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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

Reactivity of purified IgG antibodies against L. infantum chagasi antigen (LiPA). 10 μg of affinity-purified IgGs from CVL dogs (●) and negative dogs (■) was added to each well (from 10 μg to 0.625 μg/well) of microtiter plates coated with 50 μg/mL LiPA. The reaction was detected using a peroxidase conjugated anti-dog IgG antibody (1 : 2000). Absorbance values at 492 nm were means of duplicates.
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fig1: Reactivity of purified IgG antibodies against L. infantum chagasi antigen (LiPA). 10 μg of affinity-purified IgGs from CVL dogs (●) and negative dogs (■) was added to each well (from 10 μg to 0.625 μg/well) of microtiter plates coated with 50 μg/mL LiPA. The reaction was detected using a peroxidase conjugated anti-dog IgG antibody (1 : 2000). Absorbance values at 492 nm were means of duplicates.

Mentions: For the screening of phage-borne peptides from libraries, IgGs purified from sera of thirty-eight naturally infected and sixteen uninfected dogs were used. Levels of anti-LiPA antibodies were previously examined in sera samples collected one week before phage display experiments (data not shown). After fractionation of the pool of sera of infected or noninfected animal through Protein A-Sepharose 4B column, a dose-dependent reactivity of the purified IgGs towards LiPA was observed in an ELISA format (Figure 1).


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)

Reactivity of purified IgG antibodies against L. infantum chagasi antigen (LiPA). 10 μg of affinity-purified IgGs from CVL dogs (●) and negative dogs (■) was added to each well (from 10 μg to 0.625 μg/well) of microtiter plates coated with 50 μg/mL LiPA. The reaction was detected using a peroxidase conjugated anti-dog IgG antibody (1 : 2000). Absorbance values at 492 nm were means of duplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Reactivity of purified IgG antibodies against L. infantum chagasi antigen (LiPA). 10 μg of affinity-purified IgGs from CVL dogs (●) and negative dogs (■) was added to each well (from 10 μg to 0.625 μg/well) of microtiter plates coated with 50 μg/mL LiPA. The reaction was detected using a peroxidase conjugated anti-dog IgG antibody (1 : 2000). Absorbance values at 492 nm were means of duplicates.
Mentions: For the screening of phage-borne peptides from libraries, IgGs purified from sera of thirty-eight naturally infected and sixteen uninfected dogs were used. Levels of anti-LiPA antibodies were previously examined in sera samples collected one week before phage display experiments (data not shown). After fractionation of the pool of sera of infected or noninfected animal through Protein A-Sepharose 4B column, a dose-dependent reactivity of the purified IgGs towards LiPA was observed in an ELISA format (Figure 1).

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