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Research Article

Efficacy of Sandwich and Dot-ELISA in Diagnosis of Fascioliasis using a Pair of Polyclonal Antibodies Against Cathepsin L Antigen in Naturally Infected Sheep

Tarek M. Diab, Ibrahim Rabia, Wafaa E.L. Komy and Azza E.L. Amir
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The present study was designed to prepare a pair of polyclonal antibodies (PAbs) (IgG4 A7 and IgG4 B12) against Fasciola gigantica Cathepsin L. (CL) and used them in development of sandwich ELISA and dot-ELISA to detect Circulating Fasciola Antigens (CFA) in serum samples collected from a total of 157 sheep. The first PAb was used as antigen capturing Ab and the second as horse reddish peroxidase-conjugated antigen detecting Ab. After slaughtering, gross inspection of liver and parasitological stool examination, sheep were divided into Fasciola positive group (97 sheep), other helminthic infection group (30 sheep) and healthy control group (30 sheep). Fasciola antigens detected in serum of sheep by ELISA showed 94.8% sensitivity and 95% specificity. Dot-ELISA sensitivity was found to be 98.9% and specificity was 98.3%. In conclusion, Dot-ELISA gives better sensitivity and specificity than sandwich ELISA and easy to perform method for the rapid diagnosis of fascioliasis.

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Tarek M. Diab, Ibrahim Rabia, Wafaa E.L. Komy and Azza E.L. Amir, 2011. Efficacy of Sandwich and Dot-ELISA in Diagnosis of Fascioliasis using a Pair of Polyclonal Antibodies Against Cathepsin L Antigen in Naturally Infected Sheep. Research Journal of Parasitology, 6: 90-100.

DOI: 10.3923/jp.2011.90.100

Received: August 05, 2011; Accepted: November 01, 2011; Published: December 15, 2011


Fascioliasis, a chronic disease caused by helminthes, is a major cause of morbidity, mortality and decreased productivity in cattle, sheep and goats, resulting in serious economic losses in Egypt (Awad et al., 2004; El-Shazly et al., 2005). In addition, Nile Delta is considered one of the endemic areas in the world for human fasciolosis (Haseeb et al., 2002; Safar et al., 2005; Curtale et al., 2007; Mas-Coma et al., 2005, 2009).

Cathepsins are the main proteases present in the Excretory Secretory (ES) products of Fascioloa. Cathepsin L. (CL) is the major consitutent (68%) of total cathepsins obtained from ES products (Dalton et al., 2003). CL plays an important role in parasite survival, tissue digestion and escaping from immune evsion (Dalton et al., 2003; O’Neill et al., 2001). Therefore, it may be used in vaccination and diagnosis (Kuk et al., 2005).

Coprological diagnosis of fasciolosis is useful in patent infections but it is not capable of detecting early infections. The pathology of immature fasciolosis is manifested as early as 3 weeks postinfection, whereas parasitological diagnosis is impossible until the 8th-12th week when eggs are detected in the faeces (Rokni et al., 2003; Ghosh et al., 2005; Kumar et al., 2008). However, the serodiagnostic methods for the detection of antibodies are quite sensitive to detect the infection in the early stages but it did not discriminate previous exposure from current infection (Espino and Finlay, 1994; Dixit et al., 2004; Mas-Coma et al., 2005). Moreover, Fasciola shares antigens with other parasites such as S. mansoni increasing the possibility of cross-reactions when antibody detection assay systems were used for diagnosis of infection (Espino and Finlay, 1994). Alternatively, antigen-detection assays has several advantages over other methods as it can identify animals with pre-patent infection and give a more accurate indication of current infection and the parasite load rather than past infection (Zheng et al., 1990).

This study aimed to evaluate the diagnostic efficacy of a pair of polyclonal antibodies (PAbs) raised against Fasciola CL antigen for detection of circulating Fasciola antigen using sandwich ELISA and dot-ELISA in serum samples of sheep naturally infected with Fasciola.


Animal groups: This study was carried out in sheep (n = 157) slaughtered in a local abattoir located in Giza, Egypt. Blood and faecal samples were obtained from sheep brought in for slaughtering. Based on gross inspection, gold standard method, of sheep liver’ after slaughter in abattoir, Fasciola-infected and -uninfected sheep were selected. Parasitological examination of stool samples using direct smear method, Kato-Katz concentration technique (Martin and Beaver, 1968) and formal-ether sedimentation (Mansour-Ghanaei et al., 2003) was performed. Accordingly, sheep were classified into three groups, Fasciola-infected group (n = 97), other parasites infected group (n = 30) including schistosomiasis (n = 10), echinococcosis (n= 10), anclystomiasis (n = 10) and ascariasis and negative control group (n = 30). The collected blood from each sheep was centrifuged at 760 g at 4°C for 10 min and the obtained serum samples were stored at-20°C until analysis.

Purification of cathepsin L. : The standard protocol described by Coles and Rubano (1988) was followed with certain modifications to purify cathepsin L from F. gigantica regurgitant released in vitro (Dixit et al., 2002). Briefly, viable flukes were incubated in 500 mL RPMI-1640 culture medium, pH 7.3 containing 2% (w/v) glucose, 30 mM HEPES and 25 mg L-1 gentamycin. After 3-4 h incubation at 37°C, the culture medium (containing in vitro released regurgitate) was concentrated to a volume of 10 mL in an Amicon 8400 concentrator using an Amicon YM3 membrane (3 kDa molecular mass cut-off, Amicon) and applied to a Sephacryl S200HR gel filtration column (19x42 cm) equilibrated in 0.1 M Tris HCI, pH 7, at 4°C. The column was eluted with 0.1 M Tris HCI, pH 7 and 5 mL fractions were collected. Each fraction was assayed for Cathepsin L. activity using the fluorogenic substrate Tos-Gly-Pro-Arg-NHMec at a final concentration of 10 pM in 0.1 M glycine/HCl, pH 7. The Sephacryl S200 fractions containing Cathepsin L. activity were pooled and applied to a 50 mL QAE Sephadex column (2.5x10.0 cm) equilibrated in 0.1 M Tris/HCl, pH 7. The QAE Sephadex column was washed with 300 mL 0.1 M Tris/HCI, pH 7, then proteins eluted with 150 mL 75 mM NaCl in 0.1 M Tris/HCl, pH 7, followed by 250 mL 400 mM NaCl in 0.1 M Tris/HCI, pH 7. Five milliliter fractions (180 fractions in total) were collected and assayed for Cathepsin L. activity using the fluorogenic substrate Tos-Gly-Pro-Arg- NHMec. The Cathepsin L. activity was pooled then concentrated to 20 mL on an Amicon 8400 concentrator using a YM3 membrane. The concentrate was then diluted with distilled water to a volume of 100 mL and re-concentrated to a final volume of 10 mL to obtain a NaCl concentration of approximately 80 mM. The concentrated Cathepsin L. was stored as 1 mL aliquots at -80°C.

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE): SDS-PAGE was performed according to Laemmli (1970) as follows; 10 ug/well of each isolated batch of Cathepsin L. antigen in 20 μL of sample buffer, were electrophoresed on 12% SDS-PAGE. The run was continued until the sample reached the bottom of the gel. The protein bands in polyacrylamide gel were visualized by staining the gel with coomassie brilliant blue R-250 (Sigma).

Preparation of hyper-immune anti-Fasciola serum: Two kilogram New Zealand rabbits were immunized intramuscularly four times, 2-week intervals, with 1 mL of 1 mg of purified Cathepsin L. antigen diluted in 500 μL PBS and 500 μL of complete Freun’s adjuvant in the 1st immunization and 500 μg of purified Cathepsin L. antigen diluted in 500 μL PBS and 500 μL of incomplete Freund adjuvant in the subsequent immunizations. Three days after the last immunization dose, the rabbits were humanly sacrificed and the sera of each rabbit was collected separately and stored at-20°C until used (Espino et al., 1987).

Purification and labeling of polyclonal antibodies (pAb): Classing and subclassing of pAb isotypes was done by indirect ELISA using a panel of anti-mouse immunoglobulin peroxidase conjugates (goat anti-mouse IgM, IgG, IgG1, IgG2a, IgG2b, IgG3, IgG4 and IgA (Sigma). The produced subclasses of IgG pAbs were purified by ammonium sulfate treatment then by ion exchange chromatography (Goding, 1986). One used as capture pAb (IgG4 A7; 1st rabbit) and the other pAb (IgG4 B12; 2nd rabbit) was labeled by horse-radish peroxidase (Sigma) and used as a conjugated pAb (Tijssen and Kurstak, 1984).

Identification and Characterization of anti-ES products pAbs: Identification and characterization of the chemical nature of pAbs was performed using indirect ELISA by testing the reactivity of pAbs against the antigen before and after treatment with 20 mM sodium periodate (Sigma) and 4% trichloroacetic acid (Sigma) (Woodward et al., 1985).

Detection of circulating antigens in sheep sera by sandwich ELISA: The following sandwich-ELISA was adopted using a pair of pAbs against Cathepsin L., one as antigen capturing (IgG4 A7) and the other as antigen detecting antibody (IgG4 B12). The optimal dilutions of pAbs were determined by a checkerboard titration using a negative- and positive-control serum samples in each plate. For each step, 100 μL wellwas added unless mentioned otherwise. Polystyrene microtiter plates (Thomas Scientific, USA) were sensitized overnight at room temperature with purified IgG4 A7 pAb (10 μg mL-1 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 (FCS) (Sigma) diluted in PBS/T, pH 7.4. After 2 h incubation at 3°C, the plates were emptied by suction. Undiluted serum was added and the plates were incubated for 1 h at 3°C. After thorough washing as described above, peroxidase-conjugated IgG4 B12 pAb (10 μg mL-1 of PBS/T) was added. The plates were incubated for 2 h at 37°C and washed with PBS/T. The substrate O-phenylenediamine dihydrochloride (Sigma) was added and the plates were incubated for 30 min 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 (A492) of the plates was read using a microplate ELISA reader (Bio-Rad, Richmond CA, USA). 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 the cut off value were considered negative while those readings greater than the cut off value were considered positive.

Detection of circulating Fasciola antigens in serum by Dot-ELISA: Dot-ELISA was conducted as described earlier (Pappas, 1988; Rokni et al., 2006). Briefly, 2 μg/5 μL of IgG4 A7 was dotted on nitrocellulose membrane discs and allowed to be dried thoroughly. The discs were placed into flat bottom micro plate wells. Non-specific binding sites were blocked by adding 100 μL of 2.5% FCS tris buffer solution containing 0.5% Tween 20 (TBS/T) to each well. Blocking solution was then aspirated off and antigen disks were washed by shaking (three times, 10 min each) with PBS with 0.05% Tween 20 (Riedel de Haen, AG, Seelze, Hanover, Germany) in TBS (vol/vol). Five microliters of serum samples were added to each disk and incubated for 1 hr at room temperature. The serum samples were removed and washings were conducted as described above. The washing solution was removed and 2 μg/5 μL of horseradish peroxidase-labeled IgG4 B12 conjugate diluted in PBS/T were added to each well and incubated for 1 hr at room temperature. The conjugate was removed and other washings were conducted as mentioned before. Ten mls of DAB substrate (diamino benzidine tetrahydrocholoride) (Sigma) was added to the nitrocellulose membrane and incubated for 2 min at room temperature. The development of a deep brown colour dot on disks when compared with negative serum control was considered to be evidence of positivity. Colour development in controls was negligible or completely absent.

Statistical analysis: Data were expressed as mean±standard deviation. Standard diagnostic indices including sensitivity, specificity, positive and negative predictive values (PPV and NPV) were calculated as described by Galen (1980). Correlations between ova count and ELISA OD reading in serum samples were performed using Pearson correlation coefficient. SPSS computer program (version 12 windows) was used for data analysis.


Fasciola worms were detected in 97 sheep by gross inspection of the liver of sheep after slaughter in abattoir and used as a gold standard method. Parasitological examination of stool by Kato-Katz concentration technique for detection and counting of Fasciola eggs was performed for all sheep (n = 157). Fasciola eggs were detected in 78 out of 97 sheep (80.4%) with egg count ranging from 18-80 eggs g-1 stool.

A pair of pAbs against CL antigen was used in the present study. Isotypic analysis of pAbs revealed that they were of the IgG4 subclass with a κ light chain. The specificity of the selected pAbs against different parasite antigens (S. mansoni, Echinococcus granulosus, Ancylostoma duodenale and Ascaris lumbricoides) was determined by ELISA. Both pAbs were strongly reactive against Fasciola antigen and non reactive with other parasite antigens. The enzyme-linked immunoelectrotransfer blott (EITB) technique revealed that the 2 selected PAbs recognized 31 and 50 kDa bands of electrophorsed ES antigen. Binding of the two pAbs, IgG4 A7 and IgG4 B12, to CL antigens coated plates were strongly inhibited by treatment with 4% trichloroacetic acid (54.5 and 62%, respectively) and slightly inhibited by 20 mM sodium periodate (9.0 and 12%, respectively) denoting that they are glycoprotein in nature.

From the standard curve, the lower detection limit of ELISA assay was 4 ng/mL. In serum, the cutoff was 0.401 and CL antigen was detected by ELISA in 92 out of 97 Fasciola-infected sheep leading to overall sensitivity 94.8%. The specificity of the assay was determined as the sum of results of negative control group and other parasites group, 27 out of the 30 other parasites sheep group and all negative controls had undetectable circulating Fasciola antigen leading to overall specificity 95% (57/60). The positive and predictive values of ELISA assay were 96.8% (92/95) and 84.4% (27/32), respectively (Fig. 1, Table 1).

The optimal criteria for conducting Dot-ELISA were detected as 1 μg of antigen per dot, Fasciola antigen was detected in 96 out of the 97 Fasciola infected sheep sera leading to sensitivity of 98.9%.

Table 1: Sensitivity and specificity of the two techniques for detection of circulating Fasciola antigen in naturally Fasciola infected sheep
PPV: positive predictive value; NPV: Negative predictive value.
Fig. 1: Level of Fasciola CL 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

Fig. 2: Application of Dot-E LISA for detection of circulating Fasciola antigen in different studied groups. Lane A-B: Negative control group. Lane C-D: Other parasites group. Lane E-H: Fasciola-infected group

Fig. 3: Correlation between Fasciola egg count/g stool and OD reading of ELISA (r = 058; p<0.01) in serum samples of Fasciola gigantica infected sheep

All negative control sheep and 29 out of the 30 other parasites group sheep had undetectable Fasciola antigen and the overall specificity of the assay was 98.3% (59/60). Only one case of Schistosoma showed false positive reactions in this serum titer. The positive and negative predictive values of the assay were 98.9% (96/97) and 98.3% (29/30), respectively (Table 1, Fig. 2).

In Fasciola-infected sheep, a positive correlation was recoded between ova count/gm stool and ELISA OD reading in serum samples (r = 0.58, p<0.01) (Fig. 3).


Herein, purification of F. gigantica CL antigen by alcohol precipitation method followed by ion-exchange chromatography led to a removal of host components and an increase in antigen purity and hence an increase in both the sensitivity and specificity of ELISA and dot-ELISA assays. Electrophoretic separation on 12% gel resolved CL antigen into 1 protein band with the molecular weight of 27 kDa. Affinity chromatography has been shown to be a very effective tool for isolation of candidate diagnostic and vaccine molecule (Fagbemi et al., 1995, 1997; Yadav et al., 2005; Kumar et al., 2008).

In this study the sensitivity and specificity of sandwich ELISA was 94.8 and 95%, respectively. Cathpsin L. was found to be a promising antigen used in the diagnosis of F. Hepatica and F. gigantica infections during early prepatency in sheep, cattle and buffaloes (Dixit et al., 2002, 2004) and has given a satisfactory level of sensitivity, though several workers have reported varying degrees of cross-reactivity of CP antigen with other related helminths. Cross-reactivity of F. gigantica 28 kDa CP with Schistosoma bovis, Paramphistomum microbothrium and Dicrocoelium hospes in cattle has been reported by Fagbemi and Goubadia (1995). Cross-reactivity of native F. hepatica Cathepsin L. with sera from cattle infected with Dictyocaulus viviparus was observed by Cornelissen et al. (1999). Cornelissen et al. (2001) reported a specificity of 75.3% in naturally infected cattle using F. hepatica recombinant Cathepsin L. protease as antigen. However, O’Neill et al. (1998) observed no cross-reactivity of Cathepsin L. antigen with sera from human patients with S. mansoni, hydatidosis, cysticercosis and Chagas disease by using IgG4 ELISA. Carnevale et al. (2001) found that recombinant pro-Cathepsin L. was 100% specific in the detection of human F. hepatica infection. A commercially available ELISA kit based on the fas-2 antigen, CL1 (Levieux and Levieux, 1994) for the serodiagnosis of F. hepatica, by Institute Pourquier, Montpellier, France, has been found 98.2 and 98.3% sensitive and specific, respectively, in cattle and 96.9 and 99.4% sensitive and specific, respectively, in sheep (Molloy et al., 2005). Yadav et al. (2005) used purified Cathepsin L. CP for detection of anti-F. gigantica antibody in experimentally infected sheep and buffaloes using ELISA, Western blotting and dipstick ELISA and reported that it detected infection as early as 4 weeks post-infection.

Dot-ELISA has been considered as one of the valuable methods in diagnosis of different parasitological diseases including fasciolosis (Hassan et al., 2002; Intapan et al., 2003), toxoplasmosis (Elsaid et al., 1995), schistosomiasis (Schaefer et al., 1995; Mansour et al., 2009), hydatidosis (Hadighi et al., 2003) and cysticercosis (Liu et al., 1996). In this study, the sensitivity and specificity of dot-ELISA were 98.9 and 98.3%, respectively. In fact, all previous studies used dot-ELISA for screening anti-Fasciola antibodies in both ruminant and human but not in detecting Fasciola antigen as in this study. In this manner, Intapan et al. (2003) used F. gigantica 27 kDa (FG 27) as a target antigen in dot-ELISA and found that the sensitivity, specificity and accuracy were 100, 97.4 and 98.2%, respectively. Dalimi et al. (2004) used F. gigantica partially purified antigen in Fasciola infected patients in dot-ELISA test and reported 94.2% sensitivity and 99.4% specificity. Also, Rokni et al. (2006) used ESP as a target antigen in F. hepatica infected patients in dot-ELISA and found that the sensitivity and specificity were 96.8% and 96.1%, respectively. Antigen detection assays have an apparent advantage over antibody detection assays in that antigenemia implies active infection (Dixit et al., 2004; Mas-Coma et al., 2005) and decreasing the possibility of cross reaction with other parasites due to sharing the same epitopes (Espino and Finlay, 1994). The improved specificity in the present study could be attributed to the use of a pair of pAbs of IgG4 isotype as antigen capturing and binding antibodies, in this manner, Hassan et al. (2002) conducted dot-ELISA used for measurement of different anti-Fasciola IgG isotypes and reported that IgG2 and IgG4 demonstrated the highest specificity (>99%), followed by IgG1 (90%) and the least specific test was obtained with detection of IgG (85%). Levieux and Levieux (1994) concluded that detection of anti-Fasciola isotypes especially IgG4 was very specific for accurate diagnosis of fasciolosis. Also, Wongkham et al. (2005) developed ELISA using recombinant F. gigantica CL1 antigen to detect specific immunoglobulin G (IgG) subclass antibodies (IgG1, IgG2, IgG3 and IgG4) in human fasciolosis and reported that IgG4 enhanced both specificity and accuracy of ELISA test.

Generally, the difference in specificities could also be attributed to the different target antigens used and the different protocols used in preparation and purification of these antigens and finally the type of antibody used (Cornelissen et al., 1992; Dumenigo et al., 2000; Neyra et al., 2002). Also, polyparasitosis was considered an important factor that may decrease the specificity especially when an animal harbour two or three parasitic infections at the same time raising the possibility of cross-reaction.

Dot-ELISA has several advantages over sandwich ELISA, i.e., nitrocellulose papers spotted with antigen are stable for at least three months at-20°C, all incubation steps are performed at room temperature and the results can be read with the naked eye, thus an expensive spectrophotometer is not required. The test is applicable to diagnose in the field setting as well as in laboratories that are not well equipped. The dot–ELISA is simpler and allows testing of multiple samples at the same time (Intapan et al., 2003; Wongkham et al., 2005; Rokni et al., 2006).

It is concluded that, dot ELISA is an easier, more rapid, sensitive and specific test for diagnosing fascioliasis and cheaper than sandwich ELISA, especially in case of light infection, denoting that it could detect the infection at earlier stages. Besides, it is a more feasible test in the field.

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