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 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.


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.
Alifrangis
Michael
M
Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology and Institute of Public Health, University of Copenhagen, Denmark. malif@biobase.dk
Christensen
Inge T
IT
Jørgensen
Flemming S
FS
Rønn
Anita M
AM
Weng
Jimmy E
JE
Chen
Ming
M
Bygbjerg
Ib C
IC
Sirawaraporn
Worachart
W
Palarasah
Yaseelan
Y
Koch
Claus
C
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000913:Antibodies, Protozoan / Q000097:blood / Q000276:immunology; D000953:Antigens, Protozoan / Q000737:chemistry / Q000276:immunology*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000276:immunology; D011817:Rabbits; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000276:immunology*
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000939:Epitopes; D007106:Immune Sera; D013762:Tetrahydrofolate Dehydrogenase