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Evaluation of an ELISA for canine leishmaniasis immunodiagnostic using recombinant proteins
Author(s) -
De SOUZA C. M.,
SILVA E. D.,
ANO BOM A. P. D.,
BASTOS R. C.,
NASCIMENTO H. J.,
Da SILVA JUNIOR J. G.
Publication year - 2012
Publication title -
parasite immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.795
H-Index - 75
eISSN - 1365-3024
pISSN - 0141-9838
DOI - 10.1111/j.1365-3024.2011.01334.x
Subject(s) - affinity chromatography , ehrlichia canis , canine leishmaniasis , biology , antigen , virology , leishmania , immunoassay , serology , microbiology and biotechnology , leishmania infantum , leishmaniasis , recombinant dna , canis , leishmania chagasi , visceral leishmaniasis , western blot , antibody , immunology , parasite hosting , biochemistry , enzyme , paleontology , world wide web , computer science , gene
Summary The present work describes the isolation and purification of two Leishmania chagasi (= syn. Leishmania infantum ) recombinant proteins, rLci2B and rLci1A, and their use in the development of an immunoassay for the diagnostic of canine leishmaniasis. After protein expression and cell disruption, rLci2B was purified by immobilized metal affinity chromatography followed by size exclusion chromatography, whereas rLci1A, expressed as an inclusion body, was treated with urea and purified by anion‐exchange chromatography. Homogeneities were ascertained by denaturing gel electrophoresis (MW rLci2B = 46 370; MW rLci1A = 88 400), isoelectric focusing (pI rLci2B = 5·91; pI rLci1A = 6·01) and Western blot. An indirect ELISA was developed using the purified antigens rLci2B and rLci1A and a leishmaniasis canine serum panel ( n = 256). The ELISA showed 100% sensitivity and 95% specificity for rLci2B and 96% sensitivity and 92% specificity for rLci1A. The purified proteins did not present cross‐reactivity with sera from dogs infected with Trypanosoma caninum, Babesia canis and Ehrlichia canis . Cross‐reaction was verified with sera from dogs infected with Leishmania brasiliensis (11·7% for rLci2B and 2·9% for rLci1A). Based on ELISA results, it is suggested the use of rLci2B and rLci1A as antigens in an alternative serological assay for diagnostic of canine leishmania.