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Immunodiagnosis of canine visceral leishmaniasis using mimotope peptides selected from phage displayed combinatorial libraries.

Toledo-Machado CM, de Avila RA, NGuyen C, Granier C, Bueno LL, Carneiro CM, Menezes-Souza D, Carneiro RA, Chávez-Olórtegui C, Fujiwara RT - Biomed Res Int (2015)

Bottom Line: Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays.Each individual peptide or a mix of them was reactive with infected dogs serum.The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitologia, ICB, Universidade Federal de Minas Gerais, CP 486, Belo Horizonte 31270-901, MG, Brazil.

ABSTRACT
ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL). The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL.

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Related in: MedlinePlus

Comparison of ELISA reactivity of canine sera against individual phage-display selected peptides, the pool of peptides, the L. infantum chagasi antigen (LiPA), and reactivity in the EIE-LVC kit. ELISA was performed in different groups of dogs (negative/control group, CVL group, and TC/T. cruzi group). Cut-off was determined according to ROC curves.
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fig3: Comparison of ELISA reactivity of canine sera against individual phage-display selected peptides, the pool of peptides, the L. infantum chagasi antigen (LiPA), and reactivity in the EIE-LVC kit. ELISA was performed in different groups of dogs (negative/control group, CVL group, and TC/T. cruzi group). Cut-off was determined according to ROC curves.

Mentions: In order to evaluate the antigen-specific dog antibody response against synthetic peptides, an ELISA was optimized to obtain the best signal-to-noise ratio and to develop a reproducible and robust assay capable of capturing antibodies over a biologically relevant assay range. The EIE-LVC kit was used as a reference. ELISA cut-off values for peptides 5, 6, and 11 and pooled peptides (peptides 5+6+11) as antigens were 0.488, 0.698, 0.594, and 0.410, respectively. In similar conditions, the cut-off values were 0.546 and 0.104, for L. infantum chagasi antigen and EIE-LVC kit, respectively. The peptides were initially tested on sera of 38 dogs with L. infantum chagasi (CVL) (Figure 3, Table 1). Antibodies against peptides 5, 6, and 11 and pooled peptides were detected in 100% of the serum samples from these dogs. In order to evaluate specificity (Sp), serum samples from negative control dogs and samples from dogs infected by E. canis, B. canis, and T. cruzi were tested. Peptides 5 and 11 demonstrated an excellent specificity value (100.00%) slightly better than the 97.10% obtained for peptide 6 and pooled peptides, which showed 2 false positive results in the T. cruzi group (Figure 3 and Table 1). Comparative analysis using either the crude antigen or the reference EIE-LVC ELISA demonstrated also high sensitivity (100.00% and 94.74%, resp.). However, these tests showed a poorer specificity (76.81% and 68.12%, resp.) due to the large number of false negative results in negative and T. cruzi group.


Immunodiagnosis of canine visceral leishmaniasis using mimotope peptides selected from phage displayed combinatorial libraries.

Toledo-Machado CM, de Avila RA, NGuyen C, Granier C, Bueno LL, Carneiro CM, Menezes-Souza D, Carneiro RA, Chávez-Olórtegui C, Fujiwara RT - Biomed Res Int (2015)

Comparison of ELISA reactivity of canine sera against individual phage-display selected peptides, the pool of peptides, the L. infantum chagasi antigen (LiPA), and reactivity in the EIE-LVC kit. ELISA was performed in different groups of dogs (negative/control group, CVL group, and TC/T. cruzi group). Cut-off was determined according to ROC curves.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4325972&req=5

fig3: Comparison of ELISA reactivity of canine sera against individual phage-display selected peptides, the pool of peptides, the L. infantum chagasi antigen (LiPA), and reactivity in the EIE-LVC kit. ELISA was performed in different groups of dogs (negative/control group, CVL group, and TC/T. cruzi group). Cut-off was determined according to ROC curves.
Mentions: In order to evaluate the antigen-specific dog antibody response against synthetic peptides, an ELISA was optimized to obtain the best signal-to-noise ratio and to develop a reproducible and robust assay capable of capturing antibodies over a biologically relevant assay range. The EIE-LVC kit was used as a reference. ELISA cut-off values for peptides 5, 6, and 11 and pooled peptides (peptides 5+6+11) as antigens were 0.488, 0.698, 0.594, and 0.410, respectively. In similar conditions, the cut-off values were 0.546 and 0.104, for L. infantum chagasi antigen and EIE-LVC kit, respectively. The peptides were initially tested on sera of 38 dogs with L. infantum chagasi (CVL) (Figure 3, Table 1). Antibodies against peptides 5, 6, and 11 and pooled peptides were detected in 100% of the serum samples from these dogs. In order to evaluate specificity (Sp), serum samples from negative control dogs and samples from dogs infected by E. canis, B. canis, and T. cruzi were tested. Peptides 5 and 11 demonstrated an excellent specificity value (100.00%) slightly better than the 97.10% obtained for peptide 6 and pooled peptides, which showed 2 false positive results in the T. cruzi group (Figure 3 and Table 1). Comparative analysis using either the crude antigen or the reference EIE-LVC ELISA demonstrated also high sensitivity (100.00% and 94.74%, resp.). However, these tests showed a poorer specificity (76.81% and 68.12%, resp.) due to the large number of false negative results in negative and T. cruzi group.

Bottom Line: Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays.Each individual peptide or a mix of them was reactive with infected dogs serum.The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitologia, ICB, Universidade Federal de Minas Gerais, CP 486, Belo Horizonte 31270-901, MG, Brazil.

ABSTRACT
ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL). The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL.

Show MeSH
Related in: MedlinePlus