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.
Highlights
Hemagglutination-inhibition (HI) and neutralization are used to evaluate vaccines against influenza virus A (H5N1); poor standardization leads to interlaboratory variation of results
Assay variability limits comparison of candidate influenza virus subtype H5N1 vaccines in different clinical trials, posing challenges for licensure, if specific seroprotective titers are required as endpoints [3,4,5]
We assessed the reproducibility of neutralization and hHI tests for influenza virus A (H5N1) and evaluated the suitability of a standard for detection of antibody
Summary
Serum Samples We used 14 serum samples (coded A–N) from persons who had received nonadjuvanted or adjuvanted splitproduct vaccine derived from reassortant clade 1 virus We calculated the percentage of endpoints of replicate tests for identical serum samples A and L that differed >2-fold or >4-fold for each antigen and assay in each laboratory. We compared differences between hHI and neutralization GMTs for 07/150 by different laboratories by using a paired nonparametric Wilcoxon signed-rank test for each antigen separately. We compared differences among overall (for all laboratories) mean GMT for all serum samples by using a paired nonparametric Wilcoxon signed-rank test for each antigen separately; e.g., for NIBRG-14, the difference between the overall mean GMT for hHI and neutralization was calculated for each sample, and these differences were compared with zero. Using the Wilcoxon signed-rank test for each antigen separately, we compared these differences with zero
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