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.
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.
Giraldo
M
M
Department of Biochemistry and Immunology, UFMG, 31270-910 Belo Horizonte, MG, Brazil.
Cannizzaro
H
H
Ferguson
M A
MA
Almeida
I C
IC
Gazzinelli
R T
RT
D000208:Acute Disease; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood / Q000276:immunology; D000953:Antigens, Protozoan / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000276:immunology; D002847:Chromatography, Agarose; D002908:Chronic Disease; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology*; D007075:Immunoglobulin M / Q000097:blood / Q000276:immunology*; D013058:Mass Spectrometry / Q000379:methods; D014122:Toxoplasma / Q000254:growth & development* / Q000276:immunology / Q000302:isolation & purification; D014123:Toxoplasmosis / Q000276:immunology / Q000469:parasitology*
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D007074:Immunoglobulin G; D007075:Immunoglobulin M