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I I I I I I I I I I I I I I I I I I- II counts - NSB counts Percent Bound = MB counts _ NSB counts x100 where: counts = Radioactive counts per minute of either a standard curve or unknown tube; MB = Radioactive counts per minute of maximum binding tube; and NSB = Nonspecific binding (See below). A computer program, contained in the counter, automatically determined the relative percent bound for each standard concentration and plotted a log-Iogit graph of the standard curve (% 'bound -v- standard concentrations). The program then examined the percent bound for each unknown sample and interpolated the concentration of the sample from the standard curve graph. Total T4 content of individual samples .was determined by expanding the T4 content of 25 J.LI subsample to the total 500 J.LI sample. and converting this concentration to total ng equivilent T 4. This number was divided by the weight of the fish to provide the relative T 4 concentration of each fish in nglg body weight. The mean i: SEM were calculated for fish collected on the same sample. date and plotted -v- age (days from fertilization), using Statview II (Brainpower. Inc) on an Apple Mcintosh II ex microcomputer. 2.3 Quality Assurance Procedures Accuracy level of the assay was determined by: (1) Blind analysis of quality control standards at high. medium and low concentrations. After obtaining results from our assay. we received information about the actual concentration from Diagnostic Products. Inc. These samples were inserted in duplicate at the front. middle and end of our assay. Mean measured concentrations (i:SD) were then compared to actual concentrations to determine assay reliability. (2) Nonspecific binding (NSB). i.e., radioactivity-that sticks to the test tube but not bound to binding sites, was determined by 5