The development of a multiplex serological assay for avian influenza based on Luminex technology

Avian influenza (AI) is an infectious disease in birds with enormous impact on the poultry sector. AI viruses are divided into different subtypes based on the antigenicity of their surface proteins haemagglutinin (HA) and neuraminidases (NA). In birds, 16 HA subtypes and 9 NA subtypes are detected in different combinations. Traditional serological methods for the subtyping of AI antibodies are labour-intensive and have to be performed for each HA and NA subtype separately. This study describes the development of a multiplex serological assay for subtyping AI antibodies in poultry sera using Luminex xMAP technology. This multiplex assay allows the detection of all AI serotypes in one single assay. For all HA and NA subtypes, recombinant proteins were purified and coupled to colour-coded magnetic bead sets. Using the Luminex MAGPIX device, binding of serum antibodies to the antigens on the bead sets is detected by fluorescent secondary antibodies, and the different bead sets are identified. The results of the multiplex assay were compared with that of the traditional singleplex assays. We show that serotyping using the novel multiplex serological assay is consistent with the results of the traditional assays in 97.8% of the reference sera and in 90.8% of the field sera. The assay has a higher sensitivity than the traditional assays, and requires a smaller sample volume. Therefore, the assay will allow complete AI-serotyping in small volumes of field sera, which will improve the monitoring of AI subtypes circulating in poultry significantly.