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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

(a) Enrichment of phage binding after three rounds of panning. 50 μL of a 1010 transduction unit phage suspension collected after each round of panning was added to wells of microtiter plates coated with 5 μg/mL anti-LiPA IgG. Phages were detected using a peroxidase conjugated anti-M13 antibody (1 : 3000). Values of absorbance at 492 nm are means of duplicates. (b) Reactivity of individual clones isolated after panning 3. 50 μL of a 1010transduction unit phage suspension isolated after the third round of panning was added to each well of microtiter plates coated with 1 μg/well of anti-L. infantum chagasi IgGs (●) and anti-T. cruzi IgGs (■). Phages were detected using a peroxidase conjugated anti-M13 antibody (1 : 3000). Values of absorbance at 492 nm are means of duplicates. (c) The aminoacid sequences of the three selected peptides.
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fig2: (a) Enrichment of phage binding after three rounds of panning. 50 μL of a 1010 transduction unit phage suspension collected after each round of panning was added to wells of microtiter plates coated with 5 μg/mL anti-LiPA IgG. Phages were detected using a peroxidase conjugated anti-M13 antibody (1 : 3000). Values of absorbance at 492 nm are means of duplicates. (b) Reactivity of individual clones isolated after panning 3. 50 μL of a 1010transduction unit phage suspension isolated after the third round of panning was added to each well of microtiter plates coated with 1 μg/well of anti-L. infantum chagasi IgGs (●) and anti-T. cruzi IgGs (■). Phages were detected using a peroxidase conjugated anti-M13 antibody (1 : 3000). Values of absorbance at 492 nm are means of duplicates. (c) The aminoacid sequences of the three selected peptides.

Mentions: In order to identify peptides that bind to anti-LiPA antibodies, four different phage libraries were screened. A significant enrichment of phage binding to the target antibodies was obtained after three rounds of panning (Figure 2(a)). One hundred and ninety-eight phage clones were randomly picked from the third round of selection, and twelve clones were selected based on their reactivity (absorbance at 492 nm ≥ 1.0) against IgGs from L. infantum chagasi infected dogs (data not shown). Afterwards, the twelve clones were checked by ELISA for their ability to bind to anti-T. cruzi dog IgGs. Clones 5, 6, and 11 were further selected due their high reactivity against Leishmania-specific IgGs and without cross-reactivity with IgGs from dogs with Chagas disease (Figure 2(b)).


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)

(a) Enrichment of phage binding after three rounds of panning. 50 μL of a 1010 transduction unit phage suspension collected after each round of panning was added to wells of microtiter plates coated with 5 μg/mL anti-LiPA IgG. Phages were detected using a peroxidase conjugated anti-M13 antibody (1 : 3000). Values of absorbance at 492 nm are means of duplicates. (b) Reactivity of individual clones isolated after panning 3. 50 μL of a 1010transduction unit phage suspension isolated after the third round of panning was added to each well of microtiter plates coated with 1 μg/well of anti-L. infantum chagasi IgGs (●) and anti-T. cruzi IgGs (■). Phages were detected using a peroxidase conjugated anti-M13 antibody (1 : 3000). Values of absorbance at 492 nm are means of duplicates. (c) The aminoacid sequences of the three selected peptides.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4325972&req=5

fig2: (a) Enrichment of phage binding after three rounds of panning. 50 μL of a 1010 transduction unit phage suspension collected after each round of panning was added to wells of microtiter plates coated with 5 μg/mL anti-LiPA IgG. Phages were detected using a peroxidase conjugated anti-M13 antibody (1 : 3000). Values of absorbance at 492 nm are means of duplicates. (b) Reactivity of individual clones isolated after panning 3. 50 μL of a 1010transduction unit phage suspension isolated after the third round of panning was added to each well of microtiter plates coated with 1 μg/well of anti-L. infantum chagasi IgGs (●) and anti-T. cruzi IgGs (■). Phages were detected using a peroxidase conjugated anti-M13 antibody (1 : 3000). Values of absorbance at 492 nm are means of duplicates. (c) The aminoacid sequences of the three selected peptides.
Mentions: In order to identify peptides that bind to anti-LiPA antibodies, four different phage libraries were screened. A significant enrichment of phage binding to the target antibodies was obtained after three rounds of panning (Figure 2(a)). One hundred and ninety-eight phage clones were randomly picked from the third round of selection, and twelve clones were selected based on their reactivity (absorbance at 492 nm ≥ 1.0) against IgGs from L. infantum chagasi infected dogs (data not shown). Afterwards, the twelve clones were checked by ELISA for their ability to bind to anti-T. cruzi dog IgGs. Clones 5, 6, and 11 were further selected due their high reactivity against Leishmania-specific IgGs and without cross-reactivity with IgGs from dogs with Chagas disease (Figure 2(b)).

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