7766

• UCREFRP Catalog Number: 7766
• Author: Scholz, A. T., et al.
• Title: Notes on the Development and Measurement of Egg and Larval Thyroxine Concentration in Flannelmouth Suckers ‎(
• USFW Year: 1992.
• Copyright Material: NO

and 25 psi. . The dried sample was stored at OOC until T 4 content was
assayed approximately one week later.

2.2 T4 RBdloimmunoBssy

On the day radioimmunoassays (RIA) were perlormed, samples were
resuspended in 250 IJ.I 95% ETOH and 250 IJ.I 0.11 M sodium barbital (pH
8.6). Each 500 IJ.I sample was vortexed for 15 seconds after each solution
was added. The sample was then transferred into a plastic eppendorl snap
cap vial and centrifuged at 3000 rpm for 10 minutes using an Eppendol1
Model 5415 C refrigerated centrifuge at 4.0oC. Each sample was assayed
in duplicate for T4 content using a coat-a-count T4 RIA kit (Diagnostic
Products, Inc.). In this procedure 25 IJ.I of an unknown sample and 1 ml of
radiolabelled T4 (1251-T4) were added to a tube coated with antibodies
(Ab) that contained T4 receptors. Thus, each tube contained the same
amount of 1251-T4 but different amounts of unknown T4. The procedure is
based on competative binding i.e., 1251-T4 and unkown T4 compete for Ab
binding sites and bind in proportion to their relative concentrations.
Hence 1251-T4 will be displaced from binding sites if the unknown sample
contains more T4 than if the unknown sample contains less T4. Actual
concentrations of unknown samples were determined by preparing a
standard curve with known concentrations of T 4, ranging from 0 to 24
ng/dl. Twentyfive IJ.I aliquots of 0,1,4,10,16 and 24 ng/dl standard curve
concentrations and 1 ml of 1251-T4 were added to Ab coated tubes and
subjected to the same assay procedures as unknown flannel mouth sucker
samples.

Unknown and standard curve tubes were vortexed for 5 seconds, then
. incubated for 1 h 0 370C to allow time for T4 and 1251-T4 to compete for
binding sites on the Ab. This is a reversible binding reaction. Equilibrium
is attained in less than 1h. The T4 and 1251-T4 bind in proportion to the
relative concentration according to the Law of Mass Action. Non-bound or
1ree' T4 and 1251-T4 was discarded by pouring the liquid into a
radioactive waste container and then blotting the remaining liquid out of
the tubes. Radioactivity of the 'bound' fraction remaining in the Ab coated
tubes was counted for 1 minute using a programmable Cobra ac Model
85002 Auto-gamma Counter (Packard Instrument, Co.).

Maximum binding (MB) of radioactivity was determined from the
standard curve tubes containing 0 ngldl T 4. Percent bound of each of the
remaining standard curve and unknown samples was calculated from the
equation:

4

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