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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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Related in: MedlinePlus

Selection of the gp75 mimotope. a Immunoblotting showing the specific recognition of anti-gp43 and anti-gp75 mAbs against exoantigen proteins. The purified anti-gp43 mAb was used at 1:50 dilution. The anti-gp75 mAb was diluted at 1:400 (lane 1), 1:200 (lane 2), 1:100 (lane 3) or 1:50 (lane 4). b Monitoring of specific phages to anti-gp75 mAb during three rounds of biopanning. c Binding assay showing that phage displaying P13 is a putative mimotope to anti-gp75 mAb. Phages presenting P9, P13, P18, P19, P25 and P36 peptides were incubated with 5E7C or 17C mAbs (negative control). Phages that bound to those antibodies were recovered through infection in E. coli ER2738. After incubation in LB IPTG/Xgal medium, they were quantified. It is observed that only the P13 phage clone specifically bound to mAb 5E7C when compared with anti-gp43 mAb (P < 0.05). Statistical analysis were performed with Student’s t test (P < 0.05)
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Fig1: Selection of the gp75 mimotope. a Immunoblotting showing the specific recognition of anti-gp43 and anti-gp75 mAbs against exoantigen proteins. The purified anti-gp43 mAb was used at 1:50 dilution. The anti-gp75 mAb was diluted at 1:400 (lane 1), 1:200 (lane 2), 1:100 (lane 3) or 1:50 (lane 4). b Monitoring of specific phages to anti-gp75 mAb during three rounds of biopanning. c Binding assay showing that phage displaying P13 is a putative mimotope to anti-gp75 mAb. Phages presenting P9, P13, P18, P19, P25 and P36 peptides were incubated with 5E7C or 17C mAbs (negative control). Phages that bound to those antibodies were recovered through infection in E. coli ER2738. After incubation in LB IPTG/Xgal medium, they were quantified. It is observed that only the P13 phage clone specifically bound to mAb 5E7C when compared with anti-gp43 mAb (P < 0.05). Statistical analysis were performed with Student’s t test (P < 0.05)

Mentions: The specificity of anti-gp75 (5E7C) and anti-gp43 mAbs (17C) was checked as shown in Fig. 1a. Figure 1b shows the enrichment of the phages after selection cycles. The amount of phages recovered was 4.2 × 1011 and 8.9 × 1011 pfu/mL in the first and second rounds (Fig. 1b), respectively. After the third round, in which more stringent conditions were used, 1.8 × 1011 phage clones were obtained. These results indicate the presence of clones bearing the peptide sequence recognized by 5E7C.Fig. 1


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)

Selection of the gp75 mimotope. a Immunoblotting showing the specific recognition of anti-gp43 and anti-gp75 mAbs against exoantigen proteins. The purified anti-gp43 mAb was used at 1:50 dilution. The anti-gp75 mAb was diluted at 1:400 (lane 1), 1:200 (lane 2), 1:100 (lane 3) or 1:50 (lane 4). b Monitoring of specific phages to anti-gp75 mAb during three rounds of biopanning. c Binding assay showing that phage displaying P13 is a putative mimotope to anti-gp75 mAb. Phages presenting P9, P13, P18, P19, P25 and P36 peptides were incubated with 5E7C or 17C mAbs (negative control). Phages that bound to those antibodies were recovered through infection in E. coli ER2738. After incubation in LB IPTG/Xgal medium, they were quantified. It is observed that only the P13 phage clone specifically bound to mAb 5E7C when compared with anti-gp43 mAb (P < 0.05). Statistical analysis were performed with Student’s t test (P < 0.05)
© Copyright Policy
Related In: Results  -  Collection

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

Fig1: Selection of the gp75 mimotope. a Immunoblotting showing the specific recognition of anti-gp43 and anti-gp75 mAbs against exoantigen proteins. The purified anti-gp43 mAb was used at 1:50 dilution. The anti-gp75 mAb was diluted at 1:400 (lane 1), 1:200 (lane 2), 1:100 (lane 3) or 1:50 (lane 4). b Monitoring of specific phages to anti-gp75 mAb during three rounds of biopanning. c Binding assay showing that phage displaying P13 is a putative mimotope to anti-gp75 mAb. Phages presenting P9, P13, P18, P19, P25 and P36 peptides were incubated with 5E7C or 17C mAbs (negative control). Phages that bound to those antibodies were recovered through infection in E. coli ER2738. After incubation in LB IPTG/Xgal medium, they were quantified. It is observed that only the P13 phage clone specifically bound to mAb 5E7C when compared with anti-gp43 mAb (P < 0.05). Statistical analysis were performed with Student’s t test (P < 0.05)
Mentions: The specificity of anti-gp75 (5E7C) and anti-gp43 mAbs (17C) was checked as shown in Fig. 1a. Figure 1b shows the enrichment of the phages after selection cycles. The amount of phages recovered was 4.2 × 1011 and 8.9 × 1011 pfu/mL in the first and second rounds (Fig. 1b), respectively. After the third round, in which more stringent conditions were used, 1.8 × 1011 phage clones were obtained. These results indicate the presence of clones bearing the peptide sequence recognized by 5E7C.Fig. 1

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