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. 2009 Aug;15(8):1252-9.
doi: 10.3201/eid1508.081754.

Reproducibility of serologic assays for influenza virus A (H5N1)

Iain Stephenson  1 , Alan Heath, Diane Major, Robert W Newman, Katja Hoschler, Wang Junzi, Jacqueline M Katz, Jerry P Weir, Maria C Zambon, John M Wood
Affiliations

Reproducibility of serologic assays for influenza virus A (H5N1)

Iain Stephenson et al. Emerg Infect Dis. 2009 Aug.

Abstract

Hemagglutination-inhibition (HI) and neutralization are used to evaluate vaccines against influenza virus A (H5N1); however, poor standardization leads to interlaboratory variation of results. A candidate antibody standard (07/150) was prepared from pooled plasma of persons given clade 1 A/Vietnam/1194/2004 vaccine. To test human and sheep antiserum, 15 laboratories used HI and neutralization and reassortant A/Vietnam/1194/2004, A/turkey/Turkey/1/2005 (clade 2.2), and A/Anhui/1/2005 (clade 2.3.4) viruses. Interlaboratory variation was observed for both assays, but when titers were expressed relative to 07/150, overall percentage geometric coefficient of variation for A/Vietnam/1194/2004 was reduced from 125% to 61% for HI and from 183% to 81% for neutralization. Lack of reduced variability to clade 2 antigens suggested the need for clade-specific standards. Sheep antiserum as a standard did not reliably reduce variability. The World Health Organization has established 07/150 as an international standard for antibody to clade 1 subtype H5 and has an assigned potency of 1,000 IU/ampoule.

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Figures

Figure 1
Figure 1
Intralaboratory reproducibility showing proportion (%) of replicate assays differing by >2-fold (A, B) and >4-fold (C, D) by horse hemagglutination-inhibition and neutralization assays for each participating laboratory for all serum samples (A, C) and postvaccination serum samples (B, D). Laboratory 11 did not return hemagglutinin-inhibition result.
Figure 2
Figure 2
Range of hemagglutination-inhibition (HI) and neutralization titers to clade 1 homologous NIBRG-14 virus in postvaccination serum sample F: the number of laboratories reporting specific titer dilution of absolute titers and titers relative to 07/150. A) Absolute horse HI titers, B) absolute neutralization titers, C) titers relative to 07/150 horse HI titers, D) titers relative to 07/150 neutralization titers.
Figure 3
Figure 3
Range of hemagglutination-inhibition (HI) and neutralization titers to clade 2.2 and 2.3 heterologous NIBRG-23 and IBCDC-RG5 viruses in postvaccination serum sample F: the number of laboratories reporting specific titer dilution of absolute titers and titers relative to 07/150. A) Absolute horse HI titers, B) absolute neutralization titers, C) titers relative to 07/150 horse HI titers, D) titers relative to 07/150 neutralization titers.
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