Using structural analysis to generate parasite-selective monoclonal antibodies.
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.
Kron
Michael A
MA
Department of Medicine, Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. mkron@mcw.edu
Cichanowicz
Sam
S
Hendrick
Angela
A
Liu
Aizhuo
A
Leykam
Joseph
J
Kuhn
Leslie A
LA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D017178:Brugia malayi / Q000276:immunology*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal