Antigen Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Reston Ebola Virus Nucleoprotein

DOI: 10.1128/CDLI.10.4.552-557.2003

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FIG. 1.
IFA using HeLa cells expressing rNP of EBO-R (A to C) or EBO-Z (D-F) and using Vero cells infected with EBO-S (G to I). The reactivities of Res2-6C8 (A, D, and G), Res2-1D8 (B, E, and H), and 3-3D (C, F, and I) to NP of EBO-R, EBO-Z, and EBO-S are shown. Res2-6C8 and Res2-1D8 reacted to EBO-R NP, but not to the NPs of EBO-Z and EBO-S. The MAb 3-3D reacts to the NPs of all three Ebola virus species.
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FIG. 2.
Reactivities of Res2-6C8 and Res2-1D8 to the truncated EBO-R rNPs in IgG ELISA. Res2-6C8 recognizes the amino acid residues between aa 636 and 639 (sequence, DPDI). Res2-1D8 recognizes the amino acid residues between aa 636 and 643 (sequence, DPDIGQSK). The mean OD value ± SD in the IgG ELISA is shown.
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FIG. 3.
Reactivity of MAbs Res2-6C8 (□), Res2-1D8 (▵), and 3-3D (♦) to the rNPs of EBO-R and EBO-Z in the antigen capture ELISA. His-EBO-R-NP (A) and His-EBO-Z-NP (B) were used as antigens. The mean ± SD of four assays is shown.

TABLE 1.

Primers used for epitope mapping recognized by Res2-6C8 and Res2-1D8

Primer	Sequence (5′ to 3′)^a	Template of reaction
R635f	CAAGGATCCGAAGACCCTGATATC	pGEX-ΔNP_631-739
R636f	TTGGGATCCGACCCTGATATCGGTCAATC	cDNA of EBO-R
R637f	TTGAATGGATCCCCTGATATCGGT	pGEX-ΔNP_631-739
R638f	AATGAAGGATCCGATATCGGTCAA	pGEX-ΔNP_631-739
Mu639f	TGAAGACCCTGATTAAGGTCAATCAAAGT	pGEX-ΔNP_631-739
Mu639r	ACTTTGATTGACCTTAATCAGGGTCTTCA	pGEX-ΔNP_631-739
Mu640f	AGACCCTGATATCTAACAATCAAAGTCTA	pGEX-ΔNP_631-739
Mu640r	TAGACTTTGATTGTTAGATATCAGGGTCT	pGEX-ΔNP_631-739
Mu641f	CCCTGATATCGGTTAATCAAAGTCTATGC	pGEX-ΔNP_631-739
Mu641r	GCATAGACTTTGATTAACCGATATCAGGG	pGEX-ΔNP_631-739
Mu642f	TGATATCGGTCAATAAAAGTCTATGCAAA	pGEX-ΔNP_631-739
Mu642r	TTTGCATAGACTTTTATTGACCGATATCA	pGEX-ΔNP_631-739
Mu643f	TATCGGTCAATCATAATCTATGCAAAAAT	pGEX-ΔNP_631-739
Mu643r	ATTTTTGCATAGATTATGATTGACCGATA	pGEX-ΔNP_631-739
Mu644f	CGGTCAATCAAAGTAAATGCAAAAATTAG	pGEX-ΔNP_631-739
Mu644r	CTAATTTTTGCATTTACTTTGATTGACCG	pGEX-ΔNP_631-739

↵ a BamHI restriction site is underlined. Introduced stop codon is in boldface.

TABLE 2.

Comparison of the epitopes of Res2-6C8 and Res2-1D8

Ebola virus species^a	Amino acids corresponding to epitope of:
Ebola virus species^a	Res2-6C8	Res2-1D8
EBO-R, 1996	₆₃₆DPDI₆₃₉	₆₃₆DPDIGQSK₆₄₃
EBO-R, 1989/1990	₆₃₆DPDI₆₃₉	₆₃₆DPDIGQSK₆₄₃
EBO-Z, 1976	₆₃₆NQDS₆₃₉	₆₃₆NQDSDNTQ₆₄₃
EBO-S, 1976	₆₃₆EALP₆₃₉	₆₃₆EALPINSK₆₄₃

↵ a GenBank accession no.: EBO-R, 1996, AB050936; EBO-R, 1989/1990, AF522874; EBO-Z, 1976, AF086833; and EBO-S, 1976, AF173836.

TABLE 3.

Detection of EBO-R NP from clinical specimens

Sample	Monkey no.	Titer by antigen capture ELISA^a			Result by:
Sample	Monkey no.	Res2-6C8	Res2-1D8	3-3D^b	RT-PCR^c	IHC^d
Serum	2612	160	40	20	+	+
	2866	320	320	160	+	ND
Liver	2877	40	<10	10	ND^e	ND
	2882	160	40	40	+	+
Spleen	2877	320	160	80	ND	ND
	2882	80	20	<10	ND	+
	2885	160	40	<10	+	ND

↵ a Antigen titer in the specimens. The antigen titer in the ELISA was determined as the reciprocal of the highest dilution showing a positive reaction. The titer was determined by a single test. The cutoff OD value was 0.2.
↵ b Ebola virus species cross-reactive MAb (29).
↵ c RT-PCR with primers RES-Nf2 and RES-Nr2.
↵ d IHC, immunohistochemistry for the detection of Ebola virus NP (14).
↵ e ND, not done.