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

Abstract

Pituitary pars intermedia dysfunction (PPID) is a neurodegenerative disease of senior horses. Loss of dopaminergic inhibition of the melanotropes of the pars intermedia leads to increased concentrations of pro-opiomelanocortin (POMC)-derived peptides. Diagnosis is challenging due to pre-analytical variables, such as sample storage, handling, and time to analysis. Our objective was to develop an ELISA for ACTH measurement, which could ultimately form the basis for a stall-side equine ACTH test. We selected 2 ACTH-specific monoclonal antibodies, CBL57 and EPR20361-248, based on the recognition of separate epitopes, strong and rapid color change, and minimal background interference, including no cross-reactivity with themselves, each other, and the test reagents. CBL57 was chosen as the detection antibody (or secondary antibody). EPR20361-248, functionalized on superparamagnetic iron oxide beads, was chosen as the capture antibody (or primary antibody) to bind ACTH in plasma. The incorporation of magnetic beads marks the initial stage in establishing a platform that could potentially be utilized in the field, similar to other stall-side tests. The concentrations of antibodies, magnetic beads, and incubation durations were optimized. Our immunoassay detected unglycosylated rat recombinant ACTH. Further studies are ongoing to optimize and validate our assay using equine plasma and serum samples.

Keywords: ACTH; ELISA; equine Cushing disease; horses; pituitary pars intermedia dysfunction.

Figure 1.

Investigation of detection and capture antibody cross-reactivity. Experiment without magnetic beads using 1 mg/mL of each antibody and varied ACTH concentrations. When no ACTH is present, there is minimal signaling, suggesting that antibodies do not cross-react with each other. Error bars denote SD between duplicates at each ACTH concentration.

Figure 2.

Investigation of analytical sensitivity. Preliminary data suggest the assay’s limit of detection is 1–10 pg/mL. Error bars denote SD between duplicates at each ACTH concentration.

Figure 3.

Investigation of linearity. Annotated blue dots are the averages of duplicates at each ACTH concentration. The trendline (dashed line) was determined using Excel spreadsheet trendline function and the corresponding equation of the line and R value. Linearity of assay is established at 0–40 pg/mL.