A novel epitope-blocking ELISA for specific and sensitive detection of antibodies against H5-subtype influenza virus hemagglutinin

Sączyńska, Violetta; Florys-Jankowska, Katarzyna; Porębska, Anna; Cecuda-Adamczewska, Violetta

doi:10.1186/s12985-021-01564-6

Table 1 Sera used for H5 EB-ELISA development and preliminary validation

From: A novel epitope-blocking ELISA for specific and sensitive detection of antibodies against H5-subtype influenza virus hemagglutinin

Chickens	Immunogen	Serum description	Samples (N)	HI titer	Origin
Anti-H5 HA positive (1): reference antisera against LPAIVs of the H5 subtype
Used in evaluation of diagnostic sensitivity 1 (Dse 1)
SPF	H5N1 AIV, inactivated	One batch (#1)	1	1:512 with H5N1 AIV according to the certificate	x-OvO Ltd.^1a
SPF	H5N2 AIV, inactivated	Three batches (#1, #2 and #3)	3	1:256 or 1:512 with H5N2 AIV according to the certificates	x-OvO Ltd.^1a
SPF	H5N3 AIV, inactivated	Three batches (#1, #2 and #3)	3	1:512 with H5N3 AIV according to the certificates	x-OvO Ltd.^1a
SPF	H5N9 AIV, inactivated	Two batches (#1 and #2)	2	1:512 or 1:256 with H5N9 AIV according to the certificates	x-OvO Ltd.^1a
Positive controls (PC) used for repeatability determination
SPF	H5N2 AIV, inactivated	Batch #3 (weak PC)	1	1:512 with H5N2 AIV according to the certificate	x-OvO Ltd.^1a
SPF	H5N3 AIV, inactivated	Batch #1 (strong PC)	1	1:512 with H5N3 AIV according to the certificate	x-OvO Ltd.^1a
Anti-H5 HA positive (2): experimental antisera against HA from H5N1 HPAIV
Used in evaluation of diagnostic sensitivity 2 (Dse 2)
Commercial layers, Rossa 1 line	rH5-E. coli adjuvanted with alum	From 69 chickens at 8, 9, 10 or 11 weeks of age; 1 or 2 sampling time points per chicken	115 (115)	1:8–1:512 with H5N2 AIV, determined at IBA	IBA^2a
Anti-H5 HA negative (1): sera of various origin
Used in determination of cutoff value and diagnostic specificity (Dsp)
SPF layers, White Leghorn line	None	From 10 chickens at 9 or 11 weeks of age; 1 or 2 sampling time points per chicken	18	Negative AI status	DPD NVRI³
Commercial layers, Rossa 1 line	None	From 30 chickens at 7, 8, 9, 10 or 11 weeks of age; 3–5 sampling time points per chicken	130 (63)	< 1:8 with H5N2 AIV, determined at IBA	IBA^2b
Commercial broilers, Ross 308 line	None	From 42 chickens at 3, 5, 5 ½, 6 or 7 weeks of age; 1–4 sampling time points per chicken	61 (33)	< 1:8 with H5N2 AIV, determined at IBA	IBA^2b
Negative control (NC) used for repeatability determination
Different strains and sex	None	Normal chicken serum, 1 batch (NC)	1	< 1:8 with H5N2 AIV, determined at IBA	Abcam⁴
Anti-H5 HA negative (2): reference antisera against LPAIVs of the non-H5 subtypes
Used in evaluation of analytical specificity (Asp)
SPF	AIVs: H1–H4, H6–H12 and H14–H16 inactivated	One batch of each antiserum	20	1:128–1:2048 with homologous AIVs according to the certificates	x-OvO Ltd.^1b
SPF	H13N6 AIV, inactivated	Two batches (#1 and #2)	2	1:128 or 1:1024 with H13N6 AIV according to the certificates	x-OvO Ltd.^1b

¹Certified by Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe; Legnaro, Padova, Italy) and purchased from x-OvO Ltd. (Dunfermline, Scotland, UK). Details of reference antisera against (a) H5 and (b) non-H5 subtype AIVs are provided in Additional file 1: Tables S2 and S3, respectively
²Collected in (a) the test vaccine and (b) control chicken groups during immunization studies with H5 HA protein produced in bacteria (rH5-E. coli) at the Institute of Biotechnology and Antibiotics (IBA; Warsaw, Poland). The number of samples described previously with HI titers [10] are provided in brackets. Details of the vaccine antigen and experimental antisera against H5 HA are presented in Additional file 1: Tables S1 and S4, respectively
³Obtained from the Department of Poultry Diseases, National Veterinary Research Institute (DPD NVRI; Puławy, Poland)
⁴(Cambridge, England, UK), Cat. No. b7477

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