Using structural analysis to generate parasite-selective monoclonal antibodies. Protein Sci 2008 Jun;17(6):983-9
Date
04/16/2008Pubmed ID
18411421Pubmed Central ID
PMC2386738DOI
10.1110/ps.073429808Scopus ID
2-s2.0-44349164577 (requires institutional sign-in at Scopus site) 4 CitationsAbstract
Diagnosis of eukaryotic parasitic infection using antibody-based tests such as ELISAs (enzyme-linked immunosorbent assays) is often problematic because of the need to differentiate between homologous host and pathogen proteins and to ensure that antibodies raised against a peptide will also bind to the peptide in the context of its three-dimensional protein structure. Filariasis caused by the nematode, Brugia malayi, is an important worldwide tropical disease in which parasites disappear from the bloodstream during daylight hours, thus hampering standard microscopic diagnostic methods. To address this problem, a structural approach was used to develop monoclonal antibodies (mAbs) that detect asparaginyl-tRNA synthetase (AsnRS) secreted from B. malayi. B. malayi and human AsnRS amino acid sequences were aligned to identify regions that are relatively unconserved, and a 1.9 A crystallographic structure of B. malayi AsnRS was used to identify peptidyl regions that are surface accessible and available for antibody binding. Sequery and SSA (Superpositional Structural Analysis) software was used to analyze which of these peptides was most likely to maintain its native conformation as a synthetic peptide, and its predicted helical structure was confirmed by NMR. A 22-residue peptide was synthesized to produce murine mAbs. Four IgG(1) mAbs were identified that recognized the synthetic peptide and the full-length parasite AsnRS, but not human AsnRS. The specificity and affinity of mAbs was confirmed by Western blot, immunohistochemistry, surface plasmon resonance, and enzyme inhibition assays. These results support the success of structural modeling to choose peptides for raising selective antibodies that bind to the native protein.
Author List
Kron MA, Cichanowicz S, Hendrick A, Liu A, Leykam J, Kuhn LAAuthor
Michael Kron MD Director, Professor in the Medicine department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAnimals
Antibodies, Monoclonal
Brugia malayi
Cell Line
Enzyme-Linked Immunosorbent Assay
Female
Humans
Immunohistochemistry
Male
Mice
Mice, Inbred BALB C
Models, Molecular
Molecular Sequence Data
Nuclear Magnetic Resonance, Biomolecular
Protein Conformation
Surface Plasmon Resonance
