CA1073351A - Competitive thyroid hormone radio assay in a single tube - Google Patents
Competitive thyroid hormone radio assay in a single tubeInfo
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- CA1073351A CA1073351A CA269,943A CA269943A CA1073351A CA 1073351 A CA1073351 A CA 1073351A CA 269943 A CA269943 A CA 269943A CA 1073351 A CA1073351 A CA 1073351A
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- 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
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Abstract
THYROID HORMONE ASSAY
ABSTRACT OF THE DISCLOSURE
A "single tube" thyroid hormone assay is provided whereby endogenous thyroid hormone is initially separated from a serum sample with an acid reagent and thereafter a tracer amount of radioactive labelled thyroid hormone is allowed to competi-tively bind with antibodies for thyroid hormone. The resulting free hormone (not bound to antibody) is separated from the hormone bound to such antibodies by contacting the solution with an aqueous solution of water-soluble sulfate salt containing a minor but effective amount of added animal serum to cause precipitation of the hormone bound to the thyroid hormone binding protein ma-terial. Thereafter, either the free hormone in supernatant fluid or the bound hormone that has precipitated is counted with a scintillation counter.
ABSTRACT OF THE DISCLOSURE
A "single tube" thyroid hormone assay is provided whereby endogenous thyroid hormone is initially separated from a serum sample with an acid reagent and thereafter a tracer amount of radioactive labelled thyroid hormone is allowed to competi-tively bind with antibodies for thyroid hormone. The resulting free hormone (not bound to antibody) is separated from the hormone bound to such antibodies by contacting the solution with an aqueous solution of water-soluble sulfate salt containing a minor but effective amount of added animal serum to cause precipitation of the hormone bound to the thyroid hormone binding protein ma-terial. Thereafter, either the free hormone in supernatant fluid or the bound hormone that has precipitated is counted with a scintillation counter.
Description
~0~3351 BACKGRQU~D OF THE INVENTION
This invention relates to diagnostic tests for deter-mining the level of thyroid hormone within a body fluid. In another aspect, this invention relates to an improved test for measuring total thyroid hormone in a sample of serum. In another aspect, this invention relates to a novel radioimmun-oassay for thyroid hormone.
Various diagnostic tests are known for determining thyroid function. These tests include the basal metabolism test, the thyroid uptake test, various colorimetrlc and chemical procedures for determining the level of thyroxine iodine in the blood and a test commonly referred to as the T-3 uptake test which measures the unsaturated binding capacity of thyrobinding globulin and other thyroxine binding proteins within a serum sample. Perhaps the most commonly used test available is the diagnostic test which utilizes radioisotopically labelled hormone to determine the level of thyroid hormone thyroxine (C15HllI4N04)present in serum. This test, commonly referred to as T-4 assay, measures the total quantity of hormone within a sample of blood serum. The most commonly used T-4 assay which determines the level of thyroxine within a sample of blood ~erum, utilizes the technique of competitive protein binding. To carry out the T-4 assay, it is necessary first to release the thyroid hormone thyroxine from endogenous thyroxine binding proteins present in a serum sample. After this, a known quantity of thyroxine binding -;~
This invention relates to diagnostic tests for deter-mining the level of thyroid hormone within a body fluid. In another aspect, this invention relates to an improved test for measuring total thyroid hormone in a sample of serum. In another aspect, this invention relates to a novel radioimmun-oassay for thyroid hormone.
Various diagnostic tests are known for determining thyroid function. These tests include the basal metabolism test, the thyroid uptake test, various colorimetrlc and chemical procedures for determining the level of thyroxine iodine in the blood and a test commonly referred to as the T-3 uptake test which measures the unsaturated binding capacity of thyrobinding globulin and other thyroxine binding proteins within a serum sample. Perhaps the most commonly used test available is the diagnostic test which utilizes radioisotopically labelled hormone to determine the level of thyroid hormone thyroxine (C15HllI4N04)present in serum. This test, commonly referred to as T-4 assay, measures the total quantity of hormone within a sample of blood serum. The most commonly used T-4 assay which determines the level of thyroxine within a sample of blood ~erum, utilizes the technique of competitive protein binding. To carry out the T-4 assay, it is necessary first to release the thyroid hormone thyroxine from endogenous thyroxine binding proteins present in a serum sample. After this, a known quantity of thyroxine binding -;~
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protein (generally thyroid hormone bindincJ globulin) and a tracer quantity of radioactively labelled thyroid hormone are added to -the thyroxine obtained from the sample. The thyro-xine including the endogenous thyroxine obtained from thesample and the radioactively labelled thyroxine compete for binding sites on the known quanti-ty of thyroxine binding pro-tein. After the competitive binding step/ the free or unbound thyroxine is separated from the thyroxine bound to the thyro-xine binding proteins and the relative quantity of thyroxine in the original sample is determined by counting the radio-activity of either the free thyroxine or the bound thyroxine in a scintillation well counter.
Recently the competitive protein binding assays for thyroid hormone have been modified to include the use of spe-cific antibodies as the thyroxine bi,nding protein instead of the thyroxine binding globulins. Such procedures are called radioimmunoassays of thyroid hormone.
In general, the methodology of -the T-4 competitive protein binding assay and the radioimmunoassay is quite simi-lar. In both procedures, it is necessary first to releasethyroxine from endogenous thyroxine binding proteins present in the serum and to alter -the thyroxine binding proteins in such a way that their further participation in the reaction is prohibited. In the conventional T-4 competitive protein binding assays, this has been accomplished by initially treating the serum sample with organic solvents such as ethyl alcohol to denature the thyroid hormone binding protein. Such a procedure is described in U.S. Paten-t No. 3,666,854. Other chemical methods have been utilized which include the treatment of the sample with inor~anic chemicals such as alkaline solutions.
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However, the use of alkaline extraction solutions as cornmerclal test kit components is not desirable because such solutions exhi~it instabili-ty during storage and use. rlore specifically, such solutions rapidly absorb CO2 w~ich results in a lowering o~ the pH thereo~. Another method includes the treatment o~ the sample with.an acid solution to effect separation between the thyroid hormone and the thyroid hormone binding protein and thereafter contacting the solution with an inorganic crystal-line sorbent such as magnesium silicate which sorbs the free ;~
normone only. Such a method is descri.bed in U.S. Patent
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protein (generally thyroid hormone bindincJ globulin) and a tracer quantity of radioactively labelled thyroid hormone are added to -the thyroxine obtained from the sample. The thyro-xine including the endogenous thyroxine obtained from thesample and the radioactively labelled thyroxine compete for binding sites on the known quanti-ty of thyroxine binding pro-tein. After the competitive binding step/ the free or unbound thyroxine is separated from the thyroxine bound to the thyro-xine binding proteins and the relative quantity of thyroxine in the original sample is determined by counting the radio-activity of either the free thyroxine or the bound thyroxine in a scintillation well counter.
Recently the competitive protein binding assays for thyroid hormone have been modified to include the use of spe-cific antibodies as the thyroxine bi,nding protein instead of the thyroxine binding globulins. Such procedures are called radioimmunoassays of thyroid hormone.
In general, the methodology of -the T-4 competitive protein binding assay and the radioimmunoassay is quite simi-lar. In both procedures, it is necessary first to releasethyroxine from endogenous thyroxine binding proteins present in the serum and to alter -the thyroxine binding proteins in such a way that their further participation in the reaction is prohibited. In the conventional T-4 competitive protein binding assays, this has been accomplished by initially treating the serum sample with organic solvents such as ethyl alcohol to denature the thyroid hormone binding protein. Such a procedure is described in U.S. Paten-t No. 3,666,854. Other chemical methods have been utilized which include the treatment of the sample with inor~anic chemicals such as alkaline solutions.
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However, the use of alkaline extraction solutions as cornmerclal test kit components is not desirable because such solutions exhi~it instabili-ty during storage and use. rlore specifically, such solutions rapidly absorb CO2 w~ich results in a lowering o~ the pH thereo~. Another method includes the treatment o~ the sample with.an acid solution to effect separation between the thyroid hormone and the thyroid hormone binding protein and thereafter contacting the solution with an inorganic crystal-line sorbent such as magnesium silicate which sorbs the free ;~
normone only. Such a method is descri.bed in U.S. Patent
3,776,698. Generally, when acid extractions have been utilized in competitive protein binding assays, the precipitated endogenous proteins are physically removed from the resulting endogenous thyroid horrnone prior to proceeding with subsequent steps in the test procedure which occur at higher pH's. This is due mainly to the fact that acid precipitated or denatured proteins are known to be resolubilized or renatured in solutions of higher pH. Thus acid denaturation is believed to be "reversible".
Indeed a commercially available so-called normalized T-4 type 20 test sold under the trademark oE "Quantisorb-125" by Abbott ~aboratories utilizes this above described phenomenon of re-naturation of acid denatured proteins as an essential step thereof.
Another approach for releasing the thyroxine from r the endogenous thyroxine binding protein is by heat denatura-tion. Heat denaturation of the serurl~ protein provides good recovery of the thyroid hormone with extraction efficiencies approach-ing about 100%. However, this method is time-consuming in that it ~1 .
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iO~3351 requires a minimum of 15 minutes in a boiling ~ater bath for complete destruction of binding proteins.
Likewise, when carrying out tlle radioimmunoassay for thyroxine, it is firs~ necessary to release the thyroxine from endogenous thyroxine binding proteins present ~ithin the serum sample and to alter the thyroxine binding proteins in such a way that their further participation in the reaction is pro-hibited. In the radioim~unoassay, this is most commonly accom-plished to a degree by blocking the thyroxine binding sites on endogenous proteins with chemicals such as 8-anilino-l-napllt]lalen sulfonic acid, diphenylhydantoin, salicylates, or thimerosal.
l~o~ever, the use of such blocking agents in radioimmulloassay has posed problems. For example, it has been shown that the quantity of these blocking agents necessary to occupy all sites may be related to the level of endogenous thyrobinding protein whicl can vary significantly in individual serum samples.
Thus, in both the conventional T-4 competitive protein binding assay and the radioimmunoassay, the thyroxine must initially be extracted from native proteins and thereafter bound to specific ~roteins, and then the free hormone separated from the protein bound hormone. The final separation has been accomplished by sorption of the free hormone such as to resins~
charcoal or inorganic crystalline sorbents. The conventional resins include ion exchange resins such as the ion exchanger having strongly basic amino or quaternary ammonium ~roups such as disclosed in U.S. Paten~ No. 3,414,383. These organic ion ~xchange resins can be either in loose form or incorporated in polyurethyane sponges as disclosed in U.S. Patellt No. 3,206,602, - 1~7335~
or enclosed in porous bags or the like. Other conventional methods include a selective sorption of the Eree hormone by charcoal which has been coated with suitable proteins or other polymers, or the use of molecular sieves such as Sephadex.
The use of the inorganic crystalline sorbent materials are dis- -;
closed in U.S. Patent ~o. 3,666 j854 and U.S. Patent No. 3,776, 698. In general, the methods which rely on sorption systems to separate the free hormone from the hormone bound to the protein are dependent upon protein concentration in the assay system, and some systems may require adjustment of the protein level for valid results. In addition, some of the sorbents such as the resins are temperature sensltive necessitating correction oE assay values obtained in working conditions where the temperatures are variable. In addition, the sorbents are in general time dependent and careful timing during the sorption process is necessary in order to obtain reproducible results.
Antibody-bound hormone may be separated from the free fraction by precipitation of the specific protein. One common approach used to precipitate the bound fraction is the double antibody technique which, due to a second incubation period,iis time-consuming. Another means of sepaxation is chemical precip-itation of the bound fraction with either high molecular ~eight polymers or salts. In order to precipitate the minute amounts of gamma globulin present, exogenous gamma globulin is conven-tionally added to the assay system prior to separation of the bound and free fractions.
A problem which has been encountered is the phenomenon of non-specific binding occurring during the assay. Non-speciEic binding stated simply is the binding of the free hormone including * Trade Mark 6 ' ' . :.
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~3351 the free radioactivel~ labelled hormone with naterials other than the thyrobinding protein in the conventional T-4 assay or the antibody in the radioimmunoassay. The inability to assess the degree of non-specific binding occurring in the absence of the hinding protein is a disadvantage of those assay systems in which binding protein and radioactively labelled hormone are employed as a single reagent.
In general, a thyroid hormone assay is needed which will initially extract all or substantiallv all of the tilyroid hormone from the endogenous serum sample in a reproducible manner, will not rely on blocking agents or solid sorbents to separate the bound from the free hormone after -the competitive ~inding step, but wil.l effect -the separations quickly and efficiently in a reproducible manner wherein a low but repro-ducible anount of non-specific binding will occur during each test and which can be carried out in a single test tu~ without the requirement of intermedia-te decanting of test solution(s) thereform.
SHORT STATE~IENT OF THE INVENTION
According to the invention there is provided a method of measuring the level of thyroid hormone in a sample of serum containing endogenous thyroid hormone and endogenous thyroid hormone binding protein com?rising: (a) admixing said serum sample with an effective amount of an aqueous acid solu-tion sufficient to separate said thyroid hormone from said thyroid hormone binding protein; (b) adjusting the pH of the resulting mixture to a value in the range of from about 7.0 to about 8.5 in the absence of blocking asents while adding thereto a known amoun-t of radioacti.vely labelled thyroid hormone and a known amount of thyroid hormone binding antibodies and allow-ing the resulting solution to equilibrate- (c) thorouqhly ad-mixing the resulting equilibrated mixture from step (b) wi-th ~ _ 7 _ ~oq33sl an aqueous solution of a water soluble sulfate which contains sufficient sulfate such that the resulting mixture has a sul-fate concentration between about 20 and 30 weight percent to thereby result in precipi-tation of said thyroid hormone binding protein material containing thyroid hormone bound thereto and leave free thyroid hormone in the solution; (d) separating the precipitated material from said solution; and (e) counting with a scintillation counter one of (1) the free radioactively labelled thyroid hormone in said resulting solution, and ~2) the bound radioactively labelled thyroid hormone bound to said thyroid hormone binding antibodies in said precipitate.
In accordance with one embodiment of the subject in-vention, a radioimmunoassay for thyroid hormone is provided which includes the initial extraction of endogenous thyroid hormone from sample serum with an acid reagent to result in an effective inactivation of endogenous thyroid hormone binding proteins, the subsequent adjustment of the pH to a high level suitable for competitive binding (without removing the endoge-nous thyroid hormone binding protein therefrom) and the addition of a tracer quantity of radioactively labelled thyroid hormone and a known - 7a -C~
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quantity of thyroid hormone antibodies and thereafter allowing the resulting solution to equilibrate and competitive binding to occur, and then separation of the bound moiety from the ~ree moiety by the addi~ion of an aqueous sulfate salt solution whic~
causes precipitation of the bound moiety; and thereafter counting the prccipitated bound moiety or ~]~e supernatant containing the free ~oiety in a scintillation well counter. This procedure is carried out in a single tube without intermediate decanting steps.
In accordance with another embodiment of the subject invention, I have found that the separation of free from bound thyroid hormone fractions which rcsnlts ~rom the competitivc binding step in a thyroxine assay syst.em can be efficiently and reproducibly effected by a manner whereby the non-specific bind ing in the system is monitored at a lo~ constant level if the bound fraction is precipitated with an aqueous sulfate salt solution to which a minor but effective amount of added animal serum has been previously added.
DETAILED DESCRIPTION OF T}IE INV~lTIO~
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The assay of the subject invention was developed in an effort to provide a "single tube" thyroid hormone assay which would not utilize blocking agents nor an external solid phase sorption step and require a minimum of manipulation by the ~.
laboratory technician.
First, I have discovered tha~ endogcnous thyroid hormone binding protein can be initially inactivatcd and thereby scparated from endogenous thyroid hormone by acid treatment~ and : I .; ' "" '~, ' , .
3~1 thereafter the pl-l of the resulting mixture can be raised and thyroid hormone antibodies added thereto to competitively bind :
with the endogenous thyroid hormone without interference from the endogenous protein. This is quite surprising in view of the fact that acid inac-tivated thyroid hormone binding protein is known to renature at higher pH's and interfere with competi- ~.
tive binding between thyroid hormone and exogenous thyroid hormone binding protein by actually entering into the competitive binding reaction. Thus, I have found that a radioimmunoassay for thyroid hormone can be carried out immediately after acid separation of endogenous thyroid hormone from endogenous thyroid hormone binding protein after a single upward pH adjustment of ;^~
the mixture and without first having to remove the endogenous thyroid hormone bindiny protein from the mixture.
Secondly, as stated above, most sorbents are dependent upon the total protein concentration in the mixture such that slight variations in the protein concen-tration of the sample can affect the percentage uptake of the sorbent. In addition, an effective thyroxine radioimmunoassay must have a low reproducible non-specific binding of the thyroid hormone which in essence is a low but uniform degree of binding of the thyroid hormone to constituents other than the antibody.
The thyroid hormone radioimmunoassay of the subject invention utilizes a salt precipitation step to separate the bound hormone from the free hormone in solution. Carrier or adjuvant proteins are conventionally added to such mixtures before the addition of sulfate in order to effect and expedite precipitation. However, I have found that the adjuvant protein can be mixed with the sulfate prior to in-troduc-tion to the assay ,~ ' ' ~i ~. .
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mixture. Using this combination as a precipitant, I have found that a constant, reproducible non~specific binding as well as an acceptable spread between the hypothyroid and hyperthyroid samples will result. Furthermore, the use of this combination as precipitant permits the use of a wide range of volumes of the serum sample, and allows the assav of serum samples containing a wide range of protein concentration.
The subject invention will be described in detail in relation to radioimmunoassay for thyroxine, even thou~h one having ordinary skill in the art can easily adapt my novel pro-cedure to assay triiodothyronine and other thyronines.
Before the endogenous serum thyroxine can be assayed utilizing the improved salt precipitation step of the subject invention, the hormone must be efficientlv and reproducibly extracted from a serum sample. In addition, all endogenous pro-tein which could possibly bind thyroxine during the several -steps of the assay must be completely inactivated. In accordance with one embodiment of the subject invention, the endogenous thyroxine is extracted from the serum sample with an acid solu-tion. The acid solution is generally maintained at a nH within the range of from about l.0 to 2.2. The preferable pH range is from about 1.0 to about 2Ø Any stable acid solution which is nondeletrious to the thyroid hormone can be used in the scope of the subject invention. For example, aqueous solutions of HCl, H2SO4, H3PO4 and the like can be used within the scope of the subject invention. HCl is the preferred acid. These materi-als can be buffered with suitable buffering agents, e.g., salts of weak acids at a concentration of about 0.02 to 0.07 l~l. Also, the ionic strength of the acid solution can be maintained by `~l , , . "',~. :
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the presence of suitable materials such as neutral sal-ts, e.g., NaCl, KCl and the like at a concentration of 0.02 to 0.07 molar. ~, In general, at least about 10 and preferably about 20 volumes of the acid extraction reagent is combined with each volume of the serum sample. The pH of the resulting mixture should be in the range of from about 1.3 to about 3Ø Complete inactivation of the endogenous thyroxine binding proteins is accomplished immediately, even though the inactive proteins .
remain in solution. For example, the serum is added to the acid extraction solution contained within a vial, and the vial is shaken for a few seconds (generally 10 to 15 seconds) to admix thoroughly the serum and the acid extraction solution.
This will allow time for the acid solution -to break the bonds ~etween the thyroxine and the thyroxine binding protein and to inactivate completely the thyroxine binding protein.
Now that the resulting solution contains all of the endogenous thyroxine therewithin, and substantially all of the thyroxine binding protein has been deactivated, it is necessary to adjust the pH of the solution upwardly to a suitable pH in which competitive binding of the thyroid hormone and thyroid antibody can take place while adding to the solution a tracer quantity of radioactively labelled thyroid hormone (T-4) and a known,quantity of antiserum containing the thyroid hormone anti-bodies. In accordance with a preferred embodiment of this in-vention, the radioactive thvroid hormone followed by the anti-serum is added to the acid solution containing the unknown quantity of endogenous thyroid hormone. More specifically, the solution ., , ~ .
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contailling the radioacti~e thyroxine can cor.l~rise a solution having a plll~ithin thc range of about 7.6 to 9.0 and preferal~ly about 8.3, and l~ill contaill a tracer ~uantity of radioactively labelled thyroicl 1lOT~One, and preferably a bu~fer to maintain the pll of the solution. A suitable SUCh SO1UtiO11 COmPriSeS
0.04 ~ sodium bar~ital carrier W]liC]I is adjusted to a p~l of 8.3 by the addition of hydrochloric acid ~hich contains the tracer quantity of T-~ 125I
j Any radioactive isotope of iodine, tritium, or carbon 10 !,can be used. It is preferred that a hormone be utilized l~hic]
jlis labelled Wit]l 125I. The buffered solution containing the ¦,tracer quantity o~ radioactively labclled ~hyroid hormone litllyroxine, which is at least 20 and prcfer.lbly about 40 volumcs ! greater than the serum sample, is added to the acid solution an-l llthe con~ents are thereafter thoroughly mixed by sl~aking.
~ Next, a solution containing the antithyroxine scrum ¦ is added to the resulting solution. The antiserum can contai~l i a suitable buffer such as sodium barbital and can generally havc I',a p~l within the range from about 7.6 to 9Ø The volume of tl~c I'antiserum solution added to the test mixture can be the same as ¦that used to deliver the ~adioact;~e isotope labelled thyroid hormone to the test mixture. After the thyroxine antiserum solu tion has been added to the solution containing the extracted !Ithyroid hormone and the radioactive ~uantity of thyroid hormone, I,the resulting mixture is thoroughly mixed. The resulting solu-tion will have a p~T in the range of from about 7.0 to 8.5 and preferably about 7.~-8.4 and is incubated at ~oom temperature from 30 to about 60 ~inutes to allol~ the formation of the thyroxine-antibody com~lexes. Since the antibody ~ill bind both lQ~3351 ' the radioactive thyroxine and serum thyroxine e~ually well, the amowlt of radioactive thyroxine reco~ered ~ill reflect the con-centration of thyroxine in the original sample. As is well ~nown, the antiserum used in this step should llave a high speci~
ficity for thyroxine. Furthermore, as previously noted, the inactivated thyroid hormone binding protein in the mixture sur-prisingly does not interfere with the competitive binding betl~een the thyroid hormone and the antibody.
Once the solution is equilibrated sucll that the competitive binding between the thyroxine antibodies and thyroxine is complete, the antibodics containing bound hormone are precipitated in accordance with the improved salt precipita-tion step of the subject invention. The precipitant solution whicll is used in the scope of the subject invention comprises an aqueous solution of a water soluble sulfate salt which will ef~ectively precipitate proteinaceous materials without deleteri-ously ~recipitating free hormone from solution. Examples of suitable sulfate salts which can be used are the alkali metal sulfates such as sodium and potassium sulfate, ammonium sulfate, and zinc sulfate. Because of the solubility range, availability and convenience, the most preferred salt is ammonium sulfate.
The concentration of the ammonium sulfatc can vary according to the amount of aqueous precipitant fluid which is desired to be used. Generally, the concentration in the precipitant solution should be SUCII that when it is admixed Wit]l the ~hyroxine-anti-body containing solution, the resulting concentration o~ sulfate will be in the range of about 20 to 30% by weight and pTeferably in the range of about 23 to 27% by weight and most prefcrably about 23 to 24% by ~eight. I have found that the concentration `` iO~3351 of the sulfate in the precipitant solution can conveniently range from about 30 to about 40% by weight and preferably from about 33 to 39% by weight thereof.
In addition to the sulfate, I have found it necessary to add a minor but effective amount of animal serum as adjuvant to the antibody-thyroxine containing solution at the time of ;
precipitation in order to obtain a low but reproducible order of non-specific binding and a marked distinction in thyroxine levels of hypothyroid and hyperthyroid serum samples. Further-more, the use of the above combination permits the use of a wide range of sample volume (i.e., from about 2 to about 50 microliters), and allows the assay of serum samples containing a wide range of protein concentration. In general, the amount of added serum which is necessary to accomplish this result is a volume greater than the initial volume of the serum sample being tested. Generally, any type of animal serum can be uti- `
lized as an adjuvant in this manner even though I have found some variation in the quantity of serum which must be used which is directly related to the specie of animal from which the serum is obtained. In general, when utilizing a seru,n sample of 10 microliters or less and when using bovine serum, sheep serum or human serum as the source of adjuvant protein, the volume of this added carrier protein must be greater than about 4 times the initial volume of the serum sample being tested and preferably it is greater than about 6 times the volume of the sample being tested and can be a much larger volume, e.g., from 12 to 20 times the volume of the initial sample. In essence, once the total protein in the solution reaches about 60 microliters then a constant non-specific C
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binding and an acceptable percent spread is obtained and .-~
further amounts of protein added either from the sample or otherwise will not deletriously effect the analyses. I have found that approximately twice as much rabbit serum is needed than the above bovine, sheep or human serum and about one-half as much horse serum is needed than the bovine, sheep or human serum. The serum is preferably contained within the concentra-ted sulEate precipi-tant solution which as s-tated above generally has a concentration of about 30 to 40~ by weight and preferably of about 33 to 39~ by weight.
To separate the free from the bound fractions in the test solution, a sufficient quantity of the precipitant solu-tion is added to the sample solution containing the thyroxine .~rld antibody to result in a final concentration o~ the sulfate between abou-t 20 and 30 weight percent thereof as set forth above and to result in a sufficient concentration of the adju- `
vant carrier protein within the ranges as set forth above. The ~ixture should be thoroughly admixed, for example, by covering the tube in which it is contained and inverting it from 5 to 20 times. The precipitate of the antibody containing the bound hormone together with the adjuvant proteins forms immediately at the lower sulfate concentration and the resulting mixture should be centrifuged until the precipitated proteinaceous material forms a small button within the bottom of the vessel.
Thereafter, either the free fraction in the super-natant fluid or bound fraction in the precipitate should be counted in a scintillation well counter. It is preferred to count the bound fraction. The reading of the scintillation counter is compared to the total number of counts contained ~' . " ~ , .... .. ...
in the amount of tracer radioisotopically labelled thyroid hormone which was initially added. The percent antibody bound values are thus obtained. ~he percent antibody bound values are then correlated with standard values obtained by measurin~
percent antibody bound of standard samples containing known amounts of thyroid hormone to thereby determine the amount of thyroid hormone within the sample in a manner well known in the art.
The minor but effective amount of adjuvant serum pro-tein is necessary in order to provide an assay which can uti-llze various volumes of serum and which has constant non-spe-cific binding and also has an acceptable recovery range between ;
high and low thyroxine values. I-t is believed tha-t the prin-ciple active ingredient within the serum which effects the desired but unexpected results i9 yamma globulin. Generally, most animal serum contains between about 0.5 and 2 grams gamma globulin per 100 milliliters serum while -the total protein in the serum is about 5 to 8 grams per 100 milliliters serum. It is noted at this point, while it is believed that gamma globu-lin is the active ingredient in the serum for this purpose, because of convenience and availability, it is preferred to utilize the entire serum sample. In general, when utilizing bovine, human or sheep serum in quantities of 4 times the volume of the initial serum sample or less, fluctuating, non-reprodu-cible values of non-specific binding have been determined in addition to an unacceptable percent spread between the high and low thyroxine containing serum samples. However, when using such serum in volumes greater than about 4 times the initial volume of the serum, a plateau is reached where substantially constant non-specific binding is obtained and an excellent .; :
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~LV~3351 spread in r~covery values is also obtained. This plateau is well defined at about 6 volumes greater than the volume oE
the ini-tial sample and continues with no variation with added quantities of protein up to 12 volumes and more based upon original volume of the sample serum.
The following examples are given to better facili-tate the understanding of this invention and to show some specific preferred embodiments of the subject invention and are therefore not intended to limit the scope of the claimed 10 invention. !, EX~lPLE I
This example illustrates -the assay reproducibility of the novel thyroxine assay of the subject invention. The specific assay comprised the use of the following reactant solutions-(a) The extractant solution consisted of a solution containing ~.025 N HCl; and 0.05 ~ KCl;
(b) The T-4 I reagent solution contained a tracer quantity of radioiso-tope T-4 in a 0.04 molar sodium barbital solution which contained sufficient HCl to render a pH thereof of 8.3;
(c) The T-4 antiserum contained thyroxine antibodies (formed in a xabbit) and con-tained within O~r);l ~1 sodium barbital solution which had been adjusted with HCl to render a final pH of 8.3-' I
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,. . .. ..
. ~d) The precipitant solution consisted of an aqueous solution containing 37% by weight ammonium sulfate and containing bovine serum at a concentration of 4 volumes .
percent;
(e) The standards utilized in running the t~st were as follows:
1 microgram thyroxine per deciliter 6 micrograms thyroxine per deciliter 12 micrograms thyroxine per deciliter ~
1~ micrograms thyroxine per cleciliter .~:
Tlle above solutions ~ere uti.lized to assay fro~en pools of hypothyroid serum, normal serum and hyperthyroid serum by 11 different technicians, The test procedure utilized included initially adding 10 microlit.ers o a serum sample ~or standaTd as the case may be~
to 200 microliters of the extraction solution, ~fter this step, . the resulting solution was thoroughly admixed and then 400 microliters of the T~ 25I reagent were added to the resulting solution and the solution agitated. After this, 400 microliters of the T-~ antiserum solution we~e added and the resultillg solu-tion thoroughly admixed, At this ~oint, the solu~ion was incubated at room temperature for 45 or 60 minu~es, and at the end of the incubation period, the precipitate in the sulfate solution was resuspended and 2 milliliters of *his suspension were added to each tube containing the test solution. This resulted in 80 microllters of adjuvant serum bein~ added to each origi.nal 10 microliter sample and a final sulfate concentration . lt~q335~
in each test solution of 23,7% by ~Jeight. The tubes ~ere cappcd and each tubc inverted gently about 10 times, Then within 20 minutes after the addition of tile precipitant, the tubes ~.~ere centrifu~ed for 10 minutes at 1000-1500 gravities or 2000-2500 rpm and witllin 3 hours the supernatant was discarded and the rcsultant button of precipitan counted in a scintillation well counter. The results of the runs are set forth in Table 1 below:
'~
Table _e um Pool l-ly~othyroid Norr,lal l5ypcrt]lyroid Number of Observations 21a 317 210 Number of Days 16 15 16 Number of ~ots 2 2 2 Number of Technicians 12 ll 12 ~5ean (~g/dl) 3.2 7.9 14.1 Standard Deviation (~g/dl)0.28 0.30 0.47 Coefficient of Variation (~) 8.8 3.8 3.3 As sho~n from the table, the reproducibility of tlle assay is excellent.
~73351 EXAIPLE II
_ . .
The linearity of the radioimmunoassay of the subject invention is demonstrated in this example utilizing -the assay solutions described in Example I above. SpeciEically, the endogenous thyroxine from 3 serum pools was initially measured using the procedure set for-th in Example I above. Thereafter, quantities of 5, 10/ 15 and 20 micrograms per deciliter of crystalline thyroxine were added to 4 samples, respectively, from each of the 3 serum pools and the resulting samples were assayed in accordance with the procedure set forth in Example I
to illus-trate the uniformity of the percent recovery obtained from running the -test. The results are set forth in Table 2 below:
Measured Added Predicted Measured Recovered P~ecovered Serum Endogenous Exogenous Total Total Exogenous Exogenous _Pool T4(~lg/dl) T4(~g/dl) T4(~g/dl) T4(~g/dl) T4(Jg/dl) Percent 1 3.3 5 8.3 8.3 5.0 100 13.3 13.4 10.1 101 13.3 18.~ 15.1 101 23.3 23.6 20.3 102 2 3.6 5 8.6 8.7 5.1 102 13.6 13.5 9.9 99 18.6 18.8 15.2 101 23.6 24.0 20.4 102 3 3.0 5 8.0 7.9 4.9 98 13.0 12.9 9.9 99 18.0 18.1 15.1 101 23.0 23.0 20.0 100 The above results illustrate excellent recovery of the added T-4 and linearity of the tes-t system.
, ~33Sl E,~AMPLE III
This example is presented to illustrate the constant reproducible plateau of non-specific binding of the hormone to the antibody which is re~ched when the precipitant solution contains bovine serum in excess of 4 volumes per volume of the initial serum sample. In each instance, a solution containing a known quantity of thyroid hormone ~a 10 microliter sample), a tracer quantity of radioactively labelled thyroid hormone, and a known quantity of antiserum wére contacted with different precipitated solutions. Furthermore, the non-specific binding for each such precipitant solution was determined by running . -each tes-t but with the antiserum eliminated from the buffered antiserum solution. The precipitant solution was varied bo-th in concentration of the ammonium sulfate and the carrier protein as illustrated in the table below and the non-specific binding of three different serum standards were tested by eliminating the antiserum from the antiserum buffer solution as described in Example III. The results are set forth in Table 4.
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Indeed a commercially available so-called normalized T-4 type 20 test sold under the trademark oE "Quantisorb-125" by Abbott ~aboratories utilizes this above described phenomenon of re-naturation of acid denatured proteins as an essential step thereof.
Another approach for releasing the thyroxine from r the endogenous thyroxine binding protein is by heat denatura-tion. Heat denaturation of the serurl~ protein provides good recovery of the thyroid hormone with extraction efficiencies approach-ing about 100%. However, this method is time-consuming in that it ~1 .
,, ., ' '~'' :' . .
iO~3351 requires a minimum of 15 minutes in a boiling ~ater bath for complete destruction of binding proteins.
Likewise, when carrying out tlle radioimmunoassay for thyroxine, it is firs~ necessary to release the thyroxine from endogenous thyroxine binding proteins present ~ithin the serum sample and to alter the thyroxine binding proteins in such a way that their further participation in the reaction is pro-hibited. In the radioim~unoassay, this is most commonly accom-plished to a degree by blocking the thyroxine binding sites on endogenous proteins with chemicals such as 8-anilino-l-napllt]lalen sulfonic acid, diphenylhydantoin, salicylates, or thimerosal.
l~o~ever, the use of such blocking agents in radioimmulloassay has posed problems. For example, it has been shown that the quantity of these blocking agents necessary to occupy all sites may be related to the level of endogenous thyrobinding protein whicl can vary significantly in individual serum samples.
Thus, in both the conventional T-4 competitive protein binding assay and the radioimmunoassay, the thyroxine must initially be extracted from native proteins and thereafter bound to specific ~roteins, and then the free hormone separated from the protein bound hormone. The final separation has been accomplished by sorption of the free hormone such as to resins~
charcoal or inorganic crystalline sorbents. The conventional resins include ion exchange resins such as the ion exchanger having strongly basic amino or quaternary ammonium ~roups such as disclosed in U.S. Paten~ No. 3,414,383. These organic ion ~xchange resins can be either in loose form or incorporated in polyurethyane sponges as disclosed in U.S. Patellt No. 3,206,602, - 1~7335~
or enclosed in porous bags or the like. Other conventional methods include a selective sorption of the Eree hormone by charcoal which has been coated with suitable proteins or other polymers, or the use of molecular sieves such as Sephadex.
The use of the inorganic crystalline sorbent materials are dis- -;
closed in U.S. Patent ~o. 3,666 j854 and U.S. Patent No. 3,776, 698. In general, the methods which rely on sorption systems to separate the free hormone from the hormone bound to the protein are dependent upon protein concentration in the assay system, and some systems may require adjustment of the protein level for valid results. In addition, some of the sorbents such as the resins are temperature sensltive necessitating correction oE assay values obtained in working conditions where the temperatures are variable. In addition, the sorbents are in general time dependent and careful timing during the sorption process is necessary in order to obtain reproducible results.
Antibody-bound hormone may be separated from the free fraction by precipitation of the specific protein. One common approach used to precipitate the bound fraction is the double antibody technique which, due to a second incubation period,iis time-consuming. Another means of sepaxation is chemical precip-itation of the bound fraction with either high molecular ~eight polymers or salts. In order to precipitate the minute amounts of gamma globulin present, exogenous gamma globulin is conven-tionally added to the assay system prior to separation of the bound and free fractions.
A problem which has been encountered is the phenomenon of non-specific binding occurring during the assay. Non-speciEic binding stated simply is the binding of the free hormone including * Trade Mark 6 ' ' . :.
. .
.. . . ..
~3351 the free radioactivel~ labelled hormone with naterials other than the thyrobinding protein in the conventional T-4 assay or the antibody in the radioimmunoassay. The inability to assess the degree of non-specific binding occurring in the absence of the hinding protein is a disadvantage of those assay systems in which binding protein and radioactively labelled hormone are employed as a single reagent.
In general, a thyroid hormone assay is needed which will initially extract all or substantiallv all of the tilyroid hormone from the endogenous serum sample in a reproducible manner, will not rely on blocking agents or solid sorbents to separate the bound from the free hormone after -the competitive ~inding step, but wil.l effect -the separations quickly and efficiently in a reproducible manner wherein a low but repro-ducible anount of non-specific binding will occur during each test and which can be carried out in a single test tu~ without the requirement of intermedia-te decanting of test solution(s) thereform.
SHORT STATE~IENT OF THE INVENTION
According to the invention there is provided a method of measuring the level of thyroid hormone in a sample of serum containing endogenous thyroid hormone and endogenous thyroid hormone binding protein com?rising: (a) admixing said serum sample with an effective amount of an aqueous acid solu-tion sufficient to separate said thyroid hormone from said thyroid hormone binding protein; (b) adjusting the pH of the resulting mixture to a value in the range of from about 7.0 to about 8.5 in the absence of blocking asents while adding thereto a known amoun-t of radioacti.vely labelled thyroid hormone and a known amount of thyroid hormone binding antibodies and allow-ing the resulting solution to equilibrate- (c) thorouqhly ad-mixing the resulting equilibrated mixture from step (b) wi-th ~ _ 7 _ ~oq33sl an aqueous solution of a water soluble sulfate which contains sufficient sulfate such that the resulting mixture has a sul-fate concentration between about 20 and 30 weight percent to thereby result in precipi-tation of said thyroid hormone binding protein material containing thyroid hormone bound thereto and leave free thyroid hormone in the solution; (d) separating the precipitated material from said solution; and (e) counting with a scintillation counter one of (1) the free radioactively labelled thyroid hormone in said resulting solution, and ~2) the bound radioactively labelled thyroid hormone bound to said thyroid hormone binding antibodies in said precipitate.
In accordance with one embodiment of the subject in-vention, a radioimmunoassay for thyroid hormone is provided which includes the initial extraction of endogenous thyroid hormone from sample serum with an acid reagent to result in an effective inactivation of endogenous thyroid hormone binding proteins, the subsequent adjustment of the pH to a high level suitable for competitive binding (without removing the endoge-nous thyroid hormone binding protein therefrom) and the addition of a tracer quantity of radioactively labelled thyroid hormone and a known - 7a -C~
~-3226 1~3351 . .
quantity of thyroid hormone antibodies and thereafter allowing the resulting solution to equilibrate and competitive binding to occur, and then separation of the bound moiety from the ~ree moiety by the addi~ion of an aqueous sulfate salt solution whic~
causes precipitation of the bound moiety; and thereafter counting the prccipitated bound moiety or ~]~e supernatant containing the free ~oiety in a scintillation well counter. This procedure is carried out in a single tube without intermediate decanting steps.
In accordance with another embodiment of the subject invention, I have found that the separation of free from bound thyroid hormone fractions which rcsnlts ~rom the competitivc binding step in a thyroxine assay syst.em can be efficiently and reproducibly effected by a manner whereby the non-specific bind ing in the system is monitored at a lo~ constant level if the bound fraction is precipitated with an aqueous sulfate salt solution to which a minor but effective amount of added animal serum has been previously added.
DETAILED DESCRIPTION OF T}IE INV~lTIO~
. ~
The assay of the subject invention was developed in an effort to provide a "single tube" thyroid hormone assay which would not utilize blocking agents nor an external solid phase sorption step and require a minimum of manipulation by the ~.
laboratory technician.
First, I have discovered tha~ endogcnous thyroid hormone binding protein can be initially inactivatcd and thereby scparated from endogenous thyroid hormone by acid treatment~ and : I .; ' "" '~, ' , .
3~1 thereafter the pl-l of the resulting mixture can be raised and thyroid hormone antibodies added thereto to competitively bind :
with the endogenous thyroid hormone without interference from the endogenous protein. This is quite surprising in view of the fact that acid inac-tivated thyroid hormone binding protein is known to renature at higher pH's and interfere with competi- ~.
tive binding between thyroid hormone and exogenous thyroid hormone binding protein by actually entering into the competitive binding reaction. Thus, I have found that a radioimmunoassay for thyroid hormone can be carried out immediately after acid separation of endogenous thyroid hormone from endogenous thyroid hormone binding protein after a single upward pH adjustment of ;^~
the mixture and without first having to remove the endogenous thyroid hormone bindiny protein from the mixture.
Secondly, as stated above, most sorbents are dependent upon the total protein concentration in the mixture such that slight variations in the protein concen-tration of the sample can affect the percentage uptake of the sorbent. In addition, an effective thyroxine radioimmunoassay must have a low reproducible non-specific binding of the thyroid hormone which in essence is a low but uniform degree of binding of the thyroid hormone to constituents other than the antibody.
The thyroid hormone radioimmunoassay of the subject invention utilizes a salt precipitation step to separate the bound hormone from the free hormone in solution. Carrier or adjuvant proteins are conventionally added to such mixtures before the addition of sulfate in order to effect and expedite precipitation. However, I have found that the adjuvant protein can be mixed with the sulfate prior to in-troduc-tion to the assay ,~ ' ' ~i ~. .
lQ~33~
mixture. Using this combination as a precipitant, I have found that a constant, reproducible non~specific binding as well as an acceptable spread between the hypothyroid and hyperthyroid samples will result. Furthermore, the use of this combination as precipitant permits the use of a wide range of volumes of the serum sample, and allows the assav of serum samples containing a wide range of protein concentration.
The subject invention will be described in detail in relation to radioimmunoassay for thyroxine, even thou~h one having ordinary skill in the art can easily adapt my novel pro-cedure to assay triiodothyronine and other thyronines.
Before the endogenous serum thyroxine can be assayed utilizing the improved salt precipitation step of the subject invention, the hormone must be efficientlv and reproducibly extracted from a serum sample. In addition, all endogenous pro-tein which could possibly bind thyroxine during the several -steps of the assay must be completely inactivated. In accordance with one embodiment of the subject invention, the endogenous thyroxine is extracted from the serum sample with an acid solu-tion. The acid solution is generally maintained at a nH within the range of from about l.0 to 2.2. The preferable pH range is from about 1.0 to about 2Ø Any stable acid solution which is nondeletrious to the thyroid hormone can be used in the scope of the subject invention. For example, aqueous solutions of HCl, H2SO4, H3PO4 and the like can be used within the scope of the subject invention. HCl is the preferred acid. These materi-als can be buffered with suitable buffering agents, e.g., salts of weak acids at a concentration of about 0.02 to 0.07 l~l. Also, the ionic strength of the acid solution can be maintained by `~l , , . "',~. :
~335~
the presence of suitable materials such as neutral sal-ts, e.g., NaCl, KCl and the like at a concentration of 0.02 to 0.07 molar. ~, In general, at least about 10 and preferably about 20 volumes of the acid extraction reagent is combined with each volume of the serum sample. The pH of the resulting mixture should be in the range of from about 1.3 to about 3Ø Complete inactivation of the endogenous thyroxine binding proteins is accomplished immediately, even though the inactive proteins .
remain in solution. For example, the serum is added to the acid extraction solution contained within a vial, and the vial is shaken for a few seconds (generally 10 to 15 seconds) to admix thoroughly the serum and the acid extraction solution.
This will allow time for the acid solution -to break the bonds ~etween the thyroxine and the thyroxine binding protein and to inactivate completely the thyroxine binding protein.
Now that the resulting solution contains all of the endogenous thyroxine therewithin, and substantially all of the thyroxine binding protein has been deactivated, it is necessary to adjust the pH of the solution upwardly to a suitable pH in which competitive binding of the thyroid hormone and thyroid antibody can take place while adding to the solution a tracer quantity of radioactively labelled thyroid hormone (T-4) and a known,quantity of antiserum containing the thyroid hormone anti-bodies. In accordance with a preferred embodiment of this in-vention, the radioactive thvroid hormone followed by the anti-serum is added to the acid solution containing the unknown quantity of endogenous thyroid hormone. More specifically, the solution ., , ~ .
, i~7335~
contailling the radioacti~e thyroxine can cor.l~rise a solution having a plll~ithin thc range of about 7.6 to 9.0 and preferal~ly about 8.3, and l~ill contaill a tracer ~uantity of radioactively labelled thyroicl 1lOT~One, and preferably a bu~fer to maintain the pll of the solution. A suitable SUCh SO1UtiO11 COmPriSeS
0.04 ~ sodium bar~ital carrier W]liC]I is adjusted to a p~l of 8.3 by the addition of hydrochloric acid ~hich contains the tracer quantity of T-~ 125I
j Any radioactive isotope of iodine, tritium, or carbon 10 !,can be used. It is preferred that a hormone be utilized l~hic]
jlis labelled Wit]l 125I. The buffered solution containing the ¦,tracer quantity o~ radioactively labclled ~hyroid hormone litllyroxine, which is at least 20 and prcfer.lbly about 40 volumcs ! greater than the serum sample, is added to the acid solution an-l llthe con~ents are thereafter thoroughly mixed by sl~aking.
~ Next, a solution containing the antithyroxine scrum ¦ is added to the resulting solution. The antiserum can contai~l i a suitable buffer such as sodium barbital and can generally havc I',a p~l within the range from about 7.6 to 9Ø The volume of tl~c I'antiserum solution added to the test mixture can be the same as ¦that used to deliver the ~adioact;~e isotope labelled thyroid hormone to the test mixture. After the thyroxine antiserum solu tion has been added to the solution containing the extracted !Ithyroid hormone and the radioactive ~uantity of thyroid hormone, I,the resulting mixture is thoroughly mixed. The resulting solu-tion will have a p~T in the range of from about 7.0 to 8.5 and preferably about 7.~-8.4 and is incubated at ~oom temperature from 30 to about 60 ~inutes to allol~ the formation of the thyroxine-antibody com~lexes. Since the antibody ~ill bind both lQ~3351 ' the radioactive thyroxine and serum thyroxine e~ually well, the amowlt of radioactive thyroxine reco~ered ~ill reflect the con-centration of thyroxine in the original sample. As is well ~nown, the antiserum used in this step should llave a high speci~
ficity for thyroxine. Furthermore, as previously noted, the inactivated thyroid hormone binding protein in the mixture sur-prisingly does not interfere with the competitive binding betl~een the thyroid hormone and the antibody.
Once the solution is equilibrated sucll that the competitive binding between the thyroxine antibodies and thyroxine is complete, the antibodics containing bound hormone are precipitated in accordance with the improved salt precipita-tion step of the subject invention. The precipitant solution whicll is used in the scope of the subject invention comprises an aqueous solution of a water soluble sulfate salt which will ef~ectively precipitate proteinaceous materials without deleteri-ously ~recipitating free hormone from solution. Examples of suitable sulfate salts which can be used are the alkali metal sulfates such as sodium and potassium sulfate, ammonium sulfate, and zinc sulfate. Because of the solubility range, availability and convenience, the most preferred salt is ammonium sulfate.
The concentration of the ammonium sulfatc can vary according to the amount of aqueous precipitant fluid which is desired to be used. Generally, the concentration in the precipitant solution should be SUCII that when it is admixed Wit]l the ~hyroxine-anti-body containing solution, the resulting concentration o~ sulfate will be in the range of about 20 to 30% by weight and pTeferably in the range of about 23 to 27% by weight and most prefcrably about 23 to 24% by ~eight. I have found that the concentration `` iO~3351 of the sulfate in the precipitant solution can conveniently range from about 30 to about 40% by weight and preferably from about 33 to 39% by weight thereof.
In addition to the sulfate, I have found it necessary to add a minor but effective amount of animal serum as adjuvant to the antibody-thyroxine containing solution at the time of ;
precipitation in order to obtain a low but reproducible order of non-specific binding and a marked distinction in thyroxine levels of hypothyroid and hyperthyroid serum samples. Further-more, the use of the above combination permits the use of a wide range of sample volume (i.e., from about 2 to about 50 microliters), and allows the assay of serum samples containing a wide range of protein concentration. In general, the amount of added serum which is necessary to accomplish this result is a volume greater than the initial volume of the serum sample being tested. Generally, any type of animal serum can be uti- `
lized as an adjuvant in this manner even though I have found some variation in the quantity of serum which must be used which is directly related to the specie of animal from which the serum is obtained. In general, when utilizing a seru,n sample of 10 microliters or less and when using bovine serum, sheep serum or human serum as the source of adjuvant protein, the volume of this added carrier protein must be greater than about 4 times the initial volume of the serum sample being tested and preferably it is greater than about 6 times the volume of the sample being tested and can be a much larger volume, e.g., from 12 to 20 times the volume of the initial sample. In essence, once the total protein in the solution reaches about 60 microliters then a constant non-specific C
....
t7335i ~ ~
binding and an acceptable percent spread is obtained and .-~
further amounts of protein added either from the sample or otherwise will not deletriously effect the analyses. I have found that approximately twice as much rabbit serum is needed than the above bovine, sheep or human serum and about one-half as much horse serum is needed than the bovine, sheep or human serum. The serum is preferably contained within the concentra-ted sulEate precipi-tant solution which as s-tated above generally has a concentration of about 30 to 40~ by weight and preferably of about 33 to 39~ by weight.
To separate the free from the bound fractions in the test solution, a sufficient quantity of the precipitant solu-tion is added to the sample solution containing the thyroxine .~rld antibody to result in a final concentration o~ the sulfate between abou-t 20 and 30 weight percent thereof as set forth above and to result in a sufficient concentration of the adju- `
vant carrier protein within the ranges as set forth above. The ~ixture should be thoroughly admixed, for example, by covering the tube in which it is contained and inverting it from 5 to 20 times. The precipitate of the antibody containing the bound hormone together with the adjuvant proteins forms immediately at the lower sulfate concentration and the resulting mixture should be centrifuged until the precipitated proteinaceous material forms a small button within the bottom of the vessel.
Thereafter, either the free fraction in the super-natant fluid or bound fraction in the precipitate should be counted in a scintillation well counter. It is preferred to count the bound fraction. The reading of the scintillation counter is compared to the total number of counts contained ~' . " ~ , .... .. ...
in the amount of tracer radioisotopically labelled thyroid hormone which was initially added. The percent antibody bound values are thus obtained. ~he percent antibody bound values are then correlated with standard values obtained by measurin~
percent antibody bound of standard samples containing known amounts of thyroid hormone to thereby determine the amount of thyroid hormone within the sample in a manner well known in the art.
The minor but effective amount of adjuvant serum pro-tein is necessary in order to provide an assay which can uti-llze various volumes of serum and which has constant non-spe-cific binding and also has an acceptable recovery range between ;
high and low thyroxine values. I-t is believed tha-t the prin-ciple active ingredient within the serum which effects the desired but unexpected results i9 yamma globulin. Generally, most animal serum contains between about 0.5 and 2 grams gamma globulin per 100 milliliters serum while -the total protein in the serum is about 5 to 8 grams per 100 milliliters serum. It is noted at this point, while it is believed that gamma globu-lin is the active ingredient in the serum for this purpose, because of convenience and availability, it is preferred to utilize the entire serum sample. In general, when utilizing bovine, human or sheep serum in quantities of 4 times the volume of the initial serum sample or less, fluctuating, non-reprodu-cible values of non-specific binding have been determined in addition to an unacceptable percent spread between the high and low thyroxine containing serum samples. However, when using such serum in volumes greater than about 4 times the initial volume of the serum, a plateau is reached where substantially constant non-specific binding is obtained and an excellent .; :
' `? ~
~LV~3351 spread in r~covery values is also obtained. This plateau is well defined at about 6 volumes greater than the volume oE
the ini-tial sample and continues with no variation with added quantities of protein up to 12 volumes and more based upon original volume of the sample serum.
The following examples are given to better facili-tate the understanding of this invention and to show some specific preferred embodiments of the subject invention and are therefore not intended to limit the scope of the claimed 10 invention. !, EX~lPLE I
This example illustrates -the assay reproducibility of the novel thyroxine assay of the subject invention. The specific assay comprised the use of the following reactant solutions-(a) The extractant solution consisted of a solution containing ~.025 N HCl; and 0.05 ~ KCl;
(b) The T-4 I reagent solution contained a tracer quantity of radioiso-tope T-4 in a 0.04 molar sodium barbital solution which contained sufficient HCl to render a pH thereof of 8.3;
(c) The T-4 antiserum contained thyroxine antibodies (formed in a xabbit) and con-tained within O~r);l ~1 sodium barbital solution which had been adjusted with HCl to render a final pH of 8.3-' I
,. ~
. . - .. :. :..... . .. .. . .
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,. . .. ..
. ~d) The precipitant solution consisted of an aqueous solution containing 37% by weight ammonium sulfate and containing bovine serum at a concentration of 4 volumes .
percent;
(e) The standards utilized in running the t~st were as follows:
1 microgram thyroxine per deciliter 6 micrograms thyroxine per deciliter 12 micrograms thyroxine per deciliter ~
1~ micrograms thyroxine per cleciliter .~:
Tlle above solutions ~ere uti.lized to assay fro~en pools of hypothyroid serum, normal serum and hyperthyroid serum by 11 different technicians, The test procedure utilized included initially adding 10 microlit.ers o a serum sample ~or standaTd as the case may be~
to 200 microliters of the extraction solution, ~fter this step, . the resulting solution was thoroughly admixed and then 400 microliters of the T~ 25I reagent were added to the resulting solution and the solution agitated. After this, 400 microliters of the T-~ antiserum solution we~e added and the resultillg solu-tion thoroughly admixed, At this ~oint, the solu~ion was incubated at room temperature for 45 or 60 minu~es, and at the end of the incubation period, the precipitate in the sulfate solution was resuspended and 2 milliliters of *his suspension were added to each tube containing the test solution. This resulted in 80 microllters of adjuvant serum bein~ added to each origi.nal 10 microliter sample and a final sulfate concentration . lt~q335~
in each test solution of 23,7% by ~Jeight. The tubes ~ere cappcd and each tubc inverted gently about 10 times, Then within 20 minutes after the addition of tile precipitant, the tubes ~.~ere centrifu~ed for 10 minutes at 1000-1500 gravities or 2000-2500 rpm and witllin 3 hours the supernatant was discarded and the rcsultant button of precipitan counted in a scintillation well counter. The results of the runs are set forth in Table 1 below:
'~
Table _e um Pool l-ly~othyroid Norr,lal l5ypcrt]lyroid Number of Observations 21a 317 210 Number of Days 16 15 16 Number of ~ots 2 2 2 Number of Technicians 12 ll 12 ~5ean (~g/dl) 3.2 7.9 14.1 Standard Deviation (~g/dl)0.28 0.30 0.47 Coefficient of Variation (~) 8.8 3.8 3.3 As sho~n from the table, the reproducibility of tlle assay is excellent.
~73351 EXAIPLE II
_ . .
The linearity of the radioimmunoassay of the subject invention is demonstrated in this example utilizing -the assay solutions described in Example I above. SpeciEically, the endogenous thyroxine from 3 serum pools was initially measured using the procedure set for-th in Example I above. Thereafter, quantities of 5, 10/ 15 and 20 micrograms per deciliter of crystalline thyroxine were added to 4 samples, respectively, from each of the 3 serum pools and the resulting samples were assayed in accordance with the procedure set forth in Example I
to illus-trate the uniformity of the percent recovery obtained from running the -test. The results are set forth in Table 2 below:
Measured Added Predicted Measured Recovered P~ecovered Serum Endogenous Exogenous Total Total Exogenous Exogenous _Pool T4(~lg/dl) T4(~g/dl) T4(~g/dl) T4(~g/dl) T4(Jg/dl) Percent 1 3.3 5 8.3 8.3 5.0 100 13.3 13.4 10.1 101 13.3 18.~ 15.1 101 23.3 23.6 20.3 102 2 3.6 5 8.6 8.7 5.1 102 13.6 13.5 9.9 99 18.6 18.8 15.2 101 23.6 24.0 20.4 102 3 3.0 5 8.0 7.9 4.9 98 13.0 12.9 9.9 99 18.0 18.1 15.1 101 23.0 23.0 20.0 100 The above results illustrate excellent recovery of the added T-4 and linearity of the tes-t system.
, ~33Sl E,~AMPLE III
This example is presented to illustrate the constant reproducible plateau of non-specific binding of the hormone to the antibody which is re~ched when the precipitant solution contains bovine serum in excess of 4 volumes per volume of the initial serum sample. In each instance, a solution containing a known quantity of thyroid hormone ~a 10 microliter sample), a tracer quantity of radioactively labelled thyroid hormone, and a known quantity of antiserum wére contacted with different precipitated solutions. Furthermore, the non-specific binding for each such precipitant solution was determined by running . -each tes-t but with the antiserum eliminated from the buffered antiserum solution. The precipitant solution was varied bo-th in concentration of the ammonium sulfate and the carrier protein as illustrated in the table below and the non-specific binding of three different serum standards were tested by eliminating the antiserum from the antiserum buffer solution as described in Example III. The results are set forth in Table 4.
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P~ ~ ~ CO ~ .:
.: .
0 ~
~ o oo oo 1~ oo :~ o ~ ,~ ~ ~ ~ ~o h O
o~ ~ ~( :, O t_ r~ ~ ~ CO CO t~ , ,~
.,~ h ~ o h ~ r~ ~
o . ..
~1 ~ ~ ~1 :
o o ~
~) h o ~ .
~d ,~ ~ a ::
P~ E~ o ~o `D Ln U~
rl ~ ~
C~ h O
h o~o a) 'i~ ,t ~O r/
~ h ~:: o .
t:: h~rl ~ Lf) ~ L~
~) H ~1 ~1) ~1 ~ ~ ~0 ;~ O
sd O h o E~ u) P~ `D
,_ ~ ' Z a~
~t q o
4~ ~ r~ Ln O h O
o~o ~ r l r~ ~. 1.1~ ~ d` Ln O ~) h F~ O ~
~rl h rl 0~ 117Ln Ln LO
Q) ~1 h O ::
h O .
~: ~ ~D Ln LnLn Lt~
a>
~ , ~0 ~ ' ~)1~ L
E~ lo Ln Ln `D n ~;~ ' h 01 o~o ~ --Ir~ ~ .
~ rl ~ Ln t~ h ~ ~ . .
h-r ~d r~
O
h O . .
P~ ) Ln d Ir~
.r~ ~
4~ ~ o\ ~;
~
O ~ ~
a) t)OtJ) t:o ~
L r~~4 v~r~
00 _ :~ h ~ ~: h ~-- h O~r~ a) o a ;ii~
~ 33~1 E~A~IPLE IV
This example indicates the constant degree of non-specific binding and excellent spread of recovery values between hypothyroid and hyperthyroid serum samples when utili-zing the test of the subject invention with the precipitant fluid containing the minor but effective quantity of added seru~ as adjuvant. ~lore specifically, the procedure se-t forth in Example III was utilized to assay various serum standards . :
except that the concentration of the serum in the precipitant solution was varied such that the resultant test solution would carry 60, 70, 80, 90, 100 and 110 microliters of added serum which is added with the precipitant to contact the test solution containing the initial 10 microliters of the serum sample. As will be noted when the carrier protein is present in the pre-cipitant solution in quantities of 6 times the volume of initial sample or more, the value of the non-specific binding stabili zes; whereas when the quantity of the carrier protein is only 4 times that of the initial volume of the sample, the non-spe-cific binding varies in an unpredictable manner. The results are set forth in Tab~e 5 below:
,j~
iO~ 3~1 ~ ~ Ln U~ ,, ~
.~ ~ ~ , ~ o U~
h r-l F:~ ~ Ln o ~D r~ ~ r~ r- Ln ,1~ co o Ln ~ ~ n ~ ~I 0\o ~
a o r~ O ~ ~D V ~D `D ~ ~ ~ ~ l'~ ~ ~ I~) ~ ~ ~ ~ ~ ~ ~ ~
.,~ ~, . . .,.
o I o~ :
a~ ~ u~
.
Z u) ~ ~D ~D
o O Ln ~ 00 .~ ~ ~ ~ 0~ 0 Ln `D
~ h ~1 ~( ~ Lf~~
~ ~ ~ O ~1~ 0 0~00 0 LnCO~ Ln ~r~l ~ , t-drl o\'~ ~:S , .. . ... . ... . ... . ...
,~ -- O O O 0~ 0 ~ 1~ 1-- 1 O~ O`) CO C~ O~ 00 00 00 ~) t~ ~ t~
~10 ~ ~Ln ~ ~ ~ ~) .~,~ . .
O I ~ ~
g ~ CO ~` ~ . .
P~ 4 `D
. ~ a ~, 00 0 Ln ~D Ln a) ~ , Ln ~ .-1 co r~ Ln ~o o o ~ o oo oo a~ o c~ ~1 o o~
. ... . ,, . . ...... , . , . . ... , . ,~
o a~ oo OD co C~ co ~ r~ ~ ~ n ~rl O ~ Ln Ln 1~ Ln ~ ~ ~ ~ ~ ~ ~
,p~o~ , .
O ~d ~ Q) ~ ~ ~
h O ~-rl ~ Z. U) ~1 ~ ~D `D
Ln r~
~1 a) ~ t~ ~ m 1~ ~ v tv ~ vn ~ ,~
E- ~ ~ ~ J ~ Ln ~r) o- o o~
~ 0 ~ . ~ h ~ ~ o ~ ~ O ~ ~ O O O ~o o~ a~
,t o p~ v .,~. .
~ tJ~ v~ a) a) ~ ~ D o t`~v h o ~ ~
~ ~) ~ tl) vn ~: Ln ~ 01 ~ ~t h e) E~: ~a) v, ~ ~ o o Ln r-- ~ a ~: ~ ,~ t~ o ,~ o t~ o~ o o o o ~ t~
t~ ~ o~o ~ tl> ~-~ ~ ol ~~ ,0 o~ o tn tn tn 0~ O~ tn 1~1~1 .r~ O ~ ~ ),t~lt~lt~ t~l t~lt~lt~l t~P~-.~l . . n O I ~ ~ ~
h o ~ . . a~ vn P. ~. t~ D t~
~ 1 t~ ~ `D h .n . . . P~
. ~ <I ~ t~
~ Ic J- vn ~ ~
~ ~ t;s t~ t~l ~ t~ O t~O ~ t~ t~ t~O ~ t~ ~ O O O ~ ~0 tO
tv ,t 0~O ~ . . . . . . . . . . . . . . . . . .
~~ O r~ ~ t)O 0~ tO O O tn O t~O 0~ 0~
~r~ O ~ ~ ~ d t`l ~1 t~l t~ t~ t~l t`~ t`l .~`D . .
U V~
a) ~~D 0~ t`
h o p~
a) ~ o o o o P~ . . . . . E~
~ ~ ~v ~n o ~ ~D t~l ~ O
~v O h -~ r l ~1 ~_ U~
t~, 15~
~3351 EXA~I~LE V
This example is presented to illustratc the specifici.ty and sample serum protein independence of the radioimmunoassay of t]~e subject invention. Specifically, tlle endogellous thyroxine of different sample volumes of various sera containing elcvated and decreased protein levels was measured usi.ng the test pro-cedure and the reactant solutions described in Example I. The results are set forth in Table 6 below:
335~
Table 6 Serum Measured Corrected*
Volume T4 to 10~l1 Type of Serum Sample (~ g/dl) (~g/dl) Hyperthyroid 10 17.9 17.9 Total Protein, 7.3 g/dl 7-5 13.7 18.3
o~o ~ r l r~ ~. 1.1~ ~ d` Ln O ~) h F~ O ~
~rl h rl 0~ 117Ln Ln LO
Q) ~1 h O ::
h O .
~: ~ ~D Ln LnLn Lt~
a>
~ , ~0 ~ ' ~)1~ L
E~ lo Ln Ln `D n ~;~ ' h 01 o~o ~ --Ir~ ~ .
~ rl ~ Ln t~ h ~ ~ . .
h-r ~d r~
O
h O . .
P~ ) Ln d Ir~
.r~ ~
4~ ~ o\ ~;
~
O ~ ~
a) t)OtJ) t:o ~
L r~~4 v~r~
00 _ :~ h ~ ~: h ~-- h O~r~ a) o a ;ii~
~ 33~1 E~A~IPLE IV
This example indicates the constant degree of non-specific binding and excellent spread of recovery values between hypothyroid and hyperthyroid serum samples when utili-zing the test of the subject invention with the precipitant fluid containing the minor but effective quantity of added seru~ as adjuvant. ~lore specifically, the procedure se-t forth in Example III was utilized to assay various serum standards . :
except that the concentration of the serum in the precipitant solution was varied such that the resultant test solution would carry 60, 70, 80, 90, 100 and 110 microliters of added serum which is added with the precipitant to contact the test solution containing the initial 10 microliters of the serum sample. As will be noted when the carrier protein is present in the pre-cipitant solution in quantities of 6 times the volume of initial sample or more, the value of the non-specific binding stabili zes; whereas when the quantity of the carrier protein is only 4 times that of the initial volume of the sample, the non-spe-cific binding varies in an unpredictable manner. The results are set forth in Tab~e 5 below:
,j~
iO~ 3~1 ~ ~ Ln U~ ,, ~
.~ ~ ~ , ~ o U~
h r-l F:~ ~ Ln o ~D r~ ~ r~ r- Ln ,1~ co o Ln ~ ~ n ~ ~I 0\o ~
a o r~ O ~ ~D V ~D `D ~ ~ ~ ~ l'~ ~ ~ I~) ~ ~ ~ ~ ~ ~ ~ ~
.,~ ~, . . .,.
o I o~ :
a~ ~ u~
.
Z u) ~ ~D ~D
o O Ln ~ 00 .~ ~ ~ ~ 0~ 0 Ln `D
~ h ~1 ~( ~ Lf~~
~ ~ ~ O ~1~ 0 0~00 0 LnCO~ Ln ~r~l ~ , t-drl o\'~ ~:S , .. . ... . ... . ... . ...
,~ -- O O O 0~ 0 ~ 1~ 1-- 1 O~ O`) CO C~ O~ 00 00 00 ~) t~ ~ t~
~10 ~ ~Ln ~ ~ ~ ~) .~,~ . .
O I ~ ~
g ~ CO ~` ~ . .
P~ 4 `D
. ~ a ~, 00 0 Ln ~D Ln a) ~ , Ln ~ .-1 co r~ Ln ~o o o ~ o oo oo a~ o c~ ~1 o o~
. ... . ,, . . ...... , . , . . ... , . ,~
o a~ oo OD co C~ co ~ r~ ~ ~ n ~rl O ~ Ln Ln 1~ Ln ~ ~ ~ ~ ~ ~ ~
,p~o~ , .
O ~d ~ Q) ~ ~ ~
h O ~-rl ~ Z. U) ~1 ~ ~D `D
Ln r~
~1 a) ~ t~ ~ m 1~ ~ v tv ~ vn ~ ,~
E- ~ ~ ~ J ~ Ln ~r) o- o o~
~ 0 ~ . ~ h ~ ~ o ~ ~ O ~ ~ O O O ~o o~ a~
,t o p~ v .,~. .
~ tJ~ v~ a) a) ~ ~ D o t`~v h o ~ ~
~ ~) ~ tl) vn ~: Ln ~ 01 ~ ~t h e) E~: ~a) v, ~ ~ o o Ln r-- ~ a ~: ~ ,~ t~ o ,~ o t~ o~ o o o o ~ t~
t~ ~ o~o ~ tl> ~-~ ~ ol ~~ ,0 o~ o tn tn tn 0~ O~ tn 1~1~1 .r~ O ~ ~ ),t~lt~lt~ t~l t~lt~lt~l t~P~-.~l . . n O I ~ ~ ~
h o ~ . . a~ vn P. ~. t~ D t~
~ 1 t~ ~ `D h .n . . . P~
. ~ <I ~ t~
~ Ic J- vn ~ ~
~ ~ t;s t~ t~l ~ t~ O t~O ~ t~ t~ t~O ~ t~ ~ O O O ~ ~0 tO
tv ,t 0~O ~ . . . . . . . . . . . . . . . . . .
~~ O r~ ~ t)O 0~ tO O O tn O t~O 0~ 0~
~r~ O ~ ~ ~ d t`l ~1 t~l t~ t~ t~l t`~ t`l .~`D . .
U V~
a) ~~D 0~ t`
h o p~
a) ~ o o o o P~ . . . . . E~
~ ~ ~v ~n o ~ ~D t~l ~ O
~v O h -~ r l ~1 ~_ U~
t~, 15~
~3351 EXA~I~LE V
This example is presented to illustratc the specifici.ty and sample serum protein independence of the radioimmunoassay of t]~e subject invention. Specifically, tlle endogellous thyroxine of different sample volumes of various sera containing elcvated and decreased protein levels was measured usi.ng the test pro-cedure and the reactant solutions described in Example I. The results are set forth in Table 6 below:
335~
Table 6 Serum Measured Corrected*
Volume T4 to 10~l1 Type of Serum Sample (~ g/dl) (~g/dl) Hyperthyroid 10 17.9 17.9 Total Protein, 7.3 g/dl 7-5 13.7 18.3
5.0 9.4 13.8 2.5 4.7 18.8 1.672.9 17.4 Hyperthyroid, 10 13.7 13.7 Total Protein, 7.5 g/dl 7-5 10.6 14.1 5.07.4 14.8 2.53.5 14.0 1.672.2 13.2 10 Alpha Globulins, 2.1 g/dl 10 15.4 15.4 (Elevated) 7-511.5 15.3 5.07.9 15.8 2.54.1 16.4 Albumin, 5.4 g/dl (Elevated) 10 7.7 7.7 5.04.0 8.0 3.32.5 7.5 2.51.8 7.2 Gamma Globulins, 4.6 g/dl 10 7.3 7.3 (Elevated) 5.03.6 7.2 3.32.5 7.5 2.5-1.7 6.~3 ~ypothyroid 10 2.5 2.5 Total Protein, 8.2 g/dl 20 5.3 2.7 8.2 2.7 Total Protein, 4.5 g/dl 10 3.1 3.1 ~Decreased) 20 6.7 3.4 30 10.4 3.5 Albumin, 0.9 g/dl 10 3.5 3.5 (Decreased) 20 7.2 3.6 30 10.4 3.5 Feline (10 Animals - 10 1.3 1.3 3 Determinations Each) 2q 3.6 1.6 5.4 1.8 Canine (10 Animals - 10 1.4 1.4 3 Determinations Each) 20 3.0 1.5 4.8 1.6 Equine (10 Animals - 10 0.8 0.8 3 Determinations Each) 20 2.2 1.1 3 8 1.3 * These values are corrected only to the volume of 10~l1 and do not reflect a correction for non-specific binding. All values for any serum would be equal if corrected for non-specific binding.
, .1 ~
., ~335~
While this invention has been described in relation to its preferred embodiments, it is to be understood that various modifications thereof will be apparent to those skilled in the art upon reading this specification, and it is intended to cover such modifications as fall within the scope of the appended claims.
, .1 ~
., ~335~
While this invention has been described in relation to its preferred embodiments, it is to be understood that various modifications thereof will be apparent to those skilled in the art upon reading this specification, and it is intended to cover such modifications as fall within the scope of the appended claims.
Claims (20)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of measuring the level of thyroid hormone in a sample of serum containing endogenous thyroid hormone and endogenous thyroid hormone binding protein comprising:
(a) admixing said serum sample with an effective amount of an aqueous acid solution sufficient to separate said thyroid hormone from said thyroid hormone binding protein;
(b) adjusting the pH of the resulting mixture to a value in the range of from about 7.0 to about 8.5 in the absence of blocking agents while adding thereto a known amount of radio-actively labelled thyroid hormone and a known amount of thyroid hormone binding antibodies and allowing the resulting solution to equilibrate;
(c) thoroughly admixing the resulting equilibrated mixture from step (b) with an aqueous solution of a water soluble sulfate which contains sufficient sulfate such that the resulting mixture has a sulfate concentration between about 20 and 30 weight percent to thereby result in precipitation of said thyroid hormone bind-ing protein material containing thyroid hormone bound thereto and leave free thyroid hormone in the solution;
(d) separating the precipitated material from said solution;
and (e) counting with a scintillation counter one of (1) the free radioactively labelled thyroid hormone in said resulting solution, and (2) the bound radioactively labelled thyroid hormone bound to said thyroid hormone binding antibodies in said precipitate.
(a) admixing said serum sample with an effective amount of an aqueous acid solution sufficient to separate said thyroid hormone from said thyroid hormone binding protein;
(b) adjusting the pH of the resulting mixture to a value in the range of from about 7.0 to about 8.5 in the absence of blocking agents while adding thereto a known amount of radio-actively labelled thyroid hormone and a known amount of thyroid hormone binding antibodies and allowing the resulting solution to equilibrate;
(c) thoroughly admixing the resulting equilibrated mixture from step (b) with an aqueous solution of a water soluble sulfate which contains sufficient sulfate such that the resulting mixture has a sulfate concentration between about 20 and 30 weight percent to thereby result in precipitation of said thyroid hormone bind-ing protein material containing thyroid hormone bound thereto and leave free thyroid hormone in the solution;
(d) separating the precipitated material from said solution;
and (e) counting with a scintillation counter one of (1) the free radioactively labelled thyroid hormone in said resulting solution, and (2) the bound radioactively labelled thyroid hormone bound to said thyroid hormone binding antibodies in said precipitate.
2, The method of Claim 1 wherein said aqueous acid solution is an aqueous solution of HCl having a pH in the range of about 1,0 to about 3Ø
3. The method of Claim 1 further comprising an effective amount of animal serum admixed with said aqueous solution of said water soluble sulfate.
4. The method of Claim 3 wherein said animal serum is selected from bovine, human and sheep serum.
5. The method of Claim 4 wherein the volume of said animal serum added to said mixture is at least 6 times larger than the volume of said serum sample and said volume of said serum sample is up to about 10 microliters.
6. The method of Claim 5 wherein the volumetric ratio of said serum sample to said animal serum added thereto is in the range of from about 1:6 to about 1:20.
7. The method of Claim 6 wherein said volumetric ratio is in the range of from about 1:6 to about 1:12.
8. The method of Claim 6 wherein said animal serum is bovine serum.
9. The method of Claim 6 wherein said animal serum is horse serum.
10. The method of Claim 9 therein said volume of said horse serum added to said serum sample is at least twice as large as said serum sample.
11. The method of Claim 6 wherein said animal serum is rabbit serum.
12. The method of Claim 1 wherein said volume of said rabbit serum added to said sample is 8 times as large as the volume of said sample serum.
13. The method of Claim 1 wherein said sulfate solution is an aqueous solution of a sulfate selected from alkali metal sulfates and ammonium sulfates.
14. The method of Claim 13 wherein said aqueous sulfate solution is an aqueous solution of ammonium sulfate having a concentration in the range from about 30 to about 40 weight percent ammonium sulfate.
15. The method of Claim 14 wherein said resulting mixture has a sulfate concentration in the range of from about 23 to 27% by weight thereof.
16. The method of Claim 15 wherein said thyroid hormone is thyroxine.
17. The method of Claim 15 wherein said thyroid hormone is triiodothyronine.
18. In a radioimmunoassay for thyroid hormone in a serum sample having a known volume and containing an unknown quantity of thyroid hormone and thyroid hormone binding protein wherein said thyroid hormone is initially separated from said thyroid hormone binding protein and thereafter a known quantity of radioactively labelled thyroid hormone and a known quantity of thyroid hormone antibodies are added thereto and allowed to competitively bind, and thereafter, free thyroid hormone (not bound to thyroid hormone antibody) is separated from thyroid hormone bound to thyroid hormone antibody, and the amount of said unknown hormone is determined by counting with a scintillation counter one of (a) said radioactively labelled hormone bound to said thyroid hormone antibody, and (b) said radioactively labelled thyroid hormone which is not bound to said thyroid hormone antibody, the improvement comprising:
initially separating said thyroid hormone from said thyroid hormone binding protein contained in said serum sample by contacting said serum sample with an acidic solution, and thereafter adjusting the pH of said mixture to a higher value in the absence of a blocking agent and adding said known quantity of said radioactively labelled thyroid and said known quantity of said thyroid hormone antibodies thereto without first removing said thyroid hormone binding protein from said mixture.
initially separating said thyroid hormone from said thyroid hormone binding protein contained in said serum sample by contacting said serum sample with an acidic solution, and thereafter adjusting the pH of said mixture to a higher value in the absence of a blocking agent and adding said known quantity of said radioactively labelled thyroid and said known quantity of said thyroid hormone antibodies thereto without first removing said thyroid hormone binding protein from said mixture.
19. The method of Claim 18 wherein said acidic solution is an aqueous solution of HC1 having a pH of from about 1 to about 3.
20. The method of Claim 19 wherein said aqueous solution has a pH in the range of from about 1 to about 2Ø
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/654,541 US4066410A (en) | 1975-09-15 | 1976-02-02 | Thyroid hormone assay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1073351A true CA1073351A (en) | 1980-03-11 |
Family
ID=24625282
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA269,943A Expired CA1073351A (en) | 1976-02-02 | 1977-01-18 | Competitive thyroid hormone radio assay in a single tube |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPS52106794A (en) |
| AU (1) | AU508703B2 (en) |
| CA (1) | CA1073351A (en) |
| DE (1) | DE2703668C2 (en) |
| GB (1) | GB1558044A (en) |
| SE (1) | SE442918B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3044385A1 (en) * | 1980-11-25 | 1982-06-24 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR CARRYING OUT ANALYTICAL PROVISIONS AND ROTOR INSERT ELEMENT SUITABLE FOR THIS |
| KR101548284B1 (en) * | 2014-05-23 | 2015-08-28 | 주식회사 나노엔텍 | A Novel Method for Detecting an Analyte and Kits Using It |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE789215A (en) * | 1971-09-27 | 1973-03-26 | Textron Inc | BLOW MOLDING PROCESS AND APPARATUS |
-
1977
- 1977-01-18 CA CA269,943A patent/CA1073351A/en not_active Expired
- 1977-01-26 AU AU21646/77A patent/AU508703B2/en not_active Expired
- 1977-01-27 SE SE7700867A patent/SE442918B/en unknown
- 1977-01-29 DE DE2703668A patent/DE2703668C2/en not_active Expired
- 1977-02-01 GB GB4026/77A patent/GB1558044A/en not_active Expired
- 1977-02-02 JP JP981677A patent/JPS52106794A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| SE442918B (en) | 1986-02-03 |
| DE2703668C2 (en) | 1985-09-26 |
| SE7700867L (en) | 1977-08-03 |
| DE2703668A1 (en) | 1977-08-04 |
| GB1558044A (en) | 1979-12-19 |
| AU2164677A (en) | 1978-08-03 |
| JPS52106794A (en) | 1977-09-07 |
| AU508703B2 (en) | 1980-03-27 |
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