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Journal of Clinical Microbiology, April 2000, p. 1453-1460, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Fractionation of Membrane Components from Tachyzoite Forms of Toxoplasma gondii: Differential Recognition by Immunoglobulin M (IgM) and IgG Present in Sera from Patients with Acute or Chronic Toxoplasmosis

Mónica Giraldo,1,2 Hélia Cannizzaro,1,2 Michael A. J. Ferguson,3 Igor C. Almeida,3,dagger and Ricardo T. Gazzinelli1,2,*

Department of Biochemistry and Immunology, UFMG, 31270-910 Belo Horizonte, MG,1 and Laboratory of Chagas Disease, Centro de Pesquisas René Rachou, FIOCRUZ, 30190-002 Belo Horizonte, MG,2 Brazil, and Department of Biochemistry, University of Dundee, DD1 5EH Dundee, Scotland, United Kingdom3

Received 22 June 1999/Returned for modification 1 September 1999/Accepted 8 December 1999

Tachyzoite forms of Toxoplasma gondii were subjected to a sequential organic solvent extraction, which allows fractionation of membrane components according to their degrees of hydrophobicity, yielding three fractions named F1 (most hydrophobic) to F3 (least hydrophobic). Fractions F2 (80.85% specificity and 86.95% sensitivity) and F3 (89.36% specificity and 93.61% sensitivity) gave the best results, being preferentially recognized by immunoglobulin M (IgM) and IgG in sera from patients with acute and chronic toxoplasmosis, respectively. Improved scores of specificity (100%) and sensitivity (100%) were achieved when a secondary antibody against human IgG1 instead of total IgG was employed to measure the reactivity of IgG antibodies with the F3 fraction. To purify tachyzoite antigens recognized by human IgM or IgG antibodies, the F2 or F3 fraction was loaded onto an octyl-Sepharose column and eluted with a propan-1-ol gradient. The main antigen(s) recognized by IgM or IgG eluted in a single peak from the octyl-Sepharose resin loaded with either F2 (30 to 50% propan-1-ol) or F3 (15 to 35% propan-1-ol), respectively. These semipurified fractions gave improved scores when used to detect T. gondii-specific IgM (95.7% specificity and 81.8% sensitivity) or IgG (100% specificity and 93.75% sensitivity) in an enzyme-linked immunosorbent assay. Further biochemical and immunological analyses of antigens partially purified from F2 and F3 indicate that glycoinositolphospholipids are preferentially recognized by IgM, whereas proteins of approximately 30 to 40 kDa are recognized by IgG, elicited during T. gondii infection in humans.

* Corresponding author. Mailing address: Laboratory of Chagas' Disease, Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima 1715, 30190-002 Belo Horizonte, MG, Brazil. Phone: (031) 295-3566. Fax: (031) 295-3115. E-mail: ritoga{at}dedalus.lcc.ufmg.br.

dagger Present address: Department of Parasitology, Biomedical Science Institute, University of São Paulo, São Paulo 05508-900, Brazil.

Journal of Clinical Microbiology, April 2000, p. 1453-1460, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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