Avidity analysis of the human immune response to a chitin binding protein of Toxoplasma gondii.

A 30-33 kDa electroeluted fraction of T. gondii tachyzoites improved discrimination between acute and chronic phase sera when used instead of the whole tachyzoite extract in an avidity-ELISA. In order to identify the components of these fractions, crude tachyzoite antigen was fractionated by anionic exchange chromatography. The 30-33 kDa antigen cluster eluted in the not-bound fraction could account for a large proportion of the antibody response against the 30-33 kDa electroeluted fraction. According to the N-terminal sequence data, this antigen fraction is composed mainly of SAG1 and another protein with high homology to chitin binding proteins from plants.

Identification of circulating antigens, including an immunoglobulin binding protein, from Toxoplasma gondii tissue cyst and tachyzoites in murine toxoplasmosis.

Here we describe the identification of Toxoplasma gondii circulating antigens in sera of BALB/c mice experimentally infected with either the virulent RH strain, or the cystogenic WTD1 strain or with an isolate from a human patient. The circulating antigens were identified by immunoblot in tachyzoite (RH strain) and in tissue cyst (ME-49 strain) crude antigens, using antibodies produced by immunisation of BALB/c mice with homologous sera from infected animals. The most relevant tachyzoite antigen identified are in the following four clusters of 109-94, 67-57, 35-31 and 28-21 kDa. Tissue cyst-specific circulating antigens, like the 18 kDa one, were detected in sera from mice infected with the cystogenic strains. These immune sera, after depletion of tachyzoite specific antibodies, recognised three tissue cysts antigens with Mr of 120, 79 and 48 kDa, and a cluster of antigens in the range of 68-53 kDa. We produced monoclonal antibodies by fusion of myeloma cells with lymphocytes from the mouse immunised with circulating antigens from the RH strain. One of the clones (3A11/H12) obtained, secretes IgG(1) and recognises a peptide epitope from a tachyzoite 67 kDa protein. This parasite protein also binds irrelevant mouse IgG(1) as well as immunoglobulins from other species. The reactivity with non-specific antibodies was inhibited by preincubation with 2% normal mouse and goat serum, while the reaction with the monoclonal antibody 3A11/H12 was not. Furthermore, a biotinylated F(ab')(2) of an irrelevant mouse IgG(1) did not show any reactivity while the F(ab')(2) of the monoclonal antibody 3A11/H12 reacts specifically with the 67 kDa antigen suggesting that this circulating antigen is a putative Fc binding protein.

Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA.

The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF) obtained from fertile (FC) and non-fertile cysts (NFC). Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC) and NFC (PNFC), respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates) were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates.

IgG recognizing 21-24 kDa and 30-33 kDa tachyzoite antigens show maximum avidity maturation during natural and accidental human toxoplasmosis.

We describe the avidity maturation of IgGs in human toxoplasmosis using sequential serum samples from accidental and natural infections. In accidental cases, avidity increased continuously throughout infection while naturally infected patients showed a different profile. Twenty-five percent of sera from chronic patients having specific IgM positive results could be appropriately classified using exclusively the avidity test data. To take advantage of the potentiality of this technique, antigens recognized by IgG showing steeper avidity maturation were identified using immunoblot with KSCN elution. Two clusters of antigens, in the ranges of 21-24 kDa and 30-33 kDa, were identified as the ones that fulfill the aforementioned avidity characteristics.

Use of specific antibody and circulating antigen serum levels in the hydatid immunodiagnosis of asymptomatic population.

Levels of specific antibodies (Ab) and circulating antigens (CAg) were tested by ELISA in sera from 115 surgically confirmed hydatid patients, 41 individuals exhibiting other parasitic and unrelated diseases and 69 healthy subjects. Addition of CAg data to Ab detection in this sera collection increased sensitivity from 85% (only Ab) to 89% (Ab + CAg). Combination of ultrasonography with Ab and CAg serology for diagnosis of asymptomatic population in endemic areas was analyzed. One field survey (163 persons) involved both blood extraction and ultrasonography to all the population. Three people exhibited cyst images and all of them were Ab positive, while 6 Ab and 1 CAg positive individuals exhibited no cyst image. Another survey (1620 persons) involved a selection of 85 subjects for serology according to ultrasound data and record of family hydatid history. Twelve per cent exhibited no hydatid image being serologically positive and 14% were serologically negative but exhibited cyst image. Ultrasonography and serology (Ab and CAg) should be used in combination to maximize the diagnostic yield in asymptomatic population.

High performance latex reagent for hydatid serology using an Echinococcus granulosus lipoprotein antigen fraction purified from cyst fluid in one step.

A lipoprotein fraction from fertile bovine hydatid cyst fluid (FBHCF) was isolated by affinity chromatography on acrylic-heparin. The heparin-binding lipoprotein fraction (HBLF) was proved to be free from host immunoglobulins and albumin. Immunoblotting with confirmed human hydatid sera showed that the major relevant diagnostic bands in FBHCF were also present in HBLF. HBLF or FBHCF were used to sensitize polystyrene latex particles. HBLF-latex showed both higher reactivity with positive hydatid human sera and higher batch-to-batch reproducibility than FBHCF-latex. Titration of sera from 119 surgically confirmed hydatid patients, 48 with other parasitic diseases, 6 with unrelated diseases and 37 healthy subjects was performed using HBLF-latex and conventional ELISA (using FBHCF). Sensitivity and specificity were 83 and 87% for ELISA and 87 and 88% for HBLF-latex. These results indicate that, while being simpler and faster to perform, the HBLF-latex has a similar diagnostic value to that of the conventional ELISA test.