Skip to main content
Download PDF

Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA) for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool

Parasites & Vectors volume 4, Article number: 176 (2011) Cite this article

Abstract

Background

This research was carried out to develop a reliable monoclonal antibody (MoAb)-based sandwich enzyme linked immunosorbent assay (ELISA) for the diagnosis of active Fasciola gigantica infection in both serum and stool for comparative purposes.

Methods

From a panel of MoAbs raised against F. gigantica excretory/secretory antigens (ES Ags), a pair (12B/11D/3F and 10A/9D/10G) was chosen due to its high reactivity and strict specificity to F. gigantica antigen by indirect ELISA.

Results

The two MoAbs were of the IgG1 and IgG2a subclasses, respectively. Using SDS-PAGE and EITB, the selected MoAbs recognized 83, 64, 45 and 26 kDa bands of ES Ags. The lower detection limit of ELISA assay was 3 ng/ml. In stool, the sensitivity, specificity and diagnostic efficacy of ELISA was 96%, 98.2 and 97.1%; while in serum they were 94%, 94.6% and 94.3%, respectively. Moreover, a positive correlation was found between ova count in stool of F. gigantica infected patients and the OD readings of ELISA in both stool and serum samples (r = 0.730, p < 0.01 and r = 0.608; p < 0.01, respectively).

Conclusions

These data showed that the use of MoAb-based sandwich ELISA for the detection of F. gigantica coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, non-invasive technique for the diagnosis of active F. gigantica infection.

Background

Fasciola hepatica and F. gigantica are two trematode species which have an important impact on public health due to the infections they cause in humans and livestock. F. hepatica has a cosmopolitan distribution, mainly in temperate zones, while F. gigantica is found in tropical regions of Africa and Asia [1–3]. Although the majority of cases are attributed to F. hepatica, human infections with F. gigantica are also present in many countries [4–6]. In the Nile Delta of Egypt, beside the two species, a third intermediate form of Fasciola sp. has been identified [3] using molecular approaches [7].

Parasitological diagnosis of human fascioliasis is often unreliable and has low sensitivity, as parasite eggs are not found during the pre-patent period and shedding of parasitic eggs is intermittent [8–10]. Moreover, Fasciola eggs may be found in the stools of uninfected persons who have eaten raw infected liver leading to false positive diagnosis [11]. Alternatively, detection of circulating Fasciola antigen in both serum and stool was found to be more sensitive and specific [12]. The majority of methods based on antigen detection are applied to F. hepatica infection, but only few are applied to F. gigantica infection [13–15].

This research was carried out to establish a highly efficient MoAb-based sandwich ELISA to diagnose active F. gigantica infection by detecting excretory/secretory antigens (ES Ags) in both serum and stool samples of infected patients for comparative purposes.

Methods

Study Population

Patients admitted to Gastroenterology and Hepatology Department, Theodor Bilharz Research Institute (TBRI), who complained of abdominal pain, loss of body weight, dyspepsia, fever and diarrhea were subjected to parasitological stool examination on three consecutive days using merthiolate-iodine-formaldehyde concentration method [16]. The number of eggs per gram stool was determined by the modified Kato-thick smear technique [17]. Three groups were used; F. gigantica infected group where patients had the characteristic large operculated Fasciola eggs in their stool samples with no evidence of other parasitic infections (n = 50). Other parasites group (n = 60) included S. mansoni (n = 20), S. hematobium (n = 20) and Hymenolepis nana (n = 20). Control group (n = 30) were age- and sex-matched parasite-free healthy individuals.

Stool Elute Preparation and Serum Samples Collection

Aqueous elutes of a portion of each stool specimen were prepared by adding approximately 3 parts of 0.01 M phosphate-buffered saline (PBS), pH 7.2, containing 0.05% Tween 20 (PBS/T) to 1 part of stool in a centrifuge tube [18]. The mixture was homogenized and then centrifuged at 900 × g for 5 min. The supernatant was aspirated and stored at -80°C until use.

Whole blood was collected from each subject and centrifuged at 760 × g at 4°C for 10 minutes and the obtained serum samples were stored at -80°C until use.

Fasciola Excretory/Secretory (ES) Antigens

Livers of infected cattle were obtained from a local abattoir at Giza District, Egypt. Live intact F. gigantica adult worms were collected from the bile ducts and thoroughly washed at room temperature with 0.9% sodium chloride. The worms were individually incubated at 37°C in 5 ml RPMI 1640 medium, pH 7.4, supplemented with 100 U of penicillin and 100 μg of streptomycin per ml medium (Sigma Chemicals, St. Louis, USA). Following 24 h incubation, the medium was centrifuged at 1500 × g for 10 min at 4°C. The supernatants containing the ES Ags were aspirated [19] and filtered using filter paper No. 1 and 0.22 mm filter membrane (Millipore, Bedford, MA). The protein content was determined using Bio-Rad assay kit. Supernatants were stored at -20°C until use.

Development of MoAbs against ES Ags

BALB/c mice were immunized with F. gigantica ES Ags [20]. Immune splenocytes were fused with non-secreting murine myeloma cells (P3 × 63 Ag. 8) in the presence of 43% polyethylene glycol (Sigma) [21]. Hybridomas were screened for anti-Fasciola antibodies by ELISA, and highly reactive hybrids were cloned by limiting dilution using a splenocyte feeder layer. Specificity was determined by indirect ELISA [22] against S. mansoni SEA and E. granulosus antigens. MoAbs showing no-cross reactivity with other parasites were selected. Hybridoma cells were injected intraperitoneally into BALB/c mice for large-scale production of MoAbs.

Isotypic Analysis of MoAbs

Determination of MoAb isotype was done by indirect ELISA using ELISA plates coated with 20 μg/ml of F. gigantica ES Ags and a panel of goat anti-mouse peroxidase-conjugated immunoglobulins (IgM, IgG, IgG1, IgG2a, IgG2b, IgG3 and IgA, Sigma).

Characterization of Target Antigen

Sodium dodecylsulphate-polyacrilamide gel electrophoresis (SDS-PAGE) and Immunoblot was carried out as described by Laemmli [23]. F. gigantica ES antigens was fractionated on 12.5% SDS-PAGE and either stained with Commassie brilliant blue 0.05% or transferred into nitrocellulose membrane and probed with peroxidase-conjugated IgG MoAb [24, 25]. The chemical nature of MoAb-recognized epitopes was defined by testing the reactivity of target antigens by indirect ELISA before and after 20 mM sodium periodate treatment [26].

Purification and Conjugation of MoAbs

Selected MoAbs of IgG class were purified by ammonium sulfate precipitation, dialyzed against an excess of PBS and passed through a prepacked Mono-Q-HR 5/5 column (ion exchange chromatography) [27]. The antigen detecting MoAb was conjugated to horseradish peroxidase (type VI; Sigma) [28].

Detection of circulating Fasciola antigen (CFA) in both serum and stool by MoAb-based sandwich ELISA

The following sandwich-ELISA was adopted using a pair of MoAbs against ES Ags, 12B/11D/3F as antigen capturing and 10A/9D/10G as antigen detecting antibody. The optimal dilutions of MoAbs were determined by a checkerboard titration using a negative- and positive-control serum and stool samples in each plate. For each step, 100 μl/well was added unless mentioned otherwise. Polystyrene microtiter plates (Thomas Scientific, USA) were sensitized overnight at room temperature with purified 12B/11D/3F MoAb (5 μg/ml of 0.1 M carbonate buffer, pH 9.6). The plates were thoroughly washed with PBS/T (2 min/wash), and unbound sites were blocked with 200 μl/well of 2.5% fetal calf serum (Sigma) diluted in PBS/T, pH 7.4. After 2 h incubation at 37°C, the plates were emptied by suction. Undiluted serum and stool elutes were added (undiluted) and the plates were incubated for 1 h at 37°C. After thorough washing as described above, peroxidase-conjugated 10A/9D/10G MoAb (10 μg/ml of PBS/T) was added. The plates were incubated for 1 h at 37°C and washed with PBS/T. The substrate O-phenylenediamine dihydrochloride (Sigma) was added and the plates were incubated for 30 minutes in the dark at room temperature. The enzyme reaction was stopped with 50 μl/well of 8 N H2SO4. The absorbance at 492 nm wavelength (A 492 ) of the plates was read using a microplate ELISA reader (Bio-Rad, Richmond CA, USA).

Statistical analysis

Data were expressed as mean ± standard deviation. Standard diagnostic indices including sensitivity, specificity and diagnostic efficacy were calculated as described by Galen [29]. Correlations between different parameters were performed using Pearson correlation coefficient. P value greater than 0.05 was considered not significant and less than 0.01 was considered highly significant. SPSS computer program (version 12 windows) was used for data analysis.

Ethical considerations

This study was conducted on patients admitted to Theodore Bilharz Research Institute after approval of the institutional ethical committee and obtaining an informed consent from every patient.

Results

The SDS-PAGE analysis and Coomassie brilliant blue staining of F. gigantica ES Ags are shown in Figure 1. The fractionated ES Ags containing several bands ranged from 14 to 100 kDs.

Figure 1
figure 1

Fasciola ES antigens resolved from SDS-PAGE and stained by Coomassie brilliant blue 0.05%. Lane 1: High molecular weight standard. Lanes 2, 3, 4: Fasciola ES antigens from three different cultures.

Full size image

Sensitivity, Specificity and Characterization of MoAbs

From a panel of MoAbs raised against F. gigantica ES products, a pair (12B/11D/3F and 10A/9D/10G) was selected due to their high reactivity to F. gigantica antigen by indirect ELISA. Both MoAbs showed no cross-reactions with S. mansoni, S. hematobium or H. nana antigens. Isotypic analysis of the 12B/11D/3F and 10A/9D/10G MoAbs revealed that they were of IgG1 and IgG2 subclasses, respectively. The enzyme-linked immunoelectrotransfer blot (EITB) technique revealed that the two selected MoAbs recognized 83, 64, 45 and 26 kDa bands of electrophorsed ES Ag (Figure 2). The chemical nature of MoAbs-recognized epitopes was defined following 20 mM sodium periodate treatment of target antigen. Marked reduction in MoAbs' reactivity was detected (Table 1), denoting that the reactive epitopes were glycoprotein in nature.

Figure 2
figure 2

Enzyme-linked immunoelectrotransfer blot of purified Fasciola ES antigen resolved on 12.5% SDS-PAGE and probed with a pair of MoAbs against ES products. Lane 1: Low molecular weight standard. Lane 2: The 10A/9D/10G MoAb. Lane 3: The 12B/11D/3F MoAb.

Full size image
Table 1 Impact of ES antigen treatment with 20 mM sodium periodate on MoAbs' reactivity by indirect ELISA (OD = 492 nm)
Full size table

MoAb-Sandwich ELISA

From the standard curve, the lower detection limit of ELISA assay was 3 ng/ml. The cut- off value was calculated as the mean OD reading of negative controls + 2 standard deviation of the mean. The OD readings equal to or less than cut- off value were considered negative while those readings greater than the cut off value were considered positive. The specificity of the assay was determined as the sum of results of negative control group and other parasites group.

In serum, the cut-off value was 0.263 and ES Ag was detected in 47 out of 50 Fasciola-infected patients and the sensitivity of the assay was 94% while in stool elutes the cut-off value was 0.294 and 48 out of 50 Fasciola-infected patients were positive, yielding a sensitivity of 96%. All the 20 negative controls had ES Ag levels below the cut-off value in both serum and stool samples. Furthermore, 32 out of 35 serum samples and 34 out of 35 stool elutes belonging to patients harboring other parasites had undetectable ES Ag of Fasciola leading to overall specificity 94.6% (52/55) and 98.2% (54/55) in serum and stool samples, respectively. The diagnostic efficacy of the assay was 94.3% and 97.1%, respectively (Figures 3, 4).

Figure 3
figure 3

Level of Fasciola ES antigen detected in serum samples of different studied groups measured by sandwich ELISA (OD reading at 492 nm). The horizontal line represents the cut-off value of the assay (the lower detection limit). Dotted lines represent the mean OD value.

Full size image
Figure 4
figure 4

Level of Fasciola ES antigen detected in stool samples of different studied groups measured by sandwich ELISA (OD reading at 492 nm). The horizontal line represents the cut-off value of the assay (the lower detection limit). Dotted lines represent the mean OD value.

Full size image

A positive correlation was found between ova count in stools of Fasciola- infected patients and the OD readings of ELISA in both stool and serum samples (r = 0.730, p < 0.001 and r = 0.608; p < 0.001, respectively).

Discussion

In this study, a MoAb-based sandwich ELISA was employed for detection of circulating F. gigantica ES Ags in both serum and stool samples of F. gigantica infected patients. Identification of the target antigens recognized by anti-Fasciola MoAbs (12B/11D/3F and 10A/9D/10D) showed that they were glycoproteins and their MW lay in the 83, 64, 45 and 26 kDa regions. Close to our findings, Arafa et al. [30] produced monospecific antibodies against ES Ags of F. gigantica whose target antigens were recognized at 27.5, 32.5 and 55 kDa regions. They reported that cross reactivity with Schistosoma mansoni occurs at higher MW (110-120 kDa).

In the present study, our MoAbs were able to detect F. gigantica antigens level ≥ 3 ng/ml in sandwich ELISA. This could be attributed to the use of a pair of MoAbs, one as antigen capturing antibody (12B/11D/3F) which enhance the binding capacity of the other MoAb (10A/9D/10G) to the ES Ags that could help to reduce background and led to lowering the detection limit of these MoAbs. This detection limit is lower than those of Espino and Finlay [11] who detected Fasciola ES Ags in patients' stool at a concentration > 15 ng/ml. Also, Anuracpreeda et al. [31] developed a MoAb-based sandwich ELISA for detection of 28.5 kDa tegumental antigen (TA) in sera of mice experimentally infected with F. gigantica and found that the lower detection limit was 60 ng/ml for SE antigens, 16 ng/ml for whole body antigen, and 600 pg/ml for 28.5 kDa TA.

In the present study, the sensitivity and specificity of MoAb-based ELISA in serum was 94% and 95.6%, while in stool samples it was 96% and 98.2%, respectively. A few antigen detection assays have been developed for diagnosis of F. gigantica in human fluids with varied ranges of sensitivities and specificities [11, 18, 30]. The difference in specificities could be attributed to the complex composition of ES that makes this antigenic preparation not amenable to compare its performance in ELISA results reported from different groups. This may be due to the different protocols used to prepare ES [32–34] or to the variation in its composition when obtained from parasites derived from different hosts [35], which is not the case when a purified antigen is used [36].

In this study, the diagnostic efficacy of MoAb-based sandwich ELISA in stool (coproantigens) was superior to serum samples (97.1% vs 94.3%); this could be due to the fact that coproantigens offered several advantages, e.g., the levels of coproantigens are less affected by immune complex formation than circulating Fasciola antigens, coproantigens are detectable during prepatent and patent phases of infection, non-invasive and finally the nature of these antigens of being glycoprotein making the coproantigen stable under several different storage conditions which ensured its diagnostic value [11, 18, 31, 37, 38].

A positive correlation was found between ova count/gm stool of Fasciola infected patients and the OD readings of ELISA in both stool and serum samples. Other studies have demonstrated that coproantigens are correlated with Fasciola egg counts [11] and the parasite burden [31, 39, 40]. As the fecal egg count is presumably dependent on the number of flukes in the host, one can postulate that stool antigen level in patients infected with Fasciola are directly related to the number of adult parasites. Therefore, the absence of coproantigen in one of our patients may be due to a very light parasite burden and consequently undetectable levels of antigens in stools [14, 18]. On the other hand, Ubeira et al. [32] reported that there was no correlation between number of ova/gm stool and coproantigens levels measured by ELISA.

Conclusion

Our results provide evidence that detection of ES antigens in stool specimens improves and simplifies the diagnosis of F. gigantica infection in human. By using this assay, the presence of ES Ags was easily demonstrated in most of the stool specimens from patients with confirmed F. gigantica presenting a reliable, non-invasive diagnostic method for active infection.

References

  1. Torgerson P, Claxton J: Epidemiology and control. Fasciolosis. Edited by: Dalton JP. 1999, CABI Publishing, Oxon, UK, 113-149.

    Google Scholar 

  2. Mas-Coma S, Bargues M, Valero M: Fascioliasis and other plant-borne trematode zoonoses. Int J Parasitol. 2005, 35: 1255-78. 10.1016/j.ijpara.2005.07.010.

    Article  CAS  PubMed  Google Scholar 

  3. Mas-Coma S, Valero MA, Bargues MD: Chapter 2. Fasciola, lymnaeids and human fascioliasis, with a global overview on disease transmission, epidemiology, evolutionary genetics, molecular epidemiology and control. Adv Parasitol. 2009, 69: 41-146.

    Article  PubMed  Google Scholar 

  4. Ashrafi K, Valero MA, Massoud J, Sobhani A, Solaymani-Mohammadi S, Conde P, Khoubbane M, Bargues MD, Mas-Coma S: Plant-borne human contamination by fascioliasis. Am J Trop Med Hyg. 2006, 75: 295-302.

    PubMed  Google Scholar 

  5. Ashrafi K, Valero MA, Panova M, Periago MV, Massoud J, Mas-Coma S: Phenotypic analysis of adults of Fasciola hepatica, Fasciola gigantica and intermediate forms from the endemic region of Gilan. Iran Parasitol Int. 2006, 55: 249-260.

    Article  CAS  PubMed  Google Scholar 

  6. Le TH, De NV, Agatsuma T, Blair D, Vercruysse J, Dorny P, Nguyen TG, McManus DP: Molecular confirmation that Fasciola gigantica can undertake aberrant migrations in human hosts. J Clin Microbiol. 2007, 45: 648-650. 10.1128/JCM.01151-06.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Lin Ai, Chen Mu-Xin, Alasaad Samer, Elsheikha Hany, Li Juan, Li Hai-Long, Lin Rui-Qing, Zou Feng-Cai, Zhu Xing-Quan, Chen Jia-Xu: Genetic characterization, species differentiation and detection of Fasciola spp. by molecular approaches. Parasit Vectors. 2011, 4: 101-10.1186/1756-3305-4-101.

    Article  PubMed Central  PubMed  Google Scholar 

  8. Ghosh S, Rawat P, Velusamy R, Joseph D, Gupta SC, Singh BP: 27 kDa Fasciola gigantica glycoprotein for the diagnosis of prepatent fasciolosis in cattle. Vet Res Commun. 2005, 29: 123-135.

    Article  CAS  PubMed  Google Scholar 

  9. Ghosh S, Rawat P, Gupta SC, Singh BP: Comparative diagnostic potentiality of ELISA and dot-ELISA in prepatent diagnosis of experimental Fasciola gigantica infection in cattle. Indian J Exp Biol. 2005, 43: 536-541.

    CAS  PubMed  Google Scholar 

  10. Kumar N, Ghosh S, Gupta SC: Early detection of Fasciola gigantica infection in buffaloes by enzyme-linked immunosorbent assay and dot enzyme-linked immunosorbent assay. Parasitol Res. 2008, 103: 141-150. 10.1007/s00436-008-0941-4.

    Article  PubMed  Google Scholar 

  11. Espino A, Finlay C: Sandwich enzyme-liked immunosorbent assay for detection of excretory/secretory antigens in humans with fascioliasis. J Clin Microbiol. 1994, 32: 190-3.

    PubMed Central  CAS  PubMed  Google Scholar 

  12. Hillyer GV: Immunodiagnosis of human and animal fasciolosis. Fasciolosis. Edited by: Dalton JP. 1999, CABI Publishing, Oxon, U.K, 435-447.

    Google Scholar 

  13. Estuningsih ES, Widjayanti S, Adiwinata G, Piedrafita D: Detection of coproantigens by sandwich ELISA in sheep experimentally infected with Fasciola gigantica. Trop Biomed. 2004, 21 (Suppl 2): 51-56.

    Google Scholar 

  14. Mezo M, Gonzalez W, Carro C, Ubeira F: An ultra-sensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3). J Parasitol. 2004, 90: 845-52. 10.1645/GE-192R.

    Article  PubMed  Google Scholar 

  15. Mezo M, González-Warleta M, Ubeira FM: The use of MM3 monoclonal antibodies for early immunodiagnosis of ovine fascioliasis. J Parasitol. 2007, 93: 65-72. 10.1645/GE-925R.1.

    Article  CAS  PubMed  Google Scholar 

  16. Sapero JJ, Lawless DK: The MIF stain preservative technique for the identification of intestinal parasites. Am J Trop Med Hyg. 1953, 2: 613-619.

    CAS  PubMed  Google Scholar 

  17. Siongok TKA, Mahmoud AAF, Guma JH, Warren KS, Muller AS, Handa AK, Houser HB: Morbidity in schistosomiasis mansoni in relation to intensity of infection: A study of a community in Machakos, Kenya. Am J Trop Med Hyg. 1976, 25: 273-284.

    Google Scholar 

  18. Espino A, Diaz A, Perez A, Carlos M: Dynamics of antigenemia and coproantigens during a human Fasciola hepatica outbreak. J Clin Microbiol. 1998, 36: 2723-6.

    PubMed Central  CAS  PubMed  Google Scholar 

  19. Dalton JP, Heffernan M: Thiol protease released in vitro by Fasciola hepatica. Mol Biochem Parasitol. 1989, 35: 161-6. 10.1016/0166-6851(89)90118-7.

    Article  CAS  PubMed  Google Scholar 

  20. Cianfriglia M, Armellini D, Massone A, Mariani M: Simple immunization protocol for high frequency production of soluble antigen specific hybridomas. Hybridoma. 1983, 2: 451-7. 10.1089/hyb.1983.2.451.

    Article  CAS  PubMed  Google Scholar 

  21. Galfre G, Milstein C: Properties of monoclonal antibodies: Strategies and procedures. Meth Enzymol. 1981, 73: 3-46.

    Article  CAS  PubMed  Google Scholar 

  22. Demerdash Z, Mahmoud S, Shaker Z, Hassan S, Mansour M: Detection of circulating schistosome antigens in serum and urine of schistosomiasis patients and assessment of cure by a monoclonal antibody. J Egypt Soc Parasitol. 1995, 25: 471-84.

    CAS  PubMed  Google Scholar 

  23. Laemmli U: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-5. 10.1038/227680a0.

    Article  CAS  PubMed  Google Scholar 

  24. Tsang V, Peralta J, Simons A: Enzyme-Linked Immunoelectro-Transfer Blot Techniques (EITB) for Studying the Specificities of Antigens and Antibodies Separated by Gel Electrophoresis. Methods in Enzymology: Immunochemical Techniques. Edited by: Langone JJ, Vunakie HV. 1983, Academic Press, New York, 337-91.

    Google Scholar 

  25. Abdel Rahman S, Malone J, O'reilly K: Biochemical characterization and localization of Fasciola hepatica 26-28 kDa diagnostic coproantigen. Parasite Immunol. 1999, 21: 279-86. 10.1046/j.1365-3024.1999.00191.x.

    Article  CAS  PubMed  Google Scholar 

  26. Woodward M, Young W, Bloodgood R: Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J Immunol Meth. 1985, 78: 143-53. 10.1016/0022-1759(85)90337-0.

    Article  CAS  Google Scholar 

  27. Maizels RM, Robertson BD: Purification of Antigens and Antibodies. 1988, Imperial College of Science, Technology and Medicine, London, 3

    Google Scholar 

  28. Tijssen P, Kurstak P: Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays. Anal Biochem. 1984, 136: 451-7. 10.1016/0003-2697(84)90243-4.

    Article  CAS  PubMed  Google Scholar 

  29. Galen RS: Predictive values and efficiency of laboratory testing. Pediat J Clin North Am. 1980, 27: 861-69.

    CAS  Google Scholar 

  30. Arafa MS, Abaza SM, El-Shewy KA, Mohareb EW, El-Moamly AA: Detection of Fasciola-specific excretory/secretory (E/S) protein fraction band (49.5 kDa) and its utilization in diagnosis of early fascioliasis using different diagnostic techniques. J Egypt Soc Parasitol. 1999, 29 (Suppl 3): 911-26.

    CAS  PubMed  Google Scholar 

  31. Anuracpreeda P, Wanichanon C, Chawengkirtikul R, Chaithirayanon K, Sobhon P: Fasciola gigantica: Immunodiagnosis of fasciolosis by detection of circulating 28.5 kDa tegumental antigen. Exp Parasitol. 2009, 123: 334-340. 10.1016/j.exppara.2009.08.014.

    Article  CAS  PubMed  Google Scholar 

  32. Ubeira FM, Muiño L, Valero MA, Periago MV, Pérez-Crespo I, Mezo M, González-Warleta M, Romarís F, Paniagua E, Cortizo S, Llovo J, Más-Coma S: MM3-ELISA detection of Fasciola hepatica coproantigens in preserved human stool samples. Am J Trop Med Hyg. 2009, 81 (Suppl 1): 156-62.

    PubMed  Google Scholar 

  33. Cornelissen JB, de Leeuw WA, van der Heijden PJ: Comparison of an indirect haemagglutination assay and an ELISA for diagnosing Fasciola hepatica in experimentally and naturally infected sheep. Vet Quart. 1992, 14: 152-156.

    Article  CAS  Google Scholar 

  34. Dumenigo BE, Espino AM, Finlay CM, Mezo M: Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica. Vet Parasitol. 2000, 89: 153-161. 10.1016/S0304-4017(00)00206-5.

    Article  CAS  PubMed  Google Scholar 

  35. Lee CG, Zimmerman GL, Wee SH: Fasciola hepatica: Comparison of flukes from Korea and the United States by isoelectric focusing banding patterns of whole-body protein. Vet Parasitol. 1992, 42 (Suppl 3-4): 311-6.

    Article  CAS  PubMed  Google Scholar 

  36. Neyra V, Chavarry E, Espinoza JR: Cysteine proteinases Fas1 and Fas2 are diagnostic markers for Fasciola hepatica infection in alpacas (Lama pacos). Vet Parasitol. 2002, 105: 21-32. 10.1016/S0304-4017(02)00002-X.

    Article  CAS  PubMed  Google Scholar 

  37. Velusamy R, Singh BP, Sharma RL, Chanda D: Detection of circulating 54 kDa antigen in sera of bovine calves experimentally infected with F. gigantica. Vet Parasitol. 2004, 119: 187-195. 10.1016/j.vetpar.2003.09.023.

    Article  CAS  PubMed  Google Scholar 

  38. Valero MA, Ubeira FM, Khoubbane M, Artigas P, Muino L, Mezo M, Pérez-Crespo I, Periago MV, Mas-Coma S: MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica. Vet Parasitol. 2009, 159: 77-81. 10.1016/j.vetpar.2008.10.014.

    Article  CAS  PubMed  Google Scholar 

  39. Anuracpreeda P, Wanichanon C, Sobhon P: Fasciola gigantica: immunolocalization of 28.5 kDa antigen in the tegument of metacercaria and juvenile fluke. Exp Parasitol. 2009, 122: 75-83. 10.1016/j.exppara.2009.03.007.

    Article  CAS  PubMed  Google Scholar 

  40. Dumenigo B, Espino A, Finlay C: Detection of Fasciola hepatica antigens in cattle feces by a monoclonal antibody-based sandwich immunoassay. Res Vet Sci. 1996, 60: 278-9. 10.1016/S0034-5288(96)90055-7.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by internal project No. 65 D at Theodor Bilharz Research Institute and supported in part by ASTF project No. 66, Egypt.

Author information

Authors and Affiliations

  1. Department of Immunology, Theodor Bilharz Research Institute, Giza, Egypt

    Zeinab A Demerdash, Salwa H Mohamed, Faten S Mahmoud, Mona K Zoheiry, Wafaa A Mansour & Azza E El-Bassiouny

  2. Department of Parasitology, Theodor Bilharz Research Institute, Giza, Egypt

    Tarek M Diab & Ibrahim R Aly

  3. Department of Gastroenterology and Hepatology, Theodor Bilharz Research Institute, Giza, Egypt

    Mohy E Attia

Authors
  1. Zeinab A Demerdash
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tarek M Diab
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ibrahim R Aly
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Salwa H Mohamed
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Faten S Mahmoud
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mona K Zoheiry
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wafaa A Mansour
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mohy E Attia
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Azza E El-Bassiouny
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tarek M Diab.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

Both ZAD and AEE designed the study. ZAD, AEE, SHM, FSM, WAM, IRA and MKZ developed the MoAbs-based sandwich ELISA. MEA performed the clinical examinations to subjects of the study IRA and TMD collected the samples, performed the parasitological examinations and evaluated the diagnostic efficacy of the assay. TMD collected the data and performed the statistical analysis. All the authors discussed, revised, read and approved the final manuscript.

Authors’ original submitted files for images

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Demerdash, Z.A., Diab, T.M., Aly, I.R. et al. Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA) for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool. Parasites Vectors 4, 176 (2011). https://doi.org/10.1186/1756-3305-4-176

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/1756-3305-4-176

Keywords

Parasites & Vectors

ISSN: 1756-3305

Contact us