Development of an enzyme-linked immunosorbent assay for the detection of phenothiazine tranquillisers in horses.

The researchers developed an easy-to-use test to detect acepromazine, a tranquillizer drug, and its metabolites in horses up to 25 hours after administration through urine samples, with the potential to prevent misuse of these drugs at horse races and shows.

Objective of the Research

• The research aimed to develop a simple, effective method to detect the presence of the tranquillizer drug acepromazine (ACP) and its resulting metabolites in horses.
• The motivation behind the study was the need for an easy-to-implement testing method that could be used in settings such as horse races, shows, and sales to deter and detect the illicit use of tranquillizer drugs in horses.

Research Methodology

• An ACP hapten was synthesised – this is a molecule which can elicit an immune response only when attached to a larger carrier. In this case, the carrier was bovine serum albumin.
• The hapten-carrier complex was injected into a horse to generate antibodies against the ACP drug.
• A competitive enzyme-linked immunosorbent assay (ELISA) was developed. In this assay, the ACP attached to a solid phase via the enzyme lysozyme competes with any free ACP present in solution for a limited amount of antibody.
• The solutions tested included phosphate buffered saline, horse serum, and horse urine.

Key Findings

• The developed ELISA assay was able to successfully detect the presence of ACP and/or some of its metabolites in horse urine, for at least 25 hours after the administration of a given dose of the drug.
• As low a concentration as 0.24 micrograms per millilitre of ACP or its metabolites could be detected.
• Horse serum testing was ruled out due to non-specific interference, rendering it unreliable for the purposes of this assay.

Significance of the Research

• The developed test presents an advantage due to its level of detection sensitivity and the ease of performing the assay.
• The simple mechanism and effectiveness of this method make it a potentially attractive alternative to the more complex methods currently being used to screen horse urine samples in professional equine contexts.