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PCR diagnosis of toxoplasmosis in dead poetuses
Oral Sessions
I Parasitology
International
47 (Suppl.)
O-0430
(1998)
133-281
DEVELOPMENT OF SANDWICH ELBA FOR DElXCTIONOFNUCLEOTIDETRIPHOSPHATE HYDROLASEOF TOXOPLASMA GONDII
Kikuchi T, Furuta T. Kojima S Department of Parasitology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Toxoplasma got&i has a specific enzyme for nucleottde ttiphosphate hydrolase, NTPase. Since NTPase was detected in the mice infected with T. gondii as a circuiting antigen, this enzyme IS thought to be a good target antigen for diagnosis of T. gondii infection. We have made a monoclonal antibody (K-l) agamst NTPase. This antibody recognized 63 kDa protein and could reduce the NTPase activity in vitro. In this study, we tried to develop a sandwtch ELISA (SELISA) for diagnosis of T. gondii infection by using the antibody. T. gondii cysts were inoculated orally into ddY mice, and NTPase in sera was measured by ELISA at various days after infection. NTPase in sera increased at day 3 and peaked at day 7 after inoculation, and then decreased gradually. On the other hand, serum IgM antibody titers against T. gondii detected at day 7, and then reached the highest level at 21 days after infection. IgG antibody of T.gondii was detected at day 14 and then Increased gradually, reached the highest level at day 35. The cysts in bratn tissues were detected at 14 days and their numbers increased gradually throughout experiment. Appearance of cysts tn the bram tissues is seemed to be correlated wtth elevation of serum IgG antibody titers against T.gondii. From these studies, since NTPase in the sera from mtce infected with T. go&i was found earlier than the appearance of antibody against T. go&i and could be detected by the SELISASFLISA was thought to be a good diagnosis method to diagnose acuteTo.wplusnux infection.
O-0429 m
CONGENITAL TOXOPLASMOSIS : VALUE PRENATAL DIAGNOSIS AND POST-NATAL FOLLOW-UPOF NEWBORNS
E*. Gavmet MF*,
Raymond .I **, Tourtc-Schacter
O-0431
OF
C*. Dupq-Came!
MOLECULAR CLONING OF BABESIA CABALLI ANTIGEN GENES AND ELISA DEVELOPMENT C w*, Y. KAWASE’. Y. SAKO’, T. K&da*.
J*
*Labuntor) of Pdrawt~l~gy, Cochtn Hoapltal. Pdn\. France. and ** Lilbwaior! of Mwubiolog), SamcVmccnt-dc-Paul Hospital. Pat\. Fracc
E. ZWEIGARTH**. M. ONUhiA*.
Sapporo, Japan **Onderstepaort ***Equine
T. KANEMARU’**,
R. WADA*‘*.
a-d
*Graduate School of Veterinary Medicine, Hokkaido University, Vetexinary Institute, Onderstepoon, South Africa,
Research Institute, Japan Racing Association, Tochigi. Japan.
Current methods practically or experimentally
used for serodiagnosis of equine
piroplasmosis which is causedby Elabesia equi or B. coballi arecomplement fixation, indiit
flumescent antibody tests
However, besidessensitivity
, andenzyme-linked immunosorbent assay. these tests, productionofantigensfor
andspecificityof
serodiagnosis is accompanied by technical problems on specificity and sensitivity. In orderto develops recombinant antigen-based semdiagnosticmethcd infection. we have cloned and chawteri~ encoding B. cab&i
two cDNA clones @AI
for&
c&Ii
and FkA2)
proteins recognized by infected horse antibodies.
BcAl prcduct was revealed to be a JO !cDa heat shock protein (hspJ0). antibodies affinity-purified
from 8. c&z/Ii-infected
with a69 kDa protein of B. equi,
7he
horse on BcAl product reacted
Serological crossreactions between these &&-sin
species may be partly due to the reaction against hsp7Os Bc.42 with an open reading frame for a polypeptide of 212 amino acid residues with 2 possible N-glycosylation revealed to be a
antibodies aftinitypurified and 134-212 were expffsed q&infected
By homology search, this product was
with BcA2 nxombmant
protein bands of 53 and 55 kDa. immunoblotting,
sites.
amber of rlmptoq-associated protein of Bobcrio species.
in
Two fragments containing amino acid No. I-2 I2
E. coli by using a pET vector system.
both of the pr&tcu
By
did not crossrexted with serum form B.
horse and the latter product showed stronger reactxm with 8.
infected horse serum than the fomw
one.
be used as a diagnositic antigen specific for ELISA
Horse
product reacted two merozoite
cahlli-
From tbeseresults. a BcA2 productcan
B. c&d/i.
We are now developing an
with this recombinant pmduct, which may improve speciticity and
sensitivity of serodiagnosisofequinepiroplasmosis methods.
andskw~dxdizationofdiagnostic
I Parasitology
International
47 (Suppl.)
O-0430
(1998)
133-281
DEVELOPMENT OF SANDWICH ELBA FOR DElXCTIONOFNUCLEOTIDETRIPHOSPHATE HYDROLASEOF TOXOPLASMA GONDII
Kikuchi T, Furuta T. Kojima S Department of Parasitology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Toxoplasma got&i has a specific enzyme for nucleottde ttiphosphate hydrolase, NTPase. Since NTPase was detected in the mice infected with T. gondii as a circuiting antigen, this enzyme IS thought to be a good target antigen for diagnosis of T. gondii infection. We have made a monoclonal antibody (K-l) agamst NTPase. This antibody recognized 63 kDa protein and could reduce the NTPase activity in vitro. In this study, we tried to develop a sandwtch ELISA (SELISA) for diagnosis of T. gondii infection by using the antibody. T. gondii cysts were inoculated orally into ddY mice, and NTPase in sera was measured by ELISA at various days after infection. NTPase in sera increased at day 3 and peaked at day 7 after inoculation, and then decreased gradually. On the other hand, serum IgM antibody titers against T. gondii detected at day 7, and then reached the highest level at 21 days after infection. IgG antibody of T.gondii was detected at day 14 and then Increased gradually, reached the highest level at day 35. The cysts in bratn tissues were detected at 14 days and their numbers increased gradually throughout experiment. Appearance of cysts tn the bram tissues is seemed to be correlated wtth elevation of serum IgG antibody titers against T.gondii. From these studies, since NTPase in the sera from mtce infected with T. go&i was found earlier than the appearance of antibody against T. go&i and could be detected by the SELISASFLISA was thought to be a good diagnosis method to diagnose acuteTo.wplusnux infection.
O-0429 m
CONGENITAL TOXOPLASMOSIS : VALUE PRENATAL DIAGNOSIS AND POST-NATAL FOLLOW-UPOF NEWBORNS
E*. Gavmet MF*,
Raymond .I **, Tourtc-Schacter
O-0431
OF
C*. Dupq-Came!
MOLECULAR CLONING OF BABESIA CABALLI ANTIGEN GENES AND ELISA DEVELOPMENT C w*, Y. KAWASE’. Y. SAKO’, T. K&da*.
J*
*Labuntor) of Pdrawt~l~gy, Cochtn Hoapltal. Pdn\. France. and ** Lilbwaior! of Mwubiolog), SamcVmccnt-dc-Paul Hospital. Pat\. Fracc
E. ZWEIGARTH**. M. ONUhiA*.
Sapporo, Japan **Onderstepaort ***Equine
T. KANEMARU’**,
R. WADA*‘*.
a-d
*Graduate School of Veterinary Medicine, Hokkaido University, Vetexinary Institute, Onderstepoon, South Africa,
Research Institute, Japan Racing Association, Tochigi. Japan.
Current methods practically or experimentally
used for serodiagnosis of equine
piroplasmosis which is causedby Elabesia equi or B. coballi arecomplement fixation, indiit
flumescent antibody tests
However, besidessensitivity
, andenzyme-linked immunosorbent assay. these tests, productionofantigensfor
andspecificityof
serodiagnosis is accompanied by technical problems on specificity and sensitivity. In orderto develops recombinant antigen-based semdiagnosticmethcd infection. we have cloned and chawteri~ encoding B. cab&i
two cDNA clones @AI
for&
c&Ii
and FkA2)
proteins recognized by infected horse antibodies.
BcAl prcduct was revealed to be a JO !cDa heat shock protein (hspJ0). antibodies affinity-purified
from 8. c&z/Ii-infected
with a69 kDa protein of B. equi,
7he
horse on BcAl product reacted
Serological crossreactions between these &&-sin
species may be partly due to the reaction against hsp7Os Bc.42 with an open reading frame for a polypeptide of 212 amino acid residues with 2 possible N-glycosylation revealed to be a
antibodies aftinitypurified and 134-212 were expffsed q&infected
By homology search, this product was
with BcA2 nxombmant
protein bands of 53 and 55 kDa. immunoblotting,
sites.
amber of rlmptoq-associated protein of Bobcrio species.
in
Two fragments containing amino acid No. I-2 I2
E. coli by using a pET vector system.
both of the pr&tcu
By
did not crossrexted with serum form B.
horse and the latter product showed stronger reactxm with 8.
infected horse serum than the fomw
one.
be used as a diagnositic antigen specific for ELISA
Horse
product reacted two merozoite
cahlli-
From tbeseresults. a BcA2 productcan
B. c&d/i.
We are now developing an
with this recombinant pmduct, which may improve speciticity and
sensitivity of serodiagnosisofequinepiroplasmosis methods.
andskw~dxdizationofdiagnostic