Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay.
Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units.
Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan Kenya Medical Research Institute, Nairobi, Kenya.
Ferdinard
F
Adungo
Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan yufuxun@yahoo.com moritak@nagasaki-u.ac.jp.
Fuxun
F
Yu
Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.
David
D
Kamau
Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.
Shingo
S
Inoue
Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.
Daisuke
D
Hayasaka
Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.
Guillermo
G
Posadas-Herrera
Kenya Medical Research Institute, Nairobi, Kenya.
Rosemary
R
Sang
Kenya Medical Research Institute, Nairobi, Kenya.
Matilu
M
Mwau
Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan yufuxun@yahoo.com moritak@nagasaki-u.ac.jp.
Kouichi
K
Morita
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology / Q000302:isolation & purification*; D000956:Antigens, Viral / Q000032:analysis*; D003955:Diagnostic Tests, Routine / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007075:Immunoglobulin M / Q000097:blood; D007155:Immunologic Factors; D008807:Mice, Inbred BALB C; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology; D015004:Yellow Fever / Q000175:diagnosis*; D015005:Yellow fever virus / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D007075:Immunoglobulin M; D007155:Immunologic Factors; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins