A sensitive EIA for 17 beta-estradiol and progesterone in culture medium for oocyte in vitro maturation procedures.
A sensitive heterologous enzyme immunoassay (EIA) was validated to determine 17 beta-estradiol (E2) and progesterone levels, without previous extraction, in culture medium from rabbit oocytes matured in vitro with and without the addition of IGF-I. Polyclonal E2 (C902), and progesterone (C914) antibodies were raised in rabbits using 6-keto-17 beta-estradiol 6-carboxymethyloxime:BSA, and 11 alpha-hydroxyprogesterone 11 alpha-hemisuccinate:BSA. Horseradish peroxidase was used as label, conjugated to 17 beta-estradiol 3-hemisuccinate, and to progesterone 3-carboxymethyloxime. Standard dose response curves covered a range between 0 and 1 ng/well (100 microliters). The low detection limits of the technique were 1.99 pg/well for E2, and 13.21 pg/well for progesterone. Intra- and interassay coefficient of variation percentage (% CV) were < 6.3 and < 7.8 for E2 and progesterone, respectively (n = 10). The recovery rate of known E2 or progesterone concentrations added to a pool of culture maturation medium averaged 96.39%, and 98.65%, respectively. Compared with RIA, EIA values were in close agreement for E2 (n = 15, R = 0.96, P < 0.001), and progesterone (n = 15, R = 0.99, P < 0.001). Medium samples were obtained after oocyte maturation in vitro for 16 h. Use of IGF-I significantly elevated steroids production in the oocyte surrounded cumulus cells. The EIA described here is highly sensitive and specific assay, and provides a rapid, simple, inexpensive, and non-radiometric alternative to RIA for determining E2 and progesterone levels in oocyte culture medium.
Lorenzo
P L
PL
Depto. Fisiología Animal, Facultad de Veterinaria, UCM, Madrid, Spain. lorenzopl@vet.ucm.es
Illera
J C
JC
Silván
G
G
Munro
C J
CJ
Rebollar
P G
PG
Alvariño
J M
JM
Illera
M J
MJ
Illera
M
M
D000818:Animals; D000917:Antibody Formation; D000918:Antibody Specificity; D016922:Cellular Senescence / Q000187:drug effects; D003470:Culture Media; D004958:Estradiol / Q000032:analysis*; D007124:Immunoenzyme Techniques; D009865:Oocytes / Q000166:cytology / Q000187:drug effects*; D011374:Progesterone / Q000032:analysis*; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D003470:Culture Media; D011374:Progesterone; D004958:Estradiol