Excretory-secretory antigens of T. canis (TcES), T. vitulorum (TvES), Ascaris lumbricoides (AlES) were prepared by the method described in Rajapakse et al. (1992). Necator americanus larval antigen (NaL) was prepared using the Harada-Mori culture technique (Ash and Orihel, 1987). The protein content of all antigen preparations was estimated by the Bicinchoninic acid protein assay kit (Sigma Chemicals, UK).

One dimensional polyacrylamide gel electrophoresis (PAGE) was carried out using Mini-PROTEAN II Electrophoresis Cells (Bio-Rad, USA). Briefly, 10% polyacrylamide gel with an acrylamide/bis ratio of 36.5 : 1 in the presence of 10% sodium dodecyl sulphate (SDS) supplemented with Temed (Sigma, T-9281) and ammonium persulfate in Tris-HCl buffer pH 8.8, according to Laemmli (1970). Molecular weight standard mixtures (MW 15,000-150,000, Sigma M-0671) were used for calibrating the gel. The antigen was diluted in a Tris (pH 6.8) sample buffer (0.1M Tris-HCl, 2% SDS, 10% glycerol, 0.2 M 2-mercaptoethanol and 0.1% bromophenol blue) and then loaded in the gel with 20 µg protein/lane. The electrophoresis was monitored using 0.1% bromophenol blue and the current was set at 30 mA. The protein fractions were visualized by staining with 0.10% Coomassie Brilliant Blue R 250 (Sigma, B-0149).

The relative molecular weights were calculated using prestained protein of standard molecular weight according to the relative electrophoretical mobility (RM), using the following equation:

The RM values (ordinate) were related to known molecular weights of the standard proteins (abscissa) in a semi-logarithmic graph to provide a basis for interpolation of the data from proteins of the Pe-antigen.

Gels with parasite antigen were electrophoretically transferred onto nitrocellulose sheets (0.22 µm) for immunoblotting according to the procedure described by Towbin et al. (1979). The transfer was performed in a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad apparatus) for 12 h in a constant current of 35 V in a transfer buffer (Tris-glycine-methanol). The nitrocellulose papers were blocked by a blocking solution with 5% non-fat dried milk in TBS-Tween (0.01 M Tris, 0.15 M NaCl, 0.05% Tween-20) and then incubated for 90 min with primary antibodies (pool of 10 serum samples from buffalo cows and calves) diluted to 1/50 in the blocking solution and 5% normal rabbit serum in a rotating homogenizer. Subsequently, the nitrocellulose was washed 3 times (15 min each) in TBS-Tween and milk solution. Specifically bound antibodies in all filters were detected with anti-bovine alkaline phosphatase conjugate (Sigma, A-7914) diluted 1 : 30,000 in the blocking solution for 90 min. After rinsing 3 times in TBS-Tween and milk solution, the blots were incubated at room temperature for about 10 min in enzymatic substrate (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium B).

Anti-TcES rabbit serum was prepared against TcES. Two New Zealand white rabbits (3-4 months old) were used. Antigen was adjusted to a protein concentration of 2 mg/ml in PBS and was emulsified with an equal volume of Freund's incomplete adjuvant (Sigma Chemicals, USA). Each rabbit was inoculated on the axillary region of all 4 limbs intramuscularly (0.25 ml at each site) at weekly intervals for 4 weeks. Before immunization 3 ml of blood was collected from each rabbit from the ear-marginal vein. Rabbits were bled weekly before every subsequent injection for serum collection. All serum samples were stored at -20℃ until use.

Test sera came from 20 patients with a history suggestive of visceral toxocariasis and positive on the T. canis L2 larval ES antigen ELISA (TES-ELISA). Sera from 20 healthy subjects that were negative on TES-ELISA were used as negative controls.

Sera were collected from 50 cats at animal clinics, where minimum attention had been given to anthelmintic medication by their owners. Samples were collected from the cephalic vein and, after separation, stored at -20℃ until used.

All antigens, namely TcES, TvES, AlES and NaL, were subjected to SDS-PAGE in a gel containing a molecular weight marker, followed by western blot against anti-TcES rabbit serum, as well as TES-ELISA positive human serum with appropriate conjugate in order to identify the T. canis species-specific antigen fraction we named TcES-57. All SDS-PAGE and western blots were repeated at least 3 times.

Two New Zealand white rabbits (3-4 months old) were used. The gel strip containing the 57 kDa band was crushed in PBS and mixed with an equal volume of Freund's incomplete adjuvant. Each rabbit was inoculated on the axillary region of all 4 limbs intramuscularly with 0.25 ml of the mixture at each site, at weekly intervals for 4 weeks. Pre-immunization blood was collected from the marginal ear vain, sera separated and stored at -20℃. Four 4 weeks after rabbits were bled (4 ml), serum was separated and stored at -20℃. Thereafter, the rabbits were given booster immunizations weekly for one month and were bled weekly, serum collected and stored at -20℃.

The following human serum samples were used in this assay: (a) 50 TES-ELISA positives, (b) 20 TES-ELISA negatives, (c) sera from 6 parasitologically proven (eggs in feces) A. lumbricoides infected patients, (d) sera from 3 parasitologically proven (eggs in feces) hookworm infected patients. In addition, sera from 50 cats were used. The assay was optimized by checkerboard titration. The microtitration plate wells were coated with anti-TcES-57 rabbit serum diluted 1/800 in carbonate buffer, pH 9.6. A hundred µl of antigen was added to each well and plates were incubated at 4℃ overnight. Following this plates were washed 3 times in phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS-Tween) pH 7.4. After the last wash, plates were inverted and blotted onto absorbent paper to empty the wells completely. The wells were post coated with 120 µl of PBS containing 1% bovine serum albumin and 2.5% sucrose and incubated at room temperature for 1 hr. A hundred µl of TcES antigen (diluted to 10 µg/ml of protein in PBS) was placed in each well and plates incubated for 1 hr at room temperature (RT). Plates were then washed 3 times in washing buffer and emptied as before. A hundred µl of diluted serum samples were added to the test wells in duplicate and incubated for 1 hr at RT. Following this the washing step was repeated to remove unbound serum. To assay specifically bound antibodies, wells were incubated for 1 hr at RT with 100 µl of peroxidase-conjugated either anti-human or anti-cat IgG as appropriate, diluted 1 : 250, and washed as before. Then, 100 µl of enzyme substrate o-phenylenediamine dihydrochloride and 3% hydrogen peroxide solution (Sigma Aldrich Corporation, India) was added. The enzyme hydrolysis of substrate was monitored. After an appropriate interval, as determined by the rate of reaction of a reference standard serum, hydrolysis was stopped by addition of 100 µl of 3 M H₂SO₄. Optical density (OD; ELISA values) was determined photometrically at 490 nm in an ELISA reader (Minireader 11, Dynatech Laboratories Inc.). Interpretation of ELISA results was based on OD values determined for the local population as described by Iddawela et al. (2003) as follows: < 0.2, no serological evidence of toxocariasis; 0.2-0.7, compatible with past infection or current light infection; > 0.7, compatible with recent infection.

The western blot using anti-TcES rabbit serum is shown in Fig. 2. When the nitrocellulose paper bound proteins were probed with anti-TcES rabbit sera, bands were seen in TcES, TvES, AlES and NaL lanes. Eleven bands were detected in the TcES lane. Of these, 5 bands were darkly stained and the others were lightly stained. The band at 57 kDa was prominent. In the TvES lane, 5 bands were detected with a prominent band at 28 kDa. In the AlES lane, one clear band at 21 kDa was detected, and in the NaL lane, one lightly stained band was detected at 66 kDa. Western blot of TcES, TvES, AlES and NaL antigens using serum of patients with clinical toxocariasis, who were seropositive on TES-ELISA, are shown in Fig. 2. In the TcES antigen lane, the bands were very similar to that of anti-TcES rabbit serum except that a band at 28 kDa was not seen and a band at 80 kDa was darkly stained. In the AlES lane, 2 bands were seen, one at 65 kDa that was darkly stained and the other at 21 kDa. In TvES lane, 5 bands were detected with human serum and 4 of the bands were very similar to that detected with anti-TcES rabbit serum, i.e., 48 kDa, 43 kDa, 32 kDa and 28 kDa. With human serum, a band at 80 kDa was detected, while with the rabbit serum, a 73 kDa band was seen. In the NaL antigen lane, 2 bands were seen, one prominent at 54 kDa and the other band at 66 kDa. The banding pattern was similar in high and low ELISA positive serum samples except that, in high positive serum samples, the bands were more darkly stained. No bands were seen on nitrocellulose paper with the control sera (pre-immunized rabbit serum and TcES-ELISA negative serum samples, conjugate and substrate control).

A single band at 57 kDa was seen in the TcES lane while no bands were detectable in the other lanes (Fig. 3).

Of the 50 TES-ELISA positive sera tested using the antigen TcES-57 on the double sandwich ELISA, 45 (90%) were positive. The 20 TES-ELISA negative sera remained negative on the TcES-57 double sandwich ELISA as well. Sera from patients infected with A. lumbricoides and N. americanus were negative with the TcES-57 double sandwich ELISA. None of serum samples from cats were positive in sandwich ELISA with TcES-57 captive primary serum. However, when TcES captive primary serum was used, 11 serum samples were positive in ELISA.