Characterization of Novel Leishmania infantum Recombinant Proteins Encoded by Genes from Five Families with Distinct Capacities for Serodiagnosis of Canine and Human Visceral Leishmaniasis

Oliveira, Geraldo G. S.; Magalhães, Franklin Barbalho; Teixeira, Márcia Cristina Aquino; Pereira, Andréa Mendes; Pinheiro, Cristiane Garboggini Melo de; Santos, Lenita Ramires dos; Nascimento, Marília B.; Bedor, Cheila Nataly Galindo; Albuquerque, Alessandra Lima de; dos-Santos, Washington L. C.; Gomes, Yara M.; Moreira, Edson Duarte; Brito, Maria Edileuza Felinto de; Carvalho, Lain Carlos Pontes de; Neto, Osvaldo P. de Melo

doi:10.4269/ajtmh.2011.11-0102

Cited by 29 publications

(53 citation statements)

References 61 publications

(69 reference statements)

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“…Serological methods are powerful tools in CVL diagnosis, being frequently used for canine mass screening. Several Leishmania antigens have been characterized, and recombinant technology has been used for the development of new enzymatic immunoassays [16] , [28] , [29] . Given the lack of sensitivity of Brazilian recommended assays to CVL diagnosis, mainly in asymptomatic dogs [8] , [9] , the search for new antigens is still needed.…”

Section: Discussionmentioning

confidence: 99%

Novel Recombinant Multiepitope Proteins for the Diagnosis of Asymptomatic Leishmania infantum-Infected Dogs

et al. 2015

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BackgroundVisceral leishmaniasis is the most severe form of leishmaniasis. Worldwide, approximately 20% of zoonotic human visceral leishmaniasis is caused by Leishmania infantum, also known as Leishmania chagasi in Latin America. Current diagnostic methods are not accurate enough to identify Leishmania-infected animals and may compromise the effectiveness of disease control. Therefore, we aimed to produce and test two recombinant multiepitope proteins as a means to improve and increase accuracy in the diagnosis of canine visceral leishmaniasis (CVL).Methodology/Principal FindingsTen antigenic peptides were identified by CVL ELISA in previous work. In the current proposal, the coding sequences of these ten peptides were assembled into a synthetic gene. Furthermore, other twenty peptides were selected from work by our group where good B and T cell epitopes were mapped. The coding sequences of these peptides were also assembled into a synthetic gene. Both genes have been cloned and expressed in Escherichia coli, producing two multiepitope recombinant proteins, PQ10 and PQ20. These antigens have been used in CVL ELISA and were able to identify asymptomatic dogs (80%) more effectively than EIE-LVC kit, produced by Bio-Manguinhos (0%) and DPP kit (10%). Moreover, our recombinant proteins presented an early detection (before PCR) of infected dogs, with positivities ranging from 23% to 65%, depending on the phase of infection in which sera were acquired.Conclusions/SignificanceOur study shows that ELISA using the multiepitope proteins PQ10 and PQ20 has great potential in early CVL diagnosis. The use of these proteins in other methodologies, such as immunochromatographic tests, could be beneficial mainly for the detection of asymptomatic dogs.

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Section: Discussionmentioning

confidence: 99%

Novel Recombinant Multiepitope Proteins for the Diagnosis of Asymptomatic Leishmania infantum-Infected Dogs

et al. 2015

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“…A set of 12 recombinant L. infantum antigens (rLci1A, rLci2B, rLci3, rLci4, rLci5, rLci6, rLci7, rLci8, rLci10, rLci11, rLci12, rLci13) was previously selected from DNA libraries based on antibody reactivity using sera from culture-positive dogs [ 21 , 22 ]. Histidine-tagged recombinant proteins were produced after sub-cloning DNA fragments as described previously [ 21 ]. The antigens were then purified by affinity chromatography using PD-10 Desalting Workmate nickel-sepharose columns (Amersham Pharmacia Biotech AB, Sweden), in accordance with the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Multi-antigen print immunoassay (MAPIA)-based evaluation of novel recombinant Leishmania infantum antigens for the serodiagnosis of canine visceral leishmaniasis

et al. 2015

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BackgroundDomestic dogs are the principal reservoir hosts of Leishmania infantum in regions where visceral leishmaniasis (VL) is endemic. Although serologic methods are frequently used for the screening of infected dogs, antibody-based tests require further assessment, due to lack of sensitivity and specificity. In this study, we employed a multi-antigen printing immunoassay (MAPIA) to compare the antibody responses to novel recombinant proteins of L. infantum with the potential for the detection of canine VL.FindingsMAPIA strips were prepared employing 12 recombinant proteins. Antibody reactivity to these antigens was compared using a panel of sera collected from clinically asymptomatic (n = 16) and symptomatic (n = 41) culture-positive animals. Our findings showed that the canine immune response to antigen differs between dogs and depends on infection status. Using this screening assay, when five out of the 12 antigens were combined, an overall 81% detection rate of L. infantum-infected dogs was achieved.ConclusionsWe conclude that MAPIA is an effective screening tool to rapidly select multiple antigens of diagnostic utility to be used in a more sensitive point of care diagnostic test such as the Dual-Path Platform (DPP) multiplex test for the rapid detection of infected dogs.

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“…All diseases were parasitologically confirmed. The infectious agent of all diseases was specifically confirmed, and assay for VL demonstrated sensitivity and specificity above 90% [33]. L. infantum kinesin was also tested by an ELISA kit showing as non-reactive to other species of Trypanosomatidae [34,35].…”

Section: Rlci2b Antigenmentioning

confidence: 99%

A gold nanoparticle piezoelectric immunosensor using a recombinant antigen for detecting Leishmania infantum antibodies in canine serum

Ramos‐Jesus

Pontes-de-Carvalho²,

Barrouin‐Melo

et al. 2016

Biochemical Engineering Journal

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Visceral leishmaniasis (VL) is a severe systemic and infectious disease potentially fatal when undiagnosed or untreated. So far, accurate diagnosis is still a problem, especially in endemic areas and in the tracking and screening of asymptomatic dogs, which are good reservoirs and main host in urban areas. Recombinant antigens based on DNA technology have provided more reliable serological diagnostics since the specificity can be achieved easier than using whole extracts. This work reports a sensitive piezeolectric immunosensor for anti-Leishamnia antibodies based on rLci2B recombinant antigens immobilized on quartz crystal electrode. The electrode surface was assembled by a nafion film recovered by gold nanoparticles (AuNPs) to promote a greater amount of rLci2B due to the increase of surface area and stability on bonds. The immunosensor distinguished the positive VL canine serum showing a good linearity (r = 0.989; p << 0.01) and a low relative error (<5%) at 1:400 to 1:1600 serum dilutions. The system based on AuNP achieved better results regarding sensitivity and reproducibility than the cysteamine-based immunosensor without AuNP (r = 0.769; p = 0.147). The results obtained show as a promising tool for diagnosis of the VL.

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Characterization of Novel Leishmania infantum Recombinant Proteins Encoded by Genes from Five Families with Distinct Capacities for Serodiagnosis of Canine and Human Visceral Leishmaniasis

Cited by 29 publications

References 61 publications

Novel Recombinant Multiepitope Proteins for the Diagnosis of Asymptomatic Leishmania infantum-Infected Dogs

Novel Recombinant Multiepitope Proteins for the Diagnosis of Asymptomatic Leishmania infantum-Infected Dogs

Multi-antigen print immunoassay (MAPIA)-based evaluation of novel recombinant Leishmania infantum antigens for the serodiagnosis of canine visceral leishmaniasis

A gold nanoparticle piezoelectric immunosensor using a recombinant antigen for detecting Leishmania infantum antibodies in canine serum

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