Radioimmunoassay for measurement of thyroxine (T4) and triiodothyronine (T3) in blood serum

Abstract

This invention relates to a highly accurate, rapid and simple estimation of thyroxine (T 4 ) directly from blood serum and also relates to the accurate measurement of triiodo-L-thyronine (T 3 ) directly from blood serum. More specifically, the invention relates to a rapid, specific and reliable radioimmunoassay (RIA) technique for measurement of both T 4 and T 3 in unextracted serum. The method requires very small amounts of serum, e.g., 25 microliters (μl) to measure T 4 concentration in nearly all specimens representing clinical states of eu-, hypo- and hyperthyroidism, and 250 μl to measure T 3 concentrations in specimens representing most clinical states.

I claim: 
     
       1. A method of measurement of the concentration of a particular thyroid hormone selected from the group consisting of thyroxine (T 4 ) and triodo-L-thyronine (T 3 ) in unextracted human serum, which comprises: A: adding to a measured quantity of unextracted human serum, a. a blocking agent .Iadd.selected from the group consisting 8-anilino-1-napthalene sulfonic acid and 3-(4-anilino-1-naphthylazo) 2,7 naphthalene disulfonic acid, and present .Iaddend.in an amount sufficient to displace essentially all of said particular thyroid hormone to be measured from thyroxine-binding globulin (TGB),   b. buffering ions to buffer said serum to a pH of between about 6.8 to about 9.6,   c. radioactive thyroid hormone, of the type to be measured, in an amount which will give a measurable counting rate of either antibody bound or free radioactivity after reaction equilibrium has been reached as set forth in Step B below; and   d. an antibody in sufficient quantity to bind a significant quantity of said radioactive thyroid hormone in the absence of any of the particular nonradioactive thyroid hormone to be measured,     B: allowing reaction of both particular thyroid hormone, to be measured, and said radioactive thyroid hormone, with said antibody to proceed substantially to equilibrium to thereby produce antibody bound radioactive thyroid hormone;   C: separating said antibody bound radioactive thyroid hormone, to be measured from said free radioactive thyroid hormone;   D: measuring the quantity of radioactive thyroid hormone selected from antibody bound radioactive thyroid hormone and free radioactive thyroid hormone;   E: preparing a standard curve with known amounts of the particular thyroid hormone to be measured, and   F: correlating the quantity of radioactive thyroid hormone measured with a known amount of said particular thyroid hormone read from said standard curve.   
     
     
       2. The method of claim 1 wherein said thyroid hormone to be measured is thyroxine, said blocking agent is 8-anilino-1-napthalene disulfonic acid, said blocking agent being added to said human serum in quantities of at least about 4 ug/ul serum. 
     
     
       3. The method of claim 1 wherein said buffering ions are barbital ions, and said pH of said serum is about 8.6. 
     
     
       4. The method of claim 1 wherein said radioactive thyroid hormone is initially present in the order of 10,000 counts per minutes. 
     
     
       5. The method of claim 1 wherein said antibody bound radioactive thyroid hormone after reaction, gives a counting rate of from about 2000-6000 counts per minute. 
     
     
       6. The method of claim 1 wherein said antibody bound radioactive thyroid hormone, to be measured, is separated from said free radioactive thyroid hormone by precipation of said antibody bound radioactive thyroid hormone with a second antibody. 
     
     
       7. The method of claim 1 wherein said antibody bound radioactive thyroid hormone, to be measured, is separated from said free radioactive thyroid hormone by precipation of said antibody bound radioactive thyroid hormone with gamma globulin. 
     
     
       8. The method of claim 1 wherein said antibody bound radioactive thyroid hormone, to be measured, is separated from said free radioactive thyroid hormone by precipation of said antibody bound radioactive thyroid hormone with charcoal suspension. 
     
     
       9. The method of claim 1 wherein said antibody bound radioactive thyroid hormone, to be measured, is separated from said free radioactive thyroid hormone by precipation of said antibody bound radioactive thyroid hormone with polyethylene glycol. 
     
     
       10. The method of claim 1 wherein said thyroid hormone to be measured is T 4 , said blocking agent is 2.7 napthalene disulfonic acid, said blocking agent being added to said human serum in quantities of at least about 2 ug/ul serum. 
     
     
       11. The method of claim 1 wherein said thyroid hormone to be measured is T 3 , said blocking agent is 2,7 napthalene disulfonic acid and said blocking agent is added to said human serum in quantities of at least about 1 ug/ul serum.