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Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum

BACKGROUND: The aim of this study was to develop site-specific antibodies as a tool to capture Plasmodium falciparum-dihydrofolate reductase (Pf-DHFR) from blood samples from P. falciparum infected individuals in order to detect, in a sandwich ELISA, structural alterations due to point mutations in...

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Autores principales: Alifrangis, Michael, Christensen, Inge T, Jørgensen, Flemming S, Rønn, Anita M, Weng, Jimmy E, Chen, Ming, Bygbjerg, Ib C, Sirawaraporn, Worachart, Palarasah, Yaseelan, Koch, Claus
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2004
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC449723/
https://www.ncbi.nlm.nih.gov/pubmed/15193156
http://dx.doi.org/10.1186/1475-2875-3-16
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author Alifrangis, Michael
Christensen, Inge T
Jørgensen, Flemming S
Rønn, Anita M
Weng, Jimmy E
Chen, Ming
Bygbjerg, Ib C
Sirawaraporn, Worachart
Palarasah, Yaseelan
Koch, Claus
author_facet Alifrangis, Michael
Christensen, Inge T
Jørgensen, Flemming S
Rønn, Anita M
Weng, Jimmy E
Chen, Ming
Bygbjerg, Ib C
Sirawaraporn, Worachart
Palarasah, Yaseelan
Koch, Claus
author_sort Alifrangis, Michael
collection PubMed
description BACKGROUND: The aim of this study was to develop site-specific antibodies as a tool to capture Plasmodium falciparum-dihydrofolate reductase (Pf-DHFR) from blood samples from P. falciparum infected individuals in order to detect, in a sandwich ELISA, structural alterations due to point mutations in the gene coding for Pf-DHFR. Furthermore, we wanted to study the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes. METHODS: A homology model of Pf-DHFR domain was employed to define an epitope for the development of site-specific antibodies against Pf-DHFR. The homology model suggested an exposed loop encompassing amino acid residues 64–100. A synthetic peptide of 37-mers whose sequence corresponded to the sequence of amino acid residues 64–100 of Pf-DHFR was synthesized and used to immunize mice for antibodies. Additionally, polyclonal antibodies recognizing a recombinant DHFR enzyme were produced in rabbits. RESULTS AND CONCLUSIONS: Serum from mice immunized with the 37-mer showed strong reactivity against both the immunizing peptide, recombinant DHFR and a preparation of crude antigen from P. falciparum infected red blood cells. Five monoclonal antibodies were obtained, one of which showed reactivity towards crude antigen prepared from P. falciparum infected red cells. Western blot analysis revealed that both the polyclonal and monoclonal antibodies recognized Pf-DHFR. Our study provides insight into the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes.
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spelling pubmed-4497232004-07-10 Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum Alifrangis, Michael Christensen, Inge T Jørgensen, Flemming S Rønn, Anita M Weng, Jimmy E Chen, Ming Bygbjerg, Ib C Sirawaraporn, Worachart Palarasah, Yaseelan Koch, Claus Malar J Methodology BACKGROUND: The aim of this study was to develop site-specific antibodies as a tool to capture Plasmodium falciparum-dihydrofolate reductase (Pf-DHFR) from blood samples from P. falciparum infected individuals in order to detect, in a sandwich ELISA, structural alterations due to point mutations in the gene coding for Pf-DHFR. Furthermore, we wanted to study the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes. METHODS: A homology model of Pf-DHFR domain was employed to define an epitope for the development of site-specific antibodies against Pf-DHFR. The homology model suggested an exposed loop encompassing amino acid residues 64–100. A synthetic peptide of 37-mers whose sequence corresponded to the sequence of amino acid residues 64–100 of Pf-DHFR was synthesized and used to immunize mice for antibodies. Additionally, polyclonal antibodies recognizing a recombinant DHFR enzyme were produced in rabbits. RESULTS AND CONCLUSIONS: Serum from mice immunized with the 37-mer showed strong reactivity against both the immunizing peptide, recombinant DHFR and a preparation of crude antigen from P. falciparum infected red blood cells. Five monoclonal antibodies were obtained, one of which showed reactivity towards crude antigen prepared from P. falciparum infected red cells. Western blot analysis revealed that both the polyclonal and monoclonal antibodies recognized Pf-DHFR. Our study provides insight into the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes. BioMed Central 2004-06-12 /pmc/articles/PMC449723/ /pubmed/15193156 http://dx.doi.org/10.1186/1475-2875-3-16 Text en Copyright © 2004 Alifrangis et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.
spellingShingle Methodology
Alifrangis, Michael
Christensen, Inge T
Jørgensen, Flemming S
Rønn, Anita M
Weng, Jimmy E
Chen, Ming
Bygbjerg, Ib C
Sirawaraporn, Worachart
Palarasah, Yaseelan
Koch, Claus
Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum
title Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum
title_full Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum
title_fullStr Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum
title_full_unstemmed Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum
title_short Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum
title_sort homology building as a means to define antigenic epitopes on dihydrofolate reductase (dhfr) from plasmodium falciparum
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC449723/
https://www.ncbi.nlm.nih.gov/pubmed/15193156
http://dx.doi.org/10.1186/1475-2875-3-16
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