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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 IBA2a
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 IBA2b
  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 IBA2b
 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
  1. 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
  2. 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
  3. 3Obtained from the Department of Poultry Diseases, National Veterinary Research Institute (DPD NVRI; Puławy, Poland)
  4. 4(Cambridge, England, UK), Cat. No. b7477