Thyroxine (T.sub.4) radioimmunoassay

• US 4,018,883 A
• Filed: 03/10/1975
• Issued: 04/19/1977
• Est. Priority Date: 03/10/1975
• Status: Expired due to Term

• Current Assignee: Nichols Institute For Endocrinology
• Original Assignee: Nichols Institute For Endocrinology
• Inventors: Parslow, Margaret E.
• Primary Examiner(s): Padgett, Benjamin R.
• Assistant Examiner(s): Nucker, Christine M.
• Application Number:
  US05/556,930
• Time in Patent Office: 771 Days
• Field of Search: 424/1, 424/1.5, 23/230 B, 23/230.6
• US Class Current: 436/500
• CPC Class Codes: G01N 33/78 Thyroid gland hormones , e.g. T3, T4, TBH, TBG or their receptors
• CPC Class Codes: Y10S 436/804 Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10S 436/826 Additives, e.g. buffers, diluents, preservatives

Abstract

It has been discovered polyethylene glycol can be added as a separator to the other reagents of a T₄ radioimmunoassay of unextracted serum prior to introduction of the serum, without impairing the sensitivity of the measurement. A single, highly stable composite reagent is prepared to measure the concentration of T₄ in a measured quantity of human serum; the composite reagent comprises, in freeze dried form, a blocking agent in sufficient quantity to displace essentially all the T₄ in the measured quantity of serum bound to TBG, an antibody in sufficient quantity to bind a significant quantity of the T₄ in the measured quantity of serum, PEG in sufficient quantity to precipitate essentially all the antibody, and a buffering agent in sufficient quantity to inhibit binding the T₄ in the measured quantity of serum to TBP. To run a radioimmunoassay, the human serum being assayed and quantities of standard serums containing known concentrations of T₄ are added to a mixture of the composite reagent reconstituted with water and radioactive T₄ in sufficient quantity to give a measurable counting rate. After an incubation period, the antibody precipitated by the PEG, including the antibody bound T₄, is removed by centrifuging and aspirating the supernatants. The radio activity of the precipitates is then counted to correlate the T₄ in the serum being assayed with the standard serums.

Claims

1. A composite reagent for a radioimmunoassay to measure the concentration of thyroxine (T₄) in a measured quantity of human serum, the reagent comprising in the absence of non-radioactive T₄ :

a blocking agent in sufficient quantity to displace essentially all of the T₄ in the measured quantity of serum bound to thyroxine-binding globulin (TBG);

an antibody in sufficient quantity to bind a significant quantity of the T₄ in the measured quantity of serum;

polyethylene glycol (PEG) in sufficient quantity to precipitate essentially all the antibody; and

a buffering agent in sufficient quantity to inhibit binding the T₄ in the measured quantity of serum to thyroxine-binding prealbumin.

2. The reagent of claim 1, in which the blocking agent is 8-anilino-1-naphthaline-sulfonic acid (ANS).

3. The reagent of claim 2, in which the buffering agent has a pH of about 8.6.

4. The reagent of claim 3, additionally comprising a sufficient quantity of gamma globulin to provide a visual indicator.

5. The reagent of claim 4, additionally comprising radioactive T₄.

6. The reagent of claim 5, in a freeze dried form.

7. The reagent of claim 1, in which the buffering agent has a pH of about 8.6.

8. The reagent of claim 1, additionally comprising a sufficient quantity of gamma globulin to provide a visual indicator.

9. The reagent of claim 1, additionally comprising radioactive T₄.

10. The reagent of claim 1, in a freeze dried form.

11. The method of preparing a composite reagent for a radioimmunoassay to measure the concentration of thyroxine (T₄) in a measured quantity of serum, the method comprising the following steps in the order recited:

dissolving in an aqueous buffer solution having a pH of about 8.6 sufficient gamma globulin to provide a visible precipitate after centrifusion and supernatant aspiration and sufficient 8-anilino-1-naphthalene-sulfonic acid (ANS) to displace all the T₄ bound to thyroxine binding globulin in the measured quantity of serum;

slowly adding sufficient polyethylene glycol (PEG) to precipitate substantially all the gamma globulin, thereby producing a thick slurry;

adding to the slurry an antibody capable of binding a significant quantity of T₄ ; and

thoroughly mixing the recited ingredients.

12. The method of claim 11, comprising the additional step of freeze drying the mixed ingredients.

13. The method of claim 11, comprising the additional step of adding a measurable amount of radioactive T₄ to the mixed ingredients in the absence of human serum.

24. The method of claim 11, in which the gamma globulin and the ANS in the dissolving step are approximately in the ratio of 10:

1.

14. A method of measuring the concentration of thyroxine (T₄) in a measured quantity of human serum, the method comprising the steps of:

combining the following to form a mixture;

a blocking agent in a quantity sufficient to displace essentially all the T₄ in the measured amount of human serum from thyroxine binding globulin (TBG);

an antibody in sufficient quantity to bind a significant quantity of the T₄ in the measured quantity of serum;

polyethylene glycol (PEG) in sufficient quantity to precipitate essentially all the antibody; and

a buffering agent in sufficient quantity to inhibit binding the T₄ in the measured quantity of serum to thyroxine-binding prealbumin;

adding the mixture to the measured quantity of human serum and radioactive T₄ in sufficient quantity to give a measurable counting rate;

incubating the serum and radioactive T₄ with the mixture added to allow reaction of the T₄ in the serum and the radioactive T₄ with the antibody to proceed substantially to equilibrium, thereby producing antibody bound T₄, a percentage of which is radioactive;

removing the antibody precipitated by the PEG including the antibody bound T₄ from the supernatant; and

measuring the quantity of radioactive T₄ in the removed precipitate or the supernatant.

15. The method of claim 14, in which the step of adding the mixture to the measured quantity of human serum and radioactive T₄ comprises the steps of:

adding the radioactive T₄ to the mixture before adding the mixture to the measured quantity of human serum;

pipetting the measured quantity of human serum into a test tube; and

pipetting a measured quantity of the mixture including radioactive T₄ into the test tube.

16. The method of claim 15, in which the measuring step comprises the steps of:

pipetting into a plurality of test tubes measured quantities of standard human serum samples having different known concentrations of T₄ ;

pipetting into the test tubes measured quantities of the mixture including radioactive T₄ ;

incubating the standard serum samples and radioactive T₄ with the mixture added to allow reaction of the T₄ in the standard serum samples and the radioactive T₄ with the antibody to proceed substantially to equilibrium, thereby producing antibody bound T₄ a percentage of which is radioactive;

removing the antibody precipitated by the PEG including the antibody bound T₄ of the standard serum samples;

counting the radioactivity in the precipitate including the antibody bound T₄ of the measured quantity of human serum being assayed;

counting the radioactivity of the precipitate including the antibody bound T₄ of the standard serum samples; and

correlating the count of the serum being assayed with the counts of the standard serum samples.

17. The method of claim 14, in which gamma globulin is combined in the mixture in sufficient quantity to provide a precipitate visible to the human eye.

18. The method of claim 14, in which the removing step comprises the steps of:

centrifuging the serum and radioactive T₄ with the mixture added; and

aspirating the supernatants.

19. The method of claim 14, in which the blocking agent comprises 8-anilino-1-naphthalene-sulfonic acid (ANS).

20. The method of claim 14, in which the radioactive T₄ in the mixture is approximately 0.2 nanograms per 0.5 milliliters of the mixture and the radioactivity is approximately 100 millicures per milligram of T₄.

21. The method of claim 20, in which the mixture contains approximately 17.5 percent by weight of PEG and 3 percent by weight of gamma globulin.

22. The method of claim 21, in which the mixture contains the antibody in sufficient quantity to bind between 35% and 50% of the radioactive T₄.

23. The method of claim 22, in which the mixture contains as a blocking agent approximately 0.03 percent of 8-anilino-1-naphthalene-sulfonic acid (ANS) by weight.