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Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents.

Alban SM, de Moura JF, Minozzo JC, Mira MT, Soccol VT - BMC Infect. Dis. (2013)

Bottom Line: An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae.In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae.The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Engenharia de Bioprocessos e Biotecnologia, Universidade Federal do Paraná, Curitiba 81531-990, Brasil.

ABSTRACT

Background: An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated.

Methods: Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents.

Results: Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae.

Conclusions: The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

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Related in: MedlinePlus

Reactivity of phage clones by ELISA with sera of patients and controls. Microtiter plates were coated with anti-phage antibody (1:800) and incubated with 2 × 1010 phages/mL followed by the addition of sera (1:100). The detection of the reaction was carried out with peroxidase conjugated anti-human antibody and OPD as chromogen. Each cell in the table represents an individual. The analysis was performed in duplicate and the hatched cells represent positive samples. The cut-off value was calculated as the average of the optical density plus twice the standard deviation obtained with endemic controls.
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Figure 3: Reactivity of phage clones by ELISA with sera of patients and controls. Microtiter plates were coated with anti-phage antibody (1:800) and incubated with 2 × 1010 phages/mL followed by the addition of sera (1:100). The detection of the reaction was carried out with peroxidase conjugated anti-human antibody and OPD as chromogen. Each cell in the table represents an individual. The analysis was performed in duplicate and the hatched cells represent positive samples. The cut-off value was calculated as the average of the optical density plus twice the standard deviation obtained with endemic controls.

Mentions: The respective phage clones carrying the identified peptide sequences were then assessed against sera of leprosy patients and controls by ELISA (Figure 3). Reactive control samples were the same regardless of the assessed clones, which could indicate an unspecific reaction between serum and phages.


Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents.

Alban SM, de Moura JF, Minozzo JC, Mira MT, Soccol VT - BMC Infect. Dis. (2013)

Reactivity of phage clones by ELISA with sera of patients and controls. Microtiter plates were coated with anti-phage antibody (1:800) and incubated with 2 × 1010 phages/mL followed by the addition of sera (1:100). The detection of the reaction was carried out with peroxidase conjugated anti-human antibody and OPD as chromogen. Each cell in the table represents an individual. The analysis was performed in duplicate and the hatched cells represent positive samples. The cut-off value was calculated as the average of the optical density plus twice the standard deviation obtained with endemic controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585472&req=5

Figure 3: Reactivity of phage clones by ELISA with sera of patients and controls. Microtiter plates were coated with anti-phage antibody (1:800) and incubated with 2 × 1010 phages/mL followed by the addition of sera (1:100). The detection of the reaction was carried out with peroxidase conjugated anti-human antibody and OPD as chromogen. Each cell in the table represents an individual. The analysis was performed in duplicate and the hatched cells represent positive samples. The cut-off value was calculated as the average of the optical density plus twice the standard deviation obtained with endemic controls.
Mentions: The respective phage clones carrying the identified peptide sequences were then assessed against sera of leprosy patients and controls by ELISA (Figure 3). Reactive control samples were the same regardless of the assessed clones, which could indicate an unspecific reaction between serum and phages.

Bottom Line: An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae.In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae.The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Engenharia de Bioprocessos e Biotecnologia, Universidade Federal do Paraná, Curitiba 81531-990, Brasil.

ABSTRACT

Background: An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated.

Methods: Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents.

Results: Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae.

Conclusions: The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

Show MeSH
Related in: MedlinePlus