An alternate technique for isolation of Toxocara canis excretory-secretory antigens

The aim of the present study was to test the effectiveness of a sausage-casing membrane for dialysis of Toxocara excretory-secretory antigens (TES). The protein concentrated by the tested membrane was compared with that obtained using a Sigma commercial membrane, as were the protein fractions found by polyacrylamide gel electrophoresis. Standard positive and negative serum samples were evaluated in an ELISA immunoassay, and equivalent data were obtained in all steps, indicating that the sausagecasing membrane is efficient, besides being less expensive to process.


INTRODUCTION
Toxocariasis is a zoonotic disease caused by ascarid nematodes of the genus Toxocara.Human infection occurs by accidental ingestion of embryonated eggs of T. canis or T. cati, intestinal parasites of dogs and cats respectively, found in soil and sand contaminated with the faeces of these animals (Beaver, 1956).
Enzyme-linked immunosorbent assay (ELISA) has proven an excellent tool in the diagnosis of toxocariasis, used in combination with clinical and laboratory data.In Brazil, laboratory diagnosis is performed only by certain health services, because of the high cost and limited availability of commercial kits (Rubinsky-Elefant et al., 2006b).
As an antigen for serology, Toxocara excretedsecreted antigens (TES) obtained from second-stage Toxocara canis larvae have proved to be the best choice because of their specificity (De Savigny, 1975).These antigens can be obtained in vitro from larvae maintained in culture medium, free of macromolecules, which produce material with antigenic activity.The TES antigen is composed of different molecules of protein that are excreted by the larva or released from its surface.Its performance and composition can be influenced by differences in the techniques used to maintain the larvae.The top five TES molecules separated by polyacrylamide gel electrophoresis have 32, 55, 70, 120, and 400 kDa (Rubinsky-Elefant et al., 2001).The antigen production process involves a dialysis step that is required to eliminate the components of the culture medium.The dialysis membrane is obtained from laboratory supply companies, and is relatively expensive.The purpose of this study was to test the effectiveness of a low-cost commercially available sausage-casing membrane, for dialysis in the production of TES antigen.

Production of TES antigen
The TES antigen was produced according to De Savigny et al. (1979), as modified by Rubinsky-Elefant et al. (2006a).
Eggs of T. canis were obtained by dissecting adult female specimens obtained from the São Paulo Center for Zoonoses Control.To promote the development of infective larvae, eggs were incubated in 2% formalin for approximately 1 month, at 28 °C with daily stirring.After incubation, the eggs were repeatedly washed with saline and centrifuged for three minutes at 2,000 rpm to remove the formalin.Subsequently, 5% sodium hypochlorite was added for five minutes to lyse the chitin-protein layer, which was then washed with saline solution.To release the larvae, the material was placed in a flask containing glass beads, with Eagle medium plus gentamicin, at 37 °C, and subjected to gentle mechanical agitation.After a few minutes, this material was filtered in a modified Baermann apparatus, and the larvae were collected in sterile tubes and cultured in Eagle medium at 37 °C.
The excretion and secretion products of the larvae were collected weekly by drawing up the supernatant of the cultures.The protease inhibitor 0.2 M PMSF (phenylmethylsulfonyl fluoride) was added to the supernatant, and the mixture was stored at -20 ºC.Subsequently, the supernatant samples were concentrated 50 to 100 times in an Amicon device with a YM10 membrane (Amicon Company, Cleveland, USA) and divided into two aliquots of approximately 7.0 mL used to test the effectiveness of the prepared commercial sausage-casing membrane (Trip cellulose, size 34USA, Viscofan Company, Brazil) compared with the Sigma (St. Louis, CA, USA) membrane (D-9777, retention over 12,000 Da) for dialysis in distilled water.

Preparation of the sausage-casing membrane (SCM) for use in dialysis
The membrane was boiled in a solution of distilled water with 5% neutral detergent to remove hydrophilic and lipophilic dirt.After five minutes of boiling, the membrane was rinsed several times with distilled water to eliminate the detergent.

Evaluation of the sausage-casing membrane in the dialysis process
Using 7.0 mL aliquots of the antigen solution, the dialysis was carried out using either a Sigma membrane (SM) or a sausage-casing membrane (SCM), in order to compare protein retention.The protein content was estimated (Lowry, 1951) after the dialyzed material had been centrifuged at 15,000 rpm for 30 minutes and filtered through a 0.22 mm Millipore membrane.

Evaluating the effectiveness of the TESscm
The effectiveness of the antigens obtained for diagnostic purposes was also tested by ELISA, using samples of two negative serum samples control, one limiar reactive serum sample (LRS), one titred positive serum sample control, and ten serum samples from patients (diluted from 1/80 to 1/10,240-two-fold serially diluted).The serum samples were previously absorbed with antigens of Ascaris suum to test for anti-Toxocara spp.by the ELISA method (Rubinsky-Elefant, 2001).

RESULTS AND DISCUSSION
After dialysis, the protein concentrations obtained were 1.45 mg/ mL for TESsm and 2.02 mg/mL for TESscm.Therefore, the amount concentrated in TESscm was 39.3% higher than that in TESsm.
The ELISA serological test results, using plates sensitized either with TESscm or TESsm antigen, both produced equivalent data to the standard positive serum (Table I) and the serum sample from patients (Table II).
The comparative analysis between the membranes for dialysis in water showed that the sausage-casing membrane (SCM) was able to retain the main protein fractions that comprise the TES antigen.This result is evident when one observes the similarity between the absorbances of the positive serum control, and the titers of the serum obtained with plates coated with Toxocara excretory-secretory antigens TESsm and TESscm.Although serum no. 9 showed different titers in the two plates, a difference of one titer is not significant, and we can therefore conclude that the reaction was excellent.
Few public-health services in Brazil presently provide a laboratory diagnosis of toxocariasis (Figueredo et al., 2005;Paludo et al., 2007;Rubinsky-Elefant et al., 2008) However, epidemiological studies show a high prevalence of anti-T.canis in several regions of the country (Alderete et al., 2003;Damian et al., 2007;Prestes-Carneiro et al., 2008;Colli et al., 2010).In most cases, migration of infected larvae through the tissues of humans does not result in clinical manifestations (Bass et al., 1983;Shantz, 1989).However, it can cause fever, hepatomegaly, splenomegaly, respiratory symptoms, muscle pain, and damage to the eyeball (Glickman et al., 1985;Taylor et al., 1988).Complications involving asthma for example, are common in patients treated in pulmonology services in Brazil.
There are some difficulties in reaching a laboratory diagnosis of this zoonosis, such as the low sensitivity and specificity (Barros et al., 2003) and high cost (Rubinsky-Elefant et al., 2006b) of commercial diagnostic kits.Some studies have described methodological modifications that significantly improved, quantitatively and qualitatively, the obtention of T. canis larvae (Smith, 1989;Alcantara Nevesa et al., 2008).However, none of these reduced the costs involved.The sausage-casing membrane is inexpensive (approximately 100 times cheaper than the Sigma membrane).

CONCLUSION
The sausage-casing membrane was able to replace the Sigma membrane in the dialysis process, and could also be used in the stage of concentrating the TES antigen, replacing the Amicon process.Therefore, the use of sausage-casing membrane may allow the production of TES antigens at a reduced cost, and will be valuable for laboratories with limited financial resources.

TABLE I -
Absorbances of the positive serum control in the Enzyme-Linked Immunosorbent assay (ELISA) with plates coated with Toxocara excretory-secretory antigens TESsm and TESscm

TABLE II -
Titres of the serum from patients in the Enzyme-Linked Immunosorbent Assay (ELISA) with plates coated with Toxocara excretory-secretory antigens TESsm and TESscm FIGURE 1 -Toxocara excretory-secretory antigens: TESsm: Sigma membrane, or TESscm: a sausage-casing membrane, in a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) with silver staining; MW-standard marker for molecular weight (kDa).