rROP2(186-533): a novel peptide antigen for detection of IgM antibodies against Toxoplasma gondii

Abstract

Toxoplasma gondii infections are prevalent in a wide range of mammalian hosts including humans. Infection in pregnant women may cause the transmission of parasite to the fetus that makes serious problems. IgM antibodies against Toxoplasma (Toxo-IgM) have been believed to be significant indicators for both recently acquired and congenital toxoplasmosis. So far, however, there has not been any recognized protein of T. gondii that specifically reacts to IgM antibodies. Here, an antigen exclusively for detection of IgM antibodies screened by two-dimensional electrophoresis and mass spectrometry has been reported. The study identified 13 Toxoplasma proteins probed by IgG antibodies and one (rhpotry protein 2 [ROP2]) by IgM antibodies with human sera of Toxo-IgM(-)-IgG(+) and -IgM(+)-IgG(-), respectively, which had been prescreened by Toxo-IgM and -IgG commercial kits from the suspected cases. Following cloning, expression, and purification of the fragment of ROP2(186-533), an enzyme-linked immunosorbent assay with rROP2(186-533) to measure IgM and IgG antibodies was developed. As a result, 100%(48/48) of sera with Toxo-IgM(+)-IgG(-)showed positive Toxo-IgM but none of them (0%) showed positive Toxo-IgG when rROP2(186-533) was used as antigen. Neither Toxo-IgG nor Toxo-IgM antibodies were found when tested with 59 sera of Toxo-IgM(-)-IgG(+). These results indicate that rROP2(186-533) could be used as an antigen that specifically capture Toxo-IgM antibodies and may have a high potential in the serological diagnosis of both acute acquired and congenital toxoplasmosis.

FIG. 1.

Two-dimensional electrophoresis (2-DE) and immunoblot patterns of the whole soluble protein of Toxoplasma gondii RH strain. (A) CBB-stained 2-DE pattern with pH 3–10 linear strips, points marked by numbers are the common protein spots between immunoblot pattern of IgM^–-IgG⁺ and 2-DE pattern, points marked by letters are the common protein spots between immunoblot pattern of IgM⁺-IgG^–and 2-DE pattern. (B) Immunoblot map probed by sera of patients with IgM^–-IgG⁺. (C) Immunoblot map probed by patient sera with IgM⁺-IgG^–. (D) Immunoblot map probed by normal control sera of healthy donors.

FIG. 2.

SDS-PAGE analysis of rROP2_186–533. Lane M: Prestained protein ladder. Lane 1: Cell lysates from pET-28a in Escherichia coli BL21 strain. Lane 2–7: Cell lysates from pET-28a-ROP2_186–533 in E. coli BL21 strain induced with isopropyl-β-D-thiogalactoside (IPTG) for 1–6 h. Lane 8: Purification products of rROP2_186–533.

FIG. 3.

Identification of rROP2_186–533 by Western blotting probed with anti-His-tag monoclonal antibody. Cell lysates from pET28a-rROP2 in E. coli BL21 before (lane 1) and after (lane 2) IPTG induction. Lane M: Prestained protein ladder.

FIG. 4.

(A) Level of IgM antibodies against rROP2_186–533 in sera of patients. (B) Level of IgG antibodies against rROP2_186–533 in sera of patients. IgM⁺-IgG^–: Sera of patients with Toxo-IgM antibodies only. IgM^–-IgG⁺: Sera of patients with Toxo-IgG antibodies only. Leishmania: Sera of patients with Leishmania spp. Plasmodium: Sera of patients with tertian malaria. Control: healthy donors.

FIG. 5.

Comparison of the antibody titers of rROP2_186–533 in each group. ***p<0.0001 compared with healthy donors. IgM⁺-IgG^–: Sera of patients with Toxo-IgM antibodies only. IgM^–-IgG⁺: Sera of patients with Toxo-IgG antibodies only. Leishmania: Sera of patients with Leishmania spp. Plasmodium: Sera of patients with tertian malaria. Control: healthy donors.

FIG. 6.

The kinetics of IgG and IgM antibodies againt T. gondii with rROP2_186–533 and whole soluble antigen of T. gondii in enzyme-linked immunosorbent assay.