US4018883A
Expired / Lapsed
US
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Thyroxine (T4) radioimmunoassay
Assignee: Nichols Institute for Endocrinology
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Filing: 1975-03-10
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Granted: 1977-04-19
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Expires: 1995-03-10
Abstract
Abstract It has been discovered polyethylene glycol can be added as a separator to the other reagents of a T 4 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 T 4 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 T 4 in the measured quantity of serum bound to TBG, an antibody in sufficient quantity to bind a significant quantity of the T 4 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 T 4 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 T 4 are added to a mixture of the composite reagent reconstituted with water and radioactive T 4 in sufficient quantity to give a measurable counting rate. After an incubation period, the antibody precipitated by the PEG, including the antibody bound T 4 , is removed by centrifuging and aspirating the supernatants. The radio activity of the precipitates is then counted to correlate the T 4 in the serum being assayed with the standard serums.
Inventors
- 👤 Margaret E. Parslow
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 07:48
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/74; G01N33/78; Y; Y10; Y10S; Y10S436/00; Y10S436/804; Y10S436/826
Claims Analysis
Total Claims: 27
Claim 1
Independent Claim
112 words
A composite reagent for a radioimmunoassay to measure the concentration of thyroxine (T 4 ) in a measured quantity of human serum, the reagent comprising in the absence of non-radioactive T 4 : a blocking agent in sufficient quantity to displace essentially all of the T 4 in the measured quantity of serum bound to thyroxine-binding globulin (TBG); an antibody in sufficient quantity to bind a significant quantity of the T 4 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 T 4 in the measured quantity of serum to thyroxine-binding prealbumin.
Claim 2
Dependent Claim
112 words
A composite reagent for a radioimmunoassay to measure the concentration of thyroxine (T 4 ) in a measured quantity of human serum, the reagent comprising in the absence of non-radioactive T 4 : a blocking agent in sufficient quantity to displace essentially all of the T 4 in the measured quantity of serum bound to thyroxine-binding globulin (TBG); an antibody in sufficient quantity to bind a significant quantity of the T 4 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 T 4 in the measured quantity of serum to thyroxine-binding prealbumin.
Claim 3
Dependent Claim
14 words
The reagent of claim 1, in which the blocking agent is 8-anilino-1-naphthaline-sulfonic acid (ANS).
Claim 4
Dependent Claim
16 words
The reagent of claim 2, in which the buffering agent has a pH of about 8.6.
Claim 5
Dependent Claim
18 words
The reagent of claim 3, additionally comprising a sufficient quantity of gamma globulin to provide a visual indicator.
Claim 6
Dependent Claim
11 words
The reagent of claim 4, additionally comprising radioactive T 4 .
Claim 7
Dependent Claim
10 words
The reagent of claim 5, in a freeze dried form.
Claim 8
Dependent Claim
16 words
The reagent of claim 1, in which the buffering agent has a pH of about 8.6.
Claim 9
Dependent Claim
18 words
The reagent of claim 1, additionally comprising a sufficient quantity of gamma globulin to provide a visual indicator.
Claim 10
Dependent Claim
11 words
The reagent of claim 1, additionally comprising radioactive T 4 .
Claim 11
Dependent Claim
10 words
The reagent of claim 1, in a freeze dried form.
Claim 12
Independent Claim
122 words
The method of preparing a composite reagent for a radioimmunoassay to measure the concentration of thyroxine (T 4 ) 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 T 4 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 T 4 ; and thoroughly mixing the recited ingredients.
Claim 13
Independent Claim
122 words
The method of preparing a composite reagent for a radioimmunoassay to measure the concentration of thyroxine (T 4 ) 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 T 4 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 T 4 ; and thoroughly mixing the recited ingredients.
Claim 14
Dependent Claim
15 words
The method of claim 11, comprising the additional step of freeze drying the mixed ingredients.
Claim 15
Dependent Claim
28 words
The method of claim 11, comprising the additional step of adding a measurable amount of radioactive T 4 to the mixed ingredients in the absence of human serum.
Claim 16
Independent Claim
212 words
A method of measuring the concentration of thyroxine (T 4 ) 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 T 4 in the measured amount of human serum from thyroxine binding globulin (TBG); an antibody in sufficient quantity to bind a significant quantity of the T 4 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 T 4 in the measured quantity of serum to thyroxine-binding prealbumin; adding the mixture to the measured quantity of human serum and radioactive T 4 in sufficient quantity to give a measurable counting rate; incubating the serum and radioactive T 4 with the mixture added to allow reaction of the T 4 in the serum and the radioactive T 4 with the antibody to proceed substantially to equilibrium, thereby producing antibody bound T 4 , a percentage of which is radioactive; removing the antibody precipitated by the PEG including the antibody bound T 4 from the supernatant; and measuring the quantity of radioactive T 4 in the removed precipitate or the supernatant.
Claim 17
Independent Claim
212 words
A method of measuring the concentration of thyroxine (T 4 ) 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 T 4 in the measured amount of human serum from thyroxine binding globulin (TBG); an antibody in sufficient quantity to bind a significant quantity of the T 4 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 T 4 in the measured quantity of serum to thyroxine-binding prealbumin; adding the mixture to the measured quantity of human serum and radioactive T 4 in sufficient quantity to give a measurable counting rate; incubating the serum and radioactive T 4 with the mixture added to allow reaction of the T 4 in the serum and the radioactive T 4 with the antibody to proceed substantially to equilibrium, thereby producing antibody bound T 4 , a percentage of which is radioactive; removing the antibody precipitated by the PEG including the antibody bound T 4 from the supernatant; and measuring the quantity of radioactive T 4 in the removed precipitate or the supernatant.
Claim 18
Dependent Claim
74 words
The method of claim 14, in which the step of adding the mixture to the measured quantity of human serum and radioactive T 4 comprises the steps of: adding the radioactive T 4 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 T 4 into the test tube.
Claim 19
Dependent Claim
174 words
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 T 4 ; pipetting into the test tubes measured quantities of the mixture including radioactive T 4 ; incubating the standard serum samples and radioactive T 4 with the mixture added to allow reaction of the T 4 in the standard serum samples and the radioactive T 4 with the antibody to proceed substantially to equilibrium, thereby producing antibody bound T 4 a percentage of which is radioactive; removing the antibody precipitated by the PEG including the antibody bound T 4 of the standard serum samples; counting the radioactivity in the precipitate including the antibody bound T 4 of the measured quantity of human serum being assayed; counting the radioactivity of the precipitate including the antibody bound T 4 of the standard serum samples; and correlating the count of the serum being assayed with the counts of the standard serum samples.
Claim 20
Dependent Claim
26 words
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.
Claim 21
Dependent Claim
29 words
The method of claim 14, in which the removing step comprises the steps of: centrifuging the serum and radioactive T 4 with the mixture added; and aspirating the supernatants.
Claim 22
Dependent Claim
14 words
The method of claim 14, in which the blocking agent comprises 8-anilino-1-naphthalene-sulfonic acid (ANS).
Claim 23
Dependent Claim
37 words
The method of claim 14, in which the radioactive T 4 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 T 4 .
Claim 24
Dependent Claim
25 words
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.
Claim 25
Dependent Claim
27 words
The method of claim 21, in which the mixture contains the antibody in sufficient quantity to bind between 35% and 50% of the radioactive T 4 .
Claim 26
Dependent Claim
23 words
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.
Claim 27
Dependent Claim
24 words
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.
References Cited (Backward Citations)
US3911096A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1980-04-18 | 1980-10-18 | 1981-04-19 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1984-04-18 | 1984-10-18 | 1985-04-19 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1988-04-18 | 1988-10-18 | 1989-04-19 | $7700.00 | Window Closed / Lapsed |