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Synthetic peptides mimic gp75 from Paracoccidioides brasiliensis in the diagnosis of paracoccidioidomycosis.

Caldini CP, Xander P, Kioshima ÉS, Bachi AL, de Camargo ZP, Mariano M, Lopes JD - Mycopathologia (2012)

Bottom Line: There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera.These data indicate a potential use of P2 as diagnostic tool in PCM.Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-Escola Paulista de Medicina, Disciplina de Imunologia, Rua Botucatu, 862, 4º andar, São Paulo, 04023-900, Brazil.

ABSTRACT
Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. In this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. The protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients' sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. The test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.

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Analysis of antigenicity, structure and specificity of the synthetic peptides obtained by phage display. a Analysis of P2 or P5 sequences. a Kyte-Doolittle hydrophilicity plot; b the red box detaches the Eisenberg alpha amphipathic regions, the green correspond to Eisenberg beta amphipathic and the blue shows Karplus-Schulz rexible regions; c Jameson-Wolff antigenic index; and d Emini surface probability plot. Positive values correspond to the hydrophilic structure. b The reactivity of 5E7C to P2 peptide was evaluated by ELISA. The ascitic liquid containing mAb was submitted to serial dilution starting at 1:50. (Color figure online)
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Fig2: Analysis of antigenicity, structure and specificity of the synthetic peptides obtained by phage display. a Analysis of P2 or P5 sequences. a Kyte-Doolittle hydrophilicity plot; b the red box detaches the Eisenberg alpha amphipathic regions, the green correspond to Eisenberg beta amphipathic and the blue shows Karplus-Schulz rexible regions; c Jameson-Wolff antigenic index; and d Emini surface probability plot. Positive values correspond to the hydrophilic structure. b The reactivity of 5E7C to P2 peptide was evaluated by ELISA. The ascitic liquid containing mAb was submitted to serial dilution starting at 1:50. (Color figure online)

Mentions: Considering that gp75 immunopurified with 5E7C showed phosphatase activity in vitro [17], the P13 cyclic peptide (CHSSLLNPC) selected by phage display against 5E7C was aligned and analyzed with phosphatases sequences deposited in the database of P. brasiliensis genome project (http://www.broad.mit.edu/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html) (data not shown). Besides, aiming to improve the antigenic potential of the peptide selected sequence, five amino acids similar to the phosphatase sequence showing greater homology were added to the N-terminal and C-terminal, flanking the initial sequence of nine amino acids. Using this approach, two sequences of peptides were obtained: NANEAHSSLLNNALSI (P2) and DEHSRSLLNPRQETW (P5). Figure 2a shows graphic analyses of the antigenic and structural parameters of the P2 and P5 sequences. As observed, P2 peptide sequence presented highest antigenic indexes and hydrophilicity profiles and therefore was selected to be synthesized and used in subsequent experiments.Fig. 2


Synthetic peptides mimic gp75 from Paracoccidioides brasiliensis in the diagnosis of paracoccidioidomycosis.

Caldini CP, Xander P, Kioshima ÉS, Bachi AL, de Camargo ZP, Mariano M, Lopes JD - Mycopathologia (2012)

Analysis of antigenicity, structure and specificity of the synthetic peptides obtained by phage display. a Analysis of P2 or P5 sequences. a Kyte-Doolittle hydrophilicity plot; b the red box detaches the Eisenberg alpha amphipathic regions, the green correspond to Eisenberg beta amphipathic and the blue shows Karplus-Schulz rexible regions; c Jameson-Wolff antigenic index; and d Emini surface probability plot. Positive values correspond to the hydrophilic structure. b The reactivity of 5E7C to P2 peptide was evaluated by ELISA. The ascitic liquid containing mAb was submitted to serial dilution starting at 1:50. (Color figure online)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368115&req=5

Fig2: Analysis of antigenicity, structure and specificity of the synthetic peptides obtained by phage display. a Analysis of P2 or P5 sequences. a Kyte-Doolittle hydrophilicity plot; b the red box detaches the Eisenberg alpha amphipathic regions, the green correspond to Eisenberg beta amphipathic and the blue shows Karplus-Schulz rexible regions; c Jameson-Wolff antigenic index; and d Emini surface probability plot. Positive values correspond to the hydrophilic structure. b The reactivity of 5E7C to P2 peptide was evaluated by ELISA. The ascitic liquid containing mAb was submitted to serial dilution starting at 1:50. (Color figure online)
Mentions: Considering that gp75 immunopurified with 5E7C showed phosphatase activity in vitro [17], the P13 cyclic peptide (CHSSLLNPC) selected by phage display against 5E7C was aligned and analyzed with phosphatases sequences deposited in the database of P. brasiliensis genome project (http://www.broad.mit.edu/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html) (data not shown). Besides, aiming to improve the antigenic potential of the peptide selected sequence, five amino acids similar to the phosphatase sequence showing greater homology were added to the N-terminal and C-terminal, flanking the initial sequence of nine amino acids. Using this approach, two sequences of peptides were obtained: NANEAHSSLLNNALSI (P2) and DEHSRSLLNPRQETW (P5). Figure 2a shows graphic analyses of the antigenic and structural parameters of the P2 and P5 sequences. As observed, P2 peptide sequence presented highest antigenic indexes and hydrophilicity profiles and therefore was selected to be synthesized and used in subsequent experiments.Fig. 2

Bottom Line: There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera.These data indicate a potential use of P2 as diagnostic tool in PCM.Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-Escola Paulista de Medicina, Disciplina de Imunologia, Rua Botucatu, 862, 4º andar, São Paulo, 04023-900, Brazil.

ABSTRACT
Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. In this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. The protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients' sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. The test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.

Show MeSH
Related in: MedlinePlus