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

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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. 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.

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