Initial development of a rapid, portable, stall-side ELISA for the measurement of equine adrenocorticotropic hormone.

This research outlines the initial development of a rapid, portable ELISA (enzyme-linked immunosorbent assay) for measuring adrenocorticotropic hormone (ACTH) in horses. This could help in diagnosing Pituitary pars intermedia dysfunction (PPID), a common neurodegenerative disease in older horses. The researchers identified suitable antibodies and magnetic beads for the assay, with further tests and optimization in progress.

Background and Objectives

• This study aims to make diagnosing PPID more efficient by developing an ELISA for ACTH measurement which could be used at the horse’s side (stall-side), instead of transporting samples to labs and dealing with factors that can affect the results like storage and handling. PPID is a disease that affects older horses, leading to increased concentrations of certain peptides due to the loss of dopaminergic inhibition of melanotropes in a part of the pituitary gland called the pars intermedia.

Methods

• For developing the ELISA, two ACTH-specific monoclonal antibodies, CBL57 and EPR20361-248, were identified, based on their capacity to recognize separate epitopes (parts of the antigen recognized by the immune system), ability to cause strong and rapid color change, and minimal background interference, which means these antibodies do not interfere with themselves, each other, or the test reagents.
• The researchers chose CBL57 as the detection antibody, which is involved in signalling the presence of the hormone. The other antibody, EPR20361-248, was functionalized on superparamagnetic iron oxide beads and chosen as the capture antibody, which binds to the ACTH in the plasma. The use of magnetic beads forms the initial stage of creating a field-applicable test platform.
• The team had to optimize the concentrations of the antibodies and magnetic beads, as well as the duration of incubation, for the successful demonstration of the assay.

Results

• The immunoassay – a test involving the reaction of an antigen (in this case, ACTH) and an antibody – produced reliable results with unglycosylated rat recombinant ACTH. An unglycosylated protein is one that has not undergone a particular biochemical process called glycosylation, which involves adding a sugar molecule to the protein. Recombinant proteins are those produced through recombinant DNA technology.
• However, the study has not reached its conclusion as further tests for optimizing and validating the assay with equine plasma and serum samples are yet to be done.