DE2423174A1 - PROCEDURE FOR IN VITRO DETERMINATION OF THYROID FUNCTION AND DIAGNOSTIC REAGENT FOR THIS - Google Patents

Description

PATENT "N * '.' ··· .'TE

Dr .-! Πα. Η "· \ ί;. I-

Dip! .-- ii ^. OL. '"M, J

Dipl.-lr.s-FiA.W3 E. RUo

i BERLIN it

Mallinckrodt Chemical Works, St. Louis, Missouri, V.St.v.A.

Method for in vitro determination of thyroid function and

diagnostic reagent therefor

The invention / relates to a diagnostic reagent for use in the in vitro determination of the thyroid or thyroid function from a buffered aqueous suspension containing finely divided, amorphous silicon dioxide; a radioactive iodine-labeled thyroid hormone substance and a non-ionic contains surfactant.

The invention also relates to the method for in vitro determination of thyroid function, which comprises the following steps: mixing a blood serum sample with a measured one Amount of reagent; Separating the silicon dioxide from the suspension obtained; and measuring the radioactivity of the separated silicon dioxide residue or the supernatant liquid.

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The invention relates to the field of clinical diagnostic reagents and, more particularly, to reagents which contain contain a radioactive isotope of iodine labeled hormone substance and can be used for in vitro determinations of thyroid function.

It is known that the unsaturated binding capacity of ■ blood serum to measure by mixing a serum sample with a buffered solution, which a known kenge with a radioactive Iodine isotopically labeled thyroid hormone and an ion exchange resin or other solid sorbent substance such as Activated charcoal, talc, magnesium silicate and the like contains, which are able to bind excess labeled hormone, so that it separated and removed from the serum sample. The frenge of labeled hormone produced by the serum protein, mainly of thyroxine-binding globulin, can then be taken directly or indirectly using a scintillation counter to be determined. Such tests are commonly known in the art as T-3 tests.

In the most common forms of this test, the solid sorbent material is added after the serum sample and the marked hormone solution has been combined, in which case the function of the sorbent on binding the excess, i.e., free or unbound Mormon is restricted, so that it can be separated from the liquid serum sample containing the protein-bound hormone.

Another form of this test (USrPS 3 507 618) uses the. labeled hormone initially bound to an ion exchange resin; in this form the test depends on the desorption of the Hormone from the ion exchange resin carrier. This method is satisfactory provided the loaded resin is freshly made and carefully kept dry until the time of its actual use, and provided that all times and temperatures are carefully standardized

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controlled and precisely regulated.

A major disadvantage with this method is that with over time, and especially in the presence of moisture, the labeled hormone becomes permanently bound to the resin and therefore not available for binding by blood serum protein. The combination of a labeled hormone and an exchange resin is therefore wholly unsuitable for use in a complete and completely sealed liquid Reagent that can be manufactured and stored for future use.

When carrying out the method according to the invention, a measured amount of the blood serum to be tested is mixed with the novel Reagent of the invention mixed, the amorphous silicon dioxide separated from the liquid part of the sample e.g. by centrifugation, after which the radioactivity of either the solid silica residue or the supernatant liquid below Is determined using a suitable conventional scintillation counter.

The procedure is based on the desorption of the labeled hormone from the silicon dioxide carrier through certain proteins in the blood serum, namely the thyroxine-binding globulin. In contrast to desorption methods used in the past the present process is essentially complete and independent of time and temperature and the reagent of the invention completely completed.

In the diagnostic reagent of the invention that is with one radioactive isotope labeled hormone is detachably bound to the amorphous silicon dioxide, which together with the nonionic surfactant serves as a solid carrier. Thus, when serum is mixed with the reagent, those in the thyroxine-binding Globulin of the serum present unsaturated binding sites labeled hormone from the carrier until the binding

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capacity is practically sutted. The excess labeled hormone remains bound to the silica carrier. This transition takes place almost instantaneously at normal room temperatures; the process is therefore practically independent of time and temperature. As soon as it becomes expedient, the fixed, that excess labeled hormone-containing silicon dioxide from the liquid part of the sample, which is the protein-bound Hormone, i.e. the practically saturated thyroxine-binding globulin, is separated. This is done easily and quickly by centrifugation accomplished. As the total amount of labeled hormone initially present in the reagent is known or can easily be determined - the amount of such a hormone that has been absorbed by the serum protein can be counted either the supernatant liquid or the separated solid silica using a conventional one Scintillation counter can be determined.

Numerous nonionic surfactants suitable for use in the present invention are suggested in the Kirk-Othmer Encyclopedia of Chemical Technology, 2nd Edition, Vol. 19 "pp. 531-554". Among these, the following can be mentioned as particularly suitable: polyoxyethylene alcohols, polyoxyethylene sorbitan fatty acid esters, nonylphenoxy poly (ethyleneoxy) ethanol and sorbitan fatty acid esters.

The reagent of the present invention is in the form of a buffered one aqueous suspension, which can be packaged and stored in analysis units, just for a single test sufficient; the container in which the method of the invention is carried out may be a test vial (vial) of the The type and size commonly used in conjunction with a conventional scintillation counter.

The labeled hormone can either be L-thyroxine (T-4) or L-Triiodothyronine (T-3) be the one with a radioactive isotope of iodine as marked 1-125 or 1-131 ». Such hormones

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are well known and will be with most if not in all in vitro methods of measuring thyroid function applied.

The following examples illustrate the invention.

example 1

A suspension of 20 g of microfine precipitated silica ( ^u Quso G-32 "manufactured by Philadelphia Quartz Go.) Was suspended in 2,000 ml of a buffer pH 7.3 (j; 0.1), the solution of which had the following composition:

l ¹ ris (hydroxymethyl) aminomethane 48.4 g

HCl. . 29.8 ml

Water filled up to 2 liters

To this suspension was added an aqueous solution containing 1.0 g of nonylphenoxypolyethyleneoxy) ethanol ("Igepal 00-880" manufactured by Applied Science Laboratories, Inc.). The suspension was mixed thoroughly and then sufficient T-3 »marked 1-131 was added to obtain between 50,000-100,000 cpm / 3 ml in a conventional counter tube. The suspension was then dispensed into 3.0 ml /: E ^{1 vials (vials).}

Example 2

The reagent vials described in Example 1 were prepared in the following manner for the determination of thyroxine binding capacity of blood serum protein used:

1. 0.1 ml of patient serum was added to a reaction vial, give. 0.1 ml of control serum was also added to a reaction vial. ·

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2. The entire contents of the vials were mixed by several times Tip over or place in a vortex mixer for 10 seconds.

3. The vials were left at room temperature for 5 minutes.

4-, The vials were set at 250 rpm for 5 minutes or until that Adsorbent "caked., Centrifuged.

5. The supernatant liquid was decanted, discarded and drain the tube 1 down on a paper towel.

6. The vials of patient serum and control serum samples were counted and the index calculated.

Index = net (patient) cpm

Net (control) cpm

Example 3

Example 1 was repeated, with the exception that polyoxyethylene (2O) oleyl ether, available under the trade designation "BHIJ 98" from Atlas Chemical Industries, Inc. as a nonionic surfactant was used.

example

Example 1 was repeated except that polyoxyethylene (20) sorbitan monolaurate ₁ available under the trade designation "Tween 20" from Atlas Chemical Industries, Inc. was used as the nonionic surfactant.

example

Example Λ was repeated, with the exception that sorbitan monolaurate, available under the trade name "Span 20"

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from Atlas Chemical Industries, Inc., as a nonionic surfactant Funds was used.

The means and methods described in Examples 1-5 were used for a wide range of serum samples and the results were extremely favorable Compare in precision and accuracy with the results obtained using other, previously known, however much more time consuming methods were obtained.

In light of the above description it will be seen that according to the invention several goals are achieved and other beneficial results are obtained.

As various changes to the above procedure and the Means or masses can be made without deviating from the scope of the invention, all information in the above should be Description should only be understood as illustrative, but in no way restrictive.

Patent entitlements

409849/0806

Claims (4)

Claims Diagnostic reagent for use in in * -vitro determination thyroid function from a, buffered aqueous suspension, which finely divided, amorphous silicon dioxide; a hormone substance associated with radioactive iodine is labeled; and contains a nonionic surfactant. 2. A diagnostic reagent according to claim 1 characterized in _t that these Schxlddrüsenhormonsubstanz L-thyroxine and / or L-triiodothyronine is. 3 Diagnostic reagent according to Claim 1 or 2, characterized in that that the nonionic surface-active agent is selected from the group: polyoxyethylene alcohols, polyoxyethylene sorbitan fatty acid esters, Nonylphenoxypoly (ethylene- • oxy) ethanols and sorbitan fatty acid esters. 4. method for in vitro determination of thyroid function, characterized by the steps of: mixing a blood serum sample with a measured amount of a reagent according to any one of claims 1 to 3; Separating the silicon dioxide from the suspension obtained; and measuring radioactivity the separated silicon dioxide residue or the supernatant liquid. Dr οRo / He 409849/0806