Classifications

• • 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
  • Y10S436/804—Radioisotope, e.g. radioimmunoassay
• • 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

• Thyroxine which is released in response to nervous or hormonal stimuli.
• Thyroxine enters the circulatory system and acts either directly upon the cell or indirectly upon other hormonal systems.
• Abnormal activity of the thyroid is a common malady in humans. In hypothyroidism, the body has decreased thyroid activity which is manifest by such diseases as cretinism and myxedema. Hyperthyroidism is a state of excessive thyroid activity in which one becomes nervous, develops an increased pulse rate and sometimes goiter.
• Thyroid deficiency was associated with a reduced metabolic rate as early as 1895 and several systems based on basal metabolic rate were devised for estimating thyroid activity. However, such systems were not reliable, so more direct and precise methods were sought.
• iodine was discovered in thyroid extract but the relationship between blood iodine level and thyroid function was not firmly established until I933. This led to the use of protein-bound iodine as a means of estimating thyroid function, and by 1955 the FBI test was standard for checking thyroid activity. It soon became apparent that this test was influenced by the administration of other iodine containing compounds to the patient.
• BEI butanol-extractable iodine
• T-4 analysis occurred in 1959 when Galton et al., Biochem. J. 72, 310 (1959) liberated T-4 from serum protein by hydrolysis and separated it from other iodine compounds by passage through a resin column. Later, Pileggi et al., J. Clin. Endocr. Met. 21, 1272 (1961) developed this column chromatographic procedure into a clinically usable method.
• U.S. Pat. No. 3,471,553 set forth still another column chromatography T-4 assay in which an anionexchange resin is adjusted to an alkaline pH of about 12 to dissociate the thyroxine from its albumin and globulin carriers.
• the diluted serum solution is then poured onto the resin wherein proteins, amino acids, thyroxine, iodotyrosine and inorganic iodine are adsorbed.
• Successive washes with acetate buffer isopropyl alcohol and acetic acid remove serum proteins, iodothyronines and some organic iodine compounds.
• Further treatment of the resin with 50 percent acetic acid at a pH of 2 quantitatively removes T-4.
• the present invention for determining T4 is based on the competitive protein binding principle which is a modified form of saturation analysis.
• the T-4 to be determined is mixed with a detenninate sample of T-4 labeled with a trace amount of radioactive isotope.
• a binding agent is added which will bind a definite number of molecules. Since the binding agent cannot distinguish between the labeled and unlabeled molecules, they compete with each other on an equal basis for the binding sites. These molecules are uniformly mixed so that the binding agent will bind them in the same ratio as that existing in the free or unbound state.
• the ratio becomes smaller and fewer labeled molecules are bound by the binding agent, leaving more of the labeled molecules in the free state.
• the crosslinked dextran gel column acts as the secondary binding agent for the unbound or free T-4, whereas the primary binding agent is a thyroxine (T-4) binding protein.
• T-4 thyroxine
• a measured amount of serum is mixed with some T-4 labeled with radioactive iodine on top of the column. Both the column and T4 mixture are at pHl2 to 13. At this pH, the T-4 binding serum proteins such as prealbumin, albumin and thyroxine binding globulin are completely dissociated from T4. As the mixture flows down the column, the T-4 is bound by the dextran gel. The serum proteins are washed away with a barbital buficr at a pH of 8.6 which automatically adjusts the pH of the column to that of the buffer.
• the pH is such that the transfer of the T-4 from the column to the primary binding agent is facilitated.
• the primary binding agent dissolved in barbital buffer is then added. Equilibrium is quickly established between the two binding agents and the primary binder is washed away carrying a portion of the T-4 with it.
• the dextran gels employed in the column are crosslinked with various amounts of epichlorohydrin as described in U.S. Pat. No. 3,042,667 and have a water regain of from I to 5 grams per gram of dry gel product.
• Such gels are produced commercially by Pharmacia of Uppsala, Sweden and sold under the trade name of Sephadex in various molecular weight ranges and sieve sizes.
• Sephadex G-lO has a water regain of one gram per gram of dry gel
• Sephadex G-l5 has a water regain of 1.5 grams per gram of dry gel
• Sephadex 6-25 has a water regain of 2.5 grams per gram of dry gel
• Sephadex G50 has a water regain of 5 grams per gram of dry gel.
• Sephadex G-25 is preferred.
• the dextran gel column employed in this invention is prepared by suspending 500 grams of dry gel in two liters of distilled water and allowing it to hydrate overnight. Fines are removed by slurrying the gel in 0.1 N sodium hydroxide for about 5 minutes, allowing it to settle for 15 minutes and then drawing off the supernatant by suction. The process is repeated three times and the gel is suspended in 4,400 milliliters of 0.1 N sodium hydroxide. Four milliliters of this suspension is placed in a six milliliter plastic syringe barrel having a diameter of 13 millimeters and a length of 66 millimeters.
• Each barrel is prefitted with a bottom closure means, for example, a removable cap, and a detergent treated sintered polyethylene retaining disc about l.5 millimeters thick and having a diameter of 13 millimeters is pressed coaxially to the bottom of the plastic barrel.
• a bottom closure means for example, a removable cap
• a detergent treated sintered polyethylene retaining disc about l.5 millimeters thick and having a diameter of 13 millimeters is pressed coaxially to the bottom of the plastic barrel.
• the suspension is stirred and permitted to settle free of air bubbles after which another detergent-treated sintered polyethylene disc, like the first-mentioned disc, is inserted into the syringe barrel and pushed coaxially into firm contact with the gel.
• About 1.5 milliliters of sodium hydroxide solution remains above the upper disc.
• the upper end of the syringe is closed with a new polyethylene cap. This procedure provides a column containing about 450 milligrams of gel.
• the T-4 test herein contemplated is performed by utilizing the gel column thus prepared as follows:
• the gel column can be made alkaline by potassium hydroxide or ammonium hydroxide if desired.
• Human a-globulin can be replaced with an equivalent amount of human serum, bovine serum or bovine gamma globulin as the primary binding agent.
• Aqueous alkaline solutions buffered to a pH of from 8 to 10 with sodium phosphate or tris (hydroxymethyl) amino methane can be used at concentrations from 0.01. to 0.2 molar, but an aqueous barbital solution is preferred, since it facilitates better quantitation when used to dissolve the human a-globulin or other binding agents.
• Another variation of the present invention involves determining the radioactivity of the solutions before addition to the gel column and after elution therefrom rather than determining the radioactivity of the column itself. Percent retention is then determined by a difference calculation. However, for the sake of efficiency, it is preferable to determine the radioactivity of the column before and after elution.
• Assays for T-4 by the column methods of the prior art should not be confused with the present method which involves saturation analysis using a radioactive tracer.
• a column was utilized only to separate contaminating iodines prior to analysis of iodine. In certain cases, this was done by using an ion exchange resin column at an alkaline pH, and iodine analysis was performed on the T-4 recovered from the column by measuring the effect of iodine on the ceric-arsenious acid reaction.
• the saturation analysis method herein disclosed is a direct determination of thyroxine, rather than the indirect measurement of thyroxine as iodine.
• a process for the in vitro determination of thyroxine in serum comprising:
• aqueous alkaline solution is a barbital buffer having a pH of 8.6.

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• Endocrinology (AREA)
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• Urology & Nephrology (AREA)
• Hematology (AREA)
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• Cell Biology (AREA)
• Biotechnology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
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• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Investigating Or Analysing Biological Materials (AREA)

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Cited By (17)

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Citations (2)

• 1970
  • 1970-06-29 US US51005A patent/US3659104A/en not_active Expired - Lifetime
• 1971
  • 1971-05-26 ZA ZA713408A patent/ZA713408B/xx unknown
  • 1971-06-14 IL IL37046A patent/IL37046A/xx unknown
  • 1971-06-22 HU HUII65A patent/HU166424B/hu unknown
  • 1971-06-26 ES ES392664A patent/ES392664A1/es not_active Expired
  • 1971-06-28 CA CA116,818A patent/CA955159A/en not_active Expired
  • 1971-06-28 GB GB3027671A patent/GB1351836A/en not_active Expired
  • 1971-06-28 SE SE7108334A patent/SE392346B/xx unknown
  • 1971-06-28 FR FR7123530A patent/FR2100008A5/fr not_active Expired
  • 1971-06-28 DE DE2132112A patent/DE2132112C3/de not_active Expired
  • 1971-06-28 JP JP4640271A patent/JPS5331399B1/ja active Pending

Patent Citations (2)

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