Monoclonal Antibodies Identify NewToxoplasma gondiiSoluble Antigens

Abstract

In order to characterize Toxoplasma gondii antigens, we have produced a panel of monoclonal antibodies specific for the parasite. A total of 22 hybridomas were derived from the spleen cells of mice immunized either with a 100,000 g supernatant of a sonicate from the RH strain (called F3), or chronically infected with the Wiktor or the 76K strain. Except for one hybridoma producing an IgM, all the hybridomas derived from mice immunized with F3 produced IgG1 antibodies while those obtained from chronically infected mice produced antibodies belonging to the IgG2b, IgG2a and IgM subclasses. Western-blot analysis showed that the panel of monoclonal antibodies defines at least 7 distinct antigens or antigen families. An antigen of apparent Mw 25 kD present exclusively in the 100,000 g supernatant of the T. gondii sonicate was recognized by the majority of monoclonal antibodies derived from mice immunized with the F3 fraction. Two other antigens of apparent Mw 27 kD and 29 kD present in the soluble and insoluble fractions of the sonicate were also identified. Monoclonal antibodies against the previously described 21 kD and 31 kD surface antigens and belonging to the IgG2a but also to the IgG1 subclasses were able to mediate lysis of the parasite in the presence of human non immune serum. The 22 monoclonal antibodies did not identify antigenic differences between the two independently isolated RH and Wiktor strains.

Phenotypes Associated with Knockouts of Eight Dense Granule Gene Loci (GRA2-9) in Virulent Toxoplasma gondii

“…Parasites were isolated from freshly lysed cultures and resuspended in Laemmli buffer. Proteins were separated by 13% SDS-PAGE (non-reduced conditions), transferred to nitrocellulose membranes and detected using the following primary antibodies: mAb TG17.179 anti-GRA2 (1:15,000) [ 16 ], mAb T6.2H11 anti-GRA3 (1:10,000) [ 52 ], rabbit anti-GRA4 (1:10,000) [ 53 ], mAb TG17.113 anti-GRA5 (1:5,000) [ 16 ], rabbit anti-GRA6 (1:20,000) [ 53 ], mAb BATO 214 anti-GRA7 (1:15,000) [ 54 ], mAb 3.2 anti-GRA8 (1:10,000) [ 55 ], rabbit anti-GRA9 (1:2,500) [ 56 ], rabbit anti-actin (1:10,000) [ 57 ] or mAb TG054 anti-SAG1 (1:15,000) [ 58 ] (antibodies purchased from the Biotem company, Apprieu, France or kindly provided by L. D. Sibley, Washington University School of Medicine, Saint-Louis, MO; D. Jacobs, Innogenetics-Fujirebio Europe N.V., Ghent, Belgium; G.E. Ward, University of Vermont College of Medicine, Burlington, VT; W. Daübener, Heinrich Heine Universität, Düsseldorf, Germany).…”

A GRA1 DNA Vaccine Primes Cytolytic CD8+T Cells To Control AcuteToxoplasma gondiiInfection

“…The chemiluminescent compound Supersignal (Pierce, Rockford, Ill.) was used as the substrate according to the manufacturer's instructions. For detection of native GRA1 in T. gondii IPB-M-and IPB-G-derived TLA, a monoclonal antibody (MAb) against GRA1, MAb BATO 35 (36), was used at a dilution of 1:1,000, and pools of sera from three seropositive pVR1020-GRA1-vaccinated C3H mice or pVR1020-vaccinated C3H mice, diluted 1:300, were also used. These primary antibodies were incubated overnight at 4°C with the membrane strips.…”

Evaluation of Recombinant Dense Granule Antigen 7 (GRA7) ofToxoplasma gondiifor Detection of Immunoglobulin G Antibodies and Analysis of a Major Antigenic Domain

“…MAb BATO 214 directed to GRA7 (24) was obtained from ascites fluid. A MAb directed to an epitope in the mouse tumor necrosis factor (mTNF) leader peptide was also available and was purified from culture supernatant.…”