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

EVALUATION OF A SYNTHETIC PEPTIDE DERIVED FROM A LEISHMANIA
CYTOSOLIC FACTOR AS NEW DIAGNOSTIC MARKER TO DETECT
Leishmania INFECTED DOGS
LEOPOLDO FERREIRA MARQUES MACHADO (1) ; Adriana Canavaci Martins (1)
; Frederico Crepaldi Nascimento (1) ; Mariana Silva dos Santos (1) ; MIGUEL A.
CHÁVEZ-FUMAGALLI (2) ;; ALEXANDRE BARBOSA DOS REIS (3) EDUARDO
ANTONIO FERRAZ COELHO (2) ; ANA PAULA FERNANDES (1) .
(1) Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia,
Universidade Federal de Minas Gerais; (2) Departamento de Patologia Clínica,
Colégio Técnico. Universidade Federal de Minas Gerais; (3) Laboratório de
ImunoPatologia, Departamento de Análises Clínicas, Escola de Farmácia,
UFOP.
Introduction: Visceral leishmaniasis (VL) is the more severe form of
Leishmaniasis and is usually fatal if untreated. Dogs are the main peridomestic
reservoirs and an important step during the parasite life cycle and human
infection. Those animals may develop symptoms or remain asymptomatic for
long periods although both can transmit infection. Therefore, it is essential to
develop effective diagnostic markers to detect symptomatic and asymptomatic
dogs. Moreover, the study of new proteins or peptides may contribute to
discover the role of in immune responses. The Leishmania cytosolic factor gene
has been involved to visceralization of parasites. Thus, the aim of this work is to
evaluate the sensibility of predicted peptides during antibody detection by
ELISA. Methods and Results: The protein sequence was submitted to epitope
prediction and a synthetic peptide (P35) was obtained. The selected synthetic
epytopes were used for antibody detection by ELISA. The assays used sera
from known negative control, clinically asymptomatic and symptomatic dogs,
PCR positive/serologic negative control, and T. cruzi-infected dogs. The ELISA
reaction conditions were optimized to determine the concentrations of each
antigen and serum dilutions. ELISA plates were previously coated with the
synthetic peptide and incubated with sera. The binding of antibodies was
detected using anti-canine IgG peroxidase conjugated and the absorbance was
read at 492 nm. The cut-off was determined using mean absorbance of
negative samples plus two standard deviations, to discriminating positive from
negative results. The results of the assays using the synthetic peptide (P35)
and the peptide derived from the A2 antigen (A2-P40), which was previously
tested for diagnosis of canine visceral leishmaniasis, were comparatively
analyzed. The P35 peptide detected as positive 8% of sera in the control group;