1. Animal Biotechnology Centre, G.B. Pant University of Agriculture &
Technology, Pantnagar, India.
Abstract
Low
cost, affordable early pregnancy diagonsis kit is very deserving for livestock
management and dairy business. This study proposes to develop a kit based on
enzyme immunoassay (EIA). New Zealand white rabbits primed with 11-α-OH
progesterone hemisuccinate-BSA conjugate were boosted with progesterone antigen
for crude antisera production. Progesterone antibody was purified using an
immuno affinity chromatography column packed with 6-amino hexanoic acid
sepharose 4B beads conjugated with progesterone carboxy methyl oxime and coated
on nitrocellulose paper dipsticks. The 11-α-OH progesterone
hemisuccinate-penicillinase conjugate solution was purified using Sephadex G-50
size exclusion column. All conjugates were prepared using carbodiimide
reaction. Optimum pH and temperature for penicillinase enzyme highest activity
and iodine concentration for starch iodine substrate solution were determined.
Checker board anlysis and titration were performed to optimize antibody,
conjugate and milk dilutions for kit. Further kit was validated with randomly
selected 20 cow milk samples for pregnancy diagnosis. Chromatography data
showed peaks for fractions containing highly purified progesterone antibody and
progesterone penicillinase conjugate respectively. Optimum dilutions of
antibody, conjugate and milk samples were 1:20, 1:40, 1:3 (or 1:4) respectively
to differentiate between 1 (non pregnant) and 5 (pregnant) ng/ml of
progesterone concentration. Standard conditions of kit optimum function are pH
7.0 and 35 ºC. Penicillin solution was stable at 4 ºC for 3 weeks. Excellent
correlation was observed for pregnancy diagnosed by developed kit to detect
progesterone concentration in 20 milk samples and conventional rectal palpation
method. Developed low cost dipstick EIA kit can be used for early pregnancy
diagnosis successfully.
Keywords: Low cost, dipstick immunoblot assay, EIA kit bovine pregnancy diagnosis,
diagonostic kit
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Publication
Ms. Ref. No.: A0101001
Reviewer 1 Reviewer 2
Received: 15082015
Accepted: 14042016
Article in Press: 15042016
Article in Press: 15042016