Characterization and epitope mapping of monoclonal antibodies to the nucleocapsid protein of severe acute respiratory syndrome coronavirus.
The sudden emergence of severe acute respiratory syndrome (SARS) at the end of 2002 resulted in 774 reported deaths from more than 8000 cases worldwide. As no effective vaccines or antiviral agents are available, the most effective measure to prevent the expansion of a SARS epidemic is the rapid diagnosis and isolation of SARS patients. To establish specific diagnostic methods, we generated nine clones of monoclonal antibodies to nucleocapsid protein (NP) of SARS-coronavirus (SARS-CoV). On immunofluorescent antibody assay and Western blotting analysis, none of the monoclonal antibodies showed cross-reactivity to authentic and recombinant NPs of human coronavirus (HCoV) 229E strain. To determine the region on the NP molecule where the monoclonal antibodies bind, we generated four truncated recombinant NPs and analyzed the reactivity between monoclonal antibodies and truncated NPs. Two monoclonal antibodies reacted with a truncated NP covering from amino acid residues 111 to 230, and seven reacted with another truncated NP covering from amino acid residues 221 to 340. Epitope mapping analysis indicated that monoclonal antibody SN5-25 recognized the amino acid sequence Q(245)TVTKK(250) On SARS-NP. Within the epitope, Q245, T246, V247, K249, and K250 appeared to form an essential motif for monoclonal antibody SN5-25 to bind. The information about binding sites and epitopes of monoclonal antibodies may be useful for the development of new diagnostic methods for SARS and for analyzing the function of N protein of SARS-CoV.
Kariwa
Hiroaki
H
Laboratory of Public Health, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan. kariwa@vetemed.hokudai.ac.jp
Noda
Hiroshi
H
Nakauchi
Mina
M
Ishizuka
Mariko
M
Hashiguchi
Kazuaki
K
Hashimoto
Shingo
S
Yoshii
Kentaro
K
Asano
Atsushi
A
Agui
Takashi
T
Kogaki
Hiroyuki
H
Kurano
Yoshihiro
Y
Uchida
Yoshiaki
Y
Fujii
Nobuyuki
N
Okada
Masahisa
M
Takashima
Ikuo
I
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000914:Antibodies, Viral / Q000737:chemistry; D000918:Antibody Specificity; D015153:Blotting, Western / Q000662:veterinary; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D028941:Coronavirus 229E, Human; D000086462:Coronavirus Nucleocapsid Proteins; D003429:Cross Reactions; D018604:Epitope Mapping; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D006801:Humans; D051379:Mice; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis / Q000662:veterinary*; D014709:Vero Cells
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins