A dipstick immunoassay to rapidly measure serum oestrone sulfate concentrations in horses

Abstract

A dipstick, competitive immunoassay for rapidly measuring serum oestrone sulfate (OS) concentrations in horses was developed to distinguish mares 100 or more days pregnant from non-pregnant animals. 6-Ketoestrone 6-carboxymethyloxime conjugated to bovine serum albumin (oestrone CMO-BSA) was ‘dotted’ 25 mm from the bottom edge of 45 5 mm strips of polyester-film-supported cellulose nitrate membrane, pore size 3 m. The strips were blocked, dried and a 15 5-mm cellulose absorbent sink attached 10 mm from the top of each strip. The manufactured dipsticks were stored with desiccant at room temperature until used. A monoclonal antibody recognizing OS was coated onto uniform, blue-dyed polystyrene microspheres (mean diameter, 0.31 m) by adsorption. After blocking, several washes and resuspension by sonication, the antibody-coated microspheres were stored at 4˚C. The concentrations of oestrone CMO-BSA dotted onto the dipsticks and OS antibody coated onto the microspheres were optimized to produce a test that allowed maximum discrimination between the concentrations of OS found in serum of mares 100 or more days pregnant (i.e. >30 ng OS mL ^–1 ) relative to those found in non-pregnant mares (i.e. <10 ng OS mL ^–1 ). To perform the dipstick test, 30 L of carrier buffer, 10 L of OS antibody-coated microspheres and 10 l of OS standard or serum sample were pipetted into a microwell and mixed. A dipstick was placed in the solution. All the liquid migrated up the dipstick into the absorbent sink within 15–20 min leaving a blue dot where the OCMO-BSA had been placed. The intensity of colour of the blue dot, which correlated inversely with the concentration of OS in the standard or serum sample, was assessed visually and by computer image analysis. An OS concentration less than 5 ng mL ^–1 produced a deep blue dot, 20 ng/ml a light blue dot and a concentration greater than 50 ng mL ^–1 a very faint blue dot, or none at all. Serum samples from 42 non-pregnant mares and 40 mares over 100 days pregnant were analysed by the dipstick test. All the serum samples from non-pregnant mares produced dipsticks with deep blue dots that ranged in intensity from 20 to 38 colour intensity units, equivalent to OS concentrations less than 7 ng mL ^–1 . Sera from all the pregnant mares generated dipsticks with either faint blue dots or none at all (i.e. £=5 colour intensity units, equivalent to OS concentrations >40 ng mL ^–1 ). It is concluded that this novel, rapid dipstick immunoassay offers a practical, alternative means of analysing serum OS concentrations in horses, and enables mares that are 100 or more days pregnant to be distinguished from those that are not pregnant.