Radioimmunoassay method for triiodothyronine and thyroxine

• US 3,928,553 A
• Filed: 02/25/1974
• Issued: 12/23/1975
• Est. Priority Date: 03/23/1972
• Status: Expired due to Term

• Current Assignee: Charles S. Hollander
• Original Assignee: Charles S. Hollander
• Inventors: Hollander, Charles S.
• Primary Examiner(s): Padgett, Benjamin R.
• Assistant Examiner(s): Nucker, Christine M.
• Application Number:
  US05/445,115
• Time in Patent Office: 666 Days
• Field of Search: 23/23B 250/303 424/1,12
• 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 436/825 Pretreatment for removal of interfering factors from sample
• CPC Class Codes: Y10S 436/826 Additives, e.g. buffers, diluents, preservatives

Abstract

This invention relates to a radioimmunoassay method for triiodothyronine or thyroxine or triiodothyronine and thyroxine present in unextracted serum containing thyroxine binding prealbumin and thyroxine binding globulin which comprises: A. ADDING TO UNEXTRACTED SERUM, A SUFFICIENT AMOUNT OF A BINDING INHIBITORY AGENT TO SUBSTANTIALLY INHIBIT BINDING OF TRIIODOTHYRONINE OR THYROXINE OR TRIIODOTHYRONINE AND THYROXINE TO THE THYROXINE BINDING PREALBUMIN; B. ADDING TO THE SERUM AN AMOUNT OF AN ISOTOPICALLY LABELLED TRIIODOTHYRONINE OR THYROXINE OR TRIIODOTHYRONINE AND THYROXINE SUFFICIENT FOR DETECTION; C. ADDING TO THE SERUM, A SUFFICIENT AMOUNT OF A BINDING INHIBITORY AGENT TO SUBSTANTIALLY INHIBIT BINDING OF TRIIODOTHYRONINE OR THYROXINE OR TRIIODOTHYRONINE AND THYROXINE TO THE THYROXINE BINDING GLOBULIN; D. ADDING TO THE SERUM AN AMOUNT OF ANTISERUM CONTAINING ANTIBODY FOR TRIIODOTHYRONINE OR THYROXINE OR TRIIODOTHYRONINE AND THYROXINE WHICH IS APPROXIMATELY PROPORTIONATE TO THE TITER; E. INCUBATING THE SERUM AT A TEMPERATURE AND FOR A PERIOD OF TIME WHICH IS SUFFICIENT TO PROVIDE SUBSTANTIAL EQUILIBRATION OF THE ANTIBODY BOUND AND UNBOUND TRIIODOTHYRONINE OR THYROXINE OR TRIIODOTHYRONINE AND THYROXINE; F. SEPARATING THE UNBOUND TRIIODOTHYRONINE OR THYROXINE OR TRIIODOTHYRONINE AND THYROXINE FROM THE BOUND TRIIODOTHYRONINE OR THYROXINE OR TRIIODOTHYRONINE AND THYROXINE WHILE SUBSTANTIALLY PREVENTING RE-EQUILIBRATION OF ANTIBODY BOUND TRIIODOTHYRONINE OR THYROXINE OR TRIIODOTHYRONINE AND THYROXINE; AND G. MEASURING THE ADSORBED UNBOUND TRIIODOTHYRONINE OR THYROXINE OR TRIIODOTHYRONINE AND THYROXINE AND THE BOUND TRIIODOTHYRONINE OR THYROXINE OR TRIIODOTHYRONINE AND THYROXINE.

Claims

1. A RADIOIMMUNOASSAY METHOD FOR TRIIODOTHYRONINE OR THYROXINE OR TRIIDODTHYRONINE AND THYROXINE PRESENT IN UNEXTRACTED SERUM CONTAINING THYROXINE BINDING PRE-ALBUMIN AND THYROXINE BINDING GLOBULIN WHICH COMPRISES:

A. ADDING TO UNEXTRACTED SERUM, A SUFFICIENT AMOUNT OF A BINDING INHIBITORY AGENT TO SUBSTANTIALLY INHIBIT BINDING OF TRIIODOTHHYRONINE OR THYROXINE OR TRIIODOTHYRONINE AND THYROXINE TO THE THYROXINE BINDING PREALBUMIN;

B. ADDING TO THE SERUM AN AMOUNT OF AN ISOTOPICALLY LABELLED TRIIDOTHYRONINE OR THUROXINE OR TRIIDOHYDRORONINE AND THYROXINE SUFFICIENT FOR DETECTION;

C. ADDING TO THE SERUM, A SUFFICIENT AMOUNT OF A BINDING INHIBITORY AGENT TO SUBSTANTIALLY INHIBIT BINDING OF TRIIDOTHYRONINE OR THYROXINE OR TRIIODHYRONINE AND THYROXINE TO THE THYROXINE BINDING GLOBULIN;

D. ADDING TO THE SERUM AN AMOUNT OF ANTISERUM CONTAINING ANTIBODY FOR TRIIDOTHYRONINE OR THYROXINE OR TRIIDOTHYRONINE AND THYROXINE WHICH IS APPROXIMATELY PROPORTIONATE TO THE TITER;

E. INCUBATING THE SERUM AT A TEMPERATURE AND FOR A PERIOD OF TIME WHICH IS SUFFICIENT TO PROVIDE SUBSTANTIAL EQUILIBRATION OF THE ANTIBODY BOUND AND UNBOUND TRIIDOTHYRONINE OR THYROXINE OR TRIIDOHYRONINE AND THYROXINE;

F. SEPARATING THE UNBOUND TRIIDOTHYRONINE OR THYROXINE OR TRIIDOTHYRONINE AND THYROXINE FROM THE BOUND TRIIDOTHYRONINE OR THYROXINE OR TRIDOTHYRONINE AND THYROXINE WHILE SUBSTANTIALLY PREVENTING RE-EQUILIBRATION OF ANTIBODY BOUND TRIIDOTHYRONINE OR THYROXINE OR TRIIDOTHYRONINE AND THYROXINE;

AND G. MEASURING THE ADSORBED UNBOUND TRIIDOTHYRONINE OR THYROXINE OR TRIIDOTHYRONINE AND THYROXINE AND THE BOUND TRIIODOTHYRONINE OR THYROXINE AND THE AND THYROXINE.

2. an amount of an isotopically labelled triiodothyronine or thyroxine or triiodothyronine and thyroxine sufficient for detection;

and

3. a sufficient amount of a binding inhibitory agent to substantially inhibit binding of triiodothyronine or thyroxine or triiodothyronine and thyroxine to the thyroxine binding globulin;

b. adding to the serum an amount of antiserum containing antibody for triiodothyronine or thyroxine or triiodothyronine and thyroxine which is approximately equal to the titer;

c. incubating the serum at a temperature and for a period of time which is sufficient to provide substantial equilibration of the antibody bound and unbound triiodothyronine or thyroxine or triiodothyronine and thyroxine;

d. separating the unbound triiodothyronine or thyroxine or triiodothyronine and thyroxine from the bound triiodothyronine or thyroxine or triiodothyronine and thyroxine while substantially preventing re-equilibration of antibody bound triiodothyronine or thyroxine or triiodothyronine and thyroxine; and

e. measuring the adsorbed unbound triiodothyronine or thyroxine or triiodothyronine and thyroxine and the bound triiodothyronine or thyroxine or triiodothyronine and thyroxine.

4. The method of claim 1, wherein the binding inhibitory agent for thyroxine binding globulin is dilantin, a salicylate, methiolate, tetrachlorothyronine or 8-anilino-1-napthalene sulfonic acid.

5. The method of claim 1 wherein the isotopically labelled triiodothyronine is triiodothyronine 125I.

6. The method of claim 1 wherein the isotopically labelled thryroxine is thyroxine 131I.

7. The method of claim 1 wherein the serum is maintained at a pH of about 8 during incubation.

8. The method of claim 1 wherein incubation is carried out at about 37*C for a period of about 1 to 2 hours.

9. The method of claim 1 wherein separation of unbound from bound triiodothyronine or thyroxine or triiodothyronine and thyronine is carried out by adsorbtion of the unbound product on dextran coated activated carbon added to the incubation mixture about ten minutes after the mixture has been cooled to a temperature of about 4*C.

10. The method of claim 9 wherein the adsorbed unbound product is separated from the adsorbed product by centrifugation of filtration.

11. A radioimmunoassay method for triiodothyronine or thyroxine or triiodothyronine and thyroxine present in extracted serum in which thyroxine binding pre-albumin and thyroxine binding globulin are substantially absent, which comprises:

a. adding to the serum an amount of an isotopically labelled triiodothyronine or thyroxine or triiodothyronine and thyroxine sufficient for detection;

b. adding to the serum an amount of antiserum containing antibody for triiodothryronine or thyroxine or triiodothyronine and thyroxine which is approximately proportionate to the titer;

c. incubating the serum at a temperature and for a period of time which is sufficient to provide substantial equilibration of the antibody bound and unbound triiodothyronine or thyroxine or triiodothyronine and thyroxine;

d. separating the unbound triiodothyronine or thyroxine or triiodothyronine and thyroxine from the bound triiodothyronine or thyroxine or triiodothyronine and thyroxine; and

e. measuring the adsorbed unbound triiodothyronine or thyroxine or triiodothyronIne and thyroxine and the bound triiodothyronine or thyroxine or triiodothyronine and thyroxine.

12. A radioimmunoassay method for triiodothyronine or thyroxine or triiodothyronine and thyroxine present in unextracted serum containing thyroxine binding pre-albumin and thyroxine binding globulin whic comprises a. adding to unextracted serum a mixture comprising

13. The method of claim 12 wherein the binding inhibitory agent for thyroxine binding pre-albumin is barbituric acid, a derivative of barbituric acid or a salicylate.

14. The method of claim 13 wherein the derivative of barbituric acid is sodium barbital.

15. The method of claim 12, wherein the binding inhibitory agent for thyroxine binding globulin is dilantin, a salicylate, merthiolate, tetrachlorothyronine or 8-anilino-1-naphthalene sulfonic acid.

16. The method of claim 12, wherein the isotopically labelled triiodothyronine is triiodothyronine 125I.

17. The method of claim 12, wherein the isotopically labelled thyroxine is thyroxine 131I.

18. The method of claim 12, wherein the serum is maintained at a pH of about 8 during incubation.

19. The method of claim 12, wherein incubation is carried out at about 37*C for a period of about 1 to 2 hours.

20. The method of claim 12, wherein separation of unbound from bound triiodothyronine or thyroxine or triiodothyronine and thyronine is carried out by adsorbtion of the unbound product on dextran coated activated carbon added to the incubation mixture about ten minutes after the mixture has been cooled to a temperature of about 20 C.

21. The method of claim 20, wherein the adsorbed unbound product is separated from the adsorbed product by centrifugation or filtration.