Evaluation of an ELISA for canine leishmaniasis immunodiagnostic using recombinant proteins

Abstract

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.

A multicentric evaluation of the recombinant Leishmania infantum antigen-based immunochromatographic assay for the serodiagnosis of canine visceral leishmaniasis

“…A previous study demonstrated that rLci1A and rLci2B offer 96% and 100% sensitivity, with respective specificity rates of 92% and 95% under ELISA against sera from animals with positive parasitological test results [9]. These findings clearly indicate the potential of these selected antigens for use in CVL diagnosis.…”

“…Affinity chromatography was used to purify the rLci1A and rLci2B proteins as previously described [9]. …”

Towards a More Precise Serological Diagnosis of Human Tegumentary Leishmaniasis Using Leishmania Recombinant Proteins

“…In the last decades, an increasing number of recombinant protein candidates have been proposed to replace the crude Leishmania antigen (SLA) for the serodiagnosis of leishmaniasis [24], [31]–[34]. Presently, we decided to test the feasibility of using recombinant antigens derived from L. infantum-chagasi for the serodiagnosis of TL.…”

“…Aiming to increase ELISA specificity in the diagnosis of VL or TL, through the elimination of cross-reactive epitopes, the use of recombinant Leishmania inmunodominant antigens has been evaluated [20], [21]. Many recombinant antigens have been tested for the serodiagnosis of canine or human VL [22]–[24], however, up to date, few recombinant proteins have been described as promising for the serodiagnosis of human TL [25]. In the present study, we employed a large panel of human sera to test a series of parasite-derived recombinant proteins, using ELISA, aiming at the development of better tool for human TL serodiagnosis.…”

New Advances in the Diagnosis of Canine Visceral Leishmaniasis

“…The recombinant antigens showed no cross-reactivity with sera from dogs infected with Trypanosoma caninum, Babesia canis and Ehrlichia canis. Cross-reaction against sera from dogs infected with Leishmania braziliensis was observed for rLci1A-based ELISA (11.7%) and for rLci2B-based ELISA (2.9%) [109].…”