Evaluation of serodiagnosis of toxoplasmosis by using the recombinant nucleoside triphosphate hydrolase isoforms expressed in Escherichia coli.
The nucleoside triphosphate hydrolase (NTPase) isoforms termed, NTPase-I and NTPase-II of Toxoplasma gondii, were expressed in Escherichia coli as inclusion bodies and purified under denaturing condition. Furthermore, NTPase-I was refolded as an active form and purified under non-denaturing condition. The purified NTPase isoforms, both denatured and refolded, were tested for their usefulness as antigens for the serodiagnosis of acute toxoplasmosis in immunocompetent humans. The test was conducted by using the recombinant NTPase isoforms and comparing the enzyme linked immunosorbent assay (ELISA) absorbances with the Sabin-Feldman dye test titer. Seventy-three sera from dye test-positive patients, and 30 sera from subjects with no T. gondii infection were examined. The total positive rates in dye test positive sera were: 82% (60/73) for denatured NTPase-I; 78% (57/73) for denatured NTPase-II; and 63% (46/73) for refolded NTPase-I. For all three antigen types of recombinant NTPase, the positive rates of sera of acute toxoplasmosis suspected patients were 93% (13/14). A moderate correlation between the ELISA absorbance using these antigens and the dye test titer was observed with the correlation coefficients, 0.583 (r2) for denatured NTPase-I, 0.472 (r2) for denatured NTPase-II, and 0.604 (r2) for refolded NTPase-I in the linear regression analysis. There was no significant difference observed in the antigenicity between refolded and denatured NTPase-I, nor between the isoforms.
Nakajima-Nakano
K
K
Department of Tropical Medicine and Parasitology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan.
Makioka
A
A
Yamashita
N
N
Matsuo
N
N
Asai
T
T
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D043583:Nucleoside-Triphosphatase; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014122:Toxoplasma / Q000201:enzymology / Q000235:genetics*; D014123:Toxoplasmosis / Q000097:blood / Q000175:diagnosis*
D017931:DNA Primers; D016054:DNA, Protozoan; D007527:Isoenzymes; D011994:Recombinant Proteins; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase