[PubMed] [Google Scholar] 29. From these results, the recombinant antigen is definitely highly specific for toxocariasis and may provide more reliable diagnostic results than other methods. Toxocariasis is an important zoonosis caused by the infection of humans with ascarid nematode larvae of from dogs and from pet cats (10, 24). Once the embryonated eggs are accidentally ingested from the sponsor animal, the larvae hatch in the small intestine and migrate through the somatic organs. Lanraplenib In humans, two types of larva migrans syndromes have been recognized: visceral larva migrans (4) and ocular larva migrans (20). Recent studies suggest that the disease regularly assumes the features of a syndrome comprising chronic weakness, abdominal pain, numerous indications of allergy, and hypereosinophilia (9, 29). Toxocariasis has been proposed as a possible etiology in various neurologic syndromes (15, 28). The analysis of human being toxocariasis currently depends on immunological examinations because it is extremely hard to detect an infective larva(e) in biopsy samples. In most immunological checks, excretory-secretory antigens from second-stage larvae (TES) have been used conventionally (7, 11). Western blotting (14) and, more recently, ToxocaraCHEK (1) have been used, both of which detect immunoglobulin G against TES. However, since cross-reactivities have been reported for these procedures when the TES for some helminthic infections are used (8, 11, 13, 14), the development of an antigen more specific than TES has been attempted. Recently, we developed a recombinant second-stage larva antigen related to the 30-kDa protein of the TES secreted by infective larvae and tested its specificity as an antigen with limited numbers of serum samples from helminthiasis individuals (30). In the present study, the specificity of the recombinant antigen has been evaluated by comparing it with TES in an enzyme-linked immunosorbent assay (ELISA) using serum samples from patients infected with a wide variety of helminths. MATERIALS AND METHODS Preparations of TES and recombinant antigen. For the preparation of TES, the protocol of de Savigny was revised (6). The tradition medium (RPMI 1640) for second-stage larvae was collected every 3 to 4 4 days, pooled, and centrifuged to precipitate all debris. The producing supernatant was filtered through a 0.2-m Supor Lanraplenib Acrodisc 32 syringe filter (Gelman Sciences) into a Spectrapor dialysis tube (molecular weight cutoff, 6,000 to 8,000; Spectrum Medical Industries Inc.). The perfect solution is was dialyzed against 250 quantities of chilled sterile Lanraplenib distilled water at 4C until the phenol red disappeared. After dialysis, the supernatant was concentrated with a vacuum concentrator, reconstituted with sterile distilled water, and then kept in aliquots at ?20C. The recombinant antigen was prepared by the protocol explained previously (30). Briefly, expression of the recombinant antigen in bacteria was induced by adding isopropyl–d-thiogalactopyranoside at a final concentration of 0.4 Rabbit polyclonal to AGBL5 mM Lanraplenib Lanraplenib at 37C for 3 h. The induced cells were disrupted by sonication in 20 mM Tris-HCl, pH 8.0, containing 100 mM NaCl and 1% Triton X-100. The insoluble recombinant protein was solubilized in 8 M urea in 20 mM Tris-HCl, pH 8.0, containing 100 mM NaCl and then purified on TALON metallic affinity resin (Clontech). The recombinant antigen was eluted with 50 mM imidazole and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12). Protein content was measured from the Bradford method with a protein assay kit (Bio-Rad). The antigen was kept at ?80C until use. Human being serum samples. A total of 153 serum samples from individuals with confirmed helminthiasis verified parasitologically and/or clinically were examined. Most samples were also serologically positive for homologous parasite antigens except in two instances of capillariasis. Since the antigen from was not available, TES had to be used in these instances. Serum samples from patients infected with.