Thyroid Hormone Assay

This example illustrates the assay reproducibility of the novel thyroxine assay of the subjecting invention. The specific assay comprised the use of the following reactant solutions:

a. The extractant solution consisted of a solution containing 0.025 N HCl; and 0.05 N KCl;

b. The T-4 .sup.125 I reagent solution contained a tracer quantity of radioisotope T-4 .sup.125 I in a 0.04 molar sodium barbital solution which contained sufficient HCl to render a pH thereof of 8.3;

c. The T-4 antiserum contained thyroxine antibodies (formed in a rabbit) and contained within 0.04 M sodium barbital solution which had been adjusted with HCl to render a final pH of 8.3;

d. The precipitant solution consisted of an aqueous solution containing 37% by weight ammonium sulfate and containing bovine serum at a concentration of 4 volumes percent;

e. The standards utilized in running the test were as follows:

1 microgram thyroxine per deciliter

6 micrograms thyroxine per deciliter

12 micrograms thyroxine per deciliter

18 micrograms thyroxine per deciliter

The above solutions were utilized to assay frozen pools of hypothyroid serum, normal serum and hyperthyroid serum by 11 different technicians.

The test procedure utilized included initially adding 10 microliters of a serum sample (or standard as the case may be) to 200 microliters of the extraction solution. After this step, the resulting solution was thoroughly admixed and then 400 microliters of the T-4 - .sup.125 I reagent were added to the resulting solution and the solution agitated. After this, 400 microliters of the T-4 antiserum solution were added and the resulting solution thoroughly admixed. At this point, the solution was incubated at room temperature for 45 or 60 minutes, and at the end of the incubation period, the precipitate in the sulfate solution was resuspended and 2 milliliters of this suspension were added to each tube containing the test solution. This resulted in 80 microliters of adjuvant serum being added to each original 10 microliter sample and a final sulfate concentration in each test solution of 23.7% by weight. The tubes were capped and each tube inverted gently about 10 times.

Then within 20 minutes after the addition of the precipitant, the tubes were centrifuged for 10 minutes at 1000-1500 gravities or 2000-2500 rpm and within 3 hours the supernatant was discarded and the resultant button of precipitant counted in a scintillation well counter. The results of the runs are set forth in Table 1 below:

As shown from the table, the reproducibility of the assay is excellent.

The linearity of the radioimmunoassay of the subject invention is demonstrated in this example utilizing the assay solutions described in Example I above. Specifically, the endogenous thyroxine from 3 serum pools was initially measured using the procedure set forth in Example I above. Thereafter, quantities of 5, 10, 15 and 20 micrograms per deciliter of crystalline thyroxine were added to 4 samples, respectively, from each of the 3 serum pools and the resulting samples were assayed in accordance with the procedure set forth in Example I to illustrate the uniformity of the percent recovery obtained from running the test. The results are set forth in Table 2 below:

The above results illustrate excellent recovery of the added T-4 and linearity of the test system.

This example is presented to illustrate the constant reproducible plateau of non-specific binding of the hormone to the antibody which is reached when the precipitant solution contains bovine serum in excess of 4 volumes per volume of the initial serum sample. In each instance, a solution containing a known quantity of thyroid hormone (a 10 microliter sample), a tracer quantity of radioactively labelled thyroid hormone, and a known quantity of antiserum were contacted with different precipitated solutions. Furthermore, the non-specific binding for each precipitant solution was determined by running each test but with the antiserum eliminated from the buffered antiserum solution. The precipitant solution was varied both in concentration of the ammonium sulfate and the carrier protein as illustrated in the table below and the non-specific binding of three different serum standards were tested by eliminating the antiserum from the antiserum buffer solution as described in Example III. The results are set forth in Table 4.

This example indicates the constant degree of non-specific binding and excellent spread of recovery values between hypothyroid and hyperthyroid serum samples when utilizing the test of the subject invention with the precipitant fluid containing the minor but effective quantity of added serum as adjuvant. More specifically, the procedure set forth in Example III was utilized to assay various serum standards except that the concentration of the serum in the precipitant solution was varied such that the resultant test solution would carry 60, 70, 80, 90, 100 and 110 microliters of added serum which is added with the precipitant to contact the test solution containing the initial 10 microliters of the serum sample. As will be noted when the carrier protein is present in the precipitant solution in quantities of 6 times the volume of initial sample or more, the value of the non-specific binding stabilizes; whereas when the quantity of the carrier protein is only 4 times that of the initial volume of the sample, the non-specific binding varies in an unpredictable manner. The results are set forth in Table 5 below: Precipitant with Precipitant with Precipitant with Precipitant with Precipitant with Precipitant with Sample 60.mu.l serum 70.mu.l serum 80.mu.l serum 90.mu.l serum 100.mu.l serum 110.mu.l serum Concen- Non- N on- Non- Non- Non- Non- tration Spec. % Spec. % Spec. % Spec. % Spec. % Spec. % (.mu.g/dl) Bind. Bound .DELTA.* Bind. Bound .DELTA. Bind. Bound .DELTA. Bind. Bound .DELTA. Bind. Bound .DELTA. Bind. Bound .DELTA. 0.4 6.6 61.8 6.6 61.1 6.6 61.6 6.6 59.1 6.8 60.2 6.4 60.5 61.2 61.2 61.6 59.8 60.3 61.0 61.2 61.0 60.8 59.7 59.6 60.6 (61.4)** (61.1) (61.3) (59.5) (60.0) (60.7) 3.2 48.2 48.9 48.6 47.6 46.6 47.4 48.0 48.6 48.8 47.0 47.1 47.3 48.8 48.2 48.5 47.0 47.2 47.7 (48.3) 13.1 (48.6) 12.5 (48.6) 12.7 (47.2) 12.3 (47.0) 13.0 (47.5) 13.2 6.0 6.8 40.2 6.7 39.9 6.6 40.2 6.6 39.0 6.7 39.0 6.5 39.1 40.2 40.2 40.2 38.8 39.2 39.2 39.8 39.7 40.5 38.8 38.8 38.8 (40.1) 8.2 (39.9) 8.7 (40.3) 8.3 (38.9) 8.3 (39.0) 8.0 (39.0) 8.5 12.0 28.8 29.0 28.9 28.0 28.5 28.5 29.3 29.0 29.0 27.9 28.8 28.3 29.0 29.0 28.9 28.1 28.3 28.7 (29.0) 11.1 (29.0) 10.9 (28.9) 11.4 (28.0) 10.9 (28.5) 10.5 (28.5) 10.5 18.0 6.7 24.0 6.6 23.7 6.6 23.7 6.3 22.8 6.3 23.1 6.4 23.3 23.7 23.8 23.8 23.2 23.3 23.4 23.6 23.7 23.6 22.8 23.1 23.4 (23.8) 5.2 (23.7) 5.3 (23.7) 5.2 (22.9) 5.1 (23.2) 5.3 (23.4) 5.1 TOTAL 37.6 37.4 37.6 36.6 36.8 37.3 *Represents the differences in % Bound between **Numbers in parentheses are averages

This example is presented to illustrate the specificity and sample serum protein independence of the radioimmunoassay of the subject invention. Specifically, the endogenous thyroxine of different sample volumes of various sera containing elevated and decreased protein levels was measured using the test procedure and the reactant solutions described in Example I. The results are set forth in Table 6 below:

While this invention has been described in relation to its preferred embodiments, it is to be understood that various modifications thereof will be apparent to those skilled in the art upon reading this specification, and it is intended to cover such modifications as fall within the scope of the appended claims.