simplicity. Fig. 3. (A) Specificity testing of immunochromatographic strip. 1: Blank control; 2: M. suis, 1 mg/ml; 3: swine toxoplasma, 1 mg/ml; 4: classical swine fever virus (CSFV), 1 mg/ml; 5: porcine reproductive and respiratory syndrome virus (PRRSV), 1 mg/ml; 6: porcine parvovirus (PPV), 1 mg/ml. (B). Stability testing of immunochromatographic strip by evaluating the sensitivity and specificity. 1: Blank control; 2: M. suis, 1 mg/ml; 3: M. suis, 100 ng/ml; 4: pneumonia mycoplasma, 1 mg/ml.
weeks storage at 4 1C. This demonstrated that the strips were stable for at least 16 weeks at 4 1C. 3.6. Clinical application of the developed immunochromatographic strip A total of 350 plasma samples was detected by the developed test strip and by the ELISA kit respectively. The positive rate was 43.84% in the immunochromatographic strip test and 47.25% in the ELISA. The coincidence was only 92.78% which may be caused by the polyclonal antibody (pAb) against M. suis. In future research, the monoclonal antibodies against M. suis will be applied to improve the reliability of the developed immunochromatographic strip. M. suis is an opportunistic organism that infects pigs (Messick, 2004). Therefore, serological surveillance will be useful in determining the infection status, and it has an important role in the control of M. suis in both pigs and humans. The colloidal gold-based immunochromatographic assay was affirmed to be a rapid, simple, and sensitive method for serological surveillance in the field. Colloidal gold-based immunochromatographic assay is widely used for detecting certain antigens, such as Streptococcus suis serotype 2 (Ju et al., 2010), brevetoxins (Zhou et al., 2009), cortisol (Nara et al., 2010) and botulinum neurotoxin (Klewitz et al., 2006). In this study, we developed an immunochromatographic strip for the rapid detection of M.
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