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.
Subject
Developmental Biology,Endocrinology,Genetics,Molecular Biology,Animal Science and Zoology,Reproductive Medicine,Biotechnology
Cited by
15 articles.
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