Production of Monoclonal Antibody Against Excretory-Secretory Antigen of Fasciola hepatica and Evaluation of Its Efficacy in the Diagnosis of Fascioliasis

Abstract

Parasitological methods are not helpful for the diagnosis of fascioliasis in acute and invasive periods of the disease. Detection of coproantigens seems to be a suitable alternative approach in the diagnosis of fascioliasis. The present study aimed to develop a reliable antigen detection system, using monoclonal antibodies raised against excretory-secretory (ES) antigen of Fasciola hepatica, for the diagnosis of fascioliasis. Fasciola adult worms were collected from the bile ducts of infected animals. Species of the fluke was determined by polymerase chain reaction–restriction fragment length polymorphism (RFLP-PCR). ES antigen of F. hepatica was prepared. For production of monoclonal antibodies, mice were immunized with ES antigens of F. hepatica. Spleen cells from the immunized mice were fused with NS-1 myeloma cells, using polyethylene glycol. Hybridoma cells secreting specific antibody were expanded and cloned by limiting dilution. Moreover, polyclonal antibody was produced against F. hepatica ES antigen in rabbits. A capture enzyme-linked immunosorbent assay (ELISA) system, using produced monoclonal antibody, was designed and stool samples of infected animals along with control samples were tested by the system. The capture ELISA detected the coproantigen in 27 of 30 (90%) parasitologically confirmed fascioliasis cases, while 4 of 39 (10.25%) samples infected with other parasitic infections showed a positive reaction in this system. No positive reactivity was found with healthy control samples. Accordingly, sensitivity of 90% and specificity of 94.2% were obtained for the capture ELISA system. The results were compared with those obtained with commercial BIO-X ELISA, and a very good (kappa = 0.9) agreement was found between the commercial kit and the developed capture ELISA. Findings of this study showed that the produced monoclonal antibody has appropriate performance for the detection of Fasciola coproantigen in stool samples and can be appropriately used for the diagnosis of fascioliasis.

Introduction

Fascioliasis is one of the most important zoonotic parasitic infections considered as a neglected tropical disease.⁽¹⁾ Fasciola hepatica and Fasciola gigantica belong to the fasciolinae subfamily and are principal agents of both animal and human fascioliasis.^(1,2) Fascioliasis is not only considered to be a veterinary problem but also is a human public health problem. Increasing of cases of human fascioliasis is reported from many countries around the world and ∼17 million people are currently infected.^(2,3) To date, the highest prevalence of human fascioliasis has been described from the highlands of South America (Bolivia, Peru), China, Spain, Nile Delta in Egypt, Vietnam, and also Iran.^(1,4) Based on WHO reports, Iran is among the six countries where human fascioliasis is prevalent and considered a major health problem.⁽⁵⁾ Humans and animals can be infected by the ingestion of metacercariae-infected vegetables.⁽⁶⁾

Diagnosis of fascioliasis is challenging and the performance of currently available diagnostic tests is not satisfactory. The fluke eggs present 3 to 4 months after infection in the patient's feces. Therefore, parasitological methods are not helpful for the diagnosis of fascioliasis in the acute and invasive periods of the infection. Moreover, in the chronic phase of the disease, the parasite's egg shedding is intermittent and in some human infection cases, the worms may not reach maturity and produce eggs.⁽⁷⁾ Serological approaches, based on antibody detection, do not differentiate between current and past infection and may interfere in the diagnosis.⁽⁸⁾ So far, several serological antibody detection tests have been developed for the diagnosis of fascioliasis using different Fasciola antigens.⁽⁹⁾

In recent years, to improve the performance of serological tests for the diagnosis of fascioliasis, enormous efforts have been directed toward the excretory-secretory (ES) antigens of Fasciola.⁽¹⁰⁾ Fasciola protease antigens are one of the most important candidates that have been used in antibody detection assays. Utilizing of recombinant cathepsin L cysteine proteases for the diagnosis of human fascioliasis revealed 99.9% sensitivity and specificity.⁽¹¹⁾ In line with this study, an enzyme-linked immunosorbent assay (ELISA) system, using saposin-like protein-2 antigen, showed 87% sensitivity and 99% specificity.⁽¹²⁾ Antigen detection is an alternative serological test that has been used for the diagnosis of several parasitic infections.^(13–16) Fasciola antigen can be detected in serum or stool samples of patients only 2 months after infection.⁽¹⁷⁾ Furthermore, the antigen detection test is helpful for post-treatment follow-up of the disease.⁽¹⁸⁾ Fasciola circulatory antigens have been detected in different biological samples, including serum, saliva, or stool (coproantigens).

Detection of Fasciola coproantigens is more reliable than the other circulatory antigen. Demerdash et al. reported sensitivity of 96% and specificity of 98.2% using a monoclonal antibody in a capture ELISA system for detection of F. gigantica circulatory antigens in stool samples.⁽¹⁹⁾

Encouraging results have been obtained through the use of MM3-COPRO ELISA or its commercial version, BIO-X (BIO X Diagnostics, Belgium), for the diagnosis of human or animal fascioliasis, targeting the coproantigens in stool samples.^(17,19,20)

In comparison with circulatory antigens, the coproantigens are less affected by the immune complex. Moreover, it uses stool, which can be collected easily at several times without causing inconvenience to the patient and is collected by noninvasive techniques, reducing the risk of transmission of blood-borne infections.⁽¹⁹⁾

Considering the aforementioned points, development of a reliable and applicable antigen-based diagnostic test for the diagnosis of fascioliasis is necessary. The current study aimed to develop an appropriate antigen detection assay (capture ELISA) using monoclonal antibodies raised against F. hepatica ES antigens for the diagnosis of fascioliasis.

Materials and Methods

Sample collection

Fasciola worms were collected from the bile ducts of infected cattle and sheep from local abattoirs in Shiraz, south of Iran. Worms were washed five times with phosphate-buffered saline (PBS; pH: 7.2) to remove the exudates of bile ducts. From each infected liver, three to four worms were transferred to 70% alcohol for molecular assay and the others were transferred to PBS (pH: 7.2) for antigen preparation.

DNA extraction and determination of Fasciola species

Molecular determination of Fasciola species was carried out as previously described.⁽²¹⁾ Briefly, the anterior region (above the ventral sucker) of the adult worm was separated and homogenized with lyses buffer and proteinase K.

DNA was extracted from the homogenized tissue using the phenol–chloroform method. The ITS1 region of Fasciola was amplified using ITS1 forward; 5-TTGCGCTGATTACGTCCCTG-3, and reverse; 5-TTGGCTGCGCTCTTCATCGAC-3 primers. Restriction fragment length polymorphism–polymerase chain reaction (RFLP-PCR) was performed on PCR product using fast digest RsaI restriction enzyme to differentiate the two species of Fasciola.⁽²²⁾

Preparation of ES antigen

F. hepatica ES antigen was prepared by incubation of the adult worms in RPMI 1640 medium (one worm/mL) containing 100 U/mL of penicillin and 100 μg/mL of streptomycin at 37°C for 16–18 hours. The worms were removed and media were collected and centrifuged at 4000 g for 15 minutes. The supernatant was dialyzed against PBS (pH: 7.2), lyophilized, and stored at −20°C until use.

Production of polyclonal antibody against F. hepatica ES antigen

Two female rabbits (1.5–2 kg) were selected and were subcutaneously immunized with ES antigen of F. hepatica. Injections were performed using 500 μg of antigen and an equal volume of Freund's complete adjuvant for the first injection and Freund's incomplete adjuvant for the others, with six 7-day intervals. Seven days after the last injection, hyperimmunized serum of the rabbits was examined using the indirect ELISA test. Rabbit baseline serum (at 1:100 dilutions) was used as negative control. Rabbit IgG antibody, raised against Fasciola ES antigen, was purified using protein G affinity chromatography.

Production of monoclonal antibody against F. hepatica ES antigen

Eight female BALB/c mice (4–6 weeks old) were subcutaneously immunized with F. hepatica ES antigen. The immunization was carried out at five 7-day intervals, using 300 μg of ES antigen and an equal volume of Freund's complete adjuvant for the first injection and Freund's incomplete adjuvant for the rest of the injections. Seven days after the last injection, serum samples of hyperimmunized mice were examined for the detection of anti-ES antibodies using indirect ELISA.

Fusion was performed using myeloma cell line (NS1) and spleen cells of immunized mice. The day before the fusion, the myeloma cells were subcultured at the concentration of 3 × 10⁵ cell/mL. The spleen of the hyperimmunized mouse was removed under sterile conditions and completely homogenized. The spleen cells were isolated and red blood cells were lysed using 0.85% ammonium chloride. The spleen cells and NS-1 myeloma cells were fused using polyethylene glycol. Fused hybridoma cells were cultured in Iscove's medium containing OPI (0.15 g oxaloacetate, 0.05 g pyruvate, and 0.0082 g insulin) and HAT (100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine) in a 96-well culturing microplate. Five days after the fusion, half of the supernatant of the growing cells in each well was removed and fed fresh Iscove's OPI-HAT medium. On day 10 of the fusion, the plates were checked using an inverted microscope, and the supernatant of any well with growing hybridoma cells was screened using ES antigen of Fasciola in an indirect ELISA system. Hybridoma cells secreting anti-ES antibodies were expanded and cloned by limiting dilution.

Isotyping of monoclonal antibody

Isotypes of monoclonal antibody were determined using the Pierce Rapid Isotyping Kit (Pierce, Thermo Fisher Scientific, Rockford, IL) based on the manufacturer's instructions. Supernatant of the positive clone was diluted (1:10) using the diluent buffer provided in the kit. Diluted sample (150 μL) was added to the well of the cassette. After 5–10 minutes, the resulting colored bands were evaluated.

Purification of monoclonal antibody

IgM isotype of the monoclonal antibody was purified using both saturated ammonium sulfate and size exclusion chromatography methods. Chromatography was performed using fine polyacrylamide beads (Bio-Gel P-10) with 1500–20,000 MW fractionation range. After purification, the fraction of each peak was lyophilized and confirmed by indirect ELISA against F. hepatica ES antigen.

Labeling of monoclonal antibodies using horseradish peroxidase

Monoclonal antibodies were labeled with horseradish peroxidase (HRP) as described by Sarkari et al.⁽¹⁶⁾ Briefly, 4 mg/mL of HRP was dissolved in 200 μL of freshly prepared sodium metaperiodate (0.1 M) for 20 minutes at RT and dialyzed against sodium acetate buffer (1 Mm, pH 4.4). The pH of dialyzed solution was raised to 9.5 using 20 μL of 20 mM sodium carbonate/bicarbonate buffer (pH 9.5). The monoclonal antibody was dialyzed against 50 mM sodium carbonate/bicarbonate buffer (pH 9.5) and 8 mg of dialyzed monoclonal antibody was added to the HRP solution and mixed for 2 hours at RT. Then, 100 μL of sodium borohydride (4 mg/mL) was added to the solution and mixed for 2 hours at 4°C. The conjugated monoclonal antibody was dialyzed against PBS overnight at 4°C and stored at −20°C until use.

Collecting of stool samples

A total of 99 stool samples were collected from animals, including sheep, goat, and cattle. Fasciola-infected stool samples (n = 30) were obtained from parasitologically confirmed (by detection of Fasciola egg) samples. Moreover, 39 stool samples were collected from animals with other parasitic infections, but not Fasciola, including trichuriasis (n = 3), amebiasis (n = 9), trichostrongyliasis (n = 9), cryptosporidiosis and eimeriosis (n = 5), trchostrongyliasis and eimeriosis (n = 2), eimeriosis (n = 9), and trichuriasis and eimeriosis (n = 2). Negative control stool samples were obtained from 30 healthy animals, with no sign of parasitic infection in their stool samples when tested by parasitological methods.

Preparation of coproantigen

Cattle stool samples (2 g) or ovine stool samples (0.5 g) were homogenized with 2 mL of dilution buffer of a commercial kit and centrifuged for 10 minutes at 1000 g. Supernatant was collected and stored at −20 °C until use.

Capture ELISA using produced monoclonal antibodies raised against Fasciola ES antigens for the detection of Fasciola coproantigen

For the detection of Fasciola coproantigens, a capture ELISA system was developed. The capture ELISA was performed in Jet Biofil ELISA microplates. The plates were coated with 5 μg/mL of rabbit purified polyclonal IgG against F. hepatica in coating buffer (0.05 M carbonate/bicarbonate buffer, pH 9.5) and incubated at 4°C overnight. After washing five times with PBS containing 0.05% Tween 20, skimmed milk (5%, 100 μL/well) was added and the plates were incubated for 1.5 hours at RT. The plate was washed as before and 100 μL/well of processed stool sample was added and incubated for 2 hours. After washing as before, HRP-conjugated monoclonal antibody raised against F. hepatica ES antigen (at 1:500 dilution) was added to the plates and incubated for 1 hour. After washing as before, the plates were incubated with chromogen/substrate (100 μL/well of 0.4 mg/mL OPD, 0.3% H₂O₂ in 0.1 M citrate buffer, pH 5) for 15 minutes. Finally, the absorbance of each well was measured with ELISA reader (ELx800; Bio-Tek) at 450 and 630 nm.

Evaluation of target antigen of the produced monoclonal antibody

Immunoblotting was carried out to detect the target antigen of the produced monoclonal antibody, as previously described.⁽²³⁾ F. hepatica ES antigens were separated on 12.5% SDS-PAGE and transferred onto a nitrocellulose membrane and evaluated with the produced monoclonal antibody.

Evaluation of stool samples by commercial Bio-X F. hepatica antigenic ELISA Kit

The stool samples were tested by a commercial Bio-X F. hepatica antigenic ELISA Kit (Bio-X Diagnostics, Jemelle, Belgium) based on the manufacturer's instructions.

Statistical analysis

The diagnostic accuracy of the Fasciola antigen detection tests was evaluated by the area under the receiver operating characteristic curve. The level of agreement between different ELISA systems was analyzed using Cohen's kappa.

Results

Production of monoclonal antibody against F. hepatica ES antigen

Using an indirect ELISA system, 12 monoclonal antibodies were identified, which properly reacted with F. hepatica ES antigens. The performance of one of these monoclonal antibodies, SAG5-BC11, was superior to the rest of monoclonal antibodies in detecting of Fasciola ES antigen. Therefore, further work was focused on this monoclonal antibody. The isotype of SAG5-BC11 monoclonal antibody was IgM. Using western blotting, the SAG5-BC11 monoclonal antibody detected a 27 kDa antigen of F. hepatica ES antigens (Fig. 1).

FIG. 1.

Detection of the target Fasciola hepatica ES antigen by SAG5-BC11 monoclonal antibody. Lane 1: Protein ladder; Lanes 2–6, a 27 KDa antigen of F. hepatica detected with the SAG5-BC11 monoclonal antibody. ES, excretory-secretory.

Capture ELISA using monoclonal antibodies produced against F. hepatica ES antigens for the diagnosis of fascioliasis

The capture ELISA, using monoclonal antibody raised against F. hepatica ES antigen, detected the coproantigen in 27 of 30 (90%) parasitologically confirmed fascioliasis cases, while 4 of 39 (10.25%) samples infected with other parasitic infections, including three samples infected with trichostrongyliasis and one sample with mixed infection of cryptosporidiosis and eimeriosis, showed a positive reaction in this system. No positive reactivity was found with healthy control samples.

Accordingly, sensitivity of 90% (95% CI = 73.47%–97.89%), specificity of 94.2% (95% CI = 85.82%–98.40%), positive predictive value of 87.09% (95% CI = 69.23%–95.7%), and negative predictive value of 95.5% (95% CI = 86.8%–98.8%) were obtained for the capture ELISA system. Table 1 shows the sensitivity and specificity of the capture ELISA using SAG5-BC11 monoclonal antibody for the diagnosis of fascioliasis. The detection limit of the monoclonal antibody for detection of Fasciola ES antigen was 0.5 μg/mL. Optical density (OD) values (at 450 nm) of stool samples tested by capture ELISA using monoclonal antibody raised against ES antigen of F. hepatica are shown in Figure 2.

FIG. 2.

OD value (at 450 nm) of stool samples tested by capture ELISA using monoclonal antibody raised against ES antigen of F. hepatica. OD, optical density.

Table 1.

Sensitivity and Specificity of the Capture ELISA System, Using SAG5-BC11 Monoclonal Antibody, for the Diagnosis of Fascioliasis

Sensitivity		Specificity
	Fasciola	Amoeba	Tricho ^a	Eimeria	Trichuris	Cryptos^b and Eimeria	Tricho and Eimeria	Trichuris and Eimeria	Healthy control	Total
Positive	27	0	3	0	0	1	0	0	0	4
Negative	3	9	6	9	3	4	2	2	30	65
Total (%)	90	100	66.6	100	100	80	100	100	100	94.2

Trichostrongyliasis

Cryptosporidium spp.

ELISA, enzyme-linked immunosorbent assay.

Performance of Bio-X F. hepatica antigenic ELISA Kit in detection of coproantigens

Using Bio-X F. hepatica antigenic ELISA Kit, only 1 of 30 (3.3%) parasitologically confirmed fascioliasis cases showed negative reactivity. No cross-reactivity was detected with healthy control samples or with samples infected with other parasites. Therefore, sensitivity of 96.7% and specificity of 100% were obtained for the commercial kit for the diagnosis of fascioliasis. OD values (at 450 nm) of stool samples tested by Bio-X F. hepatica ELISA Kit are shown in Figure 3. Performance of Bio-X F. hepatica antigenic ELISA Kit and capture ELISA based on monoclonal antibody for the detection of Fasciola coproantigen are shown in Table 2.

FIG. 3.

OD value (at 450 nm) of stool samples tested by the Bio-X ELISA Kit.

Table 2.

Performance of the Capture ELISA System Using Monoclonal Antibody and Commercial ELISA Kit for the Detection of Fasciola Coproantigen

ELISA test	Sensitivity (%)	Specificity (%)	Negative predictive value (%)	Positive predictive value (%)	Validity (%)
Fasciola hepatica MAb	90	94.2	87.09	95.5	92.1
Commercial Bio-X Kit	96.67	100	100	98.5	98.3

Statistical analysis of data showed a very good agreement (kappa = 0.91) between capture ELISA using monoclonal antibodies raised against F. hepatica ES antigen and the commercial kit. The validity of the capture ELISA using monoclonal antibodies and the commercial kit was 92.1% and 98.3%, respectively.

Discussion

Parasitological diagnosis is not helpful in the active phase or the prepatent period of fascioliasis.⁽²⁴⁾ On the other hand, antibody detection assays are not able to differentiate between past and present infection. Detection of antigens, rather than antibodies, seems to be a suitable alternative approach in the diagnosis of fascioliasis. Antigen can be detected in serum samples, urine, or stool of fascioliasis patients. Up to now, attempts have been made to detect the metabolic and excretory secretory products of Fasciola in stool samples. Yet, no antigenic detection test is available for diagnosis of human fascioliasis.

The principal aim of the present study was to design a reliable and appropriate antigenic detection assay based on produced monoclonal antibodies against ES antigen of F. hepatica for the diagnosis of fascioliasis. Findings of the current study showed that the capture ELISA based on monoclonal antibodies raised against F. hepatica ES antigen has high sensitivity and appropriate specificity. Allam et al. reported a sensitivity of 96.43% and specificity of 94.87% for a capture ELISA using a polyclonal antibody raised against fatty acid-binding protein (14.5 kDa) of Fasciola for the detection of Fasciola coproantigen in human stool samples.⁽²⁵⁾ El Amir et al. reported a sensitivity of 95.5% and specificity of 92.7% for a capture ELISA system utilizing a polyclonal antibody raised against F. gigantica ES antigen for the detection of coproantigen in sheep stool samples.⁽²⁶⁾ The findings of both of these studies are consistent with the findings of the current study. Shehab et al. reported a sensitivity of 100% for an ELISA system using a polyclonal antibody raised against Fasciola ES antigen for the detection of circulatory antigens in the acute phase and sensitivity of 70% in detection of antigen in the chronic phase of human fascioliasis.⁽²⁷⁾

Considering the findings of our study, it can be concluded that detection of coproantigens can be more useful than detection of circulatory antigens for the diagnosis of chronic fascioliasis.

Arafa et al. reported a sensitivity of 91.4% and a specificity of 92.3% for a capture ELISA using a monoclonal antibody produced against 49.5 kDa antigen of F. gigantica in human stool samples.⁽²⁸⁾ Nagy et al. reported a sensitivity of 94.3% and a specificity of 98% for a capture ELISA using monoclonal antibody produced against F. gigantica ES antigen for the diagnosis of human fascioliasis.⁽²⁹⁾ These two studies are in favor of coproantigen detection in the diagnosis of fascioliasis. Ubeira et al. reported a sensitivity and specificity of 100% for a capture ELISA, using MM3 monoclonal antibody for the detection of coproantigens in human stool samples.⁽³⁰⁾ Using a monoclonal antibody against F. gigantica ES antigen, Demerdash et al. reported a sensitivity of 96% and a specificity of 98.2% for the detection of coproantigen.⁽¹⁹⁾ Sensitivity and specificity of the capture ELISA in our study were lower than those of the Ubeira and Demerdash studies.^(19,30)

From protein bands of 18–62 KDa, which have been reported in F. hepatica ES antigen,⁽²³⁾ the produced monoclonal antibody in the current study reacted with a 27 KDa antigen, which is the molecular weight range of cathepsins. These findings are in agreement with findings of a previous study that reported an appropriate performance for a 27 kDa antigen of F. hepatica for the serodiagnosis of fascioliasis.⁽³¹⁾

Monoclonal antibodies produced in the current study had cross-reactivity with a few of the parasitic infections, although the OD values of the developed capture ELISA for these samples were lower than those of positive cases, but they were above the cutoff of the system. Highly purified monoclonal antibodies may circumvent this shortcoming.

The in-house MM3-COPRO ELISA and its commercial version BIO-X (BIO X Diagnostics, Belgium) have been extensively evaluated for the detection of Fasciola coproantigens in the stool of infected human and animals with satisfactory results.^{(17,19,20,31)} More encouraging results have been recently obtained by using streptavidin-polymerized HRP in the capture ELISA system to reveal bound biotinylated monoclonal antibody MM3. These changes substantially increased the detection limit of the assay as well as its sensitivity.⁽²⁰⁾

In the present study, we used the Bio-X ELISA Kit for the detection of coproantigen where a sensitivity of 96.67% and specificity of 100% were achieved. Very good agreement was seen between the commercial kit and our developed capture ELISA based on the monoclonal antibody raised against the F. hepatica ES antigen.

The signal-to-noise ratio obtained with the in-house ELISA was relatively poor compared with the commercial Bio-X ELISA Kit or even other systems. This probably reflects the inconvenience of using IgM versus IgG monoclonal antibodies in an ELISA system for the detection of coproantigens.

Taken together, the findings of this study showed that utilization of the produced monoclonal antibody, against F. hepatica ES antigen, in the ELISA system has the appropriate performance for detection of Fasciola coproantigen in stool samples and can be used for the diagnosis of fascioliasis. Stool samples of human fascioliasis patients from endemic geographical regions of Iran should be tested with the designed capture ELISA to appropriately find out the performance of this system for the diagnosis of human fascioliasis.

Footnotes

Acknowledgments

This study was financially supported by the office of vice-chancellor for research of Shiraz University of Medical Sciences (Grant No. 6808). The results described in this article were part of a PhD student thesis of Samaneh Abdolahi Khabisi.

Author Disclosure Statement

No competing financial interests exist.

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