Aggregation-associated loss of antigenicity observed for denatured virion protein 1 of Equine rhinitis A virus in an enzyme-linked immunosorbent assay.

This research focused on the Equine rhinitis A virus (ERAV), a common respiratory infection in horses, addressing the issue of a prototype diagnostic test losing its sensitivity due to protein denaturation and aggregation. The study offers an improved way of producing relevant proteins in their natural state for diagnostic purposes.

Overview and Aims of the Research

• The research aimed to investigate a problem found with an Enzyme-linked Immunosorbent Assay (ELISA) diagnostic test for ERAV, a common horse respiratory infection.
• The ERVA detection method, particularly making use of a protein designated VP1, was found to not function properly under denatured conditions.
• The researchers therefore set out to optimize the procedure for expressing and purifying the relevant protein in its native form and verify whether denaturation was indeed causing diagnostic issues.

The Expression and Purification of rVP1

• The team managed to successfully develop a method for obtaining the full-length rVP1 protein under natural or ‘native’ conditions.
• This would ensure the protein’s structure remains intact, potentially improving the functionality and reliability of the ERAV ELISA diagnostic test.

Confirming Antigenicity Loss Due to Denaturation

• The researchers established that the denatured version of the rVP1 protein failed to provoke a reaction with ERAV antibodies within the ELISA diagnostic test.
• This confirms the suspicion that denaturation can cause a diagnostic failure, potentially leading to false negatives and unreliable testing results.

Understanding the Cause of Antigenicity Loss

• By applying Size Exclusion Chromatography (SEC), the team discovered that the denatured rVP1 protein forms large, multimeric aggregates.
• These aggregates are likely to disrupt the protein’s functional structure, thus deeming it unrecognisable to antibodies and unsuitable for diagnostic purposes.

Overall, this research provides valuable insights into the conditions necessary for successful ERAV antibody detection and steps towards optimising a reliable diagnostic test for this common and globally prevalent equine disease.