Dutch Scientists Subtype Antibodies Against Avian Influenza Virus Subtypes in Poultry Sera with xMAP® Technology

Chris Haake

Multiplexing allowed them to reduce sample volume, save money, and report results faster

Many countries in Europe and North America have reported their worst numbers of avian influenza (bird flu) in recent years. This dangerous virus—which infects food animals such as chickens, as well as dozens of wild bird species, can force farmers to cull their flocks.

One important way to monitor the avian influenza landscape is by detecting antibodies to characterize any relevant virus strains to which a flock has been exposed. Unfortunately, the diversity in influenza strains makes this very challenging. Each virus contains a specific subtype of haemagglutinin (HA) and neuraminidase (NA). For avian flu, there are 16 known HA subtypes and nine known NA subtypes, which can occur in different combinations. With traditional testing techniques, individual assays must be run for each HA and NA subtype until the correct one is identified. This is a time-consuming, tedious approach.

At Wageningen Bioveterinary Research (WBVR) in the Netherlands, scientists have adopted xMAP® Technology for a multiplex, beadbased approach to avian influenza antibody testing. The assay they created contained all 25 subtypes of HA and NA, which allowed them to generate complete serotyping information from a single reaction. Plus, multiple samples could be run at the same time for a truly scalable, high-throughput approach. The flexibility of xMAP Technology allows for simultaneous sample testing

“Getting results within a day”

Moreover, WBVR is responsible for testing the collected samples at farms throughout the year, wherein the lab then looks for the subtypes of the low pathogenic avian influenza viruses circulating throughout the Netherlands. The test, developed by a team of scientists which included veterinarian Evelien Germeraad and lab technician René Achterberg, is essential for avian influenza monitoring in poultry and in other birds. “This assay made it possible to do the subtyping of the antibodies in the poultry sera really quickly,” Germeraad says. “That process usually takes a lot of time and is expensive. Now we can get results within a day.”

Achterberg’s lab team turned to xMAP Technology when the assay they had previously used was discontinued by its manufacturer. The team was already running Luminex xMAP Technology, so that platform “was the logical successor,” Achterberg says. Developing the new test was a relatively straightforward process of coupling different variants of the HA and NA proteins to xMAP beads, and then optimizing the assay for cross-reactivity and other characteristics.

Evelien Germeraad and René Achterberg

The resulting test was a significant improvement compared to their prior methods, e.g., the hemagglutination-inhibition (HI) assay. The classical HI assay requires hundreds of microliters of serum for HA subtyping, while with xMAP Technology, only two microliters of serum is sufficient to generate comprehensive results. The workflow was reduced from at least two days to half a day. And since the chickens have limited serum, using less volume for the assay allows for repeats, if needed. The new assay also makes it possible to test many more samples at once and can increase throughput as needed.

The flexibility of xMAP Technology is another important advantage for scientists at WBVR. Since the team published their first xMAP-based avian flu assay design in 2019, they have continued to optimize it and have since added some new variants of key proteins. “That’s a great feature of Luminex assays, that you can easily add more proteins if needed,” Achterberg says. “If you use a commercial assay, you have to rely on what they include. We can include whatever we want.”

The assay now includes multiple variants for every HA and NA subtype. “Avian influenza has so many subtypes, so you really need to be able to multiplex in a test,” Germeraad says. “That’s why this assay is such a great solution.”

Learn more about Wageningen Bioveterinary Research here.

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xMAP® Technology helps deliver more data from less sample, giving you some of your time back in an increasingly busy world. Additional benefits include:

• A broad dynamic range, helping you avoid repeat testing due to out-of-range values • A compact footprint, with an embedded

PC and an easy-to-use touchscreen interface • Intuitive and flexible software • An xTENSIVE menu of commercially available kits • Custom assay support, including assay development services through the LuminexPLORE Lab

Whatever your needs are, xMAP Technology offers ways to save. Learn more about the many benefits of multiplexing with xMAP INTELLIFLEX® on our website: luminexcorp.com/INTELLIFLEX.