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Publication numberUS3816076 A
Publication typeGrant
Publication dateJun 11, 1974
Filing dateJun 9, 1972
Priority dateJun 15, 1971
Also published asDE2129553A1
Publication numberUS 3816076 A, US 3816076A, US-A-3816076, US3816076 A, US3816076A
InventorsE Backer
Original AssigneeMerck Patent Gmbh
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Process for the determination of thyroxine
US 3816076 A
Abstract
Method for the determination of thyroxine in biological fluids by adsorption on a cation exchanger, subsequent washing out of the interfering components, eluting the adsorbed thyroxine, liberating the iodine therefrom, and then carrying out the iodine determination in a conventional manner, which process is characterized by the use of an eluent, optionally together with a basic buffer, which destroys amino groups and liberates iodine from the thyroxine into the eluate.
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United States Patent [191 Backer 1 June 11, 1974 PROCESS FOR THE DETERMINATION OF THYROXINE [75] lnventor: Egbert Tobias Backer, Oegstggest,

Netherlands [73] Assignee: Merck Patent Gesellschaft mit beschrankter Haftung, Darmstadt, Germany [22] Filed: June 9, 1972 [21] Appl, No.: 261,218

[30] Foreign Application Priority Data 4/1972 Gross et a1. 23/230 B 3,659,104 3.702,821 11/1972 Fernandez 23/230 B OTHER PUBLICATIONS Sterling et al., Jour. of Clin. lnves., Measurement of Free Thyroxine Concentration in Human Serum, Vol.41, No. 5, 1962 pp. 1031-1032.

Primary ExaminerR. E. Serwin Attorney, Agent, or FirmMillen, Raptes & White 5 7 ABSTRACT Method for the determination of thyroxine in biological fluids by adsorption on a cation exchanger, subsequent washing out of the interfering components, eluting the adsorbed thyroxine, liberating the iodine therefrom, and then carrying out the iodine determination in a conventional manner, which process is characterized by the use of an eluent, optionally together with a basic buffer, which destroys amino groups and liberates iodine from the thyroxine into the eluate.

10 Claims, No Drawings l PROCESS FOR THE DETERMINATION OF THYROXINE BACKGROUND OF THE INVENTION The present invention relates to a process for the determination of thyroxine. The exact determination of the thyroxine content is of importance primarily in biological fluids, e.g., blood serum or blood plasma, for thyroid diagnostics.

Thyroxine is an iodine-containing hormone which can be synthesized only by the thyroid gland and is secreted into the blood. This hormone plays an important part in the regulation of many metabolic processes. in pathological cases, the thyroxine content in the blood serum deviates greatly from that which is found under normal physiological conditions. This is the case, for example, in Basedows disease resulting from hyperfunctioning of the thyroid, resulting in an increased thyroxine content in the blood serum.

Thyroxine normally exits in the blood serum only in a very low concentration. It is customary to indicate the thyroxine concentration in 11g. of iodine per 100 ml. of serum. The normal content is approximately 3-7 micrograms of thyroxine iodine per 100 ml. of serum. In pathological cases, the content can be lowered, or also increased to up to about micrograms of thyroxine iodine per 100 ml. of serum. Thyroxine is bound, in the blood serum, almost completely to a specific carrier protein, the so-called thyroxine-binding globulin. In addition to thyroxine, the serum also contains very small amounts of other iodine compounds, such as, for example, iodide, triiodothyronine, thyroglobulin, and possibly monoand diiodotyrosine.

A number of processes for the chemical determination of thyroxine are known.

The method used most in practice is the determination of protein-bound iodine in the serum, the so-called PBI method. In this procedure, the organic iodine compounds are precipitated together with the serum proteins, and the iodine is determined after preceding alkaline incineration or wet incineration. More specific is the determination of the iodine which can be extracted with butanol (BEI method), because in this process only thyroxine and triiodothyronine, the hormone iodine in the narrower sense, are picked up. Furthermore, determination methods are known utilizing, for isolating the hormone iodine, thin-layer chromatography, gel filtration, or ion exchangers.

However, all conventional methods for thyroxine determination exhibit considerable disadvantages. A great disadvantage in the PBI and BE] methods is that the incineration must be conducted in an expensive incinerator and the average time for the analysis of a blood sample is very long (approximately 20 hours). A further essential disadvantage of the PBI and BE] methods-is that no distinction is made between thyroxine and iodine-containing radiopaque agents or medicines. in patients who were subjected to radiological examination or therapy with an iodine-containing agent, the actual concentration of the thyroxine in the blood serum cannot be determined because these methods in all cases involve the liberation and determination of total serum iodine. In this connection, it is to be noted that iodine-containing contrast agents can reach a concentration in the blood serum which is many hundred times higher than that of thyroxine. It can take years for the entire elimination of these contrast agents from the blood circulation. During this time, when using the PBI method as well as with the use of the BE! method, one i always obtains erroneous, elevated thyroxine values.

Serum thyroxine determinations involving a thinlayer chromatography or gel filtration step have the disadvantage that they are very difficult to process and thus are not feasible as routine determinations in the clinical laboratory.

A process for the determination of serum thyroxine by means of an anion exchanger has been described in U.S. Pat. No. 3,471,553 and in Clinical Chemistry 14 (1968) 339. The serum sample is first brought to a pH of 12-13 by means of a strong base, and introduced into an anion exchange column. During passage through the column, the thyroxine is bound to the ma trix. The exchange column is then washed out with several reagents in order to remove the residual serum components and any iodine-containing contrast agents or medicines which may be contained in the serum. Thereafter, the thyroxine is eluted with 50 percent strength acetic acid. The strong adsorption of thyroxine on the matrix requires the high acetic acid concentration in the eluent. Either bromine-saturated water or a solution of KBr and KBTOg in water is then added to the thyroxine-containing eluate, thus producing free bromine in the acidic eluate. The bromine liberates the iodine from the thyroxine molecule, which can then be determined in a conventional manner.

In spite of easy manipulation, this method yet entails grave disadvantages. The serum specimen cannot be introduced into the column immediately, but rather must first be adjusted to a specific pH value. Concentrated acetic acid isnecessary for the elution of the thyroxine, and free bromine is needed for liberating the iodine, which results in inconvenience in handling due to the obnoxious odor and possible injury to health. Another disadvantage is that the iodine is not liberated quantitatively from thyroxine and consequently the thus-obtained measuring results are not accurate.

Another method is known for the determination of thyroxine which utilizes a cation exchanger. See Clin. Chim. Acta 20 (1968), 155. The serum sample is introduced into a cation exchange column and then passes through the column, during which step the thyroxine is bound to the matrix. The column is washed out with various reagents in order to remove the remaining components of the serum and any iodine-containing contrast agents or medicines which may be present in the serum. After elution of the thyroxine with ammonia and chloric acid incineration, the thus-formed iodine is determined in the usual manner. Although this method is specific to thyroxine iodine, it has considerable disadvantages. The 5N ammonia employed for the elution must again be eliminated from the eluate before it is possible to effect the liberation of the iodine with chloric acid under high temperatures. Special apparatus are needed for this purpose and it is impossible to avoid obnoxious odors and possible damage to health.

It has now been found that the above-described disadvantages of the heretofore conventional methods for thyroxine determination can be avoided by the use of a novel eluent.

SUMMARY OF THE INVENTlON In its process aspect, this invention relates to a process for the determination of thyroxine in biological fluids in which the thyroxine is adsorbed on a cation exchanger, interfering components are washed from and then adsorbed thyroxine is eluted from the cation exchanger, the iodine is liberated from the eluted thyroxine and an iodine determination of the liberated iodine is then conducted, which comprises using eluting the thyroxine with an eluent, optionally together with a basic buffer, which decomposes the thyroxine and liberates iodine from the thyroxine into the eluate.

In its composition aspect, this invention relates to an agent for the determination of thyroxine in biological fluids, comprising a thyroxine adsorbing cation exchanger, optionally a salt solution, a basic buffer, an eluent which destroys amino groups and liberates iodine from thyroxine, an iodine determination reagent and a standard solution.

DETAILED DISCUSSION In the process of this invention, the biological fluid is introduced, without preceding pH adjustment or treatment with base, and thus in undiluted form, onto a column of a cation exchanger. After washing out proteins and any interfering components, such as, e.g., exogenous iodine-containing compounds, the adsorbed thyroxine is removed from the exchanger by decomposing the thyroxine by destroying the amino group, and the iodine is liberated into the eluate and can be determined directly. Thus, no reagent need be added to the eluate in order to liberate the iodine.

Thus, for the first time, a simple method is provided for the routine determination of hormonal iodine (thyroxine and triiodothyronine), within a relatively short period of time (about 3 hours) and without the use of concentrated bases or specialized equipment. Accordingly, the novel method requires less than half the time necessary for the conventional methods. A further advantage is that there are no obnoxious odors and no health-impairing reagents involved in the determination.

For the thyroxine determination according to the process of the present invention, cation exchangers in their H form are utilized. Suitable are cation exchanger synthetic resins, e.g., cross-linked polyvinyl derivatives, for example, polystyrenes, polyacrylates and polymethacrylates, and polycondensed products, e.g., phenolic resins having acidic groups, e.g., -SO -COO", -O, PO -PO H, -AsO or -SeO and cross-linked polysaccharides. Preferred are polystyrene cation exchangers, particularly those having sulfonic acid groups, exhibiting preferably a particle size of -400 mesh, especially 200-400 mesh. The degree of cross-linking preferably ranges from2 to 12 percent, and the exchange capacity is preferably in a range of 0.6 2.5 meq./ml., preferably being I meq./ml.

When a serum or other biological sample, normally one which has not been subjected to a pretreatment, is allowed to pass through such an ion exchange column, the thyroxine contained in the sample is bound to the column, partially nonionically and partially ionically, by its amino groups. Most of the serum components, including any other inorganic iodides which may be present, are not adsorbed on the column during this step. After the serum is passed through the column, any residual interfering components are removed by washing the column, e.g., with a basic buffer, optionally after a preceding washing step with a salt solution.

The optional washing step with a salt solution is advantageous in order to remove the proteins contained in the sample and other components which are not bound to the ion exchanger, and to neutralize the ion exchange resin. Suitable salt solutions are all aqueous solutions of salts which cannot be attacked by oxidation and which do not interfere with the final iodine determination, such as, for example, sodium chloride, potassium chloride, potassium dihydrogen phosphate, potassium-sodium tartrate, sodium acetate, sodium citrate, sodium oxalate, sodium sulfate, calcium chloride, etc. The concentration of the salt solution is proferably 0.01 to 5M, depending on the solubility of the selected salt. Particularly advantageous are 0. l-l M sodium chloride solutions.

In the subsequent washing-out step, preferably with a basic buffer, the last traces of the proteins and any interfering iodine-containing medicines are removed from the column. It is advantageous to wash first with a salt solution so that the acidic ion exchanger is neut'ralized. In such a case, a small volume and a low concentration of the basic buffer will suffice without there being an essential weakening of the buffer capacity thereof. Suitable basic buffers are those capable of maintaining a pH of about 7.5 9.0, which are inert with respect to oxidizing agents, and which do not interfere with the iodine determination. Particularly advantageous are inorganic basic buffer systems, e.g., borate and sodium bicarbonate buffer, in a concentration of about 0.05 1M, preferably 0.2 0.6M, especially a borate buffer of approximately 0.4M, pH 8.6.

Suitable eluents for the process of this invention are those comprising an oxidizing agent which is effective in a basic medium and which is capable of destroying amino groups by oxidation, thereby liberating iodine from the thyroxine molecule, e.g., hypobromite or hypochlorite. Bromine water can also be used as the eluent. However, agents without troublesome odors, e.g., hypobromite or hypochlorite, are preferred. An alkali hypobromite or alkali hypochlorite solution, e.g., sodium or potassium hypobromite or hypochlorite, is particularly advantageous.

It is now possible, for the first time, to remove the thyroxine from the cation exchanger without the use of a concentrated base. In accordance with the present invention, a solution of hypobromite, particularly in a weakly basic medium, is the preferred eluent. This solution is obtained, for example, by mixing equivalent amounts of sodium hypochlorite in dilute sodium hydroxide solution and potassium bromide, and diluting the thus-produced hypobromite solution with the buffer system utilized for the washing-out step. The concentration of the eluting solution employed can vary widely but preferably is relatively dilute, e.g., less than 0.1M, preferably 0.0075 0.02 hypobromite, preferably about 0.015M.

The thus-obtained, weakly basic eluate is allowed to react until all of the iodine has been liberated from the thyroxine. To expedite this reaction, the eluate is preferably heated, e.g., to about 50-l00 C. In order to shorten the reaction time, the mixture is suitably heated in a water bath, the temperature of the eluate being maintained at approximately C. When this is done, the reaction is terminated within a few minutes. At the same time, any residual organic impurities are also destroyed.

When using hypobromite as the eluent, which is preferred, free bromine does not evolve at any time during the elution or during the treatment of the eluate. Also with regard to this factor, the process of the present invention is superior to the conventional procedures for thyroxine determination by means of ion exchangers in which bromine or a mixture of bromide/bromate is added to the protein-free acid eluate. In addition to the instability of the aqueous bromine solutions, the extremely poisonous bromine always escapes into the atmosphere during these conventional procedures.

The colorimetric determination of the iodine liberated from thyroxine is effected according to conventional procedures. Preferably, the conventional cerium(lV) arsenite reaction is utilized as described, for example, in Clin. Chim. Acta 5 (1960), 301. In general, the eluate, heated for example in a water bath, is mixed with an acidified arsenite solution and thennostated for about minutes for temperatur adaption at about 2540C., preferably at 28 C. Subsequently, an acidic cerium(lV) ammonium sulfate solution is added thereto and, after a specific period of time, preferably about 25 minutes, the light absorption is measured at 420 nm. or 436 nm.

Since, in the process of this invention, the iodine to be determined is liberated quantitatively from the thyroxine, an iodate solution can be utilized as the standard. This is a special advantage, compared to the conventional methods wherein thyroxine standard solutions are employed. The disadvantages of thyroxine standard solutions are due to the insolubility of thyroxine in water, decrease of activity and its undefined iodine content. It is advantageous in determining the standard to treat the columns with the eluent.

The agent of this invention for the determination of thyroxine content of biological fluids, by means of which the above-described procedure can be carried out, contains the aformentioned reagents. Preferably, the required reagents are utilized in theform of a test kit. This novel combination of reagents is characterized in that it contains a. a cation exchanger as described above, preferably one or more units, preferably filled glass or plastic tubes suitable for use as a column, each in an amount sufficient to adsorb all the thyroxine in-a typical serum sample, e.g., about 1-5, preferably 2 3 ml.

b. (optional) an inert (with respect to the thyroxine and eluent) soluble ionic salt, preferably as an aqueous solution, e.g., about 3 10 ml. per unit of cationic exchanger;

c. a basic buffer solution, e.g., about 5 ml. per unit of cationic exchanger;

(1. eluent solution, e.g., about 5 15 ml. per unit of cationic exchanger;

e. (optional) iodine determination reagent; and

f. (optional) standard iodine solution. In its preferred embodiment, (b) and more preferably (e) and (f) are present inthe reagent test kit. However, since some users will have ready access to (e) and (f) components, they need not be included in the test kit in order for such users to conduct thyroxine analyses.

A preferred reagent kit contains lM sodium chloride, e.g., lM, solution (optional); borate buffer, e.g., 0.4M, preferably about pH 8.6; potassium bromide solution and a sodium hypochlorite solution, both preferably about 1M, sodium hydroxide solution, preferably about 0.1M; and preferably also an indicator reagent for the measurement of iodine content, e.g., arsenous acid and cerium-ammonium sulfate and a standard iodine determination solution, e.g., a potassium iodate solution. The solutions can also be present in the test kit in the form of concentrates, i.e., to be diluted prior to use, e.g., 10 to fold.

The thyroxine determination according to the process of the present invention can also be effected in automatic analyzers. For this purpose, the thyroxinecontaining eluates of various specimens are fed, via a sample collector, to an analyzing system wherein arsenite solution and cerium-ammonium sulfate solution are successively added to the individual samples. After mixing and elapse of the reaction time, the reaction solution is introduced into a colorimeter and measured.

The process of this invention can also be utilized in the quality control of drugs, such as, for example, in the activity determination of dried and pulverized thyroids. This activity determination is of great practical importance. Whereas, by means of the conventional methods, the entire organically bound iodine is determined during this step, it is possible with the novel process to determine selectively the biologically active components (thyroxine and triiodothyronine).

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

EXAMPLE 1 A microcolumn filled with a cation exchanger (polystyrene with sulfonic acid groups, cross-linked with divinylbenzene, particle size 200-400 mesh, degree of cross-linking 2 percent) is charged with 0.5 ml. of serum, and the latter is allowed to pass through. Then, the column is washed out first with 5 ml. of lM NaCl solution and thereafter with a total of 10 ml. of 0.4M borate buffer (pH 8.6). After the washing-out step, two elutions are conducted, with respectively 5 ml. of a 0.015M solution of hypochlorite/KBr in 0.4M borate buffer. (The elution can also be effected in one stage with 8 ml'. of this eluting solution.) The eluate is heated in a water bath having a temperature of 100 C. for 8 minutes and is then cooled to room temperature.

Thereafter, the iodine liberated in the eluate is determined in the usual manner, for example as follows: The eluate is mixed with 1 ml. of 0.16N arsenite solution and brought to a constant temperature in a 28 C. water bath for 10 minutes. Then, 1 ml. of 0.0l7N cerium sulfate solution is added to the thermostated solution, mixed therewith, and thereupon the mixture is immediately returned to the water bath. Exactly 25 minutes after adding the cerium sulfate solution, the absorption of the sample is measured at 420 nm. against water.

In each determination, in parallel measurements, the blank column value, the blank reagent value, and two different standard solutions are measured. The standard reference values are determined by adding an iodate solution of a known concentration to the eluate of a blank column value and then conducting the iodine determination. The columns for obtaining the blank value and the standard reference values are each treated with the same reagents as the analysis sample.

EXAMPLE 2.

' A microcolumn filled with cation exchanger (polystyrene basis with sulfonic acid groups, particle size 50-140 mesh) is charged with 0.5 ml. of plasma, and the latter is allowed to pass therethrough. Then, the column is washed out first with ml. of 0.5M sodium acetate solution and thereafter with a total of ml. of 0.1M sodium bicarbonate solution. After this step, the elution is effected twice with respectively 5 ml. of a 0.015M solution of hypochlorite/KBr in 0.1M sodium bicarbonate solution, The eluate is heated for 5 minutes in a water bath of a temperature of 100 C. and subsequently cooled to room temperature.

The colorimetric determination is thereupon conducted analogously to Example 1.

ln repetitions of the above determination, solutions of the following salts are utilized in place of-the sodium acetate solution: potassium chloride, potassium dihydrogen phosphate, potassium-sodium tartrate, sodium citrate, sodium sulfate, sodium oxalate, and calcium chloride. The washing-out step with the use of these salt solutions can optionally be omitted, or it can be effected with 2- or 3-molar solutions.

ln place of hypochlorite/KBr as the eluent, bromine water is utilized in a further modification of the abovedescribed modes of operation.

EXAMPLE 3 mg. of dried and pulverized thyroid is hydrolyzed in a borate buffer with trypsin and erepsin and, after acidification, an aliquot is charged into a microcolumn filled with cation exchanger. After washing out the i0- dide, monoiodotyrosine, and diiodotyrosine with borate buffer, the adsorbed triiodothyronine and thyroxine are eluted with a total of 8 ml. ofa 0.015M solution of hypochlorite/KBr in 0.4M borate buffer. The liberation and subsequent determination of the iodine are effected analogously to Example 1.

EXAMPLE 4 5. 110 m1. 0.017N cerium-ammonium sulfate solution 6. 5 ml. standard solution (0.1 mg. KlO /l.) 7. 5 ml. standard solution (0.2 mg. KlO /l.) Solution 2 is diluted with 1.000 ml. of twice-distilled water. Shortly before use, 1 ml. of solution 3 is diluted with 100 m1. of the ready-for-use solution 2. All other solutions are ready for use.

EXAMPLE 5 A reagent combination for about 25 thyroxine determinations contains, in addition to a corresponding number of microcolumns filled with cation exchange resin, the following reagent solutions:

1. 50 m1. 3M sodium chloride solution 2. 2 X 250 ml. 0.4M borate buffer 3. 5 ml. 1M potassium bromide solution 4. 5 ml. 1M sodium hypochlorite solution in 0.1M sodium hydroxide solution 5. 50 m1. 0.16N arsenite solution 6. 50 ml. 0.017N-cerium-ammonium sulfate solution 7. 5 ml. standard solution (0.1 mg. KlO /l.)

8. 5 ml. standard solution (0.2 mg. KlO /1.)

Solution 1 is diluted with ml. of twice-distilled water. Shortly prior to use, respectively 1 ml. of solutions 3 and 4 are combined and diluted with 100 m1. of solution 2. All other solutions are ready for use. Optionally, solution 3 can be omitted or replaced by a 1M sodium bromide solution.

EXAMPLE 6 2 ml. of 0.15M NaCl solution and 0.5 ml. of serum are successively introduced into a microcolumn filled with cation exchanger (polystyrene with sulfonic acid groups cross-linked with divinylbenzene, particle size 200-400 mesh, degree of crosslinking 2 percent) and allowed to pass therethrough. Then, the column is washed out with 5 ml. of 0.15M NaCl solution and thereafter with a total of 10 ml. of 0.4M borate buffer (pH 8.6). After the washing-out step, the elution is conducted twice with respectively 5 ml. of a.0.015M solution of hypochlorite/KBr in 0.4M borate buffer. (The elution can also be carried out in one step with 8 ml. of this eluting solution.) The eluate is heated for 8 minutes in a water bath of 100 C. and then cooled to room temperature.

Thereafter, the iodine liberated in the eluate is determined in a conventional manner, for example as follows: The eluate is mixed with 1 ml. of 0.22N arsenite solution and brought to a constant temperature for 10 minutes in a 37 C. water bath. Then, 1 ml. of 0.045N cerium sulfate solution is added to the thermostated solution, mixed therewith, and the mixture immediately returned into the water bath. Exactly 25 minutes after adding the cerium sulfate solution, the absorption of the sample is measured at 436 nm. against water.

EXAMPLE 7 4. 3.75 ml. 1M sodium hypochlorite solution in sodium hydroxide solution .'25 ml. 0.22N arsenite solution 6. 25 ml. 0.045N cerium-ammonium sulfate solution 7. 5 ml. standard solution (0.845 mg. KIO /l.)

8. 5 ml. standard solution (1.69 mg. KlO 1.)

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

What is claimed is:

1. ln a process for the determination of thyroxine in biological fluids which comprises the successive steps of adsorbing the thyroxine on a cation exchanger, washing the cation exchanger, eluting the adsorbed thyroxine therefrom and measuring iodine liberated from the eluted thyroxine, the improvement which comprises applying a sample of the biological fluid to a column of the cation exchanger and eluting the adsorbed thyroxine from the column with an eluent which decomposes the thyroxine adsorbed on the column and liberates iodine into the eluate.

2. A process according to claim 1 wherein the eluent comprises a basic buffer.

3. A process according to claim 1 wherein the eluent comprises a solution of a hypobromite or hypochlorite.

4. A process according to claim 3 wherein the eluent comprises a basic buffer.

5. A process according to claim 4 wherein the buffer is a borate buffer.

6. A process according to claim 3 wherein the eluent comprises sodium. hypobromite or sodium hypochlorite.

7. A process according to claim 3 wherein the molarity of the hypobromite solution is 0.0075 0.02M.

8. A process according to claim 7 wherein the molarity is about 0.015M.

9. A process according to claim 1 wherein the eluate is heated.

10. A process according to claim 1 wherein an iodine determination of the eluate is conducted photometrically, employing the cerium(IV) arsenite reaction.

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US3389968 *Jul 7, 1966Jun 25, 1968Huis Clinical Lab IncMethod of determining thyroxin iodine in blood serum
US3659104 *Jun 29, 1970Apr 25, 1972Univ Jerusalem TheMethod of measuring serum thyroxine
US3702821 *Dec 14, 1970Nov 14, 1972Bio Chem Procedures IncSerum thyroxine anion exchange resin
Non-Patent Citations
Reference
1 *Sterling et al., Jour. of Clin. Inves., Measurement of Free Thyroxine Concentration in Human Serum, Vol. 41, No. 5, 1962 pp. 1031 1032.
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US4034071 *Jan 26, 1976Jul 5, 1977Allegheny General HospitalImmunoassay procedures
US4311690 *Jun 14, 1979Jan 19, 1982Damon CorporationTest set and method for the determination of free hormones
Classifications
U.S. Classification436/500, 436/825, 436/826, 436/531
International ClassificationG01N33/78
Cooperative ClassificationG01N33/78, Y10S436/825, Y10S436/826
European ClassificationG01N33/78