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1 Department of Medicine, Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA
2 Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA
3 College of Human Medicine, Michigan State University, East Lansing, Michigan 48824, USA
4 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA
5 Macromolecular Structure, Sequence, and Synthesis Facility, Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA
6 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA
7 Department of Computer Science and Engineering, Michigan State University, East Lansing, Michigan 48824, USA
8 Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan 48824, USA
9 Quantitative Biology Initiative, Michigan State University, East Lansing, Michigan 48824, USA
(RECEIVED January 4, 2008; FINAL REVISION March 12, 2008; ACCEPTED March 13, 2008)
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 Å 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 IgG1 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.
Keywords: aminoacyl-tRNA synthetase; filariasis; surface plasmon resonance; nuclear magnetic resonance; conformational determinacy; Brugia malayi; epitope prediction
Reprint requests to: Michael A. Kron, Department of Medicine, Biotechnology and Bioengineering Center, Medical College of Wisconsin, 8701 Watertown Plank Road, P.O. Box 26509, Milwaukee, WI 53226, USA; e-mail: mkron{at}mcw.edu; fax: (414) 456-6568.
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.073429808.
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