US3714344A - Method for determining thyroxine in blood serum and reagent therefor - Google Patents

Method for determining thyroxine in blood serum and reagent therefor Download PDF

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Publication number
US3714344A
US3714344A US00865785A US3714344DA US3714344A US 3714344 A US3714344 A US 3714344A US 00865785 A US00865785 A US 00865785A US 3714344D A US3714344D A US 3714344DA US 3714344 A US3714344 A US 3714344A
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thyroxine
blood serum
radioactive
solution
serum
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US00865785A
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J Brown
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Mallinckrodt Inc
Mallinckrodt Chemical Works
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Mallinckrodt Chemical Works
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    • G—PHYSICS
    • G21—NUCLEAR PHYSICS; NUCLEAR ENGINEERING
    • G21H—OBTAINING ENERGY FROM RADIOACTIVE SOURCES; APPLICATIONS OF RADIATION FROM RADIOACTIVE SOURCES, NOT OTHERWISE PROVIDED FOR; UTILISING COSMIC RADIATION
    • G21H5/00—Applications of radiation from radioactive sources or arrangements therefor, not otherwise provided for 
    • G21H5/02—Applications of radiation from radioactive sources or arrangements therefor, not otherwise provided for  as tracers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/825—Pretreatment for removal of interfering factors from sample
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/826—Additives, e.g. buffers, diluents, preservatives

Definitions

  • T-4 The serum protein, thyroxine-binding globulin
  • T-4 radioactive T-4, that is T-4 containing radioactive iodine such as iodine-125, is added to a solution containing barbital buffered TBG, essentially all of the T-4 will be bound to the TBG.
  • the radioactive T-4 will be displaced from the TBG in proportion to the amount of stable T-4 that is added. If another T-4 binding agent, such as an ion-exchange resin, is now added to this system, it will bind the radioactive T-4 that has been displaced from the TBG.
  • another T-4 binding agent such as an ion-exchange resin
  • the degree of radioactive T-4 displacement and hence the amount of stable thyroxine added to the TBG can be determined by removing the ion-exchange resin from the system and comparing the radioactivity now emitted by the TBG solution with its original radioactivity.
  • the radioactivity of the TBG solution decreases with each successive increase in added stable T-4.
  • diluted blood serum is customarily used as the source of TBG. It has not been the practice to subject it to any further treatment.
  • the serum T-4 In order to determine T-4 in blood serum, the serum T-4 must first be separated from the binding proteins. This is accomplished by denaturing the proteins with alcohol. The denatured proteins release most (approximately 80 percent) of their bound T-4 which is removed in the alcoholic supernatant after centrifugation.
  • this alcoholic extract is evaporated to avoid possible errors introduced by alcohol in the test mixture. Generally, 30 to 120 minutes are required to complete this step, depending upon the number of samples tested.
  • the present invention is thus directed to the method of determining the thyroxine content of blood serum using radioactive thyroxine as a tracer compound which comprises the steps of adding to a known quantity of a reagent consisting essentially of a solution containing thyroxine-binding globulin and radioactive thyroxine, an alcoholic extract of a sample of blood serum whose thyroxine content is to be determined and an ion-exchange resin, maintaining the globulin solution in contact with the resin for a predetermined BRIEF DESCRIPTION OF THE DRAWING
  • FIG. 1 is agraph depicting an illustrative standard curve obtained by plotting the radioactivity of treated thyroxine-binding globulin samples against amounts of stable thyroxine.
  • control samples are prepared.
  • 0.3 ml. of an ethanolic solution containing a known amount (e.g., 0, and i2 nanograms) of stable thyroxine are added.
  • a strip of ion-exchange resin membrane is then added to each test vial.
  • the'ion-exchange resin may be either anion-selective or cation-selective; Many such resins are available in the form of membranes.
  • a commercially available anion-selective resin suitable for the purposes of thisinvention is designated AR-ill (manufactured by Ionics Incorporated, -Watertown, Massachusetts).
  • the resin uptake of radioactive thyroxine is a function of rotation time, it is essential that the rotation time be the same for both the unknown and control samples.
  • the resin strip is removed with a forceps and discarded.
  • the radioactivity of each vial is then counted and recorded as the post-count. A minimum of 10,000 counts per minute will insure an error of less than 2 percent.
  • the pre-count is also advantageously determined by counting an unused vial containing the TBG-radioactive T-4 solution. The precount/post-count ratio is calculated for each of the samples.
  • the values for the standard samples are then plotted against the amount of added T-4, a straight line is drawn through the points, and the thyroxine content of the unknown serum samples is read from the resulting graph (see FIG. 1). Since the alcohol extracts approximately 80 percent of the T4 from the serum, the value read from the graph should be divided by the extraction efficiency (approximately 0.8) to give the actual thyroxine content of the serum.
  • This corrected value then represents the amount of T-4 (nanograms) in each 0.1 ml. of serum which is numerically equivalent to the number of ugm. of T-4 per 100 ml. (ugm. of patient serum.
  • the special TBG solution is conveniently prepared by passing the blood serum through a column containing a suitable anion-exchange resin which is capable of adsorbing the thyroxine but not the serum protein.
  • a suitable procedure for preparing the special TBG solution is as follows. To 137 m1. of serum is added 3280 ml. of a pH 8.6 barbital buffer solution containing 0.64 percent sodium chloride, 0.23 percent barbital sodium, and 0.6 percent barbital. A small amount of radioactive thyroxine (approximately 0.24 millicuries) is also added to serve as a tracer for determining the efficiency of the extraction.
  • An extraction column for this purpose is prepared by adding a sufficient amount of resin to a suitable glass column to provide a bed volume of 1,200 ml. and a flow rate of approximately 25 ml. per minute.
  • a strongly basic quaternary ammonium type resin is suitable.
  • a commercially available resin of this kind designated IRA-400 manufactured by the Rohm and Haas Company
  • IRA-400 is satisfactory, but other anion-selective or cation-selective resins may be used instead.
  • the resin is added to the column as a water slurry and a layer of water is always maintained above the resin.
  • the column is connected to a reservoir containing the serum solution to be extracted, and flow of the serum through the column is begun. Periodically a 1 ml. sample of the eluate is removed and its radioactivity counted to measure the efficiency of the extraction.
  • the radioactivity of the eluate should be percent or less that of the stock solution.
  • the extracted serum solution is then preferably passed through a 0.22 micron filter. More (approximately 0.30 millicuries) radioactive thyroxine is then added to the eluate and the latter is diluted with six times its volume of the aforementioned buffer solution.
  • the resulting solution should have a pH of 8.6.
  • a useful kit consists of a number of 4 ml. test vials made of glass and provided with screw caps.
  • the vials should include 4 cc. of the above TBG solution and should be matched for radioactivity.
  • the standard samples should contain known amounts of stable thyroxine (e.g., 0 and 12 nanograms) in 0.3 ml. of alcohol.
  • a container of alcohol from the same source used to prepare the control samples should also be supplied.
  • the kit should also include the required ionexchange resin, the latter preferably being in the form of membrane strips.
  • the other necessary equipment such as syringes, radiation counter and rotator are standard laboratory equipment.
  • the method for determining the thyroxine content of blood serum using radioactive thyroxine as a tracer compound which comprises the steps of adding to a known quantity of a reagent consisting essentially of a solution containing thyroxine-binding globulin and radioactive thyroxine,
  • said reagent comprises a buffered solution of radioactive thyroxine and blood serum from which most of the naturally occurring thyroxine has been extracted.
  • the method for preparing a reagent for use in determining the thyroxine content of blood serum which comprises passing diluted blood serum through a column containing an ion-exchange resin to extract most of the naturally occurring thyroxine from the serum, and adding radioactive thyroxine to the resulting eluate.
  • a reagent for use in determining the thyroxine content of blood serum which comprises a buffered solution of radioactive thyroxine and blood serum from amount of thyroxine-l l 25 to act as a tracer, two control samples containing a known amount of thyroxine dissolved in a predetermined volume of alcohol, a quantity of alcohol from the same source used to prepare the control sample, and a plurality of resin membrane strips whose dimensions permit them to fit loosely in the test containers.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
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  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
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  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • High Energy & Nuclear Physics (AREA)
  • Food Science & Technology (AREA)
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  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)
US00865785A 1969-05-01 1969-10-13 Method for determining thyroxine in blood serum and reagent therefor Expired - Lifetime US3714344A (en)

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US82109769A 1969-05-01 1969-05-01
US86578569A 1969-10-13 1969-10-13

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US (1) US3714344A (de)
JP (2) JPS5617620B1 (de)
BE (1) BE749772A (de)
CH (1) CH526781A (de)
DE (1) DE2021968A1 (de)
FR (1) FR2047211A5 (de)
GB (1) GB1314263A (de)
IL (1) IL34387A (de)
IT (1) IT943545B (de)
NL (2) NL7006455A (de)
SE (1) SE378679B (de)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3911096A (en) * 1972-06-23 1975-10-07 Professional Staff Ass Of The Radioimmunoassay for measurement of thyroxine (T{HD 4{B ) and triiodothyonine (T{HD 3{B ) in blood serum
US3929981A (en) * 1974-02-27 1975-12-30 Squibb & Sons Inc Method for determining thyroid function
US3947564A (en) * 1973-05-29 1976-03-30 Bio-Rad Laboratories Radioactive determination of serum thyroxine
US3966897A (en) * 1973-04-02 1976-06-29 Marine Colloids, Inc. Medium for use in bioassay and method of using same
US4282002A (en) * 1979-09-06 1981-08-04 Akzona Incorporated Sensitized sheep stroma immunoassay for rheumatoid factor
USRE32098E (en) * 1972-06-23 1986-03-25 Research And Education Institute, Inc. Radioimmunoassay for measurement of thyroxine (T4) and triiodothyronine (T3) in blood serum
US5359093A (en) * 1992-03-30 1994-10-25 Abbott Laboratories Reagents and methods for the detection and quantification of thyroxine in fluid samples
US5593896A (en) * 1992-03-30 1997-01-14 Abbott Laboratories Reagents and methods for the detection and quantification of thyroxine in fluid samples
US6242261B1 (en) 1992-02-10 2001-06-05 University Of Saskatchewan Assessment of ion availability in heterogeneous media using ion-exchange membranes
US20080020486A1 (en) * 2004-11-25 2008-01-24 Veolia Eau - Compagnie Generale Des Eaux Case For Sampling Water And Capturing Substances That Generate Orders And/Or Tastes Present In Water

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4111656A (en) * 1975-06-26 1978-09-05 Mallinckrodt, Inc. Radioimmunoassay methods for the determination of l-triiodothyronine and thyroxine
US4289748A (en) * 1979-05-31 1981-09-15 United States Of America Ultrasensitive enzymatic radioimmunoassay method
US11768689B2 (en) 2013-08-08 2023-09-26 Movidius Limited Apparatus, systems, and methods for low power computational imaging

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3414383A (en) * 1965-08-09 1968-12-03 Canadian Patents Dev Determination of thyroxine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3414383A (en) * 1965-08-09 1968-12-03 Canadian Patents Dev Determination of thyroxine

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3911096A (en) * 1972-06-23 1975-10-07 Professional Staff Ass Of The Radioimmunoassay for measurement of thyroxine (T{HD 4{B ) and triiodothyonine (T{HD 3{B ) in blood serum
USRE32098E (en) * 1972-06-23 1986-03-25 Research And Education Institute, Inc. Radioimmunoassay for measurement of thyroxine (T4) and triiodothyronine (T3) in blood serum
US3966897A (en) * 1973-04-02 1976-06-29 Marine Colloids, Inc. Medium for use in bioassay and method of using same
US3947564A (en) * 1973-05-29 1976-03-30 Bio-Rad Laboratories Radioactive determination of serum thyroxine
US3929981A (en) * 1974-02-27 1975-12-30 Squibb & Sons Inc Method for determining thyroid function
US4282002A (en) * 1979-09-06 1981-08-04 Akzona Incorporated Sensitized sheep stroma immunoassay for rheumatoid factor
US6242261B1 (en) 1992-02-10 2001-06-05 University Of Saskatchewan Assessment of ion availability in heterogeneous media using ion-exchange membranes
US5359093A (en) * 1992-03-30 1994-10-25 Abbott Laboratories Reagents and methods for the detection and quantification of thyroxine in fluid samples
US5593896A (en) * 1992-03-30 1997-01-14 Abbott Laboratories Reagents and methods for the detection and quantification of thyroxine in fluid samples
US20080020486A1 (en) * 2004-11-25 2008-01-24 Veolia Eau - Compagnie Generale Des Eaux Case For Sampling Water And Capturing Substances That Generate Orders And/Or Tastes Present In Water

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DE2021968A1 (de) 1970-11-05
IL34387A (en) 1973-04-30
NL7417037A (nl) 1975-04-29
CH526781A (de) 1972-08-15
BE749772A (fr) 1970-10-30
FR2047211A5 (de) 1971-03-12
JPS5617620B1 (de) 1981-04-23
NL7006455A (de) 1970-11-03
SE378679B (de) 1975-09-08
IL34387A0 (en) 1970-06-17
GB1314263A (en) 1973-04-18
JPS5542704B1 (de) 1980-11-01
IT943545B (it) 1973-04-10

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