Abstract

ENZYME LINKED IMMUNOSORBENT ASSAY FOR 17?, 20?-DIHYDROXY-4-PREGNEN-3-ONEENZYME LINKED IMMUNOSORBENT ASSAY FOR 17?, 20?-DIHYDROXY-4-PREGNEN-3-ONE

Vasundhara, Abbas Ali Mahdi and Tulsidas G. Shrivastav*

ABSTRACT

In lower vertebrates like fishes, maturation of oocyte is elicited by 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-diOH-P) which is universally known as Maturation Inducing Steroid (MIS). MIS acts on certain unrevealed receptors on the membrane of oocytes that initiates several maturation promoting factors (MPF) present in the cytoplasm of the oocytes. 17α, 20β-diOH-P is a key mediator hormone in fishes and the estimation of this steroid would help the ichthyologists to improve further the quality and quantity of aquaculture to fulfil the demand of the food market in current scenario. An Enzyme Linked Immunosorbent Assay (ELISA) system that is specific and sensitive to estimate the levels of the 17α, 20β-diOH-P in serum has been developed using antibody raised against 17α, 20β-diOH-P-3-CMO- BSA as an immunogen and 17α, 20β-diOH-P-3-CMO-HRP as an enzyme conjugate. The developed assay was further evaluated for analytical variables. The sensitivity was detected in terms of lowest detection dose (LDD) and effective dose (ED50) which is calculated as 0.13 ng/mL and 2.48 ng/mL respectively. The assay was specific showing less than 0.025% of cross reaction with 66 analogous steroids. The recovery of externally spiked 17α, 20β-diOH-P in fish pooled serum was estimated and found to be in the range of 88 to 101.22%. On the other hand, inter-assay and intra-assay coefficient of variation (CV%) were found to be in the range of 4.79 to 9.31% and 1.2 to 9.08 % respectively. The developed assay is not only sensitive but specific enough that has correlated well with the commercially available kit and has shown r = 0.99 (n = 50).

Keywords: ELISA, 17?, 20?-diOH-P, oocyte, MIS, fish.