Evaluation of Dot-ELISA and enzyme-linked immuno-electrotransfer blot assays for detection of a urinary hydatid antigen in the diagnosis of cystic echinococcosis.

BACKGROUND: Several serological assays are used for detection of a hydatid antigen in serum for diagnosis of cystic echinococcosis (CE). However, it requires technical expertise and is associated with the risk of acquiring blood-borne infections. Of late, interests have been shifted to other body fluids like urine, saliva, tear drops as alternate specimens in the diagnosis of CE. AIM: The aim of the study was to evaluate the dot enzyme-linked immunosorbent assay (Dot-ELISA) and electro-immunotransfer blot (EITB) for detection of a hydatid antigen in the urine for diagnosis of CE.
MATERIALS AND METHODS: 100 ml of urine samples were collected from the patients with confirmed CE (n=30), patients with suspected CE (n=30), patients with other diseases (n=30) and healthy controls (n=30). A hydatid antigen in urine was detected by Dot-ELISA and EITB using only polyclonal antibodies raised against a complete homogenate hydatid (CHH) antigen in rabbits. RESULTS AND
CONCLUSIONS: The Dot-ELISA using polyclonal antibodies showed a sensitivity of 53.33% and specificity of 96.66%, whereas EITB showed a sensitivity of 46.66%. The Dot-ELISA and EITB employing polyclonal antibodies showed no significant difference in sensitivity (P=0.426). Hence, the Dot-ELISA being a simple procedure can be used for detection of a hydatid antigen in urine for diagnosis of CE.

INTRODUCTION

Cystic echinococcosis (CE), a zoonotic disease is asymptomatic in majority of cases. Diagnosis of CE by a serological assay could not differentiate between past or current infection and there are frequent occurrences of false negatives in surgically confirmed cases of CE.[12] Moreover, collection of serum requires technical expertise and disposable syringes and these methods are associated with the risk of acquiring blood-borne infections.

Recently, other body fluids like urine, saliva, and tear drops are used for the diagnosis of parasitic infections. Of these specimens, urine is increasingly being used as a specimen alternate to blood for the diagnosis of many parasitic infections.[3]

MATERIALS AND METHODS

Study groups

Samples were collected from the patients with surgically confirmed and ultrasound-proven CE (n=30), from presumptive CE (n=30), from patients with other diseases (n=30), and healthy controls. The informed consent was obtained from all human adult participants.

Specimens

Serum

0Five milliliters of venous blood was collected from patients and controls under aseptic precautions and allowed to clot. The serum was separated and stored in duplicate at –20°C till use. Sodium azide (0.05 mol/l) was used as preservative.

Urine

50 to 100 ml of urine samples were collected from each of the patient in sterile glass vials using aseptic techniques as well as controls. Urine samples from each patient were concentrated by ammonium sulfate precipitation.[4] The urine sample (50-100 ml) was precipitated by 100% saturation of ammonium sulfate. The precipitate was collected by centrifugation at 3000 rpm at 4°C in a cooling centrifuge for 30 min. The precipitate was then dialyzed against phosphate buffered saline (PBS) pH 7.2 at 4°C overnight. The clear dialysate was stored at –20°C till use.

Detection of hydatid antigens in serum and urine

Antigen source

A surgically removed human hydatid cyst was collected aseptically and checked for sterility. A complete homogenate of a hydatid antigen was prepared and used.

The hydatid antigens in the serum and urine samples were detected using the Dot-enzyme linked immunosorbent assay (Dot-ELISA) and electro-immunotransfer blot (EITB) with polyclonal antibodies. Polyclonal hydatid antibodies were raised against a complete homogenate hydatid (CHH) antigen as per the method.[5]

Dot-ELISA using polyclonal hydatid antibodies

The Dot-ELISA was evaluated by using polyclonal hydatid antibodies for the detection of antigens circulated in the serum and urine of the cases of CE and controls. The assay was performed to optimize the Dot-ELISA conditions to know the concentration of the coating antibody to be dotted and the dilution of serum and urine to be used in the assay. Based on checker board titration, optimum concentration of the coating antibody used was 2 μg/strip.[6]

The procedure of Dot ELISA is as follows:

A 2 μl (2 μg/strip) of affinity purified rabbit anti-hydatid IgG was dotted on the nitrocellulose membrane (NCM). The strips were air-dried for 30 min and washed with PBS-Tween three times. Then it is blocked by PBS 7.2 containing 2% BSA by incubating for 3 h at 37°C in a rocker shaker. Wash the strips with PBS-T three times and 100 μl of 1:10 dilution of the patient sera and 1:5 dilution of concentrated urine was added and incubated for 1.5 h at 37°C with constant shaking. The strips were washed with PBS-T three times and incubated with rabbit anti-hydatid IgG conjugated to peroxidase (in-house preparation) at a dilution of 1:400 (determined by checker board titration) with PBS 7.2 containing Tween-20 (0.1%) for 1 h at 37°C in dark with constant shaking.

In-house preparation of rabbit anti-hydatid IgG coupled to horse radish peroxidase:

Coupling of rabbit anti–hydatid IgG to horse radish peroxidase

A 2 ml of affinity purified anti-hydatid rabbit IgG was dialyzed overnight against sodium carbonate bicarbonate buffer pH 9.5. Then, a fresh 0.2 M solution of sodium meta-periodate (NaIO4) was prepared and then added as 0.1 ml of this solution to HRPO (2 mg in 0.5 ml double distilled water). The mixture immediately turned green. This mixture is kept on a shaker at room temperature for 20 min. It was then dialyzed against 1.0 mM sodium acetate buffer pH 4.4 at 4°C for 5 h with 4-5 changes. The pH was raised to 9.0-9.5 by adding 10 μl of 0.2 M sodium carbonate bicarbonate buffer pH 9.5 and immediately 500 μg (2 ml) of anti-hydatid rabbit IgG was added. This mixture was kept on a shaker for 2 h at room temperature. 100 μl of freshly prepared sodium borohydride solution was added and kept at 4°C. This conjugate was dialyzed overnight against 0.1 M borate buffer pH 7.4 at 4°C with 2-3 changes. Finally, bovine serum albumin (BSA) at a final concentration of 5 mg/ml was added and distributed in vials and stored at –20°C.

Determination of optimal dilution

The flat-bottomed microtiter plate was coated with a known concentration of 5 μg/well (a 100 μl volume) of the CHH antigen in PBS 7.2 per well and kept overnight at 4°C. The plates were washed with PBS-Tween 20 and non-specific sites were blocked with a blocking solution (0.15 M PBS 7.2 with 2% BSA) for 2 h at 37°C. The plates were washed three times with PBS-Tween 20. A 100 μl of optimally diluted positive and negative serum 1:1000 in PBS-Tween 20 was added in each well in duplicate and incubated at 37°C for 1.30 h. After washing, a 100 μl of doubling dilution (100,200,400,800,1600,2400) of rabbit anti-hydatid IgG-HRPO conjugate diluted in PBS-Tween 20 is added and incubated at 37°C for 1 h. Final washing was followed by addition of 100 μl of o-phenylenediamine (OPD) substrate and incubated at room temperature in the dark. The reaction was stopped by adding 50 μl of 2 M sulphuric acid. The OD was read using the ELISA reader at 450 nm (Labsystems, Finland). The conjugate in highest dilution which gave a maximum difference between antibodies coated and control wells was considered optimal dilution.[7]

The strips were washed and incubated with 3 mg of 3, 3′ diaminobenzidine (DAB) (Sigma, USA) in 5 ml of PBS containing 0.1% Tween-20 and 5 μl H2O2 for 15-20 min at 37°C in dark under constant rocking for the development of color. The reaction was stopped by washing with double distilled water. A brown-colored spot appeared within 2-5 min indicates the positive reaction.

EITB

EITB was done using polyclonal antibodies for detection of the hydatid antigen in serum and urine as follows:

Serum and urine samples were prepared at the concentration of 1:10 and 1:1 in sample dilution buffer and separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and denaturing conditions in a homogenous gel (12%).[8]

The separated serum and urine antigens were blotted onto NCM by using the blotting apparatus (Pharmacia Biotech, USA). The transfer was done at constant volt (100 volts) for 1 h transfer time and blocked by PBS 7.2 containing 2% BSA by incubating for 3 h at 37°C under constant rocking.[9]

The NCM strips were washed three times with PBS-T and incubated with 5 ml of 1:100 dilutions (in PBS-T) of polyclonal hydatid antibodies for 1.5 h at 37°C under constant rocking to detect any hydatid antigen.

After washing NCM strips three times, the goat anti-rabbit-IgG-HRP conjugated secondary antibody (Bangalore Genei, India) at a dilution of 1:1000 was used as per the manufacturer's instruction and incubated for 1 h at 37°C in dark under constant rocking in plastic tray.

The NCM strips were washed and incubated with substrate solution (6 mg of DAB) from Sigma, USA in 10 ml of PBS containing 0.05% Tween-20 and 10 μl H2O2) for 15-20 min at 37°C in dark under constant rocking.

The reaction was stopped by washing with double distilled water. The membrane was dried by soaking in between folds of filter paper and observed for reactive antigenic bands.

Control serum and urine concentrates and known complete hydatid antigens were also processed simultaneously for validity of the test.

Statistical analysis

Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and efficiency of the diagnostic methods were calculated as per the standard method.[10] Statistical analysis for test of significance and P-value calculation were done in Microsoft Excel sheet, Epi info 2001 and SPSS software.

RESULTS

Dot-ELISA

The results of Dot-ELISA using polyclonal antibodies for detection of a hydatid antigen in serum and urine of patients with CE and controls were shown in Table 1.

Table 1

Detection of a hydatid antigen in serum and urine of cystic echinococcosis cases and controls by dot-ELISA

EITB using polyclonal hydatid antibodies for the detection of a hydatid antigen in the serum and urine from CE patients and controls were provided in the Tables 2 and 3.

Table 2

Detection of a hydatid antigen in serum of cystic echinococcosis cases and controls by EITB

Table 3

Detection of a hydatid antigen in urine of cystic echinococcosis patients and controls by EITB

A total of five antigenic peptides of Mr 92, 40, 35, 30, and 24 kDa in serum samples and antigenic peptides of Mr 41, 35, 24, 14, and 10 kDa in urine were demonstrated by the EITB. Of these, three antigenic peptides each of serum (namely 92, 30, and 24 kDa) and urine (namely 24 kDa, 14 kDa and 10 kDa) were found to be of diagnostic value [Figures 1 and 2].

Figure 1

EITB for detection of a hydatid antigen in serum for cases and controls

Figure 2

EITB for detection of a hydatid antigen in urine for cases and controls

Comparative evaluation of Dot-ELISA and EITB

The sensitivity of serological test for the presence of antigen in the serum and in the urine for diagnosis of CE is compared using Dot-ELISA and EITB showed no significant difference (P=0.426) for detection of a hydatid antigen [Tables 4 and 5].

Table 4

Diagnostic evaluation of dot-ELISA for detection of a hydatid antigen in serum and urine of cases of cystic echinococcosis and controls

Table 5

Diagnostic evaluation of EITB for detection of a hydatid antigen in serum and urine of cases of cystic echinococcosis and controls

DISCUSSION

For the past two decades, urine specimen was increasingly considered as a good clinical sample for diagnosis by detecting specific urinary antigens in many parasitic diseases.[3] The presence of the hydatid antigen in the urine for diagnosis of CE was first reported from this laboratory by CIEP and Co-A using polyclonal antibodies.[1112]

The Dot-ELISA being a simple, rapid microassay which needs minute volume of reagents to perform, the test has a potential for the detection of a hydatid antigen in the serum. In the present study, the Dot-ELISA using polyclonal antibodies was able to detect circulating antigens in 18 of 30 confirmed CE cases. The sensitivity of the assay was found to be 60%. Result of the study was in agreement with the study by Romia et al., 1992. Low sensitivity was attributed to the small amounts of circulating antigens and/or immune complexes formation.[13]

The successful detection of urinary antigen may depend on kidney leakiness but it is not clear if it is free or complexed antigen that is being detected. The nature of antigen detected is of considerable interest. This antigenuria could result from a few sequestrated parasites that are dead or dying, may be secreted/excreted metabolic products of the parasites or could be produced by deformed parasite induced by chemotherapy.[14]

It has been found that it was necessary to concentrate excreted parasitic antigens in the urine samples to impart sensitivity, as antigens were less likely to be demonstrated in untreated urine specimens.[15] In the present study, the urine samples were concentrated successfully by the ammonium sulfate precipitation method for detection of the hydatid antigen for the diagnosis of CE.

The present study was first to standardize and evaluate the Dot-ELISA using polyclonal antibodies to detect parasite-derived antigens in the urine of patients actively infected with CE. The assay showed a sensitivity of 53.33%. Result of the present study showed a higher sensitivity when compared with the sensitivity of CIEP (29.68%)[11] and Co-A (47.5%)[12] for detection of the hydatid antigen in urine.

The Dot-ELISA detected an antigen in the urine from 7 of 30 surgically confirmed and ultrasound-proven CE patients whose serum were negative for the hydatid antigen by the Dot-ELISA using polyclonal antibodies. However, the assay did not detect antigen in 9 of 30 urine samples of surgically confirmed and ultrasound proven CE patients whose serum were positive for the hydatid antigen. The sensitivity of Dot-ELISA for serum hydatid antigens (60%) was nearly comparable with the urinary hydatid antigen (53.33%).

In the present study, EITB was used to analyze the circulating hydatid antigen in the serum and to the best of our knowledge, the first attempt made to detect the presence of the hydatid antigen in the serum for diagnosis of CE.

The results of EITB revealed five antigenic peptides of Mr 92, 40, 35, 30, and 24 kDa as immunodominant peptides. Of these, three antigenic peptides (namely 92, 30, and 24 kDa) were found to be non-reactive with individuals having other parasitic diseases.

In EITB, the Mr 24 kDa was recognized as the most common antigens not only by serum but also in urine of majority of surgically confirmed and ultrasound-proven CE patients. The presence of more specific and highly sensitive 24 kDa in the serum shows that the hydatid-specific serum antigen (24 kDa) was secreted from the parasite and reinforces the hypothesis that smaller macromolecules can be transported through a hydatid cyst wall.[16]

EITB also revealed a high-molecular-weight antigenic polypeptide of Mr 92 kDa in 5 of 30 (16.66%) CE confirmed patients sera. The sensitivity of Mr 92 kDa from serum and 14 kDa and 10 kDa from urine was lower and their possible presence might be due to the strain variations of Echinococcus granulosus which needs further study for validation. Such strain variations in E. granulosus strains of different animal hosts has also been observed by Rafiei and Craig.[17]

Results of the present study detected only low-molecular-weight polypeptides (24 kDa, 14 kDa and 10 kDa) in the urine by EITB. High-molecular-weight proteins were not observed in the present study which might be due to limitation in the renal filtration barrier or due to alteration in the structural conformation of the protein. Further studies are needed to determine whether hydatid specific antigen in urine appeared because of its smaller size and positive charge, passes the renal filtration more easily than high molecular weight antigens.

The EITB assay was not able to detect the hydatid antigen in urine of all the confirmed CE cases. As suggested by Gottstein,[18] some factors which have to be considered in the interpretation of this finding are (i) intact hydatid cyst may release small amount of antigen in circulation. This fact was supported by the presence of macromolecules such as host albumin and immunoglobulin (Ig) in 20% of cyst under in vitro conditions and this may pass only in to parasite after fissuring of the cyst wall (ii) no antigen may be found in serum or urine (iii) antigen released from the parasite may be mainly bound to an antibody thus forming immune complexes. This was also supported by report from D’Amelio et al.,[19] where they were able to detect immune complexes in patients with echinococcosis, but they were unable to identify free antigen (iii) the level of antigen (Ag) in the urine also depends on Echinococcus strain, the localization of cysts, the cyst wall structure, the speed and type of growth of the hydatid cyst, and possible other factors may influence antigen release from parasite.

CONCLUSION

Detection of a hydatid antigen is more useful as it helps in differentiating current infection from the past and also in post-surgical follow up of CE cases. Collection of urine is a noninvasive procedure that could be adopted as a good alternate specimen for diagnosis of CE. As there is no significant difference between these assays, the Dot-ELISA being a simple procedure could be used for detection of a hydatid antigen in serum and/or urine.

The EITB technique is a sophisticated procedure which needs highly skilled personnel, sophisticated instruments and expensive reagents to carry out the assay, is highly suitable for adaptation in a well-equipped reference laboratory for confirming the diagnosis of CE.