Serodiagnosis of recently acquired Toxoplasma gondii infection with a recombinant antigen

Abstract

A portion of a cDNA encoding a 35-kDa antigen from Toxoplasma gondii was cloned into the CKS expression vector and expressed in Escherichia coli. By using the enzyme-linked immunosorbent assay (ELISA), the recombinant protein (rP35 antigen) was examined for reactivity with immunoglobulin G (IgG) antibodies in the sera of pregnant women. Of these women, 41 had a toxoplasma serologic profile suggestive of recently acquired T. gondii infection (Sabin-Feldman dye test [DT] titers from 1:256 to 1:32,000, positive IgM ELISA titers from 2.3 to 9.7, positive IgA ELISA from 1 to >28, and acute patterns in the differential agglutination [AC/HS] test) (group I), and 50 women had a toxoplasma serologic profile suggestive of infection acquired in the distant past (low DT titers from 1:16 to 1:512, negative IgM ELISA titers from 0 to 0.8, and chronic patterns in the AC/HS test) (group II). The classification of acute or chronic profile was based on the individual's clinical history as well as the combination of the results of the toxoplasma serological profile. An additional group (group III) was composed of sera from 50 women who were seronegative for T. gondii antibodies in the DT. The results revealed that whereas 85.3% of women in group I had IgG antibodies that reacted with the rP35 antigen, only 8% of women in group II had IgG antibodies that reacted with the same antigen. In immunoblots, the rP35 antigen was recognized by IgG antibodies in a pool of sera from individuals with a toxoplasma serologic profile compatible with acute infection but not in a pool of sera from individuals with a serologic profile characteristic of a chronic infection. These results reveal that IgG antibodies against the P35 antigen are produced during the acute stage of the infection but are uncommon in the latent or chronic phase of the infection. Thus, the rP35 antigen may be a useful serologic marker to differentiate between recently acquired infection and that acquired in the more distant past.

FIG. 1

Immunoblots with rP35 protein (A) and immunoblots with the CKS control preparation (B). The rP35-CKS proteins had an approximate molecular size of 54 kDa (panel A, arrow). The CKS protein had an approximate molecular size of 34 kDa (panel B, arrow). In panel A, lane 1 was stained with amido black only and lane 2 shows reactivity of the rP35 with a monoclonal antibody to the CKS protein. Lanes 3, 4, and 5 show reactivity of the rP35 with pooled sera from individuals in group I, II, or III, respectively. Only antibodies in sera of group I individuals reacted with the rP35 protein. In panel B, lane 6 was stained with amido black and lane 7 with a monoclonal antibody to the CKS protein. Lanes 8, 9, and 10 show the reactivity of the CKS preparation with pooled sera from individuals in group I, II, or III, respectively. Antibodies in the sera of individuals in these groups did not react with the CKS protein, but weak cross-reactions occurred with other proteins in the CKS preparation (indicated with an arrow and a star).

FIG. 2

ELISA results in 41 sera from pregnant women with a serologic profile consistent with a recently acquired infection with T. gondii (group I). The hatched columns represent OD₄₉₀ results with the rP35 preparation, and the clear columns are results with the CKS control preparation.

FIG. 3

ELISA results of 50 sera from pregnant women with serologic profile consistent with a chronic infection (group II). The hatched columns represent OD₄₉₀ results obtained with the rP35 preparation, and the clear columns represent results obtained with the CKS control preparation.

FIG. 4

ELISA results with rP35 protein in the sera from 41 pregnant women with serologic profile consistent with recently acquired infection with T. gondii (group I) after subtraction of the results of the same sera with the CKS control preparation. The horizontal lines represent the cutoff values of 0.014 (mean plus two standard deviations) and 0.019 (mean plus three standard deviations) obtained from the results noted with sera of group II individuals as described in Results.

FIG. 5

ELISA results with rP35 protein in the sera of 50 pregnant women with a serologic profile consistent with chronic infection (group II) after subtraction of the results of the same sera with the CKS control preparation. Only four sera had results higher than the cutoff value of 0.014, and only one had results higher than the cutoff value of 0.019.