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

A dipstick, competitive immunoassay for rapidly measuring serum oestrone su lfate (OS) concentrations in horses was developed to distinguish mares 100 or more days pregnant from non-pregnant animals. 6-Ketoestrone 6-carboxymet hyloxime conjugated to bovine serum albumin (oestrone CMO-BSA) was 'dotted' 25 mm from the bottom edge of 45 x 5 mm strips of polyester-film-supported cellulose nitrate membrane, pore size 3 mum. The strips were blocked, drie d and a 15 x 5-mm cellulose absorbent sink attached 10 mm from the top of e ach strip. The manufactured dipsticks were stored with desiccant at room te mperature until used. A monoclonal antibody recognizing OS was coated onto uniform, blue-dyed polystyrene microspheres (mean diameter, 0.31 mum) by ad sorption. After blocking, several washes and resuspension by sonication, th e antibody-coated microspheres were stored at 4 degreesC. The concentration s of oestrone CMO-BSA, dotted onto the dipsticks and OS antibody coated ont o the microspheres were optimized to produce a test that allowed maximum di scrimination between the concentrations of OS found in serum of mares 100 o r 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 < mu>L of carrier buffer, 10 muL of OS antibody-coated microspheres and 10 mu 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 dipst ick into the absorbent sink within 15-20 min leaving a blue dot where the O CMO-BSA had been placed. The intensity of colour of the blue dot, which cor related inversely with the concentration of OS in the standard or serum sam ple, was assessed visually and by computer image analysis. An OS concentrat ion less than 5 ng mL(-1) produced a deep blue dot, 20 ng/ml a light blue d ot 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 fr om non-pregnant mares produced dipsticks with deep blue dots that ranged in intensity from 20 to 38 colour intensity units, equivalent to OS concentra tions less than 7 ng mL(-1). Sera from all the pregnant mares generated dip sticks with either faint blue dots or none at all (i.e. 15 colour intensity units, equivalent to OS concentrations >40 ng mL(-1)). It is concluded tha t this novel, rapid dipstick immunoassay offers a practical, alternative me ans 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 p regnant.