Double-antibody sandwich ELISA using biotinylated antibodies for the detection of Echinococcus granulosus coproantigens in dogs

Acta Tropica 95 (2005) 9–15

Double-antibody sandwich ELISA using biotinylated antibodies for the detection of Echinococcus granulosus coproantigens in dogs Aitziber Benito 1 , David Carmena ∗ Department of Immunology, Microbiology and Parasitology, Faculty of Pharmacy, University of the Basque Country, Vitoria, Spain Received 16 July 2004; received in revised form 5 February 2005; accepted 8 March 2005 Available online 28 April 2005

Abstract Here we present the diagnostic evaluation of an improved double-antibody sandwich ELISA for detecting Echinococcus granulosus antigens in dog faecal samples (coproantigens). A purified rabbit IgG fraction against protoscolex excretory–secretory products was used as primary antibody, and the same fraction conjugated with biotin as secondary antibody. In order to validate the sandwich ELISA, intra- and inter-assay precision, linearity, and recovery percentages were calculated. The diagnostic evaluation of the method was carried out by investigating faecal samples from 37 dogs naturally infected with E. granulosus, 15 Echinococcus-free dogs infected with Taenia spp., 82 dogs with non-taeniid helminths and 66 dogs free of helminth infections. An overall sensitivity of 78.4% and specificity of 93.3% were determined. Positive and negative predictive values were 72 and 95%, respectively, and the diagnostic efficiency was 90.5%. In addition, the sandwich ELISA detection limit was estimated in 5.12 ng ml−1 . These results are highly satisfactory, allowing the use of this methodology in surveillance and control programs for intestinal echinococcosis in dogs. © 2005 Elsevier B.V. All rights reserved. Keywords: Echinococcus granulosus; Intestinal echinococcosis; Diagnosis; Coproantigens; Excretory–secretory antigens; Biotin

1. Introduction Echinococcosis caused by cestodes of the genus Echinococcus is widely distributed all over the world, ∗ Corresponding author at: MRC Clinical Sciences Centre, Membrane Transport Biology Group, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. Tel.: +44 208 383 8270; fax: +44 208 383 8337. E-mail address: [email protected] (D. Carmena). 1 Present address: R&D Department, TEMA, P.O. Box 3189, 1080 Vitoria, Spain.

0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2005.03.005

and recent reports demonstrate its spreading over previously echinococcosis-free geographical areas (Eckert and Deplazes, 2004). Detection of E. granulosus in dogs is essential to determine its prevalence in a region or country, and to develop surveillance and control programs for the infection (WHO, 2001). Diagnosis of intestinal infections with E. granulosus can be performed using arecoline hydrobromide as a purgative in dogs, but this technique has a highly variable sensitivity, is labour intensive, costly, biohazardous and some dogs suffer undesired side-effects (Wachira

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A. Benito, D. Carmena / Acta Tropica 95 (2005) 9–15

et al., 1990). On the other hand, coprological exams have low sensitivity since excretion of the eggs occurs sporadically. In addition, eggs of Echinococcus and Taenia species cannot be differentiated by light microscopy, although a species-specific diagnosis is now possible with a PCR test based on copro-DNA detection (Abbasi et al., 2003). Finally, autopsy of dogs and examination of the small intestine is considered the reference method for the detection of the adult stage of E. granulosus, but this ethically questionable and laborious procedure is not suitable for mass screening. As an alternative for the diagnosis of E. granulosus in dog populations, different immunological methods have been developed. ELISAs for detecting circulating antibodies have been extensively used by several groups (Jenkins and Rickard, 1986; Gasser et al., 1993; Benito et al., 2001). However, the results showed variable sensitivities, ranging from 40 to 90% (Gasser et al., 1993, 1994; Jenkins et al., 1990). These studies also demonstrated that 25–60% of the sera from infected dogs did not show significant levels of specific antibodies (Jenkins et al., 1990; Gasser et al., 1994), and that cross-reactivity with other parasite species may occur (Gasser et al., 1988). In addition, because antibody levels can remain high for a long time even when the infection has finished, serological techniques are not reliable in determining the current infection status of the dog (Deplazes et al., 1992). In the last few years, diagnosis of the intestinal E. granulosus infection in dogs has been mainly based on the detection of parasite antigens in faecal samples (coproantigens) by ELISA (Allan et al., 1992, Deplazes et al., 1994; Sakai, 1996; Ahmad and Nizami, 1998; Jenkins et al., 2000). This technique considerably improves both diagnostic sensitivity and specificity, permits the detection of the parasite during the prepatent period and shows the current status of the infection (Fraser and Craig, 1997). Results obtained in different geographical regions confirm the usefulness of this assay for epidemiological studies (Baronet et al., 1994; Sakai et al., 1995; Palmer et al., 1996; Malgor et al., 1997; el-Shehabi et al., 2000; Christofi et al., 2002). In this paper we present the diagnostic evaluation of an improved double-antibody sandwich ELISA for the detection of coproantigens in dogs infected with E. granulosus. For this purpose, the rabbit IgG fraction anti-excretory–secretory products of protocoleces was

purified and conjugated to biotin, in order to enhance the assay sensitivity.

2. Materials and methods 2.1. Faecal samples Two hundred stools from dogs were used to evaluate the diagnostic sensitivity and specificity of the sandwich ELISA. Faecal samples were classified as follows. Group 1: 37 samples from farm dogs naturally infected with E. granulosus (G1 sheep strain) in the Chubut Province, Argentina. These samples were diagnosed by identifying worms following arecoline purgation, and were kindly provided by Dr. Eduardo Fern´andez, Laboratorio de Control de Patolog´ıas Prevalentes, Hospital Zonal de Trelew, Chubut, Argentina; group 2: 15 samples from Echinococcus-free dogs infected with Taenia spp.; group 3: 82 samples from dogs infected with non-taeniid helminths, including Dipylidium caninum, Toxocara canis, Toxascaris leonina, Ancylostoma caninum, Uncinaria stenocephala and Trichuris vulpis and group 4: 66 samples from helminth-free dogs. Dog faecal samples from groups 2, 3, and 4 were obtained from the Council Animal Rescue Mission of Vitoria (Spain), as part of an epidemiological study to evaluate the prevalence of intestinal helminths in dogs ´ from the province of Alava (Benito et al., 2003). Dogs were diagnosed by post mortem intestinal examination. 51.8% of these dogs were males, and 48.2% were females. In relation with their origin, 60.5% of the dogs were from urban areas, 33% were from rural areas and 6.5% from bordering provinces (unknown environment). The age of the dogs ranged between 1 and 12 years. Faecal samples were collected from the rectum and mixed at 1:2 ratio (v/v) with PBS buffer containing 5% formaldehyde and 5 mM EDTA. Samples were shaken vigorously until slurry was formed, and this was centrifuged at 3500 × g for 20 min. Supernatants were aliquotted and stored at −20 ◦ C. 2.2. Protoscolex excretory–secretory antigens (PxES-Ag) Intact E. granulosus cysts from sheep livers were collected from the abattoir of La Guardia (La Rioja,

A. Benito, D. Carmena / Acta Tropica 95 (2005) 9–15

Spain). To obtain excretory–secretory products, protoscoleces with viability higher than 90% were selected. Viability was assessed by morphological appearance, flame cell motility and general contractile movements (Howell, 1986). Protoscoleces were cultured in PBS complemented with 10% glucose, 100 U ml−1 penicillin and 100 ␮g ml−1 streptomycin at 37 ◦ C in 5% CO2 , which promoted parasite survival for several days (Carmena et al., 2002). Every 8 h the medium was removed and replaced with fresh medium. Protein recovery from the media was achieved by using Ultrafree 15 filters with a 5 kDa pore diameter membrane (Millipore, Bedford, USA). EDTA (5 mM) and PMSF (2 mM) were added, and the PxES products were aliquotted and stored at −20 ◦ C. Protein concentration was determined by the bicinchoninic acid method (Sigma–Aldrich, Dorset, UK). 2.3. Reference positive control Faecal supernatants from three helminth-free dogs at autopsy were pooled and homogenized. PxES-Ag were added at a final concentration of 20 ␮g ml−1 , and the mix was used as a reference positive control in the sandwich ELISA assay. 2.4. Hyperimmune rabbit serum anti-PxES-Ag Polyclonal anti-PxES-Ag immune serum were obtained according to Gallart et al. (1985). Primary subcutaneous injection was carried out using PxES-Ag at 0.2 mg ml−1 concentration. Boosters at the same concentration were administered every two weeks. Titration of the antisera was performed by ELISA, as described by Nieto and Carbonetto (1989), using PxES-Ag as solid phase.

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2.6. Biotinylation of the purified IgG fraction The purified IgG fraction from the rabbit immunoserum was conjugated to biotin as described by Bayer and Wilchek (1980), in order to use it as secondary antibody in the sandwich ELISA assay. With this method, the intensity of the binding signal is considerably amplified, improving the sensitivity of the assay. 2.7. Double-antibody sandwich ELISA ELISA assays were carried out as described by Deplazes et al. (1992) and Allan et al. (1992), with some modifications. Briefly, polystyrene 96-well miTM crotitre plates (MaxiSorp , Nunc, Roskilde, Denmark) were coated with 30 ␮g ml−1 of the purified IgG anti-PxES-Ag fraction, and incubated for 3 h at 37 ◦ C. Blocking was performed with PBS-0.05% Tween 20 overnight at 4 ◦ C. Faecal supernatants were assayed at 1:2 dilution in PBS in duplicate, and incubated for 1 h at 37 ◦ C. In each plate, the reference positive control was included in serial dilutions ranging from 1:50 to 1:6400. Biotinylated IgG anti-PxES-Ag fraction was used as secondary antibody at 1:250 dilution in PBS-5% pre-immune rabbit serum for 1 h at 37 ◦ C. Then, peroxidase-conjugated EstrAvidin® (Sigma) was added at 1:1000 dilution in PBS for 1 h at 37 ◦ C. Binding was visualized with 5-aminosalicilic acid. The reaction was stopped by adding 25 ␮l/well NaOH 1N, and the absorbance value was measured at 450 nm.

3. Results 3.1. Validation of the sandwich ELISA

2.5. Purification of polyclonal IgG from the anti-PxES-Ag rabbit serum Rabbit immunoserum was dialyzed against a 20 mM di-sodium hydrogen phosphate solution pH 7, using a Spectra/Por® dialysis tubing (Spectrum Medical Industries, CA, USA). The IgG fraction was immunoprecipitated using a Hitrap® Protein A HP column (Pharmacia Biotech, Uppsala, Sweden), lyophilized, and stored at 4 ◦ C. Protein concentration was determined by the bicinchoninic acid method (Sigma–Aldrich).

3.1.1. Precision Intra- and inter-assay variation of the sandwich ELISA was investigated by assaying two positive controls (controls A and B: faecal supernatants with high and low PxES-Ag concentrations, respectively) 20 times within a series of measurements and in 20 different runs. CVs varied from 6.5% to 11.4% within a series of measurements, and the CVs regarding inter-assay precision from 7.2 to 9.3% (Table 1).

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A. Benito, D. Carmena / Acta Tropica 95 (2005) 9–15

Table 1 Validation of the sandwich ELISA for the detection of Echinococcus granulosus coproantigens (precision of the assay) Intra-assay precision n Mean PxES-Ag (␮g ml−1 ) S.D. CV (%) a

Inter-assay precision

Aa

Ba

Aa

Ba

20 1.228

20 0.448

20 1.252

20 0.428

0.144 11.4

0.030 6.5

0.020 7.2

0.032 9.3

Positive controls.

3.1.2. Linearity and recovery measurements To determine the accuracy of the sandwich ELISA, we used the linearity of dilution of positive controls method (Daly et al., 1991). Three different positive faecal supernatants (with high, medium and low PxES-Ag concentrations, respectively) were assayed undiluted and after two-fold, and four-fold dilution. The results are shown in Table 2, indicating a satisfactory linearity of the sandwich ELISA assay. From the obtained concentrations, recovery percentages were calculated. For supernatant 1, recovery ranged from 90 to 108.3%, for supernatant 2 from 91.6 to 111.1%, and for supernatant 3 from 93.8 to 100.4%.

Fig. 1. Receiver operating characteristic curve of the sandwich ELISA for the detection of Echinococcus granulosus coproantigens. Each point represents the relationship between sensitivity and specificity (considering autopsy results as gold standard) for the mean ± different S.D. values (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5) from the faecal supernatants of helminth-free dogs. The best cut-off point (arrowed) is the one located nearest to the top left of the curve. Table 3 Summary of the diagnostic evaluation of the sandwich ELISA with positive faecal supernatants (dogs infected with E. granulosus) and negative supernatants Echinococcus-free dogs infected with Taenia spp. (n = 15), dogs with non-taeniid helminths (n = 82) and dogs free of helminth infections (n = 66) Autopsy results

3.2. Diagnostic evaluation of the sandwich ELISA A receiver operating characteristic (ROC) curve was constructed in order to facilitate the optimisation of the ELISA cut-off value (see Fig. 1). The cut-off value was defined as the mean absorbance of the faecal supernatants from helminth-free dogs plus two standard deviations. This value corresponds to a PxES-Ag concentration of 0.24 ␮g ml−1 . The sensitivity of the assay at this cut-off point was 78.4%, and the specificity was 93.3% (Table 3). Sandwich

ELISA + ELISA −

Positive (n = 37)

Negative (n = 163)

Total (n = 200)

29 (78.4%) 8 (21.6%)

11 (6.7%) 152 (93.3%)

40 (20%) 160 (80%)

Autopsy has been considered as reference method.

ELISA results with the different faecal supernatants tested, expressed in ␮g ml−1 of detected PxES-Ag, are shown in Fig. 2. PxES-Ag concentrations detected in the positive faecal samples from dogs infected

Table 2 Validation of the sandwich ELISA for the detection of Echinococcus granulosus coproantigens. Linearity and recovery percentages Dilution

1:1 1:2 1:4

Supernatant 1

Supernatant 2

Supernatant 3

TV

OV

Rec%

TV

OV

Rec%

TV

OV

Rec%

0.012 0.006 0.003

0.013 0.005 0.002

108.3 90 90

0.36 0.18 0.09

0.33 0.2 0.086

91.6 111.1 95.5

1.296 0.648 0.324

1.301 0.651 0.304

100.3 100.4 93.8

TV: theoretic values of antigen (␮g ml−1 ); OV: observed values of antigen detected (␮g ml−1 ); Rec%: recovery percentage.

A. Benito, D. Carmena / Acta Tropica 95 (2005) 9–15

Fig. 2. Sandwich ELISA for the detection of Echinococcus granulosus coproantigens. Mean optical densities have been plotted against the different study groups. Dotted line represents cut-off point at mean ± 2 S.D. from the supernatants of helminths-free dogs. Solid lines represent group means.

with E. granulosus ranged from 0.24 to 24 ␮g ml−1 , and eight samples (21.6%) were negative in the ELISA assay. Only three faecal supernatants from dogs with non-taeniid helminths (3.6%) and eight samples from helminth-free dogs (21.6%) were positive in the sandwich ELISA. No false-positives were found among the 15 samples from dogs infected with Taenia spp. Positive and negative predictive values were 72 and 95%, respectively, and the diagnostic efficiency was 90.5%. In addition, the sandwich ELISA detection limit was estimated in 5.12 ng of PxESAg ml−1 .

4. Discussion Development of ELISA assays for the detection of parasite antigens in faecal samples (coproantigens) has brought important improvements on the diagnosis of intestinal echinococcosis in dogs: (i) positive ELISA results were obtained during the prepatence period as early as 7 days p.i. in experimental infections (Ahmad and Nizami, 1998); (ii) ELISA values decrease to negative levels 2–4 days after the elimination of Echinococcus worms (Jenkins et al., 2000); and (iii) ELISA results correlated well with the worm burden in the dog intestine (Craig et al., 1995; Ahmad and Nizami, 1998). In this paper, we present a double-antibody sandwich ELISA for the detection of E. granulosus

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excretory–secretory products in faeces for the immunodiagnosis of intestinal echinococcosis in dogs. The assay sensitivity has been enhanced by utilizing the rabbit purified IgG fraction anti-PxES-Ag conjugated to biotin as secondary antibody. Avidin/biotin-based assays are very useful tools in parasite immunology, and the system has been widely used to improve the sensitivity of ELISA, western-blot and immunohistochemistry techniques. The method has been successfully assayed in the immunodiagnosis of different human parasitosis, such as schistosomiasis (Salah et al., 2000), filariasis (Ramaprasad and Harinath, 1995), and trichinellosis (Nunez et al., 2000). As far as we know, this is the first time that the avidin/biotin system has been used in the immunodiagnosis of intestinal echinococcosis in dogs. Furthermore, we used ES-Ag from protoscoleces as this antigenic source has been demonstrated to higher sensitivity values than those obtained with somatic antigens (Fraser and Craig, 1997; Deplazes et al., 1999). The ES-Ag used for this study was obtained from the first 50 h of culture of protoscoleces, as parasites maintained for this time exhibit a high survival rate (>85%), assuring minimal contamination with somatic products (Carmena et al., 2002). In an attempt to improve the ELISA sensitivity, faecal supernatants were assayed at 1:2 dilutions, considerably more concentrated than those used by other authors (Deplazes et al., 1992; Ahmad and Nizami, 1998). The risk of infection for the personnel that manipulate the samples has been minimised by treating the faeces with 5% formaldehyde, which has not altered the native structure of the coproantigens (Allan et al., 1992). In addition, 5 mM EDTA was added in order to avoid antigen degradation by enzymatic activities. With regard to the validation of the sandwich ELISA, the values obtained with the intra- and interassay precision and the recovery measurements demonstrated the reproducibility of the technique. After analyzing the ROC curves, the cut-off value was established at a PxES-Ag concentration of 0.24 ␮g ml−1 . At this level, the overall sensitivity of the assay was 78.4% and the average specificity was 93.3%. Specificities ranging from 91 to 100% have been reported by several groups (Allan et al., 1992; Deplazes et al., 1992, 1994; Craig et al., 1995; Malgor et al., 1997; el-Shehabi et al., 2000). Some of these authors found a close association between the par-

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A. Benito, D. Carmena / Acta Tropica 95 (2005) 9–15

asite burden and the diagnostic sensitivity of the assay, in both experimental and natural infections. Deplazes et al. (1994) obtained a sensitivity of 29% when dogs harboured less than 100 worms, but this value reached 92% when dogs had infections with more than 100 worms. In that study, the overall sensitivity was 63%. Similarly, el-Shehabi et al. (2000) found a sensitivity of 87.5% in dogs with burdens less than 20 worms, but of 100% when dogs were infected with more than 100 worms. These results demonstrate that the amount of ES-Ag detected in faeces by ELISA is directly proportional to the worm burden in the dog gut (Craig et al., 1995; Ahmad and Nizami, 1998). According to this consideration, ELISA false negative results obtained in our study may correspond to faecal samples from dogs infected with a low number of worms. Interestingly, no false positive reactions have been detected when faecal supernatants from dogs infected with Taenia spp. were analyzed. This genus is considered the main source of antigenic cross-reactivity in the Echinococcus immunodiagnosis (Christofi et al., 2002). Finally, the limit detection of the sandwich ELISA was set at 5.12 ng of PxES-Ag ml−1 of supernatant, appreciably lower than 13.5 ng ml−1 reached by Allan et al. (1992) using somatic antigens, or 7 ng ml−1 reported by Malgor et al. (1997) using the monoclonal antibody EmA9 from E. multilocularis. It is important to take into consideration that sensitivity and specificity values vary in function of the selected cut-off value, depending on the kind of study to carry out. For epidemiological purposes, elevated sensitivities are highly desirable, since positive samples can be confirmed afterwards using more specific techniques, like PCR (Fraser and Craig, 1997; Deplazes et al., 1999). In summary, we described here a double-antibody sandwich ELISA for the detection of E. granulosus excretory–secretory products in dog faecal samples. The accuracy of the assay has been improved using a biotinylated secondary antibody and purified PxES-Ag. The combined levels of sensitivity and specificity achieved are highly satisfactory, taking into account the large panel of faecal samples that were tested. These features made this technique an especially valuable tool for the immnunodiagnosis of intestinal echinoccocosis in dogs, allowing its use in surveillance and control programs of this zoonosis.

Acknowledgements The authors are grateful to Prof. Jorge A. Guisantes for the opportunity to carry out this work at the Laboratory of Parasitology, Faculty of Pharmacy, University of the Basque Country, Spain. The authors thank Dr. Sonja Kock and Dr. David Guiliano (Department of Biological Sciences, Imperial College London, UK) for their critical revision of the manuscript. This work was financially supported by a grant from the Health Department of the Basque Government, Spain. Miss Aitziber Benito was a recipient of a PhD studentship from de Ministry of Education and Science, Spain.

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