Deglycosylation of Excretory-Secretory Antigens of the Second-Stage Larvae of Toxocara cati Improves Its Efficacy in the Diagnosis of Human Toxocariasis

Background Toxocariasis is an important zoonotic infection, especially in tropical areas. One of the significant challenges in the serodiagnosis of human toxocariasis is the cross-reaction of Toxocara antigens with other parasites due to their relatively similar glycan structures. Removing the glycan structure from Toxocara excretory-secretory (TES) antigens may increase the efficacy of these antigens in the diagnosis of toxocariasis. The current study aimed to assess the efficacy of deglycosylated Toxocara cati excretory-secretory (dTES) antigens for the serodiagnosis of human toxocariasis. Methods Toxocara ES antigens were prepared from T. cati second-stage larvae and deglycosylated using sodium hydroxide (NaOH). The TES antigens, along with the dTES antigens, were used in an ELISA as well as a western blotting system for the detection of anti-Toxocara antibodies. Sera samples collected from 30 confirmed cases of toxocariasis, 30 patients with other diseases, and 30 healthy subjects were evaluated by both systems. Results The sensitivity of TES and dTES ELISA for the diagnosis of human toxocariasis was 96.67% (95% CI = 82.78–99.92) and 93.33% (95% CI = 77.93–99.18), respectively, while the specificity of dTES (88.33%; 95% CI = 77.43–95.18) increased significantly compared to the TES (80.00%; 95% CI = 67.67–89.22). The sensitivity of both antigens was 100% (95% CI = 88.43–100) by the western blotting system. Moreover, the specificity of TES and dTES antigens was 95% (95% CI = 86.08–98.96) and 98.33% (95% CI = 91.06–99.96), respectively, when using the western blotting system. Conclusion Results of the current study indicate that the chemical removal of the glycan epitopes of T. cati ES antigens significantly reduces cross-reactivity rates with other parasitic infections. Considering the findings of the present study, the dTES antigens seem to be suitable antigens for the serodiagnosis of human toxocariasis.


Introduction
Toxocara canis and Toxocara cati are two common nematodes in dogs and cats. Toxocariasis, a neglected cosmopolitan infection, is caused by the infective larvae (L2) of T. canis and T. cati [1]. Te high infection of Toxocara in dogs and cats, as the defnitive hosts of the parasite, and the increasing presence of these popular animals close to the human settlements (whether as pets or strays) are the two main facts for the widespread worldwide distribution of toxocariasis [2,3]. Findings of meta-analysis studies indicate that the seroprevalence trend of toxocariasis is increasing worldwide, especially in tropical countries such as Iran [4][5][6][7]. On the other hand, based on a recent meta-analysis study in Iran, the mean prevalence rate of toxocariasis in dogs and cats is estimated at 24.2% and 32.6%, respectively [8].
According to the life cycle of the parasite, the transmission routes of toxocariasis are the accidental ingestion of infective eggs from contaminated soil, vegetables, or the consumption of raw or undercooked meat of paratenic hosts, such as chicken, pigs, and cow, containing encapsulated infective larvae [9][10][11]. Te migration of Toxocara larva through the bloodstream to various organs of the human body, including the liver, lungs, retina, and even the central or peripheral nervous system causes failure or dysfunction in the involved organs. Human toxocariasis has been presented as visceral larva migrans (VLM), ocular larva migrans (OLM), neurotoxocariasis, and covert toxocariasis. Covert toxocariasis is the most common form of the disease, with only general clinical or paraclinical manifestations such as hypereosinophilia, hyperbillirubinemia, and pneumonia (e.g., fever, shortness of breath, and cough) [1]. Terefore, the symptoms of toxocariasis are not always so obvious for the fnal diagnosis.
Accurately diagnosing of toxocariasis is always one of the most difcult issues for clinicians. Te best method for diagnosis of toxocariasis is the identifcation of Toxocara larvae in the tissue biopsy, but since this method is extremely invasive, the serological methods such as an enzyme-linked immunosorbent assay (ELISA) and western blotting using Toxocara excretory-secretory (TES) antigens, along with clinical symptoms and laboratory fndings (hypereosinophilia and high levels of specifc IgE), are considered reliable methods for the diagnosis of toxocariasis [12,13]. Te sensitivity and specifcity of the serological methods difer due to the type of the antigen used for diagnosis. According to diferent studies and regardless of the type of antigen used, the range of sensitivity and specifcity in the serodiagnosis of human toxocariasis by ELISA has been reported to be between 60% and 100% and 36% and 100%, respectively [1].
Since the currently available assays have cross-reactivity with some helminths that have antigenic components similar to TES antigens, the development of a reliable serological assay with high sensitivity and specifcity seems necessary [14][15][16]. Te structure of TES antigens is composed of highly immunogenic glycoproteins that act as the important components in stimulating the host's immune response against the larval stage [17].
Te secreted glycoproteins of Toxocara larvae are mainly characterized by mucin and lectin structures [18]. Te molecular weights of TES glycoprotein fractions range from 15 to 400 kDa, and some of them have already been characterized [12,19,20]. Te fractions of 32 and 120 kDa are rich in N-glycans and O-glycans, respectively [21][22][23]. It has also been documented that the fraction of 70 kDa of TES antigens is glycosylated [24].
Analysis of antigenic components of helminths shows that glycoprotein or glycolipid is the predominant antigenic structure in these parasites [25]. Previous studies indicated that the similar epitopes of helminth antigens, such as glycan structures, act as a signifcant source of cross-reactivity in the serodiagnostic methods, especially among similar phylogenetic parasites [16,[26][27][28][29][30][31]. One of the main reasons for the false positivity in serodiagnosis of toxocariasis is the similarity of some epitopes of Toxocara spp. with other helminths [30,32]. A study by Roldan et al. [16] showed that deglycosylation of TES antigens could minimize the cross-reactivity with phylogenetically related parasites and subsequently improve the serodiagnosis of toxocariasis, especially in terms of specifcity.
As mentioned before, the prevalence of T. cati is higher than that of T. canis in Iran. On the other hand, the presence of cats as a pet is more prevalent among Iranians. Terefore, it seems that T. cati has a more signifcant role in human toxocariasis, at least in Iran. Te present study aimed to evaluate the efcacy of deglycosylated TES (dTES) antigens in the diagnosis of human toxocariasis, using ELISA and western blotting methods.

Production of T. cati Excretory-Secretory (TES) Antigens.
TES antigens were prepared from the T. cati second-stage larvae (L2) as described by Zibaei et al. [19] with a few modifcations. Briefy, T. cati female worms were obtained from the intestine of necropsied stray cats (Shiraz, Iran). Te female worms were placed in a 2.5% formalin/ringer solution for approximately four weeks at 25°C until T. cati eggs were embryonated. Embryonated eggs were decoated by sodium hypochlorite solution (6-14% active chlorine) at room temperature (RT) for 30 min and then were washed three times with sterile phosphate-bufered saline (PBS) to eliminate the efect of chlorine. After washing, over 90% of the eggs were hatched by an ultrasonic water bath for 5 seconds. Te released larvae were collected using a cell strainer (40 μm) under aseptic conditions. Te recovered larvae were cultivated in the RPMI 1640 medium (Sigma-Aldrich Chemie GmbH, Germany) containing L-gluta MAX, HEPES 25 mM, sodium bicarbonate, penicillin/ streptomycin (100 IU, 100 μg/ml) and amphotericin B (2.5 μg/mL) with 5% CO 2 at 37°C. Te culture supernatant containing the TES antigens was collected and then replaced with fresh medium every week. Te TES antigen was dialyzed against distilled water overnight at 4°C. Te dialyzed TES antigens were lyophilized and their protein content was determined by the NanoDrop 2000°C spectrophotometer (Termo Scientifc) based on the manufacturer's instructions.

Production of Deglycosylated T. cati Excretory-Secretory
(dTES) Antigens. Chemical deglycosylation of TES antigens through β-elimination was performed according to the method described by Roldan et al. [16]. Briefy, the TES antigens were treated with 100 mM sodium hydroxide (NaOH) at 37°C for 4 h. After that, the reaction was neutralized by adding an equimolar concentration of hydrochloric acid (HCl). Te chemical deglycosylation procedure was investigated using carbohydrate and protein staining methods, followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) of dTES antigens.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE).
Te dTES antigens were separated according to molecular weight using SDS-PAGE on a 15% (w/v) polyacrylamide gel. In brief, dTES antigens were diluted in 2X sample bufer (1% 2-mercaptoethanol, 1% SDS, 16% 1 M Tris pH 6.8, 20% glycerol, and 0.2% bromophenol blue) and incubated at 100°C for 10 minutes. Te prepared antigens were separated by SDS-PAGE using a Mini-PROTEAN III apparatus (Bio-Rad Laboratories, Hercules, CA). Te samples were run at a voltage of 60 V for 30 min and then with a constant voltage of 90 V until the tracking dye (bromophenol blue) reached the bottom of the gel. A marker with known molecular weights ranging from 10 to 245 kDa (SinaClon, Iran) was also used in each run.

Carbohydrate and Protein
Staining. To analyze the protein and carbohydrate structures in the dTES antigens, SDS-PAGE gels were stained with Coomassie Brilliant Blue R-250 and periodic acid-Schif (PAS), respectively. Te Coomassie blue staining was performed for the detection of protein based on the procedure of Wang et al. [33] with a few modifcations. Te polyacrylamide gel was fxed in a fxing solution (10% v/v acetic acid, 10% v/v methanol, and 40% v/v ethanol) for 1 h at 4°C. Te fxed gel was immersed in a stainless-steel tank containing the staining solution (0.125% Coomassie Brilliant Blue R-250, 5% acetic acid, and 45% ethanol) and gently shaken for about 4 h at RT in the dark. Finally, the gel was put into the destaining solution (5% acetic acid and 45% ethanol) for 1 h. Ten it was replaced with the same fresh solution every hour until the background of the gel was destained.
Te periodic acid-Schif (PAS) staining was applied to ensure the absence of carbohydrate residuals in the dTES antigens according to the method described by Roldan et al. [16]. In brief, the gel fxation was performed using 25% isopropanol and 10% acetic acid overnight at 4°C. After washing with 7.5% acetic acid for 15 min, the polyacrylamide gel was soaked in 4% periodic acid for 1 h at 4°C, and then put into the undiluted Schif reagent for 1 h at 4°C in darkness. Te gel was immersed three times in 0.5% potassium metabisulphite for 10 min and washed with 7.5% acetic acid.

Serum Samples.
Tirty serum samples were collected from toxocariasis patients, diagnosed based on the serological method using the commercial ELISA kit (NOVALISA ™ T. canis IgG, NOVATEC Immundiagnostica GmbH, Germany). Moreover, 60 serum samples were obtained as controls from 30 healthy individuals and 30 patients with other diseases, including ascariasis (n � 3), strongyloidiasis (n � 4), trichostrongylosis (n � 3), hydatid cyst (n � 4), fascioliasis (n � 4), visceral leishmaniasis (n � 3), toxoplasmosis (n � 3), malaria (n � 3), and patients with fever of unknown origin (FUO) (n � 3). All serum samples were tested using the commercial ELISA kit to confrm that the control samples were negative. In addition, the samples of other diseases were confrmed by at least one of the following methods: stool examination (ascariasis, strongyloidiasis, trichostrongylosis, and fascioliasis), blood smear (malaria), and serological tests (visceral leishmaniasis and toxoplasmosis). Te hydatid cyst patients were diagnosed by the combination of two methods, computed tomography scan and a serological assay. FUO patients were also confrmed by clinical and paraclinical fndings.

Enzyme-Linked Immunosorbent Assay (ELISA).
Te serum samples were evaluated for the detection of anti-Toxocara IgG antibodies by ELISA, using TES and dTES antigens [34]. Te 96-well fat-bottom microplates (Nunc, Roskilde, Denmark) were coated with 5 μg/ml of TES or dTES antigens (100 μl/well) in 0.05 M carbonate-bicarbonate bufer (coating bufer, pH 9.6) and incubated overnight at 4°C. Te plates were washed three times with PBS containing 0.05% Tween 20 (PBST) (washing bufer); after, each of the wells was blocked with 3% skimmed milk in PBST (blocking solution) and incubated for 2 h at RT. Te plates were then washed as before, and diluted serum samples (1 : 100 in PBST) were added to each well (100 μl/well) and incubated for 1 h at RT. Ten, horseradish peroxidase-conjugated antihuman IgG (Sigma-Poole, UK) at 1 : 4000 dilution in PBST was added to the wells and incubated for 1 h at 37°C. After washing the plates three times, a chromogen/substrate (0.4 mg/ml OPD (o-phenylenediamine dihydrochloride), 0.3% H 2 O 2 in 0.1 M citrate bufer, pH 5.6) was added to the plate. Ten, the enzymatic reaction was stopped, after 30 min, by the addition of 1 M sulfuric acid (100 μl). Finally, the optical density (OD) of serum samples was determined at a wavelength of 492 nm using an automatic microplate reader (ELX800, BioTek, USA). Te cut-of value was calculated by the mean OD of the negative controls plus two standard deviations. To ensure the experiment results, all sera were tested twice.

Western Blotting.
After separating the protein fractions of TES and dTES antigens by SDS-PAGE, the proteins were transferred to a nitrocellulose (NC) membrane (Schleicher and Schuell BioScience, Germany) for western blotting, using a constant voltage of 30 V overnight at 4°C. Ten, the transfer of the protein fractions from the acrylamide gel to NC was confrmed by Ponceau S staining [19]. Te NC membranes containing the protein fractions were cut into strips with the same width (3 mm) and blocked with 5% skimmed milk in PBST for 2 h. Te strips were washed three times (each for 5 min) with PBST and then incubated with diluted serum samples (1 : 100 in blocking bufer) for 2 h. After washing as before, horseradish peroxidase-conjugated antihuman IgG (at 1 : 4000 dilution in PBST) was added to each strip and incubated for 2 h. Te NC strips were washed three times in PBST and incubated with diaminobenzidine tetrahydrochloride (DAB) chromogenic substrate prepared in 50 mM Tris-HCl, pH 7.6 in the presence of 0.1% H 2 O 2 for 5 min. Finally, the enzymatic reaction was stopped using tap water. Te serum samples of toxocariasis as positive control and healthy person as negative control, along with a standard protein molecular weight marker, were used in each run.

Statistical Analysis.
Te ROC (receiver operating characteristic) curve analysis was applied to evaluate the sensitivity and specifcity of TES and dTES antigens in the detection of anti-Toxocara spp. IgG antibodies. Te Kappa index was also used to assess the diagnostic agreement between the tests. All statistical analyses were performed Journal of Tropical Medicine 3 using SPSS software (ver. 20), and a value of less than 0.05 was considered statistically signifcant.

Ethical Considerations.
Ethical approval was obtained from the ethics committee of the Shiraz University of Medical Sciences (code: IR.SUMS.REC.1400.240). All participants completed and signed the informed consent form before being involved in the study.

Chemical Deglycosylation of TES Antigens.
Tree fractions with low molecular weight, including 15, 26, and 38 kDa, were detected from TES antigens by SDS-PAGE using 15% gel and Coomassie blue staining (Figure 1(a)). After deglycosylation of the TES antigens with 100 Mm NaOH for 4 h at 37°C, the dTES antigens revealed only one protein band with a molecular weight of 15 kDa (Figure 1(a)). After treatment of the TES antigens with NaOH in the mentioned condition, the PAS staining of SDS-PAGE gel showed that the carbohydrate structure of the TES antigens had been removed successfully. Terefore, bands at the dTES lane were diminished (Figure 1(b)).
Out of 60 control serum samples, TES antigens had the false positive reaction in 12 cases and dTES antigens in seven cases ( Figure 2). Table 1 shows the results of ELISAd using TES and dTES antigens, and Table 2 shows the sensitivity, specifcity, positive predictive value, and negative predictive value of ELISA using TES and dTES antigens in the diagnosis of toxocariasis.
Te kappa agreement rate of 0.864 was estimated between ELISAs using TES and dTES antigens, and an almost perfect agreement was seen. In addition, the kappa agreement coefcient between the ELISA of TES and dTES compared to the commercial ELISA kit was obtained 0.702 and 0.807, respectively.
Samples from healthy subjects did not yield any reaction with the fractions of TES and dTES antigens (Table 1). Western blotting using TES antigens was carried out with sera of 30 patients with other diseases, and reactivity was seen in 3 serum samples, including ascariasis (with 15 and 26 kDa fractions), trichostrongylosis (with 15 kDa fraction), and fascioliasis (with 38 kDa fraction) ( Figure 5(a)). However, the specifcity of western blotting using dTES antigens was signifcantly improved, and only one serum sample of ascariasis patients reacted against the single fraction (15 kDa) of dTES antigen ( Figure 5(b)). In Table 2, the sensitivity, specifcity, positive predictive value, and negative predictive value of western blotting using TES and dTES antigens were shown. When the results of western blotting using TES antigens were compared with those of western blotting using dTES antigens, a high degree of agreement was observed (kappa � 0.952). Kappa analysis on the results of western blot showed an acceptable agreement of 0.902 for TES and 0.924 for dTES antigens compared to the commercial ELISA kit.

Discussion
Diagnosis of human toxocariasis is based on the detection of anti-Toxocara antibodies by serological methods such as ELISA and western blotting [12,35]. Te main antigens currently employed for the diagnosis of human toxocariasis are TES antigens, but the glycan structures of TES antigens cause cross-reactivity in the serodiagnosis of human toxocariasis [16,26,31]. Tis cross-reaction is problematic in regions where other parasitic diseases are endemic. It has been documented that cross-reactivity in the serodiagnosis of parasitic infections is attributed to reactivity with glycan structures shared among the parasite's antigens [36,37]. Chemical deglycosylation of antigens through β-elimination is a method that can eliminate such glycan structures and may improve specifcity in the diagnosis of parasitic diseases. Lorenzo et al. [27] reported that the glycan structures of Anisakis simplex antigens are the source of cross-reactivity, and this cross-reaction can be reduced by β-elimination of O-glycans from these antigens.
In this study, three fractions of ES antigens of T. cati (15,26, and 38 kDa) were identifed by SDS-PAGE. Zibaei et al. [19] detected a range of 20 to 150 kDa fractions of ES antigens of T. cati by SDS-PAGE. In the study of Rolden et al. [16], fve fractions of ES antigens of T. canis (32,45,55,70, and 120 kDa) were indicated by SDS-PAGE. In general, the diferences in the detected fractions of TES antigens by SDS-PAGE may be due to the species of Toxocara (T. cati and T. canis), the developmental stage of the parasite (egg, larva, and adult), the preparation conditions of the antigen, and the type of antigen (ES and crude).
Deglycosylation can be done by two methods: enzymatic or chemical method. Enzymatic deglycosylation is the most common method of removing glycan structures, but the high cost of used enzymes is the major disadvantage of this method [38]. Whereas, chemical deglycosylation is a simpler and cheaper method and NaOH is one of the most efective used chemical reagents that release O-glycans and N-glycans from the glycoproteins at diferent concentrations [16,39]. Terefore, chemical deglycosylation of TES antigens is simpler, efcient, and practical. In the current study, chemical deglycosylation of ES antigens of T. cati secondstage larvae was performed according to Rolden et al. [16], who investigated the deglycosylation of TES antigens with NaOH at diferent concentrations, temperatures, and times. Te best conditions for deglycosylation were obtained with 100 mM NaOH for 4 h at a temperature of 37°C. In our study, after deglycosylation of the TES antigens in the mentioned conditions, no glycan structures were detected by PAS staining. Among the three detected fractions of TES antigens (15,26, and 38 kDa) by SDS-PAGE, only a protein structure with a molecular weight of 15 kDa was identifed after chemical deglycosylation of the TES antigens, whereas, Rolden et al. [16] detected a fraction of approximately 26 kDa. Te detection of fractions with a smaller molecular weight after deglycosylation is probably due to the hydrolysis of fractions with a higher molecular weight or their fragmentation into smaller subunits. Page and Maizels [40] detected a polypeptide with a molecular weight of about    15 kDa after the deglycosylation of the 120 kDa fraction of TES antigens. Lorenzo et al. [27] reported that the degradation rate of the protein core during treatment with NaOH mainly depends on the nature of the glycoprotein. In the present study, the 15 kDa fraction of dTES antigens was not characterized, so it is not known that this fraction is the same as the 15 kDa fraction of TES antigens. However, the improvement of specifcity of the western blotting method after the deglycosylation process indicates that the obtained fraction with a molecular weight of 15 kDa can be a residual component of other hydrolyzed glycoproteins in this study.
In this study, we evaluated the usefulness of ELISA using dTES antigens for the diagnosis of toxocariasis and compared the results with ELISA using TES antigens. Our Table 2: Sensitivity, specifcity, positive predictive value, and negative predictive value of ELISA and western blotting using Toxocara excretory-secretory (TES) and deglycosylated Toxocara excretory-secretory (dTES) antigens for serodiagnosis of human toxocariasis.    fndings showed that the dTES-based ELISA was not able to detect the anti-Toxocara antibodies in the serum of two toxocariasis patients, which means the elimination of the glycan structures of TES antigens reduced the sensitivity of the dTES-based ELISA compared to the TES-based ELISA. However, when ELISA was performed on the serum samples of healthy controls and patients with other diseases, high specifcity was seen for the dTES-based ELISA compared to the TES-based ELISA. Tree samples from the healthy controls and four samples from patients with other diseases (ascariasis, trichostrongylosis, strongyloidiasis, and fascioliasis) gave a positive reaction by the ELISA using the dTES antigens, and overall, the dTES-based ELISA demonstrated satisfactory specifcity compared to the TES-based ELISA for the diagnosis of human toxocariasis. In the study by Roldan et al. [16], the sensitivity of ELISA using the TES and dTES antigens was 100%, while the specifcity of dTES-based ELISA (97.7%) was signifcantly higher than TES-based ELISA (94.1%). Generally, it is expected that the specifcity of ELISA using dTES antigens will increase due to the elimination of glycan structures that cause cross-reactivity. Te results showed that the dTES antigens provide higher specifcity for the serodiagnosis of toxocariasis. Variation in sensitivity and specifcity of ELISA can be justifed by the diferences in Toxocara species used for producing TES antigens, as well as the type and number of examined sera.
ELISA is a standard and common method for the serodiagnosis of human toxocariasis, but western blotting can be considered a confrmatory method due to its higher sensitivity and specifcity [12]. Terefore, western blotting is a better method for diagnosis of toxocariasis compared to ELISA, since specifc proteins are separated from a mixture of proteins in the western blotting method, and the ability of antibody detection increases by the specifc antigens [41]. In this study, as expected, the sensitivity and specifcity of western blotting increased for both antigens (TES and dTES) compared to ELISA. We found three fractions (15,26, and 38 kDa) of TES antigens as the immunodominant proteins, with 30 toxocariasis cases reacting with at least one fraction. In addition, all sera of toxocariasis patients reacted against a single fraction with a molecular weight of 15 kDa of dTES antigen. Terefore, TES and dTES antigens were able to detect anti-Toxocara spp. antibodies in all sera of toxocariasis patients, and the sensitivity of western blotting using both antigens was 100%. Diferent studies have shown that western blotting decreases the cross-reactivity and thus increases the specifcity in the serodiagnosis of human toxocariasis compared to ELISA [41]. We performed western blotting using TES and dTES antigens on sera from patients with other parasitic infections, such as ascariasis, strongyloidiasis, trichostrongylosis, hydatid cyst, fascioliasis, visceral leishmaniasis, toxoplasmosis, and malaria, for evaluation of the specifcity of TES and dTES antigens. Sera from patients with ascariasis and trichostrongylosis reacted with the 15 kDa fraction of TES antigens. However, only one serum from patients with ascariasis was reacted with the single fraction of dTES antigen (15 kDa), and the specifcity of western blotting using the dTES antigens was signifcantly improved. Although fractions with high molecular weight were not identifed in this study, fractions with low molecular weight (15,26, and 38 kDa) had acceptable diagnostic performance. In agreement with our results, several studies reported that fractions of Toxocara spp. ES antigens with low molecular weight are more specifc and are often not recognized by nonspecifc IgG antibodies [13,16,19,42,43].
Te study by Watthanakulpanich et al. [44] regarding the performance of IgG subclass antibodies (IgG1-4) in serodiagnostic assays of human toxocariasis showed that the IgG3 subclass was more specifc in detecting the TES antigens than other subclasses, and reduces the false positivity. Te IgG2 subclass gave the greatest sensitivity, since it recognises carbohydrate epitopes of TES antigens. Considering the study mentioned above, the detection of anti-Toxocara IgG3 antibodies using dTES antigen may reduce the cross-reactivity rate and improve the test specifcity.

Conclusion
To the best of our knowledge, this is the frst report on the use of deglycosylated Toxocara cati excretory-secretory (dTES) antigens in the serodiagnosis of toxocariasis. Our study demonstrated that the elimination of glycosidic components, as epitopes responsible for cross-reactivity, improves the specifcity of ELISA and western blotting systems for the serodiagnosis of human toxocariasis. Te use of dTES antigens with a larger sample size and more various helminth infections is suggested for further confrmation of the fndings of this study. In addition, the simultaneous use of deglycosylated ES antigens of T. canis and T. cati may improve the diagnostic efcacy of ELISA or western blotting for the serodiagnosis of human toxocariasis.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Disclosure
Tis study was the subject of the Ph.D. dissertation for Mr. Ali Pouryousef.