THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

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(2) Procg.ssjnq, gf. incuÞ¡rjgI, fp.djg
The frozen 24 h incubation medium and tÍssue fragments were
thawed and centrlfuged at 3,000 x g. The supernatant was further
centrif,uged at 50,000 x g for 30 min and the c.lear supernatant
was frozen at -20 C until use.
(3) Radioimmunoassa.y for opl content
The oPL content in the media was determined by the doub.le
antibody radÌoimmunoassay as described previously.
(4) Determination of the s.yntheSis and Secretion of tipl
The newly synthesized opl was determined by measuring the
Íncorporation of 3H-leucine into opl molecule. The quantities of
3H-oPL in the medium was determined by inrmunoprec i pi tati on using
specific antiserum to opl prepared in rabbits as described previous'ry.
(i) Method of determination of the equivalence zone for opl antiserum.
The equivalence zone for maximal precipitatíon of radio_
active oPL was determined as fo ows. 0.r mr of cord opl in various
concentration ( 10 to l0O,000ng/ml) was added to test tubes containing
300 ul of phosphate buffered saline(pgS) , pH 7.6,25 ul opL antiserum,
and 20,000 cpm of l2SI-opL. The finaì volume was approximately 525 ul .
The mixture was then ìncubated for I h at 37 C, and then for an.
additional 16 h at 4 C. After overnight incubation (.16 h),2 ml of
PBS were added into the reaction nixture, and then centrifuged at
3,000 x g for 30 min. The pellets were washed and re-ientrifuged twice