Detection of the circulating antigen 14-3-3 protein of Schistosoma japonicum by time-resolved fluoroimmunoassay in rabbits
© Qian et al; licensee BioMed Central Ltd. 2011
Received: 18 March 2011
Accepted: 28 May 2011
Published: 28 May 2011
Schistosomiasis remains a major public health concern that afflicts millions of people worldwide. Low levels of Schistosoma infection require more sensitive diagnostic methods. In this study, a time-resolved fluoroimmunoassay (TRFIA) was developed for detecting the signal transduction protein 14-3-3, a circulating antigen of Schistosoma japonicum.
The detection limit of 14-3-3-TRFIA was 0.78 ng/ml, with a linear measurement range from 0.78 to 800 ng/ml. The average intra-assay and inter-assay variability of this TRFIA was 8.9% and 12.2% respectively, and the mean recovery rate ranged from 92.1% to 115.5%. Within the first 21 days post-infection in rabbits, the positive rates of the 14-3-3-TRFIA were distinctly higher compared to ELISA. All these findings illustrate that 14-3-3-TRFIA has a higher detection efficacy and is a good early diagnostic method for active Schistosoma infection.
A sandwich TRFIA for detecting the circulating antigen 14-3-3 of S. japonicum has been developed, and has demonstrated to be a good potential diagnostic method for schistosomiasis.
Over the past 50 years, the ongoing national control program has made great progress in controlling schistosomiasis japonica in China, but this disease is still a major public health concern that afflicts millions of people in endemic areas [1, 2]. Definite diagnosis of the disease plays a key role in the control of schistosomiasis . Detection of Schistosoma circulating antigen is an effective approach to discriminate between previous exposure and current infection . Effective chemotherapy and other interventions, such as local environment alternation , livestock in pens, and health education, have dramatically reduced schistosome infections, and major infection in endemic areas remains on a low level [5–7]. If the level of circulating antigen in host serum is less than the detection limit of a diagnostic method, false-negative results will be obtained, which would result in some patients missing treatment. Selecting an abundant circulating antigen as target would be very helpful for developing a highly sensitive diagnostic method of schistosomiasis. The signal transduction protein 14-3-3 of S. japonicum is abundant in excretory-secretary extracts , soluble egg extracts  and adult worm extracts , and can be used for the diagnosis of acute and chronic S. japonicum infections . In order to further improve the detection sensitivity of 14-3-3, a sandwich time-resolved fluoroimmunoassay (TRFIA) was developed using a pair of monoclonal antibodies, and it could be shown that TRFIA has a higher sensitivity in detecting 14-3-3 antigen of S. japonicum than an enzyme-linked immunosorbent assay (ELISA).
Diethylenetriaminepentaacetate (DTPA), bovine serum albumin (BSA), Tris and Triton X-100 were purchased from Sigma (St. Louis, MO, USA). A PD-10 column and a sepharose CL-6B column were obtained from the Pharmacia Company (Chalfont St Giles, UK). Pure water was produced by Barnstead Equipment (Dubuque, Iowa, USA). Flat-bottomed 96-well polystyrene microtiter plates were purchased from Nunc International (Roskilde, Denmark). Eu-labeling reagent 1244-302, including N'-[p-isothiocyanatobenzyl]-diethylenetriamine-N1, N2, N3, N4-tetraacetic acid, was obtained from Perkin-Elmer (Waltham, Massachusetts, USA). Beta-NTA was synthesized in our laboratory. AutoDELFIA1235 (Perkin-Elmer, Waltham, Massachusetts, USA) was used to measure Eu3+ fluorescence in microtiter wells. An ELISA reader was purchased from Tecan Sunrise, Switzerland. All other reagents used were of analytical grade.
S. japonicum cercariae (Chinese strain), freshly released from infected intermediate host snails (Oncomelania hupensis), were provided by the Department of Snail Biology, Jiangsu Institute of Parasitic Diseases, China.
Twelve young Japanese rabbits, each weighing about 2.5 kg, were purchased from the Experimental Animal Facility of Nanjing General Hospital of Nanjing Military Command, China, and raised in the Department of Animal Experiment, Jiangsu Institute of Parasitic Diseases. All rabbits were randomly divided into Group A and Group B. Group A included 10 rabbits, each infected with 500 cercariae of S. japonicum by abdominal skin without any treatment. Group B included two rabbits, and was used as negative control without any infection or treatment. Serum samples were collected at 0, 7, 14, 21, 28 days post-infection from all rabbits and stored at -80°C for subsequent experiments. All rabbits were sacrificed at 42 days post-infection. Their worm burden and egg burden were measured. All experiments conformed to local government regulations and Chinese national laws on animal ethics.
McAb 5C6 was labeled according to previously published methods [14, 15]. The recombinant plasmid expressing the fusion protein of glutathione-S-transferase and signal transduction protein 14-3-3 was constructed and purified . The 14-3-3 calibrators were prepared by serially diluting purified 14-3-3 protein in assay buffer, ranging from 0 - 800 ng/ml [50 mmol/l Tris-HCl (pH 7.8), 0.9% NaC1, 0.2% BSA, 0.05% NaN3, 20 μmol/l DTPA, and 0.1% Tween-20].
After optimizing the test conditions, the following TRFIA procedure was adopted: McAb 5D1 was dissolved in coating buffer (0.05 M sodium bicarbonate buffer, pH 9.6) at a concentration of 10 μg/ml. One hundred μl of this solution was added to each well and the plate was incubated overnight at 4°C. After removing the antibody solution, the plate was blocked with 20 g/l BSA (200 μl/well) in assay buffer for 2 h at room temperature. After the blocking solution was removed, the plates were dried in a high vacuum, and then stored at -20°C in a sealed plastic bag with desiccant.
The procedure for 14-3-3-TRFIA was performed by using a two-step and non-competitive "sandwich-type" protocol. Briefly, 100 μl of calibrators or serum samples without dilution were added into the coated wells, and then the plate was incubated with continuous shaking at 37°C for 2 h. After four washes in washing solution [50 mmol/l Tris-HCl buffer (pH 7.8), containing 0.9% NaCl, 0.2% Tween-20, and 0.05% NaN3], 100 μl of Eu3+-5C6 conjugate diluted in assay buffer was added into each well. The plate was incubated at 37°C for 2 h. After six washes, 200 μl of enhancement solution was added into each well. The plate was shaken for 5 min before fluorescence reading. All analysis was performed by AutoDELFIA1235 software. Duplicates of each sample were run. The calibration curve was plotted and concentrations in unknown samples were measured using the Multicalc software program, employing a spine algorithm on logarithmically transformed data. The antigen levels in sera were expressed as response counts. Response counts of samples above 2.1 times of the negative controls were judged as positive.
After optimizing the assay conditions, the following ELISA procedure was adopted: McAb 5D1 was dissolved in coating buffer at a concentration of 5 μg/ml. One hundred μl of this solution was added to each well and the plate was incubated overnight at 4°C. After removing the antigen solution, the plate was blocked by adding 200 μl/well blocking solution [5% skimmed milk power in 0.02 M phosphate buffered saline with 0.05% Tween-20 (PBST), pH 7.2] at 37°C for 1 h. After the wells were emptied, 100 μl of serum sample without dilution or calibrator was added to per well and incubated at 37°C for 1 h. After four washes, 100 μl of horseradish peroxidase (HRP)-conjugated 5C6 solution diluted in PBST was added and incubated at 37°C for 1 h. The plate was washed as above and the reaction was visualized by adding 100 μl of TMB substrate solution (GenScript, China) to each well for 5 - 10 min in dark at room temperature. The reaction was stopped by adding 100 μl/well of stop solution (1 M H2SO4) and the absorbance of each well was measured at 450 nm, using an ELISA plate reader. Duplicates of each sample were run. The antigen levels in sera were expressed as A450 values. A450 values of samples that were above 2.1 times of the negative controls were considered as positive .
Regression analysis was used to display the linearity and correlations. Analysis of data was performed using SPSS 13.0 (Chicago, IL, USA).
for the calibration curve of this TRFIA, where y indicates values of the response counts (cps); x indicates concentration of calibrator (ng/ml).
for the calibration curve of 14-3-3-ELISA, where y indicates A450 values of the calibrators; x indicates concentration of calibrator (ng/ml) (The original data can be found in Additional file 1).
The calibration curve of 14-3-3-TRFIA showed a linear relationship over the concentration from 0.78-800 ng/ml. For 14-3-3-ELISA, the calibration curve was linear from 6.25-200 ng/ml before plateauing. All these data indicated that TRFIA has a better sensitivity and measurement range than ELISA.
The recovery rate was evaluated by comparing the measured and theoretical values. The recovery rates of 14-3-3-TRFIA and 14-3-3-ELISA were analyzed by adding recombinant 14-3-3 proteins at two different concentrations (20 and 100 ng/ml) to healthy rabbit serum from Group B. The mean recovery rate of TRFIA ranged from 92.1% to 115.5%, and the mean recovery rate of ELISA ranged from 94.1% to 105.5%.
and the applied acceptance criteria for immunoassays are % RSD < 20 . The average intra-assay variations of TRFIA and ELISA were 8.9% and 5.9%, respectively. Inter-assay variations of the two assays were determined by measuring the same two calibrators (20 and 100 ng/ml) on five independent days. The average inter-assay variation of TRFIA and ELISA were 12.2% and 7.4%, respectively.
The Eu3+-labeled McAb 5C6 and the HRP labeled McAb 5C6 were stable for at least half a year at -20°C, and the results of the two methods with same reagents were reproducible over the same period.
There was a strong positive relationship between the worm burden, egg burden and the levels of 14-3-3 protein within individual rabbits (The original data can be found in Additional file 4), and it could be also found that levels of 14-3-3 protein in rabbits infected with 1500 cercariae of S. japonicum were higher than in those infected with 500 cercariae of S. japonicum. (The original data are not shown).
These results proved that the detection limit and the sensitivity of TRFIA are higher than those of ELISA, and TRFIA has stronger early diagnostic potential for schistosome infection. At present, the majority of schistosomiasis patients in epidemic areas of China have low infection levels. Circulating antigen levels are probably much lower than the detection limit of ELISA, indicating the need to develop more sensitive detection methods of schistosome circulating antigens .
TRFIA is based on the use of lanthanide chelate labels with unique fluorescence properties. Lanthanide chelate shows narrow and strong emission bands around 600 nm and an exceptionally long decay time, which allows the elimination of the high background of the fluorescent labels and increases the sensitivity and specificity of detection methods . The sensitivity is further increased due to the dissociation-enhancement principle . Eu3+-chelate is the most commonly used label in time resolved fluorometry-based analysis because of its higher fluorescence yield and lower background than other lanthanide complexes . Therefore, TRFIA has the advantages of excellent sensitivity, wide measurement range and no radioactivity. A pair of monoclonal antibodies against signal transduction protein 14-3-3 of S. japonicum with highly specificity and affinity  was chosen to develop a sandwich TRFIA for detecting 14-3-3 protein. This assay combines characteristics of immunological methods and the sensitivity of a time-resolved fluorescence revelation system.
In this experiment, circulating antigen 14-3-3 protein in sera of rabbits collected post-infection was measured by TRFIA, and positive percentages of TRFIA could reach 100% after 21 days. Thus, for early diagnosis of Schistosoma infection, this TRFIA, combined with other detection methods [22, 23], could obtain more satisfying results. The value of this TRFIA for clinical diagnosis of schistosomiasis japonica should also be further assessed.
This work is supported by the National S & T Major Program (Grant No. 2008ZX10004-011), the Preventive Medicine Project of Jiangsu Provincial Bureau of Health (Grant No. YZ201030), the Natural Science Foundation of China (Grant No. 30972581), the Special Fund for Public Interest of Science and Technology Department of Jiangsu Province (Grant No. BM2007704), the Natural Science Foundation of Jiangsu Province (Grant No. BK2008110) and Key Medical Personnel Foundation of Jiangsu Province (Grant No. RC2007095).
- Zhou XN, Guo JG, Wu XH, Jiang QW, Zheng J, Dang H, Wang XH, Xu J, Zhu HQ, Wu GL, Li YS, Xu XJ, Chen HG, Wang TP, Zhu YC, Qiu DC, Dong XQ, Zhao GM, Zhang SJ, Zhao NQ, Xia G, Wang LY, Zhang SQ, Lin DD, Chen MG, Hao Y: Epidemiology of schistosomiasis in the People's Republic of China, 2004. Emerg Infect Dis. 2007, 13: 1470-1476.PubMed CentralView ArticlePubMedGoogle Scholar
- Li SZ, Luz A, Wang XH, Xu LL, Wang Q, Qian YJ, Wu XH, Guo JG, Xia G, Wang LY, Zhou XN: Schistosomiasis in China: acute infections during 2005-2008. Chin Med J (Engl). 2009, 122: 1009-1014.Google Scholar
- Zhu YC: Immunodiagnosis and its role in schistosomiasis control in China: a review. Acta Trop. 2005, 96: 130-136. 10.1016/j.actatropica.2005.07.007.View ArticlePubMedGoogle Scholar
- Yang GJ, Vounatsou P, Zhou XN, Tanner M, Utzinger J: A potential impact of climate change and water resource development on the transmission of Schistosoma japonicum in China. Parassitologia. 2005, 47: 127-134.View ArticlePubMedGoogle Scholar
- Utzinger J, Booth M, N'Goran EK, Müller I, Tanner M, Lengeler C: Relative contribution of day-to-day and intra-specimen variation in faecal egg counts of Schistosoma mansoni within and after treatment with praziquantel. Parasitology. 2001, 122: 537-544.View ArticlePubMedGoogle Scholar
- Gryseels B, Polman K, Clerinx J, Kestens L: Human schistosomiasis. Lancet. 2006, 368: 1106-1118. 10.1016/S0140-6736(06)69440-3.View ArticlePubMedGoogle Scholar
- Zhu R, Gray DJ, Thrift AP, Williams GM, Zhang Y, Qiu DC, Zheng F, Li YS, Guo J, Zhu HQ, Wu WP, Li RS, McManus DP: A 5-year longitudinal study of schistosomiasis transmission in Shian village, the Anning River Valley, Sichuan Province, the Peoples' Republic of China. Parasit Vectors. 2011, 4: 43. 10.1186/1756-3305-4-43.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu F, Cui SJ, Hu W, Feng Z, Wang ZQ, Han ZG: Excretory/secretory proteome of the adult developmental stage of human blood fluke, Schistosoma japonicum. Mol Cell Proteomics. 2009, 8: 1236-1251. 10.1074/mcp.M800538-MCP200.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang LL, Lv ZY, Hu SM, He SJ, Li ZY, Zhang SM, Zheng HQ, Li MT, Yu XB, Fung MC, Wu ZD: Schistosoma japonicum: proteomics analysis of differentially expressed proteins from ultraviolet-attenuated cercariae compared to normal cercariae. Parasitol Res. 2009, 105: 237-248. 10.1007/s00436-009-1387-z.View ArticlePubMedGoogle Scholar
- Yu CX, Zhao F, Yin XR, Xiao D, Zhang JZ, Hua WQ, Qian CY, Song LJ, Wang J: Proteomics analysis of vomit and excretory/secretory products of Schistosoma japonicum. Chin J Schisto Control. 2010, 22: 304-309. (in Chinese)Google Scholar
- Luo QL, Qiao ZP, Zhou YD, Li XY, Zhong ZR, Yu YJ, Zhang SH, Liu M, Zheng MJ, Bian MH, Shen JL: Application of signaling protein 14-3-3 and 26 kDa glutathione-S-transferase to serological diagnosis of Schistosomiasis japonica. Acta Trop. 2009, 112: 91-96. 10.1016/j.actatropica.2009.06.011.View ArticlePubMedGoogle Scholar
- Qian CY, Song LJ, Hua WQ, Yin XR, Wang J, Zhang W, Xu YL, Yu CX: Kinetics of circulating antigen 14-3-3 in sera of rabbits firstly infected with Schistosoma japonicum and treated with/without praziquantel. Parasitol Res. 2011, 108: 493-495. 10.1007/s00436-010-2112-7.View ArticlePubMedGoogle Scholar
- Qian CY, Yu CX, Song LJ, Yin XR, Wang J, Xu YL, Zhang W: Characterization and preliminary application of six monoclonal antibodies against recombinant signal protein 14-3-3 of Schistosoma japonicum. Chin J Schisto Control. 2011, 23: 65-70. (in Chinese)Google Scholar
- Zhang J, Guo JZ, Xiao HL, Zhu L, Liu HY, Zhang Y, Huang B: Simultaneous detection of different serum pepsinogens and its primary application. World J Gastroenterol. 2010, 16: 3072-3077. 10.3748/wjg.v16.i24.3072.PubMed CentralView ArticlePubMedGoogle Scholar
- Andoh T, Nagasawa H: Development of a time-resolved fluoroimmunoassay for insulins and its application to monitoring of insulin secretion induced by feeding in the barfin flounder, Verasper moseri. Gen Comp Endocrinol. 2002, 125: 365-374. 10.1006/gcen.2001.7760.View ArticlePubMedGoogle Scholar
- Yin XR, Yu CX, Qian CY, Wang J, Song LJ, Xu YL, Zhang W: Expression and Characteristics of Schistosoma japonicum signaling protein 14-3-3. Chin J Schisto Control. 2011, 23: 168-172. (in Chinese)Google Scholar
- Zhu C, Tian H, Lu L, Li Y, Feng Z, Ma L, Guan X: A study on producing monoclonal antibody with one diagnostic marker screened electrophoretically from the urine of individuals infected with Schistosoma japonicum. Diagn Microbiol Infect Dis. 2000, 38: 237-241. 10.1016/S0732-8893(00)00207-8.View ArticlePubMedGoogle Scholar
- Findlay JW, Smith WC, Lee JW, Nordblom GD, Das I, DeSilva BS, Khan MN, Bowsher RR: Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective. J Pharm Biomed Anal. 2000, 21: 1249-1273. 10.1016/S0731-7085(99)00244-7.View ArticlePubMedGoogle Scholar
- Guan X, Shi Y: Collaborative study on evaluation of immunodiagnostic assays in schistosomiasis japonica by treatment efficacy assessment. Collaboration Group. Chin Med J (Engl). 1996, 109: 659-664.Google Scholar
- Bacigalupo MA, Meroni G, Secundo F, Lelli R: Time-resolved fluoroimmunoassay for quantitative determination of ampicillin in cow milk samples with different fat contents. Talanta. 2008, 77: 126-30. 10.1016/j.talanta.2008.05.057.View ArticlePubMedGoogle Scholar
- Huang B, Xiao H, Zhang X, Zhu L, Liu H, Jin J: Ultrasensitive detection of pepsinogen I and pepsinogen II by a time-resolved fluoroimmunoassay and its preliminary clinical applications. Anal Chim Acta. 2006, 571: 74-78. 10.1016/j.aca.2006.04.053.View ArticlePubMedGoogle Scholar
- Xu J, Feng T, Lin DD, Wang QZ, Tang L, Wu XH, Guo JG, Peeling RW, Zhou XN: Performance of a dipstick dye immunoassay for rapid screening of Schistosoma japonicum infection in areas of low endemicity. Parasit Vectors. 2011, 4: 87. 10.1186/1756-3305-4-87.PubMed CentralView ArticlePubMedGoogle Scholar
- Lei JH, Liu WQ, Sun CS, Tang CL, Li MJ, Chen YL, Li YL: Detection of circulating antigen in serum of mice infected with Schistosoma japonicum by immunomagnetic bead ELISA based on IgY. Acta Trop. 2009, 111: 39-43. 10.1016/j.actatropica.2009.02.012.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.