Development of a high-throughput cell based 384-well influenza A quantification assay for interpandemic and highly pathogenic avian strains

Melicia R. Gainey, Ann M. Wasko, Jennifer N. Garver, David J. Guistino, Eric M. Vela, John E. Bigger

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DOI: 10.4236/health.2010.21006                

Abstract

Influenza remains a world wide health threat, thus the need for a high-throughput and robust assay to quantify both seasonal and avian in-fluenza A strains. Therefore, a 384-well plate format was developed for the median tissue culture infectious dose assay (TCID50) utilizing the detection of nucleoprotein by an in situ en-zyme linked immunosorbent assay (ELISA) which was optimized for sensitivity in this assay. Highly pathogenic avian influenza, A/Vietnam/ 1203/04 (H5N1), and interpandemic strains, A/ New Caledonia/20/99 (H1N1) and A/Brisbane/ 10/07 (H3N2), were quantified using this high- throughput assay. Each 384-well plate can be used to analyze ten viral samples in quadrupli-cate, eight dilutions per sample, including all necessary assay controls. The results obtained from 384-well plates were comparable to tradi-tional 96-well plates and also demonstrate re-peatability, intermediate precision, and assay linearity. Further, the use of 384-well plates in-creased the throughput of sample analysis and the precision and accuracy of the resulting titer.

Keywords

Avian Influenza; ELISA; High-Throughput Assay; Interpandemic Influenza A; TCID50

Conflicts of Interest

The authors declare no conflicts of interest.

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