Detection of IgG in sera of patients with schistosomiasis japonica by developing magnetic affinity enzyme-linked immunoassay based on recombinant 14-3-3 protein
ARTICLE
Trans R Soc Trop Med Hyg doi:10.1093/trstmh/trt097
Qin Yua, Hai Yangb, Fei Guana, Youmei Fenga, Xiangliang Yangb and Yanhong Zhub,* a
*Corresponding author: Tel: +86 27 87792147; Fax: +86 27 87792234; E-mail: [email protected]
Received 19 May 2013; revised 25 August 2013; accepted 8 October 2013 Background: Low intensity of Schistosoma infection is the current status in China after long time treatment with praziquantel, therefore more sensitive diagnostic methods are required now. In this study, a magnetic affinity enzyme-linked immunoassay (MEIA) based on the signal transduction protein 14-3-3 of Schistosoma japonicum (Sj14-3-3), was developed for detecting schistosomiasis. Methods: Sera of infected BALB/c mice were collected and analyzed with MEIA and ELISA. Both MEIA and ELISA based on Sj14-3-3 were further used to detect serum IgG in patients. Sera from 58 schistosomiasis-related patients with low-intensity infection, and 30 non-endemic negative controls, were collected to assess the assay. Six sera from paragonimiasis patients were used to analysis cross-reactions. Results: Compared with ELISA, MEIA has a higher ratio of the mean positive value to the mean negative value (P/N) at the same dilution ratio in infected mice (3.71 vs 2.45). Similar results were observed in humans, higher P/N of MEIA compared to ELISA (3.57 vs 2.68). There was no cross-reaction with the sera of paragonimiasis patients detected by both MEIA and ELISA. Conclusions: Our studies suggested that MEIA based on recombinant Sj14-3-3 protein (rSj14-3-3) had the potential for the diagnosis of schistosomiasis. Keywords: Schistosoma japonicum, rSj14-3-3, Diagnosis, Magnetic affinity enzyme-linked immunoassay, MEIA
Introduction Schistosomiasis remains a global public health problem; in total, 78 countries and territories have been listed as endemic.1 In China, approximately 240 million people are at risk of infection with schistosomiasis japonicum and 413 000 people are actively infected.2 Significant reduction in indices of infection and morbidity was achieved with large-scale treatment with praziquantel.1,3 Therefore, diagnosis of schistosomiasis in low endemicity areas is facing a new challenge.4 Traditional diagnostic assays, parasitological methods (Kato-Katz thick smears and urine filtration), often miss light intensity infections. They are insensitive, particularly in areas of low endemicity, and labor-intensive.5 Development of improved assays, especially for the purpose of monitoring the low-infected populations, is needed. Magnetic beads have been widely used for immunoassay, cell separation and tissue typing.6 The magnetic bead-based immunoassay can increase the binding surface area for immobilization
of antigen or antibody, reduce the incubation time, increase sensitivity and make manipulation easy.7 In our previous work, magnetic bead-based immunoassay against soluble egg antigen (SEA) for Schistosoma japonicum antibody detection was developed. Higher sensitivity of magnetic affinity enzyme-linked immunoassay (MEIA)-SEA than ELISA-SEA was detected.8 However, there is a cross-reactivity problem when crude extracts are used as diagnostic antigens. A defined diagnostic antigen, which can increase sensitivity and specificity of a serological assay, is required. The signal transduction protein 14-3-3 of S. japonicum (Sj14-3-3) is abundant in excretory secretary extracts, soluble egg extracts and adult worm extracts. Luo et al. reported that 14-3-3 of S. japonicum can be used for the diagnosis of acute and chronic S. japonicum infections.9 Sj14-3-3 can induce the host to produce high titer antibody, while no cross-reactivity was found in Schistosoma mansoni and Schistosoma haematobium.10 Therefore, Sj14-3-3 may be a potent protein for diagnosis of schistosomiasis japonicum.
# The Author 2013. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: [email protected].
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Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China; bCollege of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P.R. China
Q. Yu et al.
In this work, MEIA based on recombinant Sj14-3-3 (rSj14-3-3) was first used for diagnosis of schistosomiasis japonicum and comparison was made with the ELISA method.
Materials and methods Strains and plasmids The recombinant vector pET28a containing coding sequence of Sj14-3-3 was constructed and transformed in Escherichia coli BL21 (DE3). Strains of E. coli BL21 (DE3) and expression vector pET28a were stored at 2208C in our laboratory.
washed with phosphate buffer saline (PBS; 0.01M, pH 7.4). rSj14-3-3 was added into the activated magnetic beads at 378C for 2 h while rotating. Bovine serum albumin (BSA) was used to block the residual binding sites on the beads. Finally, the beads were washed with Tris-buffer containing 0.05% Tween-20 (TBST pH7.4), resuspended in 0.1M TBS (pH 7.4, with 0.1% BSA and 0.01% sodium azide), and stored at 48C until use.
Development of MEIA method
Human serum samples
ELISA method
Fifty-eight serum samples were collected from individuals characterized with an egg count of less than 100 eggs per gram (epg) in the feces, which were identified as low-intensity infection of S. japonicum following WHO recommendations (1993).11 All patients had received the repeated praziquantel chemotherapy at the endemic regions. Thirty adult healthy serum samples collected from non-endemic areas were used as negative sera. Cross-reactions were assayed with six serum samples which were infected with paragonimiasis confirmed by parasitological and serological diagnosis. Informed consent was obtained from all participants before this part of the study was conducted.
ELISA plates were coated with purified rSj14-3-3 protein. Wells were washed and blocked with 300 mL blocking buffer (TBST and BSA [1.5%], pH 7.4) at 378C for 1.5 h. Serum samples were diluted in 0.1M TBS (pH 7.4; 1:100 for human serum, 1:20 for mouse serum), added to the coated plates (100 mL/well) in duplicate. Serum samples were allowed to bind for 1 h at 258C. After washing, AP conjugated goat anti- mouse/human IgG was added and incubated at 258C for 1 h. After a final wash, plates were detected with PMP substrates, and then stopped with 0.05 mol/L Na2CO3 after 30 min. The plates were read at 550 nm with an ELISA reader (PerkinElmer, Waltham, MA, USA).
Expression and purification of recombinant Sj14-3-3
Data analysis
The recombinant vector pET28a containing coding sequence of Sj14-3-3 was expressed in E. coli BL21 (DE3). The recombinant proteins were purified by affinity of nichel nitrilotriacetic resin with His6-tag, eluted in an imidazole gradient, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined by BCA protein assay.
Statistical analysis was analyzed by the Student’s t-test using statistical software (SPSS 13.0; SPSS Inc., Chicago, IL, USA). A difference of p,0.05 was considered statistically significant. The cut-off values for MEIA and ELISA were 2 times of the mean absorbance of negative sera. P is the mean value of OD550 nm for positive test serum and N is the mean value of OD550 nm for negative test serum.
Mouse serum samples
Coupling rSj14-3-3 to magnetic beads Based on EDC (1-ethyl-3-[dimethylaminopropyl]-carbodiimide hydrochloride) conjugation method,12 rSj14-3-3 conjugated to carboxylated magnetic beads (particle size: 2 mm; Capital Bio Corporation, Beijing, China). Briefly, magnetic beads were washed three times with 0.1M pH 6.0 MES buffer (2-Morpholineethanesulfonic acid hydrate, Sigma, USA). EDC (Sigma, St. Louis, MO, USA) and NHS (N-hydroxysuccinimide; Sigma, USA) were added. The mixture was rotated at room temperature for 15 min and then
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Figure 1. Detection of the immunospecificity of sera from infected mice with rSj14-3-3 by western blot. Lane 1: mixed normal mouse serum; Lane 2: mixed mouse sera 6 weeks post-infection.
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Animal research was performed in compliance with the ‘Regulations for the Administration of Affairs Concerning Experimental Animals in Hubei Province’ prepared by the China Hubei Provincial Science and Technology Department in 2005. Eight 6-week old mice were provided by the Experimental Animal Facility of Tongji Medical College, China. Forty S. japonicum cercariae were used to challenge each anesthetized male BALB/c mouse percutaneously. Mouse sera collected prior to infection were used as normal sera. The serum samples were collected prior and 6 weeks post infection and stored at 2208C until use.
The mixture of 60 mL magnetic beads (0.2 mg of magnetic beads coated with 10 mg rSj14-3-3) and 30 mL of diluted serum sample (1:100 for human serum, 1:20 for mouse serum) was incubated at 378C for 20 min while shaking. The desired product was collected by magnetic separation and washing to remove unbound antibody with TBST. Subsequently, the second antibody of goat anti-mouse/human IgG coupled with alkaline phosphatase (AP) (Proteintech, Wuhan, Hubei, China) was added and rotated at 378C for 20 min. After washing, 100 mL substrates of phenolphthalein monophosphate (PMP) were added and rotated at 378C. The reaction was stopped after 20 min by adding 300 mL of stopping reagent (0.05 mol/L Na2CO3). The absorbance at 550 nm was measured with a Serozyme I instrument (Merck Serono, Geneva, Switzerland). Test of each serum sample was repeated three times.
Transactions of the Royal Society of Tropical Medicine and Hygiene
Results Identification of rSj14-3-3 antigenicity with western blot The immunospecificity of sera from infected mice was tested by western blotting with rSj14-3-3. rSj14-3-3 was recognized clearly and significantly by the sera from infected but not uninfected mice (Figure 1). This result indicated a specific antibody response against rSj14-3-3.
To further evaluate MEIA based on rSj14-3-3, the human sera with low-intensity infection S. japonicum were used for detecting antibody IgG. The results showed that MEIA had higher positive detection rates than ELISA (13/58 vs 10/58), and the P/N of MEIA was higher than that of ELISA at the same dilution ratio (3.57 vs 2.68; Figure 3). In addition, when using Pearson’s correlation in associating MEIA with ELISA based on rSj14-3-3, a significant correlation existed between the two assays (r¼0.618, p,0.01) (Figure 4).
Cross-reactions
To determine whether MEIA based on rSj14-3-3 could be a suitable assay for diagnosis of schistosomiasis japonica, specific IgG antibody in mouse sera infected by S. japonicum was used. The results showed that all the infected sera produced specific anti-rSj14-3-3 antibodies. From Figure 2, all the positive sera from the mice infected by S. japonicum were effectively detected by MEIA as well as by ELISA. However, the ratio of the mean positive value to the mean negative value (P/N) of MEIA was higher than that of ELISA at the same dilution ratio (3.71 vs 2.45).
No crossing-reactivity in the sera from the six patients with paragonimiasis by both MEIA and ELISA based on rSj14-3-3 were detected. Antibodies in all the sera were negative by these two methods.
Figure 2. Detection of anti-Schistsoma japonicum IgG using magnetic affinity enzyme-linked immunoassay (MEIA; top) and ELISA (bottom) based on rSj14-3-3 in eight mice sera infected by Schistsoma japonicum. Horizontal lines represent the mean value.
Figure 3. Detection of anti-Schistsoma japonicum IgG using magnetic affinity enzyme-linked immunoassay (MEIA; top) and ELISA (bottom) based on rSj14-3-3 in sera of 58 patients with low-intensity infection. Dotted line represents the cut-off value.
Discussion Both the prevalence and the severity of schistosomiasis japonicum have decreased significantly after decades of successful
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Assessment of MEIA in detection of antibody IgG in mouse sera infected by S. japonicum
Evaluation of MEIA in detection of antibody IgG in human sera with low-intensity infection S. japonicum
Q. Yu et al.
national schistosomiasis control programs, but the disease is far from eradicated.13 In low-transmission areas, there will be mostly light infections for which parasitological diagnostic techniques will likely be insensitive.14,15 Related to the loss of ‘gold standards’ in schistosomiasis diagnosis in low-endemicity areas, there is a need to confirm a sensitive diagnostic method for the cases, and give selective treatment in low prevalence areas.16,17 Highly sensitive and specific diagnosis of schistosomiasis in human is especially required in schistosome-endemic areas.18 Molecular and immunological diagnostic techniques have the merit of high sensitivity, and are promising strategies that are complementary to traditional parasitological detection. ELISA, the indirect hemagglutination assay (IHA), the circumoval precipitin test (COPT) and dye dipstick immunoassay (DDIA), commonly use complex crude antigens such as SEA and soluble adult worm antigen (SWA), which have been shown to cross-react with other parasite diseases.4,19 PCR-based methods have shown superior accuracy, sensitivity and specificity in areas with a low intensity of infection. Nonetheless, PCR remains an expensive method, although reduced costs can be obtained by methodological modifications, such as the improvement of DNA extraction methods.4 More easy, economic and accurate methods need to be developed for schistosomiasis diagnosis in endemic areas. MEIA based on rSj14-3-3 was used for diagnosis of schistosomiasis in this study. The rSj14-3-3 purified from soluble fractions of E. coli extracts was used as a diagnostic reagent for detection of antibodies against S. japonicum. MEIA based on rSj14-3-3 was applied for the detection of anti-Schistosoma antibodies in mouse sera infected by S. japonicum. The results showed that the MEIA assay had higher precision than ELISA against rSj14-3-3, although the two assays were similar in positive detection rates. The immunodiagnostic potential of MEIA based on rSj14-3-3 on human schistosomiasis was further studied. MEIA based on rSj14-3-3 was used for diagnosis of patients with repeated chemotherapy and re-infection. The results of the tests showed MEIA has the higher P/N value compared to ELISA. In this study, all patients with the intensity of infection were below 100 epg feces and had received the repeated praziquantel chemotherapy at the endemic regions. A single Kato slide may be inadequate to detect these patients. WHO had reported that when the prevalence and intensities of infection were low, miracidial hatching might be useful for detection of Schistosoma
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Authors’ contributions: Qin Yu and Hai Yang contributed equally to this work. YZ conceived the study; YZ, YF and XY designed the experiments. QY carried out the experiments; FG participated in the animal experiments; HY participated in analysis and interpretation data. All authors read and approved the final manuscript. QY, HY and YZ are guarantors of the paper. Funding: This work was supported by the National Natural Science Foundation of China [No. 30800960] and National Basic Research Program of China [2012CB932500 and 2011CB933103]. Competing interests: None declared.
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Figure 4. Correlation between optical density (OD) values of sera analyzed by magnetic affinity enzyme-linked immunoassay (MEIA) and ELISA based on rSj14-3-3 from 58 low-intensity infection individuals.
infection.20 The results are not consistent at positive patients among the epg and MEIA, but results between MEIA and ELISA are similar. Compared to ELISA, MEIA is more rapid (1–2 h) and easy to perform without sophisticated equipment. Recombinant protein as the diagnosis antigen may not be as sensitive as SEA. Acosta et al. used a panel of recombinant antigens such as 97-kDa paramyosin, 14-kDa FABP, 28-kDa GST and 22kDa TEG to detect antibodies in patients with repeated treatment and re-infection from Schistosoma endemic areas, and the results showed that IgG responses to all recombinant antigens tended to be low.21 Qian found that 60% of all infected rabbits had positive reaction with 14-3-3 protein.22 When compared with IgG levels against SEA, most of them against 14-3-3 protein were much lower. Consistent with these reports, IgG responses to rSj14-3-3 appeared to be low in this study. Maybe single recombinant antigen has lower antibody response than SEA. When Sj14-3-3 and Sj26 kDa GST combined as antigens, the sensitivity in diagnoses of both acute and chronic schistosomiasis was 94% (67/71) and 80.7% (96/119).9 Antibody production is not merely caused by the number of eggs released by the schistosome parasite, but also by the host’s ability to produce antibody against certain epitopes of the antigen.23 Studies on the different antigens for diagnosis still need to be performed to develop more sensitive and specific assays. SEA as diagnosis antigen always has the higher sensitivity, but cross-reactivity is an issue in serological assay.24 To avoid the cross-reactions, recombinant antigens were used in serologic diagnostic tests.13,25 In this study, specific antibody against S. japonicum in patients with paragonimiasis was detected. MEIA based on rSj14-3-3 showed no cross reaction with sera from paragonimiasis patients. In conclusion, MEIA based on rSj14-3-3 for detecting anti-S. japonicum antibody had a good diagnostic consistent with the results from ELISA. But MEIA has the higher detection rate, higher P/N value, and the properties of simple and rapid operation. A weakness of this study was the fact that we were unable to evaluate the discriminant capacity of the MEIA in order to validate them. Another weakness of the assay was only one diagnostic antigen used to evaluate schistosomiasis, which caused the low sensitivity of detection. Several Schistosoma specific antigens combined as diagnostic antigens may have higher sensitivity. Further studies in different patient populations, are necessary to confirm this assay. However, use of MEIA based on rSj14-3-3 may provide a new alternative method of diagnosis and more reliable results for diagnosis of schistosomiasis.
Transactions of the Royal Society of Tropical Medicine and Hygiene
Ethical approval: Approved by Medical Ethics Committee of Tongji Hospital, Tongji medical college, Huazhong University of Science and Technology, China.
References
13 Jin Y, Lu K, Zhou W et al. Comparison of recombinant proteins from Schistosoma japonicum for schistosomiasis diagnosis. Clin Vacc Immunol 2010;17:476–80. 14 Bergquist R, Johansen MV, Utzinger J. Diagnostic dilemmas in helminthology: what tools to use and when? Trends Parasitol 2009;25:151–6. 15 Doenhoff MJ, Chiodini PL, Hamilton JV. Specific and sensitive diagnosis of schistosome infection: can it be done with antibodies? Trends Parasitol 2004;20:35–9.
2 Hao Y, Zheng H, Zhu R et al. Schistosomiasis status in People’s Republic of China in 2008. Chin J Schisto Control 2009;21:451–6.
16 Gray DJ, McManus DP, Li Y et al. Schistosomiasis elimination: lessons from the past guide the future. Lancet Infect Dis 2010;10:733–6.
3 Zhou X, Guo J, Wu X et al. Epidemiology of schistosomiasis in the People’s Republic of China, 2004. Emerg Infect Dis 2007;13:1470–6.
17 Wu W, Huang Y. Application of praziquantel in schistosomiasis japonica control strategies in China. Parasitol Res 2013;112:909–15.
4 Cavalcanti MG, Silva LF, Peralta RHS et al. Schistosomiasis in areas of low endemicity: a new era in diagnosis. Trends Parasitol 2013;29:75–82.
18 Ross AG, Sleigh AC, Li Y et al. Schistosomiasis in the People’s Republic of China: prospects and challenges for the 21st century. Clin Microbiol Rev 2001;214:270–95.
5 Coulibaly JT, N’Goran EK, Utzinger J et al. A new rapid diagnostic test for detection of anti-Schistosoma mansoni and anti-Schistosoma haematobium antibodies. Parasit Vectors 2013;6:29–37. 6 Olsvik O, Popovic T, Skjerve E et al. Magnetic separation techniques in diagnostic microbiology. Clin Microbiol Rev 1994;7:43–54. 7 Hayat A, Barthelmebs L, Marty JL. Enzyme-linked immunosensor based on superparamagnetic nanobeads for easy and rapid detection of okadaic acid. Anal Chim Acta 2011;690:248–52. 8 Liu Z, Zhang L, Yang H et al. Magnetic microbead-based enzyme-linked immunoassay for detection of Schistosoma japonicum antibody in human serum. Anal Biochem 2010;404:127–34. 9 Luo Q, Qiao Z, Zhou Y et al. Application of signaling protein 14-3-3 and 26 kDa glutathione-S-transferase to serological diagnosis of Schistosomiasis japonica. Acta Trop 2009;112:91–6.
19 Zhu Y, Hua W, Xu M et al. A novel immunodiagnostic assay to detect serum antibody response against selected soluble egg antigen fractions from Schistosoma japonicum. PLOS One 2012;7:1–6. 20 WHO. Control of Schistosomiasis. Geneva: World Health Organization; 1985. 21 Acosta LP, McManus DP, Aligui GD et al. Antigen-specific antibody isotype patterns to Schistsoma japonicum recombinant and native antigens in a defined population in Leyte, the Philippines. Am J Trop Med Hyg 2004;70:549–55. 22 Qian C, Wang J, Yu C et al. Characterization of IgG responses of rabbits to Sj14-3-3 protein after experimental infection with Schistosoma japonicum. Parasitol Res 2012;111:2209–11.
10 Schechtmna D, Tarrab-Hazdai R, Arnon R. The 14-3-3 protein as a vaccine candidate against schistosomiasis. Parasite Immunol 2001; 23:213–7.
23 Angeles JM, Goto Y, Kirinoki M et al. Human antibody response to thioredoxin peroxidase-1 and tandem repeat proteins as immunodiagnostic antigen candidates for Schistosoma japonicum infection. Am J Trop Med Hyg 2011;85:674–9.
11 WHO. The Control of Schistosomiasis. Geneva: World Health Organization; 1993.
24 Tsang VC, Wilkins PP. Immunodiagnosis of schistosomiasis. Immunol Invest 1997;26:175–88.
12 Santana SD, Pina AS, Roque AC. Immobilization of enterokinase on magnetic supports for the cleavage of fusion proteins. J Biotechnol 2012;161:378–82.
25 Abdel-Hafeez EH, Kikuchi M, Watanabe K et al. Proteome approach for identification of schistosomiasis japonica vaccine candidate antigen. Parasitol Int 2009;58:36–44.
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1 WHO. Schistosomiasis: Progress Report 2001–2011 and Strategic Plan 2012–2020. Geneva: World Health Organization; 2013.
ARTICLE
Trans R Soc Trop Med Hyg doi:10.1093/trstmh/trt097
Qin Yua, Hai Yangb, Fei Guana, Youmei Fenga, Xiangliang Yangb and Yanhong Zhub,* a
*Corresponding author: Tel: +86 27 87792147; Fax: +86 27 87792234; E-mail: [email protected]
Received 19 May 2013; revised 25 August 2013; accepted 8 October 2013 Background: Low intensity of Schistosoma infection is the current status in China after long time treatment with praziquantel, therefore more sensitive diagnostic methods are required now. In this study, a magnetic affinity enzyme-linked immunoassay (MEIA) based on the signal transduction protein 14-3-3 of Schistosoma japonicum (Sj14-3-3), was developed for detecting schistosomiasis. Methods: Sera of infected BALB/c mice were collected and analyzed with MEIA and ELISA. Both MEIA and ELISA based on Sj14-3-3 were further used to detect serum IgG in patients. Sera from 58 schistosomiasis-related patients with low-intensity infection, and 30 non-endemic negative controls, were collected to assess the assay. Six sera from paragonimiasis patients were used to analysis cross-reactions. Results: Compared with ELISA, MEIA has a higher ratio of the mean positive value to the mean negative value (P/N) at the same dilution ratio in infected mice (3.71 vs 2.45). Similar results were observed in humans, higher P/N of MEIA compared to ELISA (3.57 vs 2.68). There was no cross-reaction with the sera of paragonimiasis patients detected by both MEIA and ELISA. Conclusions: Our studies suggested that MEIA based on recombinant Sj14-3-3 protein (rSj14-3-3) had the potential for the diagnosis of schistosomiasis. Keywords: Schistosoma japonicum, rSj14-3-3, Diagnosis, Magnetic affinity enzyme-linked immunoassay, MEIA
Introduction Schistosomiasis remains a global public health problem; in total, 78 countries and territories have been listed as endemic.1 In China, approximately 240 million people are at risk of infection with schistosomiasis japonicum and 413 000 people are actively infected.2 Significant reduction in indices of infection and morbidity was achieved with large-scale treatment with praziquantel.1,3 Therefore, diagnosis of schistosomiasis in low endemicity areas is facing a new challenge.4 Traditional diagnostic assays, parasitological methods (Kato-Katz thick smears and urine filtration), often miss light intensity infections. They are insensitive, particularly in areas of low endemicity, and labor-intensive.5 Development of improved assays, especially for the purpose of monitoring the low-infected populations, is needed. Magnetic beads have been widely used for immunoassay, cell separation and tissue typing.6 The magnetic bead-based immunoassay can increase the binding surface area for immobilization
of antigen or antibody, reduce the incubation time, increase sensitivity and make manipulation easy.7 In our previous work, magnetic bead-based immunoassay against soluble egg antigen (SEA) for Schistosoma japonicum antibody detection was developed. Higher sensitivity of magnetic affinity enzyme-linked immunoassay (MEIA)-SEA than ELISA-SEA was detected.8 However, there is a cross-reactivity problem when crude extracts are used as diagnostic antigens. A defined diagnostic antigen, which can increase sensitivity and specificity of a serological assay, is required. The signal transduction protein 14-3-3 of S. japonicum (Sj14-3-3) is abundant in excretory secretary extracts, soluble egg extracts and adult worm extracts. Luo et al. reported that 14-3-3 of S. japonicum can be used for the diagnosis of acute and chronic S. japonicum infections.9 Sj14-3-3 can induce the host to produce high titer antibody, while no cross-reactivity was found in Schistosoma mansoni and Schistosoma haematobium.10 Therefore, Sj14-3-3 may be a potent protein for diagnosis of schistosomiasis japonicum.
# The Author 2013. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: [email protected].
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Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China; bCollege of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P.R. China
Q. Yu et al.
In this work, MEIA based on recombinant Sj14-3-3 (rSj14-3-3) was first used for diagnosis of schistosomiasis japonicum and comparison was made with the ELISA method.
Materials and methods Strains and plasmids The recombinant vector pET28a containing coding sequence of Sj14-3-3 was constructed and transformed in Escherichia coli BL21 (DE3). Strains of E. coli BL21 (DE3) and expression vector pET28a were stored at 2208C in our laboratory.
washed with phosphate buffer saline (PBS; 0.01M, pH 7.4). rSj14-3-3 was added into the activated magnetic beads at 378C for 2 h while rotating. Bovine serum albumin (BSA) was used to block the residual binding sites on the beads. Finally, the beads were washed with Tris-buffer containing 0.05% Tween-20 (TBST pH7.4), resuspended in 0.1M TBS (pH 7.4, with 0.1% BSA and 0.01% sodium azide), and stored at 48C until use.
Development of MEIA method
Human serum samples
ELISA method
Fifty-eight serum samples were collected from individuals characterized with an egg count of less than 100 eggs per gram (epg) in the feces, which were identified as low-intensity infection of S. japonicum following WHO recommendations (1993).11 All patients had received the repeated praziquantel chemotherapy at the endemic regions. Thirty adult healthy serum samples collected from non-endemic areas were used as negative sera. Cross-reactions were assayed with six serum samples which were infected with paragonimiasis confirmed by parasitological and serological diagnosis. Informed consent was obtained from all participants before this part of the study was conducted.
ELISA plates were coated with purified rSj14-3-3 protein. Wells were washed and blocked with 300 mL blocking buffer (TBST and BSA [1.5%], pH 7.4) at 378C for 1.5 h. Serum samples were diluted in 0.1M TBS (pH 7.4; 1:100 for human serum, 1:20 for mouse serum), added to the coated plates (100 mL/well) in duplicate. Serum samples were allowed to bind for 1 h at 258C. After washing, AP conjugated goat anti- mouse/human IgG was added and incubated at 258C for 1 h. After a final wash, plates were detected with PMP substrates, and then stopped with 0.05 mol/L Na2CO3 after 30 min. The plates were read at 550 nm with an ELISA reader (PerkinElmer, Waltham, MA, USA).
Expression and purification of recombinant Sj14-3-3
Data analysis
The recombinant vector pET28a containing coding sequence of Sj14-3-3 was expressed in E. coli BL21 (DE3). The recombinant proteins were purified by affinity of nichel nitrilotriacetic resin with His6-tag, eluted in an imidazole gradient, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined by BCA protein assay.
Statistical analysis was analyzed by the Student’s t-test using statistical software (SPSS 13.0; SPSS Inc., Chicago, IL, USA). A difference of p,0.05 was considered statistically significant. The cut-off values for MEIA and ELISA were 2 times of the mean absorbance of negative sera. P is the mean value of OD550 nm for positive test serum and N is the mean value of OD550 nm for negative test serum.
Mouse serum samples
Coupling rSj14-3-3 to magnetic beads Based on EDC (1-ethyl-3-[dimethylaminopropyl]-carbodiimide hydrochloride) conjugation method,12 rSj14-3-3 conjugated to carboxylated magnetic beads (particle size: 2 mm; Capital Bio Corporation, Beijing, China). Briefly, magnetic beads were washed three times with 0.1M pH 6.0 MES buffer (2-Morpholineethanesulfonic acid hydrate, Sigma, USA). EDC (Sigma, St. Louis, MO, USA) and NHS (N-hydroxysuccinimide; Sigma, USA) were added. The mixture was rotated at room temperature for 15 min and then
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Figure 1. Detection of the immunospecificity of sera from infected mice with rSj14-3-3 by western blot. Lane 1: mixed normal mouse serum; Lane 2: mixed mouse sera 6 weeks post-infection.
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Animal research was performed in compliance with the ‘Regulations for the Administration of Affairs Concerning Experimental Animals in Hubei Province’ prepared by the China Hubei Provincial Science and Technology Department in 2005. Eight 6-week old mice were provided by the Experimental Animal Facility of Tongji Medical College, China. Forty S. japonicum cercariae were used to challenge each anesthetized male BALB/c mouse percutaneously. Mouse sera collected prior to infection were used as normal sera. The serum samples were collected prior and 6 weeks post infection and stored at 2208C until use.
The mixture of 60 mL magnetic beads (0.2 mg of magnetic beads coated with 10 mg rSj14-3-3) and 30 mL of diluted serum sample (1:100 for human serum, 1:20 for mouse serum) was incubated at 378C for 20 min while shaking. The desired product was collected by magnetic separation and washing to remove unbound antibody with TBST. Subsequently, the second antibody of goat anti-mouse/human IgG coupled with alkaline phosphatase (AP) (Proteintech, Wuhan, Hubei, China) was added and rotated at 378C for 20 min. After washing, 100 mL substrates of phenolphthalein monophosphate (PMP) were added and rotated at 378C. The reaction was stopped after 20 min by adding 300 mL of stopping reagent (0.05 mol/L Na2CO3). The absorbance at 550 nm was measured with a Serozyme I instrument (Merck Serono, Geneva, Switzerland). Test of each serum sample was repeated three times.
Transactions of the Royal Society of Tropical Medicine and Hygiene
Results Identification of rSj14-3-3 antigenicity with western blot The immunospecificity of sera from infected mice was tested by western blotting with rSj14-3-3. rSj14-3-3 was recognized clearly and significantly by the sera from infected but not uninfected mice (Figure 1). This result indicated a specific antibody response against rSj14-3-3.
To further evaluate MEIA based on rSj14-3-3, the human sera with low-intensity infection S. japonicum were used for detecting antibody IgG. The results showed that MEIA had higher positive detection rates than ELISA (13/58 vs 10/58), and the P/N of MEIA was higher than that of ELISA at the same dilution ratio (3.57 vs 2.68; Figure 3). In addition, when using Pearson’s correlation in associating MEIA with ELISA based on rSj14-3-3, a significant correlation existed between the two assays (r¼0.618, p,0.01) (Figure 4).
Cross-reactions
To determine whether MEIA based on rSj14-3-3 could be a suitable assay for diagnosis of schistosomiasis japonica, specific IgG antibody in mouse sera infected by S. japonicum was used. The results showed that all the infected sera produced specific anti-rSj14-3-3 antibodies. From Figure 2, all the positive sera from the mice infected by S. japonicum were effectively detected by MEIA as well as by ELISA. However, the ratio of the mean positive value to the mean negative value (P/N) of MEIA was higher than that of ELISA at the same dilution ratio (3.71 vs 2.45).
No crossing-reactivity in the sera from the six patients with paragonimiasis by both MEIA and ELISA based on rSj14-3-3 were detected. Antibodies in all the sera were negative by these two methods.
Figure 2. Detection of anti-Schistsoma japonicum IgG using magnetic affinity enzyme-linked immunoassay (MEIA; top) and ELISA (bottom) based on rSj14-3-3 in eight mice sera infected by Schistsoma japonicum. Horizontal lines represent the mean value.
Figure 3. Detection of anti-Schistsoma japonicum IgG using magnetic affinity enzyme-linked immunoassay (MEIA; top) and ELISA (bottom) based on rSj14-3-3 in sera of 58 patients with low-intensity infection. Dotted line represents the cut-off value.
Discussion Both the prevalence and the severity of schistosomiasis japonicum have decreased significantly after decades of successful
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Assessment of MEIA in detection of antibody IgG in mouse sera infected by S. japonicum
Evaluation of MEIA in detection of antibody IgG in human sera with low-intensity infection S. japonicum
Q. Yu et al.
national schistosomiasis control programs, but the disease is far from eradicated.13 In low-transmission areas, there will be mostly light infections for which parasitological diagnostic techniques will likely be insensitive.14,15 Related to the loss of ‘gold standards’ in schistosomiasis diagnosis in low-endemicity areas, there is a need to confirm a sensitive diagnostic method for the cases, and give selective treatment in low prevalence areas.16,17 Highly sensitive and specific diagnosis of schistosomiasis in human is especially required in schistosome-endemic areas.18 Molecular and immunological diagnostic techniques have the merit of high sensitivity, and are promising strategies that are complementary to traditional parasitological detection. ELISA, the indirect hemagglutination assay (IHA), the circumoval precipitin test (COPT) and dye dipstick immunoassay (DDIA), commonly use complex crude antigens such as SEA and soluble adult worm antigen (SWA), which have been shown to cross-react with other parasite diseases.4,19 PCR-based methods have shown superior accuracy, sensitivity and specificity in areas with a low intensity of infection. Nonetheless, PCR remains an expensive method, although reduced costs can be obtained by methodological modifications, such as the improvement of DNA extraction methods.4 More easy, economic and accurate methods need to be developed for schistosomiasis diagnosis in endemic areas. MEIA based on rSj14-3-3 was used for diagnosis of schistosomiasis in this study. The rSj14-3-3 purified from soluble fractions of E. coli extracts was used as a diagnostic reagent for detection of antibodies against S. japonicum. MEIA based on rSj14-3-3 was applied for the detection of anti-Schistosoma antibodies in mouse sera infected by S. japonicum. The results showed that the MEIA assay had higher precision than ELISA against rSj14-3-3, although the two assays were similar in positive detection rates. The immunodiagnostic potential of MEIA based on rSj14-3-3 on human schistosomiasis was further studied. MEIA based on rSj14-3-3 was used for diagnosis of patients with repeated chemotherapy and re-infection. The results of the tests showed MEIA has the higher P/N value compared to ELISA. In this study, all patients with the intensity of infection were below 100 epg feces and had received the repeated praziquantel chemotherapy at the endemic regions. A single Kato slide may be inadequate to detect these patients. WHO had reported that when the prevalence and intensities of infection were low, miracidial hatching might be useful for detection of Schistosoma
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Authors’ contributions: Qin Yu and Hai Yang contributed equally to this work. YZ conceived the study; YZ, YF and XY designed the experiments. QY carried out the experiments; FG participated in the animal experiments; HY participated in analysis and interpretation data. All authors read and approved the final manuscript. QY, HY and YZ are guarantors of the paper. Funding: This work was supported by the National Natural Science Foundation of China [No. 30800960] and National Basic Research Program of China [2012CB932500 and 2011CB933103]. Competing interests: None declared.
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Figure 4. Correlation between optical density (OD) values of sera analyzed by magnetic affinity enzyme-linked immunoassay (MEIA) and ELISA based on rSj14-3-3 from 58 low-intensity infection individuals.
infection.20 The results are not consistent at positive patients among the epg and MEIA, but results between MEIA and ELISA are similar. Compared to ELISA, MEIA is more rapid (1–2 h) and easy to perform without sophisticated equipment. Recombinant protein as the diagnosis antigen may not be as sensitive as SEA. Acosta et al. used a panel of recombinant antigens such as 97-kDa paramyosin, 14-kDa FABP, 28-kDa GST and 22kDa TEG to detect antibodies in patients with repeated treatment and re-infection from Schistosoma endemic areas, and the results showed that IgG responses to all recombinant antigens tended to be low.21 Qian found that 60% of all infected rabbits had positive reaction with 14-3-3 protein.22 When compared with IgG levels against SEA, most of them against 14-3-3 protein were much lower. Consistent with these reports, IgG responses to rSj14-3-3 appeared to be low in this study. Maybe single recombinant antigen has lower antibody response than SEA. When Sj14-3-3 and Sj26 kDa GST combined as antigens, the sensitivity in diagnoses of both acute and chronic schistosomiasis was 94% (67/71) and 80.7% (96/119).9 Antibody production is not merely caused by the number of eggs released by the schistosome parasite, but also by the host’s ability to produce antibody against certain epitopes of the antigen.23 Studies on the different antigens for diagnosis still need to be performed to develop more sensitive and specific assays. SEA as diagnosis antigen always has the higher sensitivity, but cross-reactivity is an issue in serological assay.24 To avoid the cross-reactions, recombinant antigens were used in serologic diagnostic tests.13,25 In this study, specific antibody against S. japonicum in patients with paragonimiasis was detected. MEIA based on rSj14-3-3 showed no cross reaction with sera from paragonimiasis patients. In conclusion, MEIA based on rSj14-3-3 for detecting anti-S. japonicum antibody had a good diagnostic consistent with the results from ELISA. But MEIA has the higher detection rate, higher P/N value, and the properties of simple and rapid operation. A weakness of this study was the fact that we were unable to evaluate the discriminant capacity of the MEIA in order to validate them. Another weakness of the assay was only one diagnostic antigen used to evaluate schistosomiasis, which caused the low sensitivity of detection. Several Schistosoma specific antigens combined as diagnostic antigens may have higher sensitivity. Further studies in different patient populations, are necessary to confirm this assay. However, use of MEIA based on rSj14-3-3 may provide a new alternative method of diagnosis and more reliable results for diagnosis of schistosomiasis.
Transactions of the Royal Society of Tropical Medicine and Hygiene
Ethical approval: Approved by Medical Ethics Committee of Tongji Hospital, Tongji medical college, Huazhong University of Science and Technology, China.
References
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