immunology of infectious and parasitic diseases - XXXVII Congress ...

PRODUCTION AND EVALUATION OF CHICKEN IGY ANTIBODIES
AGAINST RECOMBINANT HSP70 OF Toxoplasma gondii
PAULO VICTOR CZARNEWSKI BARENCO 1 ; MÁRIO CÉZAR DE OLIVEIRA 1 ;
POLIANA DE CASTRO MELO 1 , JAIR PEREIRA CUNHA JÚNIOR 2 ; TIAGO
WILSON PATRIARCA MINEO 2 ; NEIDE MARIA SILVA 1
1
Laboratório de Imunopatologia, ICBIM, Universidade Federal de Uberlândia,
Uberlândia, MG;
2
Laboratório de Imunoparasitologia, ICBIM, Universidade Federal de
Uberlândia, MG;
Introduction: Toxoplasma gondii presents two developmental stages in the
intermediate hosts. During stage differentiation from tachyzoite to bradyzoite,
the parasite express T.g.HSP70. The objective of this study was to produce,
purify and test IgY specific antibodies against T.g.HSP70 as an alternative
diagnostic tool for toxoplasmosis.
Methods and Results: Recombinant T.g.HSP70 was expressed as fusion
protein with glutathione-S-transferase (GST) in E. coli BL21 DE3 vector and
purified with glutathione resin followed by thrombin cleavage and LPS removal.
Chickens were immunized with 100 µg/animal of T.g.HSP70 diluted v/v with
Freund‟s complete adjuvant and boosted after 15 and 30 days with 100
µg/animal of T.g.HSP70 diluted v/v with Freund‟s incomplete adjuvant. Eggs
were collected daily and yolk was frozen at -20°C until use. For purification of
IgY antibodies, yolk was diluted 10 times in water (pH 5,0) and centrifuged. The
supernatant was pelleted with 19% sodium sulphate. The sediment was
ressupended and dialyzed against phosphate buffer saline and then frozen at -
20°C for late use. The immunofluorescence assay was used to verify the
specificity of the purified IgY antibodies. It was observed that the IgY T. gondiispecific
antibodies were able to detect T. g.HSP70 in the cytoplasm of T. gondii
RH strain.
Conclusion: These results suggest that the IgY T.g.HSP70 could be used as
an alternative diagnostic tool for toxoplasmosis.
Finantial support: FAPEMIG, CNPq and CAPES.