# Development of a Bead-Based Multiplex Fluorescent Immunoassay to Detect Antibodies against Maedi-Visna Virus in Sheep

## TL;DR

Researchers developed a new immunoassay to detect antibodies against a specific strain of Maedi-Visna virus in sheep, aiming to improve disease surveillance in Norway.

## Contribution

A bead-based multiplex immunoassay tailored for a Norwegian MVV strain was developed and evaluated for its diagnostic performance.

## Key findings

- The assay demonstrated good repeatability with a coefficient of variation below 15% for most positive samples.
- Analytical sensitivity was equal or higher than a commercial ELISA, with high specificity for each antigen tested.

## Abstract

Serological tests that detect antibodies, especially indirect ELISAs, are the most common method to diagnose Maedi-visna virus (MVV) infections in small ruminants. However, due to the high genetic heterogeneity of the virus, commercial tests might not effectively detect infected animals in all populations. Bead-based multiplex immunoassays can detect multiple analytes simultaneously, thus enabling the detection of antibodies against several MVV antigens in the same assay. In Norway, there is a national aim to eliminate MVV from the sheep population and improving the serological test performance would strengthen the surveillance of the disease. In this study, we developed a bead-based multiplex immunoassay to detect antibodies against viral epitopes in MVV-infected sheep, including antigens based on the circulating viral strain in Norway. Thus, the assay is tailored for usage in the Norwegian sheep population. Although this work shows promising results, including repeatability, analytical sensitivity, and specificity, the diagnostic characteristics must be evaluated before the assay can be implemented in the Norwegian surveillance programme.

The Maedi-visna virus (MVV) causes a persistent infection in small ruminants, and its high genetic heterogeneity affects the performance of diagnostic tests when used in different populations. Therefore, the aim of this study was to develop a bead-based multiplex immunoassay tailored to detect antibodies against a Norwegian MVV strain. We used tissue samples from 14 PCR-positive sheep from a recent MVV outbreak in Norway to sequence the viral strain and produced recombinant antigens based on sequences from one animal. The assay included commercial TM-A and recombinant Norwegian p25, p16–25 and SU5 antigens. Cut-off values for each antigen were determined using receiver operating characteristic curves on 40 ELISA-negative and 67 ELISA-positive samples from the outbreak. The intraplate and interplate repeatability were investigated by testing a quadruplicate of five samples over three days, while the analytical sensitivity (aSe) and specificity (aSp) were measured in comparison to a commercial ELISA. The repeatability showed a coefficient of variation below 15% for most positive samples. The aSe was equal or higher for the multiplex assay than the ELISA, and the aSp of each antigen was 91.7, 93.3, 95.0 and 93.3% for p25, p16–25, SU5 and TM-A, respectively. The assay shows promising results; however, further evaluations of diagnostic characteristics are necessary before implementation in the Norwegian surveillance programme.