Chimeric antigens of Toxoplasma gondii: toward standardization of toxoplasmosis serodiagnosis using recombinant products

Abstract

We have evaluated the diagnostic utility of six antigenic regions of the Toxoplasma gondii MIC2, MIC3, M2AP, GRA3, GRA7, and SAG1 gene products, assembled in recombinant chimeric antigens by genetic engineering, in order to replace the soluble, whole-cell tachyzoite extract in serological assays. Serum samples from 100 adults with acquired T. gondii infection and from 30 infants born to mothers with primary toxoplasmosis contracted during pregnancy, of whom 20 were congenitally infected, were included. Immunoglobulin G (IgG) and IgM antibodies against epitopes carried by chimeric antigens were measured by performing parallel enzyme immunoassays (recombinant enzyme-linked immunosorbent assays [Rec-ELISAs]), and the results obtained by standard commercial assays with the whole-cell Toxoplasma antigen and assays with the chimeric antigens were compared. Our results demonstrate that IgG and IgM Rec-ELISAs with individual chimeric antigens have performance characteristics comparable to those of the corresponding commercial assays. Furthermore, we show that IgM-capture assays based on chimeric antigens improve the ability to diagnose congenital toxoplasmosis postnatally compared with the ability to diagnose congenital toxoplasmosis by the use of standard assays. The use of recombinant chimeric antigens is effective in distinguishing T. gondii-infected individuals from T. gondii-uninfected individuals and shows that immunoassays based on recombinant products could provide the basis for standardized commercial tests for the serodiagnosis of toxoplasmosis.

FIG. 1.

Schematic representation of chimeric antigens EC2 and EC3. The antigenic regions of the T. gondii MIC2, MIC3, SAG1, GRA3, GRA7, and M2AP gene products that were used to construct the GST-EC2 and GST-EC3 fusion proteins are shown.

FIG. 2.

Antigenic properties of individual protein fragments within the EC2 and the EC3 chimeric antigens. The immunoreactivities of GST-MIC2, GST-MIC3, GST-SAG1, and GST-EC2 (A) or GST-GRA3, GST-GRA7, GST-M2AP, and GST-EC3 (B) with IgG antibodies from T. gondii-infected individuals were analyzed by use of whole serum or serum after depletion of antibodies against combinations of antigen fragments (MIC2/MIC3 depletion, MIC2/SAG1 depletion, etc.). w/o, without.

FIG. 3.

Immunoreactivities of the biotin-labeled chimeric antigens. (A) IgM Rec-ELISA performance with the biotin-labeled GST-EC2 and GST-EC3 antigens with Toxoplasma IgM-positive (C+) or IgM-negative (C−) serum samples (diluted 1:100) by using different antigen concentrations. (B) IgM Rec-ELISA performance with chimeric antigens (concentration, 500 ng/ml) assayed with different dilutions of a Toxoplasma IgM-positive serum. (C) Chimeric antigens (concentration, 250 ng/ml) were stored at 4°C intervals and assayed over time by the IgM Rec-ELISA. The results are expressed as the ratio of the OD measured with the Toxoplasma IgM-positive and -negative sera (OD C+ and OD C−, respectively).