Abstract
BackgroundThe 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.MethodsA 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 conclusionsSerum 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.
Highlights
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
The selection of a potential antigenic region Using SYBYL molecular modelling system, the homology model of Pf-DHFR domain was exploited for the analysis of epitope candidates and a single potential antigenic region was identified remotely located in respect to the active site of the enzyme
In order to attempt to induce the same conformation in the synthetic peptide, Tyr64 and Asn100 was replaced by Cys residues so that the synthetic peptide could form disulphide bonds and thereby presumed a loop structure similar to that predicted from the native Pf-DHFR protein
Summary
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. The identification of peptide epitopes simulating the native protein has traditionally been based on amino acid sequences or sequence motifs exposed on the outer surface of the protein structure, thereby making these peptides potential candidates as antigen epitopes. Examples of algorithms for selecting and defining properties of exposed peptide sequences include plots of hydrophilicity, hydrophobicity, external flexibility and antigenic index. These algorithms provide only crude approximations of the native structures, and antibodies raised against the selected peptides are often lacking reactivity or show low degree of crossreactivity with the native protein [2]. The information could aid in a more precise identification of antigenic epitopes as opposed to defining epitopes based solely on the primary sequence of the protein
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