Table 1 Analysis of ELISA reactivity of T-mVLPs with DENV serotype-specific mAbs.

mAb	Serotype specificity	Epitope Specificity	ELISA OD_{450 nm}
mAb	Serotype specificity	Epitope Specificity	E1 VLPs	E2 VLPs	E3 VLPs	E4 VLPs	T-mVLPs
E29	DENV-1	EDIII	1.87	0.03	0.09	0.14	1.08
3H5	DENV-2	EDIII (FG loop of LR)	0.03	2.12	0.02	0.02	1.25
8A1	DENV-3	EDIII (A strand, FG loop of LR)	0.02	0.01	0.91	0.01	0.85
E1		EDIII (C-C’ loop)	0.04	0.03	3.43	0.02	3.39
E3		EDI/II	0.13	0.03	2.90	0.02	2.04
E4		EDI/II	0.12	0.04	3.18	0.03	2.15
E29	DENV-4	EDIII (F and G strands)	0.04	0.04	0.03	2.80	2.23
E40		EDIII (A, F and G strands)	0.01	0.01	0.01	3.23	2.68
E42		E	0.03	0.02	0.02	2.10	2.70
E43		E	0.02	0.02	0.02	2.15	1.95

Indirect ELISA was performed using E1 VLPs, E2 VLPs, E3 VLPs, E4 VLPs or T-mVLPs as coating antigens. Bound VLPs were detected using DENV-1 mAb E29²⁶, DENV-2 mAb 3H5³⁵, DENV-3 mAbs E1, E3, E4³⁶ and 8A1³⁷, or DENV-4 mAbs E29, E42 and E43³⁹. Mutational studies have identified specific aa residues recognising many of these mAbs. DENV-2 mAb 3H5 binds aa residues in FG loop of EDIII-LR³⁸, DENV-3 mAb E1 binds G340 of C-C’ loop in EDIII³⁶, DENV-4 mAb E29 binds to Y377 (F strand) and H390 (G strand) in the vicinity of EDIII-LR³⁹ and DENV-4 mAb E40 binds K310 (A strand), D375, Y377 (F strand) and H390, F392 (G strand) in the vicinity of EDIII-LR³⁹. Underlined ELISA OD_{450 nm} values indicate the serotype-specificity of the mAbs used to probe T-mVLPs. Data shown are from one of two separate experiments. Note: This assay was not optimised for quantitation of the relative proportion of the different E proteins in the T-mVLPs. Differences in ELISA reactivity of a given mAb between the T-mVLPs and the cognate monovalent E VLPs may reflect subtle differences in the accessibility of the EDIII epitopes in the tetravalent versus the monovalent VLPs. However, meaningful inter-serotypic comparison of reactivity between mAbs is precluded as each binds to distinct and unique epitope(s) of different serotypes, presumably with characteristic binding affinities.

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