DE2021968A1 - Method for blood testing using radioactive indicator substances and ion exchange resin membranes - Google Patents

Description

1BERLIN33 ..B MUNICH 47

Au _B u, «ev _lk , or, as.raBe6 ₅ Dr.-Ing. HANS RUSCHKE

ΪΠ5Γ. HEINZAGU LAR '■

PATENTANWÄLTE Telegram address:

Quadrature Munich

M 285O Dr.S / G.

Mallinckrodt Chemical Works, St. Louis, Missouri, V ₀ St.A.

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Have a blood test using radioactive Indicator substances and ion exchange resin membranes

The invention relates to the field of radioactive indicator substances, spe target! the use of such substances in connection with ion exchange resins for in vitro blood analysis. \

The use of radioactively labeled substances for the in vitro determination of various diagnostically important components of the blood serum is known. For example, as will be described in more detail below, radioactive iodine-containing thyroid hormones have been used to determine the total thyroxine content of the blood serum and / or the binding capacity of the serum proteins for thyroxine Iron has been used »

These examinations are carried out with the help of a suitable Counting method the amount of bound to the serum protein

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~ 2 radioactive indicator substance determined »

Bel these determination methods, after removing the "free" unbound indicator substance using a suitable counting method, the amount of indicator substance bound to the serum protein is determined * Thyroxine, which contains a radioactive iodine isotope (hereinafter referred to as "T-4") is called Indlkatorsubstanz used to determine the ßesamtgehalt the blood serum of thyroxine _e for example, the serum proteins are first denatured and the inactive T-4 extracted with alkenol. A certain amount of radioactively labeled T-4 bound to serum protein is then added to the extract. There is an exchange between the inactive T-4 and the radioactive T-4 until, since all factors are equal, the amount of protein-bound T-4 is proportional to the amount of inactive T-4 in the original blood sample actual amount of inactive T-4 in an unknown sample can then be compared with Ko.ntrollproben _Ä which amounts known of stable T-4 were added * are determined.

For determining the thyroxine binding index (TBl) of serum proteins becomes radioactive iodine containing L-triiodothyronine (hereinafter referred to as T-3) as an indicator substance used. T-J5, like T-4, is bound by globulin, but less tightly. T-4 is therefore only in subordinate Crowd displaced by T-J3. As a result, if a Excess radiolabelled T-5 in a serum sample is given, the amount that is bound to the serum protein is directly proportional to the thyroxine binding index, and this is determined by comparing with a control sample that contains a standard serum with a known thyroxine binding index, determined

The serum protein, thyroxine binding globulin (TBO), has

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a relatively high and specific tendency to the thyroid hormone Thyroxine (Τ ~ 4) to bind »It is also known that when radioactive T-4, i.e. T-4, the radioactive iodine, like

^ J, contains, is added to a solution made with barbital contains buffered TBG, virtually all T-4 is bound to the TBG. If then inactive T-4 to this TGB solution is given, the radioactive T-4 is released by this T-4 im Ratio of the added amount of inactive T-4 replaced If then another T-4 binding reagent, such as a Ion exchange resin is added to this system these bind the radioactive T-4 displaced by the TGB *

The degree of displacement of the radioactive T-4 and so too the amount of inactive thyroxine added to the TGB can be determined by making the ion exchange resin from the system and compares the remaining radioactivity of the TGB solution with its original radioactivityβ If increasing amounts of inactive T-4 be added to solutions containing equal amounts of radioactive T-4 and the TSB treated as above, the radioactivity of the TGB solution decreases with the increasing amount of inactive T-4 added *

If you compare the radioactivity of the treated sample with the Amount of inactive T-4, a curve is obtained as shown in Fig. 1 "If now unknown Amounts of T-4 are added to the TGB solution and treated in the same way, the amount of the added T-4 can be read from the curve.

In order to carry out this determination, dilution is generally used Blood erum used as a TGB source, and it is not it has been customary to subject it to further pretreatment » throw. In order to be able to determine the T-4 in the blood serum, must first the serum T-4 from the protein to which it is bound

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is to be separated. This is achieved by denaturing the proteins with alcohol. The denatured proteins release most (almost 80) of bound T-4 and this becomes with the alcoholic supernatant after centrifugation removed"

In a known way, the alcoholic extract is evaporated to avoid possible errors caused by the alcohol in the test solution could be caused to report. Depending on the number of the samples tested are generally 30 to 120 minutes needed.

The binding capacity of blood serum for iron is determined in much the same way as the binding capacity for thyroxine using an indicator substance that contains a radioactive iron isotope, such as iron (III) ammonium citrate, certainly·

Also the use of ion exchange resins to absorb excess that is not bound to the serum protein of the sample Indicator substance is also already known »

Such ion exchangers are granular or as with exchange resin Impregnated plastic sponge has been used. Biswaves make it difficult to separate the Resin from the sample liquid is an important source of error. For example, when using granular exchange resin Resin particles that have accidentally left the sample liquid cause defects. When using sponge impregnated with exchange resin, the sponge must be carefully squeezed out and washed to the whole Recover sample fluid before correct activity measurements can be made. This severing costs v-ie4, time and represents a source of error. For the Routine testing of large numbers of blood samples will be easier, faster and more reliable.

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more casual method of absorbing and measuring the excess not bound indicator substance required.

The invention relates generally to methods for in vitro blood analysis in which a liquid sample is first produced which contains blood serum protein and a certain amount of a radioactive indicator substance that can be bound by the serum protein and an ion exchange resin, then a relatively thin one Strips of a membrane consisting essentially of an ion exchange resin are added to this sample and this membrane is left in contact with the liquid for a certain time, then the membrane is separated from the sample and the relative amount of the indicator substance bound to the serum and to the membrane is determined.

The invention also relates to a method for determination the thyroxine content in the blood serum using radioactively marked thyroxine as an indicator substance for which to a known amount of a reagent, which consists essentially of a solution, the thyroxine binding globulin and contains radioactive thyroxine, an alcoholic extract from a sample of blood serum that determines the thyroxine content and an ion exchange resin is given, this globulin solution in contact with the resin for a particular tent is left and then the exchange resin is separated and the radioactivity of the globulin solution with that of olobulin solutions is compared to those known amounts of inactive thyroxine dissolved in the same volume of alcohol of the same Origin like those contained in the alcoholic serum extract were added. The invention also includes a Specialty reagent used in performing the above procedure and equipment that enables the practical implementation of the procedure facilitated

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The serum proteins consist of globulins, albumins and prealbumins, which can bind and transport the thyroid hormone thyroxine »The indicator substance is a substance such as Thyroxine, which is bound by the substances in the serum and replaced by a radioactive isotope, such as a radioactive iodine isotrope, is marked »The indicator substance can either be a naturally occurring component of the blood or a synthetic one Be a substance that has the same properties and shows the same behavior, such as L-triiodothyroxine, which dem natural hormone txiyroxin is similar. The indicator substance can also be a substance that contains an element such as iron, which is usually also present in the blood as a protein-bound component »

The only job of the ion exchange resin is to absorb unbound indicator substance so that it can be removed from the sample. Since the membrane is very thin and strong and very specifically absorbed, it is, for example, _a quickly and easily removed with a tweezer from the sample. The amount of the indicator substance bound to the protein of the liquid sample or the non-bound indicator substance in the resin membrane can then be determined by measuring one or the other radioactivity using a conventional counter.

Since the amount of radioactive indicator substance that was originally added to the sample is known, it is sufficient if the radioactivity in the exchange resin membrane or in the liquid sample is determined. From the measured radioactivity the radioactivity in the other sample can be calculated. It is common practice to have radioactivity in the liquid sample, i.e. the radioactivity of the indicator substance bound to the serum protein.

The ion exchange resin membranes used according to the invention

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are relatively thin strips, sheets or films of one solid hydrous gel, which consists of an insoluble high molecular weight There is a framework in which dissociable cationic or anionic groups are incorporated and preferably is reinforced by a suitable fiber material. A "dissociable cationic group" is a group understood, which contains a dissociable anion that acts against the anion to be absorbed, e.g. a radioactive iodine ion, can be exchanged. Similarly, a "dissociable anionic group" contains an exchangeable cation. Many suitable resin membranes of this type are already known, for example those in US patents 2,730,768, 2,780,6o4, 2,800,445, and 2,860,097 ^ In one type of membrane, the matrix consists of a copolymer of vinyl compounds that are dissociable Cationic groups are quaternary ammonium groups and the reinforcing fabric is made up of loosely woven fibers a copolymer of vinyl chloride and acrylonitrile, which is available under the trade name "Dynel". Get practical narrow strips of this membrane are used, which are 0.1 to 1.2 mm thick.

When the exchange has taken place, the exchange membrane, to which part of the radioactive substance is then bound, can can be removed very quickly and very easily from the liquid sample, for example by removing it with a Tweezers. When removing the membrane, the adhering droplets of the liquid sample on the test tube should be wiped off will.

However, it is not necessary to rinse the membrane because the film of the liquid sample adhering to the surface not enough to significantly affect the test results and the membrane in contrast to the resin-impregnated

0 0 9 8 A ^{5/1 A 2 7 s} * ö ° * f <2lNM.

-8 sponges practically not absorbed.

Due to the solid nature of the resin membrane, there is also no risk of getting into the liquid Sample retained particles will cause incorrect results. The diagnostic tests of the invention are based that one compares blood serum samples with control samples whose values are known * It is therefore only required both the unknown and the control samples treated under exactly the same conditions. The most important conditions are the duration of treatment and the Temperature »The use of a temperature-constant bath or space is not required. The simplicity the method according to the invention facilitates the incubation of a number of unknown samples together with a control sample under exactly the same time and temperature conditions. Precise control of these factors is therefore easier than with the previously known and used methods.

The vessel for the liquid samples can consist of any suitable material, it can consist of glass or plastic and should be shaped and sized to fit into the opening of a scintillation counter.

The following examples illustrate the invention.

example 1

Determination of the total thyroxin content of blood by using

125 production of globulin-bound thyroxine - "V "

In the example below, the matrix consists of the ion exchange membrane from a copolymer of vinyl compounds, the dissociable cationic groups are quaternary Ammonium groups and the reinforcing fabric consists of loosely woven fibers under the trade name "Dynel" available copolymers of vinyl chloride and acrylic

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nitrile. A commercially available membrane of this type is the AR-111 membrane made by Ionics Incorporated, Watertown, Massachusetts, manufactured * The diaphragm has a Thickness of 0.61 mm, contains 34% by weight of resin and has a capacity of 1.8 milliequivalents per gram of dry resin. A strip of this membrane 1 cm wide and 4.5 cm long has several times that of the absorption of the free T-4 in the following tests required capacity,

In a centrifuge tube, 2 ml of 95% ethanol are added to 1 ml of blood serum and the contents are mixed well in order to denature the serum proteins. Then it is centrifuged for 4 to 5 minutes at 2500 rpm. Then 0.3 ml of the supernatant liquid is removed, transferred to a 4 ml test tube and 3 ml of a pH 8.6 barbital buffer solution containing 0.64 $ sodium chloride, 0.23 $ barbital sodium and 0.6% barbital contains, added.

A set of comparative samples, the 0, 5, 10, 15 and 20 Millimicrograms of inactive T-4 contained in 0.2 ml of ethanol, is also prepared. 3 ml of the diethylbarbituric acid buffer solution are added to each given.

To each of the test tubes thus produced, 1 ml of a Barbltal buffer solution containing 2% human serum to which T-4 is absorbed and which contains a known amount, for example 0.1 millicuries, ^{125%, is then added} the ion exchange resin membrane is also placed in each tube. The dimensions of the membrane should be such that it fits into the tube without jamming «

The higher are soaked for 1 hour at room temperature, then opened and the membrane immediately removed with tweezers removed from the tube. It is not necessary that

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- ίο -

Rinse the membrane, as the amount of adhering solution is so small that it is of no practical importance _a

The radioactivity of each tube row is then with the help measured by a scintillation counter. The radioactivity of each standard reference sample is plotted against the number of millimicrograms of the inactive thyroxine contained in it applied »

The amount, in millimicrograms, of thyroxine in the unknown serum sample or samples can then be read from the standard curve. » This value is divided by 0.80, since with ethanol approximately $ 80 of the im Serum contained thyroxine can be extracted. The corrected value then means that contained in each 0.1 ml of serum Amount of thyroxine or the number of micrograms of thyroxine contained in 100 ml of serum »

Example 2

Determination of the thyroxine binding capacity "

In the following experiment a membrane is made of the same Type used as described in Example 1 · The the following additional reagents are used:

(a) Incubation tubes each containing 3.5 ml of buffer solution and T-3 containing ^{approximately 0.1 microcurie 1} ^ ^{1 J.} The buffer solution has the following composition: An aqueous solution of 2.42 $ trihydroxymethylaminomethane, the pH of which was adjusted to 7 * 2 by adding about 15 ml of concentrated hydrochloric acid per liter of solution »

(b) Standard Control Serum - (a serum with "normal" thyroxine binding capacity)

The serum samples to be tested are obtained by about Take 5 ml of venous blood and clot it in a test tube

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leaves and removes the serum. First, the radioactivity of the 5.5 ml buffer and ¹ ^ ¹ J-T-3 containing incubation tube is determined and this value is recorded as the initial count rate. Exactly 0.5 ml of patient serum is then added to the incubation tube. For each test series, exactly 0.5 ml of control serum is added to an incubation tube in the same way as for the unknown serum sample. A strip of the exchange resin membrane is placed in each tube, then they are sealed and incubated for 2 hours at room temperature, the tubes being shaken slowly to ensure that all of the liquid sample comes into contact with the exchange membrane. After the incubation period has elapsed, the exchange resin membranes are removed from the tube with tweezers, the strips being taken out and touching the inner edge of the tubes in order to wipe off any drops of solution adhering to the strips. Both the samples of the unknown serum and the comparison samples are measured for 1 minute in a "well counter" and the value is recorded as the final count rate.

The T-3 index, a common measure of thyroxine binding capacity, is calculated as follows:

End count rate _x iqO _ of the _{amount absorbed in} serum /% 7 Start count rate ^LmKJ

Uptake of the unknown serum f ~~% 7 = τ "'- Uptake of the control serum / _

The status of an individual's thyroid function is that Degree of saturation of the T-> Binding ability of the serum proteins proportional. In a patient with a hyperactive Thyroid, for example, this degree of satiety is greater

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than in a patient with a functionally normal thyroid gland _o Correspondingly, in a hypothyroid individual, more receptors available for binding T-3 are unoccupied than in a euthyroid patient.

Example 3

For the following experiment, the same ion exchange membrane is used that is used in the. Was described in Examples 1 and 2 "In a j5 ml of a buffer solution (s" Example l) test tube containing were added about 7 MS inactive iron, one containing radioactive tracer substance about 0.1 'μο ^J Fe and 1 ml of blood serum "The iron was added as iron (III) ammonium citrate,

The serum buffer solution is mixed and 10 minutes at room temperature ditched.

Then a strip of the ion exchange membrane is placed in the tube, this is sealed and 30 minutes at room temperature incubated, shaking it slowly in order to achieve intensive contact with the exchange resin membrane reach.

After the incubation time has elapsed, the resin strip is removed and the radioactivity of the solution was measured.

The radioactivity of this tube is compared with that of a standard which contains 1 ml of water instead of the blood serum.

The LIBC of the serum sample can be calculated according to the following equation will:

default

μ _β Pe / ml serum (LIBC) -?! ™: ϊϊ £ ϊίΪ! ί! .ΡΓ2.!? ί2: -ί.ϋδ.ϊ! .ϊ !! ί- /. «.

Pe standard impulses per minute,

0098457 U 27 ^ ₀ original

The graphic representation (Pig. 1) shows a clear one Standard curve in which the radioactivity of treated thyroxine-containing Globule samples are plotted against the amount of inactive thyroxine. When using the erfindungsgeraässen described below Method, the time required to determine the thyroxine in the blood serum is significantly reduced because the There is no need to evaporate the alcoholic extract. In addition- The accuracy of the method is significantly improved by using the special TBG reagent according to the invention, since the slope of the in Pig. I curve shown is greater than that with untreated, diluted TBG reagent obtained under the same conditions.

Example 4

About 10 ml of blood are withdrawn and allowed to clot «The serum is withdrawn and 1 ml of it is added dropwise to 2 ml Pour 95% ethanol into a centrifuge tube. The content will well mixed for denaturing the serum proteins. This alcoholic mixture is then at 2500 rpm. 45 minutes centrifuged.

0, j5 ml of the supernatant liquid are withdrawn and poured into a suitable test tube is transferred, the 4 ml of the special Contains TBG solution, the preparation of which is described below.

A number of different control samples are prepared in the same way. Instead of the ethanol serum extract 0.3 ml of an ethanolic solution are added, the one known amount (e.g. 0, and 12 nanograms) of inactive thyroxine contains.

A strip of ion exchange membrane is then placed in each test tube given. Since thyroxine is amphoteric, the ion exchange resin can be both arion-selective and cation-selective

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be. Many such exchange resins can be used as membranes. A resin suitable for use in the process according to the invention is, for example, that under Names AR-IU commercially available anion-selective resin made by Ionics Incorporated, Watertown, Massachusetts, will be produced.

After adding the resin strips, close the tubes and shake for exactly 1 hour at room temperature. Usually one of the commercially available devices specially offered for this purpose is used. «Since the amount of radioactive thyroxine absorbed by the resin is a puncture of the shaking time, it is essential that the shaking time is the same for the unknown and control samples. After one hour of shaking, the resin strip is removed with tweezers and discarded. The radioactivity of each tube is measured and recorded as the final count. At 10,000 pulses per minute the error is less than $ 2. The initial count rate is advantageously determined by measuring an unused tube which contains the TBG radioactive T-4 solution. The ratio of the initial to final count rate is calculated for each sample. The values for these standard samples are plotted against the amount of T-4 added and a straight line is drawn through the measuring points. The thyroxine content of the unknown samples is read from the resulting curve (Pig · I). Since approximately 80 # of the T-4 is extracted from the serum with alcohol, the value read from the curve must be divided by the extraction efficiency (approximately 0.8) in order to obtain the true thyroxine content of the serum. This corrected value then means the T-4 amount (nanograms) in 0.1 ml of serum and corresponds numerically to the number of μg T-4 in 100 ml ^ g %) patient serum.

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The special TBG solution is advantageously produced by the blood serum is sent through a column containing a suitable ion exchange resin that absorbs the thyroxine, But not the serum protein "

A suitable method for preparing the special TBG solution is the following: 3280 ml of a pH 8.6 barbital buffer solution containing 0.64 $ sodium chloride, 0.23 $ barbital sodium and 0.6 are added to 137 ml of serum % Barbital contains «, A small amount of radioactive thyroxine is also added (about 0.24 millicuries) to serve as an indicator to determine the efficiency of the extraction.

An extraction column suitable for this purpose is prepared by adding enough ion exchange resin to a suitable glass column to give the exchange bed one volume of 1200 ml and the amount of eluate about 25 ml per minute amounts to. A strongly basic ion exchange type resin of the quaternary ammonium salts is suitable for this purpose « For example, it can be the one commercially available as * ΙίΙΑ-4θθ ", resin made by the Rohm and Haas Company was used turn around; in its place, however, other anion or cation-selective resins are used *

The resin is added to the column as an aqueous slurry, and care is taken to keep it constantly of water is covered. The column is then connected to a storage container which contains the serum solution to be extracted ^ and the serum is sent through the column. 1 ml samples of the eluate are taken at regular intervals and the radioactivity was measured to determine the efficiency of the extraction. The radioactivity of the eluate should

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The extracted serum solution is then best filtered through a 0.22 micron filter. Additional radioactive thyroxine (approximately 0.30 millicuries) is then added to the eluate with six times the volume of the buffer solution already mentioned dilute The solution obtained should have a pH of 8.6 _e

The usefulness and accuracy of the method is further increased if the investigator is provided with equipment in which all special reagents and devices are compiled * Suitable equipment consists, for example, of a number of 4 ml glass test tubes with screw caps, which .4 ml of the TBG solution described above and should be equally permeable to radioactive radiation (matched for radioaktivity). The standard samples should contain known amounts (eg 0 and 12 nanograms) of inactive thyroxine in 0.3 ml of alcohol. A supply of the same alcohol that was used to make the comparative samples should also be available. The equipment should also contain the necessary ion exchanger, preferably in the form of membrane strips. The other necessary pieces of equipment, such as pipettes, a radiation meter and a shaker, are standard equipment in a laboratory.

Claims

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Claims (1)

Claims Method for in vitro blood analysis, in which a liquid obe is produced that contains blood serum protein and a certain amount of a radioactive indicator substance that is derived from both the serum protein and an ion exchanger ..- ■. ■■■ .. ■ '■■■ "■ ι resin is absorbed, a relatively thin one for this sample Strip of a membrane consisting essentially of an ion exchange resin is given to this membrane for a certain time kept in contact with the liquid sample for a long time, the membrane then removed from the sample and the relative amounts of the the serum protein and the membrane bound indicator substance to be determined 2.) The method according to claim 1, characterized in that the Determination is carried out by measuring the radioactivity in Serum protein and / or in the membrane is determined « 3.) Method according to claim 1 or 2, characterized in that that the blood serum protein is selected from the group consisting of globulin, albumin and prealbumin 4 ») Method according to claim 1, 2 or j5 * characterized in that the radioactive indicator substance is a thyroid hormone . .:. \ .; ^: '.. · ■■■. V '-..-. : \ | ' : - 5.) The method according to claim 4, characterized in that the thyroid hormone contains radioactive ^ J or ^-5 J. 6 *) Method according to claim 4, characterized in that 12 ¹ ^ ^'::: thyroxine containing the thyroid hormone or 131 " ^ J containing L-triiodothyronine 1st. .7 ·) Process according to Claim 1, 2 or 5, characterized in that ,,, that the radioactive indicator substance contains ^% e Eiaen (IIl) artwttomiuraol1 * rat is " 019845/1427 - i8 - δ «) Method according to the preceding claims, characterized in that that the membrane is between about 0.1 and about thick. 9.) Process according to the preceding claims, characterized in that that the membrane is a solid water-containing gel which contains an insoluble, high molecular weight matrix the dissociable ionic groups are bound. 10.) The method according to claim 8, characterized in that the gel is reinforced by a fiber material. 11.) The method according to claim 8> characterized in that the dissociable ionic groups are quaternary ammonium groups are. 12.) Process according to the preceding claims, characterized in that that the membrane is kept in contact with the liquid sample at around room temperature « 13 «) Procedure for determining the thyroxine content of blood serum using radioactive thyroxine as an indicator substance for a known amount of a reagent, which consists essentially of a solution that contains thyroxine-binding globulin and radioactive thyroxine, (a) an alcoholic extract from a blood sample whose thyroxine content is to be determined and (b) an ion exchange resin is given, this globulin solution with the said If resin is brought into contact for a certain period of time, the ion exchange resin is removed from the globulin solution and the radioactivity of the globulin solution with that of globulin solutions compared to which known quantities of inactive thyroxine dissolved in the same volume of the same al- Q 0 9 8 A 5/14 2 7 ^: ■■: · ^-. 19 ^-. ■ ■.; ■ ■■■■■ kohpls, which was also contained in the alcohol extract mentioned above, was added. lH ·.) Method according to claim 15, characterized in that the reagent is a buffered solution of radioactive thyroxine and a blood serum from which most of the naturally occurring thyroxine has been extracted. 15e) Method according to claim 15 or 14, characterized in that that the radioactive thyroxine is thyroxine _J »'■ -. 1.6. ') Method according to claim 13> 14 or lfj ,. characterized in that the globulin solution with the ion exchange resin is brought into contact by opening the sealed tube containing the resin and the solution shakes " 17.) Process for the preparation of a reagent for use in the determination of the thyroxine content of blood serum, characterized in that diluted blood serum by a is added to a column containing ion exchange resin to extract most of the naturally occurring thyroxine and that the eluate obtained contains radioactive thyroxine is added » 18.) Reagent for use in determining the thyroxine content of blood serum, characterized in that it is made from a buffered solution of radioactive thyroxine and Blood serum is made up of * from which most of the naturally occurring thyroxine has been extracted. ■ '19 ·) 'reagent according to claim 18, characterized in that the radioactive thyroxine contains ¹² ^ J. 2o.) Equipment for determining the thyroxine content of Blood serum, consisting of a "buffered solution of blood 009845/1427 serum that is no more than about $ 20 the normal amount of thyroxine contains and a sufficient amount as an indicator 12S
Substance-serving thyroxine - -v, two control samples containing a known amount of thyroxine dissolved in a certain volume of alcohol, a supply of the same alcohol that was used to make the control samples, and several strips of resin membrane sized to accommodate them fit loosely into the test tubes. 21 «) Procedure for in vitro blood analysis. 22ο) Method for determining the thyroxine content of blood serum using radioactive thyroxine as an indicator substance. 23 ») Process for the production of a reagent which is used in the Determination of the thyroxine content of the blood serum used 24.) Reagent for use in determining thyroxine * content of blood serum. 25.) Equipment for determining the thyroxine content of blood serum. 0098A5 / 1427