[A new ELISA method for the diagnosis of toxoplasmosis. Assay of serum IgM by immunocapture with an anti-Toxoplasma gondii monoclonal antibody]. / Une nouvelle méthode ELISA pour le diagnostic de la toxoplasmose. Dosage des IgM sériques par immunocapture avec un anticorps monoclonal anti-Toxoplasma gondii.

The diagnosis of acute toxoplasmosis is based exclusively on the detection of IgM anti-Toxoplasma antibodies. The principal of the immunoenzymatic test reported here is the capture of serum IgM antibodies which are detected indirectly by the sequential addition of antigen and a monoclonal antibody directed against the immunodominant epitope at the surface of the tachyzoite. This test combines the sensitivity of enzyme-based assays and the specificity of monoclonal reagents and represents an important contribution to the diagnosis of Toxoplasma gondii infection.

[A new agglutination reaction for the diagnosis of the developmental stage of acquired toxoplasmosis]. / Une nouvelle réaction d'agglutination pour le diagnostic du stade évolutif de la toxoplasmose acquise.

Agglutination of acetone treated toxoplasma (AC) is different from that one of formalin fixed parasites (HS). Sera from patients with a recently acquired ("acute") infection agglutinate both HS and AC parasites suspensions as well; contrary to sera from patients with past infection ("chronic stage") in which high titers of HS agglutination are often present, while the titres of AC agglutination are lower even negative. This is markedly observed in patients with local lesions (relapsing chorioretinitis, patients with AIDS and brain abscesses). The reason might be that different membrane toxoplasma antigens may induce the synthesis of agglutinating IgG. For example, antigens 35 KD and 27 KD described by E. Handman et al. The antibody specific for 27 KD is apparently present mainly during acute infection, contrary to the antibody specific for 35 KD which might be responsible of the high HS agglutination titre in sera from patients with chronic infection. Even if these hypotheses were not confirmed in the future, comparison of the titre in the HS and AC agglutination test might actually be helpful for practical diagnosis of the stage of toxoplasma infection.

Serodiagnosis of toxoplasma infection using a purified parasite protein (P30).

The major surface protein (P30) of Toxoplasma gondii has been purified by immunoabsorption with anti-P30 monoclonal antibodies linked to a glutardialdehyde activated affinity absorbant. SDS-PAGE analysis of the eluted material followed by silver staining showed only a single band of 30,000 mol wt. Western blotting using antibodies from a rabbit immunized with purified P30 against the total Toxoplasma extract separated by SDS-PAGE again revealed an unique antigen of 30,000 daltons. The presence of repeated epitopes within P30 was confirmed by a two-site/one-antibody radiometric assay with the purified protein. Sandwich ELISA procedures with purified P30 clearly demonstrated that all 37 tested patients with acute toxoplasmosis presented significantly high levels of IgM anti-P30 antibodies. In addition, all 40 tested patients with chronic toxoplasma infection also showed high IgG anti-P30 antibody levels. These findings represent an essential step for the development of new reagents for the diagnosis of toxoplasmosis.

Use of a monoclonal antibody in a double-sandwich ELISA for detection of IgM antibodies to Toxoplasma gondii major surface protein (P30).

A double-sandwich ELISA, developed for detection of IgM antibodies to the major surface protein of Toxoplasma gondii (P30), is proposed for the diagnosis of acute acquired toxoplasmosis. The method is based on the capture of serum IgM antibodies, which are revealed indirectly by the sequential addition of a Toxoplasma extract and a beta-galactosidase-conjugated anti-P30 monoclonal antibody. All 57 patients tested with serological characteristics of recently acquired toxoplasmosis showed high levels of IgM anti-P30 antibodies. In addition, 5 out of the 24 patients with chronic toxoplasmosis and all 7 patients with a clinical acute infection in which the classical IgM serology was negative, also presented significant anti-P30 IgM antibodies. Patients with either rheumatoid factor or antinuclear antibodies were all negative. In view of its simplicity, specificity and sensitivity, this method is recommended for the current diagnosis of T. gondii infection.

Cytophilic IgE on human blood and tissue eosinophils: detection by flow microfluorometry.

Flow microfluorometry (FMF) was used to investigate the presence of cytophilic Ig (IgE or IgG) and the proportion of Fc receptor (Fc epsilon R or Fc gamma R)-bearing eosinophils among eosinophils from 21 hypereosinophilic patients. In a large majority of the cases, it was possible to detect cytophilic IgE significantly associated with serum IgE levels. Moreover, when lung and blood eosinophils were compared, the proportion of occupied Fc epsilon R was significantly increased on lung eosinophils, whereas very few cells had cytophilic IgG. This work provides further evidence that cytophilic IgE is not restricted to cells with high affinity Fc epsilon R, but can also be detected on the cell populations with low affinity IgE receptors. These findings support the view that eosinophils can act as effector cells in immediate hypersensitivity reactions and in diseases associated with increased IgE production and hypereosinophilia.

The use of monoclonal antibodies in studies of filarial parasite antigens.

The strength of the hybridoma technology in providing probes for the study of parasite immunology is obvious. The use of monoclonal antibodies should greatly expedite the task of the identification and the isolation of antigens which are relevant to host protection, of immunodiagnosis and of immunopathology. We have produced a series of monoclonal antibodies against Brugia malayi which causes lymphatic filariasis in man. One of these monoclonal antibodies (IgM isotype) can detect circulating antigens in the sera of individuals and of laboratory animals infected with B. malayi by radioimmunoprecipitation-PEG assay. This monoclonal antibody can bind to the relevant epitope of the circulating antigen or to the antigenic determinants of the immune complexes that remained exposed in the complex. A few monoclonal antibodies raised against B. malayi were selected, one of which gave positive fluorescence reaction on the surface of microfilariae. This particular monoclonal antibody showed such biological activities as conferring resistance to circulating microfilariae and inducing cell-mediated killing of microfilariae in vitro. The same monoclonal antibody was able to identify antigenic determinants (of mol. wt, 110 Kd) on the surface of B. malayi microfilariae which may be involved in effector mechanisms related to the development of transmission inhibiting immunity in lymphatic filariasis.

Different monoclonal antibodies against the component 5 specific for Trypanosoma cruzi.

Three murine monoclonal antibodies (I-35/67, II-190/30, III-160/18) produced by immunization against total epimastigote extract or component 5-enriched fractions of Trypanosoma cruzi (Tehuantepec strain) have been demonstrated to recognize the component 5 specific for T. cruzi. By immunofluorescence studies, one of these monoclonal antibodies (I-35/67) was shown to bind to the epimastigote cell surface, and the two others (II-190/30 III-160/18) were preferentially directed against internal subcellular organelles. Immunoprecipitation using these monoclonal antibodies followed by SDS-PAGE analysis, has led to identification of four molecules with apparent molecular weights of 72 Kd, 51 Kd, 43 Kd, and 24 Kd in T. cruzi epimastigotes. Potential uses of these monoclonal antibodies in serological tests and immunochemical analysis of target antigens are discussed.

[Production of monoclonal antibody anti-antigen 5 for Trypanosoma cruzi]. / Production d'un anticorps monoclonal anti-antigène 5 de Trypanosoma cruzi.

Monoclonal antibodies against Trypanosoma cruzi have been produced by fusion of Sp 2/0 myeloma cells and splenic lymphocytes from BALB/c Mice immunized with a T. cruzi antigenic fraction. Antigenic studies of these monoclonal antibodies have led to the identification of IgG 1 antibody against the component 5 specific for T. cruzi (cellular clone II 190/151). These monoclonal antibodies will give rise not only to new diagnostic and epidemiological perspectives but will also aid in achieving a better understanding of the biological role of this antigen.