Serodiagnosis of toxoplasmosis by using a recombinant form of the 54-kilodalton rhoptry antigen expressed in Escherichia coli

Abstract

A 330-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii 54-kDa rhoptry protein (ROP2) was expressed in Escherichia coli as a fusion polypeptide containing a 48-amino-acid sequence derived from phage lambda protein Cro and E. coi protein LacI followed by six consecutive histidyl residues. Metal chelate affinity chromatography provided an easy way to isolate the recombinant product in a highly purified form (>95%). When this material was used to develop an immunoglobulin G (IgG) enzyme-linked immunosorbent assay for diagnosis of toxoplasmosis, the test reached a sensitivity of 89%o. The sensitivity of the assay was similar whether the sera contained T. gondii-specific IgM or were devoid of such IgM. It was also found that ROP2 is a conserved antigen since antibodies against the recombinant antigen could be detected in mice experimentally infected with 11 independent T. gondii isolates originating from infected human tissues tested. Thus, the 54-kDa rhoptry antigen could advantageously complement other previously described T. gondii antigens for the development of more-sensitive and more-informative recombinant antigen-based tests for toxoplasmosis diagnosis.

Recombinant Antigens To DetectToxoplasma gondii-Specific Immunoglobulin G and Immunoglobulin M in Human Sera by Enzyme Immunoassay

“…The pattern of immunoreactivity observed by Western blotting with human sera varies with the immunoglobulin class and the stage of infection. Several genes have been cloned that encode T. gondii antigens, as follows: the surface antigens P22 (SAG2) (38,46) and P30 (SAG1) (3,12,24); the dense granule antigens P24 (GRA1) (4), P28 (GRA2) (35,45), P29 (GRA7) (2,9,17), P32 (GRA6) (27,47), and P41 (GRA4) (33,55); the rhoptry antigens P54 (ROP2) (31,51,56) and P66 (ROP1) (25,37); and the B10 (36), P25 (22,55), P35, and P68 antigens (25). These recombinant antigens expressed in bacteria have been used to detect antibodies to the parasite in the serum of humans and animals.…”

Expression, purification, and improved antigenic specificity of a truncated recombinant bp26 protein of Brucella melitensis M5‐90: a potential antigen for differential serodiagnosis of brucellosis in sheep and goats

“…Recombinant proteins therefore possess low antigenicity and show higher background or cross‐reactivity with control sera owing to antibodies directed against these bacterial components, which are unwanted byproducts of recombinant proteins purified from E. coli , whereas the single affinity purification procedure does not remove these components, such as LPS, completely. Thus, the purity of a recombinant protein is inadequate for use as a diagnostic antigen unless further purification procedures or additional purification steps are used 20,21. For example, imidazole, which binds to Ni‐NTA (Qiagen) and competes with His residues in the 6‐His tag, can be used at low concentrations (≤20 mM) to inhibit nonspecific binding, and nonionic detergents can be used to remove background proteins and nucleic acids 22.…”

“…The LPS content of pools treated by IMAC was reduced from greater than 800 to 2 ng/mL by the addition of 0.1% triton X‐114 to the elution buffer. In addition, the E. coli lysate should be added to the dilution buffer for Ab1 (serum) detection to reduce interference by antibodies against E. coli that may be present in the sample sera 8,20.…”

Cellular and humoral immune responses to recombinant antigens in sheep infected with Toxoplasma gondii

“…The recombinant GRA2 antigen used was the 59 Cterminal amino acids of the protein (GRA2 59aa) expressed as a fusion protein with glutathione-Stransferase (GST) (Cesbron-Delauw et al 1992). Three recombinant fragments of ROP2, each expressed as a fusion protein with a Cro-Lac1 peptide were used; the C-terminal 330 residues (Tg34AR) (Van Gelder et al 1993), the 270 N-terminal amino acids (Tg34EAV) and the 100 N-terminal amino acids (Tg34EB). Soluble T. gondii antigen (STA) was obtained following lysis of purified RH strain tachyzoites by sonication.…”

“…ESAs comprise the majority of circulating antigens during the initial stages of infection with T. gondii, and are therefore an early target for the host immune response (Hughes & van Knapen 1982). Around 80% of humans infected with T. gondii had serum antibodies directed against GRA2 59aa (Murray et al 1993), and 90% had antibodies specific for Tg34AR (Van Gelder et al 1993). In the current study, similar percentages of infected sheep had serum antibodies against each of the recombinant antigens.…”