CA1038289A - Process and device for blood examination using substances labelled with radioactive nuclides - Google Patents

Abstract

Abstract of the Disclosure A process and a device for determining hormones in liquids is described by introducing determined amounts of a solution of the radioactively labelled hormone and the antibody or the hormone-building protein and a determined amount of the liquid to be examined into a measuring vial, connecting it with an adsorption vial the inner surface of which shows adsorptive properties as to the hormone to be determined, contacting the liquid therein with the inner surface of the adsorption vial and measuring thereafter the residual activity of the liquid.

Description

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1a382~39 The present inverltion rolates to a process and a de~ice for the blood examination usin~ substances labelled with radioactive nuclides, More particularly this invention relates to a proce~s and a device for the determination Or hormones in liquids using compounds labelled with radioactivc nuclides.
The hormones, synthesized in certain glands of the body and delivered to the circulating blood, are present in very low concentration. Only the use of indicators labelled with :, .
` 10 radioactivity has permitted to perform their exact quantitative determination.
According to the chemical nature of the hormone under consideration two different methods for analysis are used.
-When having immunogenic properties, that is to say, when being r 15 able to produce suitable antibodies, the hormone is determined ~`~ by the Radioimmunoassay (RIA), when being devoid of immunogenic properties it is determined by the competitive protein binding I analysi-c. The two methods have in common that a certain ;~ amount of the hormone must be èliminated from the reaction ~i 20 solution by means of an ion exchanger or another substance uitable for this purpose, - -According to known radio-immunochemical determination methods~ for example a mixture of a known amount of a hormone unlabelled and the same one labelled with radioactivity is reacted with a specific antibody building a complex. The hormone is labelled with a suitable nuclide, such as, for example 125I 131I 14C, 3H. Between the antibodY and the hormone a reaction equilibrium is brought about, slight " ~e .
29 amounts of the hormone being unbound, With constant antibody

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concentration thi~ free part i~ directly proportional to the total amount of thc hormone added. It i5 eliminated by means of a ~uitable adsorption agent and determined. A standard curve is drawn up permitting the determination of an unknown amount of the hormone.
In the competitive protein binding analycfi~ which is also Xnown, advantage i9 taken of the fact that hormones, especially those having a very low molecular weight, are bound and transported in the blood by specific proteins, for example, ~` 10 in the case of the two thyroid hormones l`hyro~ine and ~' Triiodothyronine, by an ~-globuline, the "Thyroxine-Binding-Globuline" (TBG). These proteins can be used as primary complex forming agents for the hormone concerned, similar to -f s -the antibodies in the radioimmunochemical methods. The free - -15 hormone is separated from the solution and determined.
~ Another embodiment of this process is the determination :;~ - . .
of the capacity of the binding proteins in the serum. A
slight amount of the radioactively labelled hormone is added --to the serum in which the capacity of the binding protein for ~
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~ 20 the same hormone is to be evaluated. Between the binding ''~'f protein and the labelled hormone a reaction takes place in 1 which the labelled hormone partially replaces the bound in-- active one and partially reacts directly with the binding , .
protein. The unbound moiety is eliminated from the solution '! 25 by adsorption and is determined. This free part is reversely f~'j proportional to the free capacity of the serum protein under consideration.
sA, ~ In all these methods, the common step is the elimination ~..... . .
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determination. Hitherto, ion exchangers, such as Amberlite(R) (registered trade mark) in the form of grains or embedded into ` polyurethane sponges or as strip, have been used for this purpose.
After the incubation of the solution with the adsorbing agent all these methods require additional time-consuming steps implying sources of error. Thus, when using ion exchange grains centrifuging is necessary after incubation in ` order to permit an unobjectionable pipetting of the super-natant. Sometimes, the grains are additionally washed several . ~
~ times before the measurement. It is true that the use of .
strips and sponges is an advance upon the grains but it is complicated and time-consuming, because the strip must be kept , moist before its use and its elimination from the solution canlead to erroneous results (drops on the strip). When using the ion exchanger sponge care must be taken that the air is eliminated from the sponge before the adsorption procedure ~ and the sponge is thoroughly washed after the incubation.
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~ The present invention provides a process for determining ;~ 20 the concentration of a hormone or a protein in liquid, which ~ comprises mixing, in any order, the following components: (i) a ~;
sample of the liquid containing the hormone or protein, (ii) where '-;f~ appropriate, a known amount of a complementary substance capable of forming a complex with the hormone or protein, and (iii) a known ..
-~ 25 amount of the hormone, protein or complementary substance which has -~ been labelled with a radioactive nuclide, measuring the radioactivity ^ of the resulting liquid, contacting that liquid with the inner --; surface of an adsorption ves.sel, said inner surface being capable .~, .~ .
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. ~ ' 10382~9 of specifically adsorbing the hormone or protein and aomprising a thermoplastic material containing from 10-60% by weight of an ion exchanger, removing the liquid from the adsorption vessel and measuring the radioactivity of the removed liquid.
It is a preferred embodiment of the process of the invention to stack or screw the adsorption vial onto the measuring vial so that the liquid is brought into contact with the inner surface of the adsorption vial several times and to turn the device consisting of the measuring vial and the adsorption vial round an axis in 90 position to the cylinder axis. It is also possible to shake the device.
The process is carried out by introducing into the measuring vial the liquid to be examined which contains the hormone and the binding protein or the antibody and, optionally, additional solutions of substances as well as the radioactively c labelled hormone, putting on the adsorption vial, closing the device and measuring the radioactivity of the liquid in the measuring vial. The d~vice (measuring and adsorption vial) is '~ fixed on a rotator and turned for a definite period of time round an axis in 90 position to the cylinder axis. During this process the solution comes into contact with the inner . surface of the adsorption vial several times, the free moiety of the hormone being adsorbed and thus eliminated from the solution. After the adsorption being completed, the device is put upright to allow the liquid to flow from the adsorption vial down to the measuring vial and to leave back the hormone :' .

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. 1038Z~9 moict~ adsorl~od at thc ~urfncc. In doille 50 ~ the two moiotics are scparated. The ~ctivity still remaining in the solution is no~ measured, for examplc in a well type scintillation counter or in an automatic gamma sample changcr without taking off the adsorption ~ial. The fact that the adsorption vial need not be taken off is a great advantage because contaminations thus are avoided. Pipetting is not necessary, either.
The process of the invention advantageously allows to determine the concentration of a hormone in the blood. The TBG capacity is determined, for example~ by introducing into a measuring vial a sufficient amount of radioacti~ely labelled ` L-triiodothyronine (T3) dissolved in a suitable buffer and ~ , .
`; adding the serum to be examined. The specific activity of the T3 used can range between 10-200 mCilmg T3 which values may also be lower or higher, in principle. As buffer solution .j , .
i any system can be used which has a sufficient buffer capacity -~ at pH 5-9, for example, tris(hydroxymethyl)-amino-methane/HCl or phosphate according to Sorensen. Advantageously, the pH of the buffer solution ought to be 7, but it may also be higher or lower. The T3 reacts with the TBG whereby it is bound by - it for the greatest part. Now, the adsorption vial is stacked ,, .
on, the whole device is closed and the initial radioactivity is measured, for example, in a well type scintillation counter or -1 an automacic gamma sample exchanger. The length of the ad-.j .'.
sorption vial stacked on can, ad~antageously, vary between 1 and 6 cm, depending on the number of the adsorbing places per surface unit. The device according to the invention is fixed on a rotator, for example, allowing it to rotate over a - 2g dctermined period of time, the liquid being brought into contact . .s ~ 6-.
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1038Z~9 several tlmes witll the innor surface of the aflsorption vial.
During this process, thc T3 - labell~d and not labelled - i9 adsorbed in the adsorption vlal and so eliminated from the solution. After the adsorption boing completed, the device i8 put upright and the activity which has remained in the - solution is measured after several minutes, as it has been described.
The process of the invention can be used in corresponding manner for the absolute determination of the thyroid gland hormones Thyroxine and Triiodothyronine. For this purpose, . a TBG solution of constant capacity is necessary by means of which a standard curve is drawn up. The T~G solution can be prepared according to known processes of column chromatography.
To that solution, radloactively labelled Thyroxine (T4) is added which is bound according to the equation T4 + TBG
TBG - T4. When unlabelled Thyroxine (T4) is add.ed to that solution, a part of the labelled Thyroxine bound to the TBG is expelled and is under free form. As this amount of the ~:'L~ ' expelled labelled thyroxine is directly proportional to the unlabelled thyroxine added, a relation between the free T4 and the T4 added is established which can be illustrated diagram- -matically in the form of a standard curve.
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-- - The T4 content in the serum is determined by setting free . .-1 .
-; the thyroid gland hormones by denaturating the binding proteins ~5 with alcohol. The alcoholic extract of L-Tr~iodothyronine (T3) and Thyroxine (T4) can be evaporated to a concentrate which can .-.,~, .
, be redissolved. An aliquot~ part of the solution is given to : "~
the TBG-T4*solution in the measuring vial by pipetting, the 29 adsorption vial is put on in accordance with the invention and ..~
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10382~39 turned, for example, for l hour similar to the T3 determination.
In this process the free T4* is adsorbed in the adsorption vial and thus eliminated from the solution. The amount of T4 of the serum to be examined can be evaluated from the quotient residual radioactivity/initial radioactivity by means of the standard curve. The value read off is indicated to be T4 because the amount of T3 is very small in comparison thereto.
The process of the invention can be used in corresponding manner to determinerthe hormones by radio-immunochemical methods (Radioimmunoassay).
The present invention also provides a device, for use j in the process of the present invention, said device consisting of an J. adsorption vessel detachably connected to a measuring vessel, the measuring vessel being closed at the bottom and capable of being ~ 15 fed with the liquid to be examined, a solution of the hormone labelled with radioactivity and the antibody or the hormone-binding protein, and the adsorption vessel i$ capable of being detachable - connected to the measuring vessel at the lower extremity of the ad-sorption vessel and capable of being closed at its upper extremity, and said adsorption vessel having an inner layer, consisting of a thermoplast containing 10-60~ of ion exchanger, which has adsorptive properties in relation to the hormone to be determined.
~ The device of this invention is illustrated diagramma-s~ tically by way of example in the accompanying drawing in which is referred as follows: measuring vial (1), liquid, solution .
~ of the radioactively labelled hormone and the antibody or the - hormone binding protein to be examined (2), adsorption vial (3), :, ; closure (4), adsorptive layer (5).
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..1~3B~9 The measuring vial can be made of glass or of plastic materials, preferably of polyethylene. Its length and breadth mey vary and depend on the volumes avail~ble of the liquids to :

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be exmnined or the diameter of the well type scintillation COUllt er.
The adsorption vial consists of a thermoplast which contains 10 - 60 % by ~eight of an ion exchanger. High-pressure polyethylene is preferred which contains 20-40 ~ by weight of an ani,on exchanger carrying quaternary amino groups capable of being ionized. The length of the adsorption vials depends on the exchanger activity and is advantageously 1 to 6 ' cm.
-` 10 The adsorption ~ial is advantageously manufactured by ~ , .
'~ thermoplastic transformation of homogenised mixtures of thermo-plasts and suitable ion exchangers. Suitable thermoplasts are the polymers which are transformed at a temperature ranging -from 100C to 200 C, preferably from 1 30c to 170C. There may be used, for example: ' ,~ Polyethylene: high-pressure polyethylene having melt indices ~-~ ; (i5) of from o.3 to 70, preferably from 0.3 to ''j 10, measured according to German standard DIN
~ - . 53735 at 190C.
,~ 20 low-pressure polyethylene having melt indices :.v .
s . . (i5) of from o.3 to 30, preferably from 10 to 30, measured according to German standard DIN
53735 at 190C.
'-,' Polypropylene having melt indic,es (i2) of from 0.4 to 40, pre-. ferably from 5-30, measured according to German standard DIN 53735 E at 230 c .
~, ~ Polyoxymethylene: obtained by homo or copolymerization of -~ trioxane and formaldehyde and cyclic acetals ;~ 29 ha~ing melt indices (i2) of from 1 to 50, prefer-,, .
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` ~038Z~9 nbly from 15 to 50 measured according to German - standard DIN 53735 nt 190 C.
Polystyrenes having melt indices (i5) from 2 to 30, prcfcr~
ably from 5 to 25, measured according to DIN
` 5 -~ 53735 at 200C.
Poly(meth)acrylates having melt indices of from 0.4 to 10, preferably from 5 to 10, measured according to ASTMD 1238-62 T~
Polyvinyl chloride having K-values of from 40-80, preferably from 50-70, measured according to DIN 53726 in cyclohexanone at 25C.
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~ Polyester from dibaslc carboxylic acids and diols~ among -~ which polyester from linear aliphatic dicarbo~-ylic acids ha~ing 4 to 12 carbon atoms and ' 15 - ~,~-diols having from 2 to 8 carbon atoms, for ~' example, sebacid~acid/ethylene glycol polyesters ~ are especially suitable.
-~ Polyamides from dibasic dicarboxylic acids and diamines, the melting point of the polymers being reduced ~, 20 by the introduction of, for example, ether, .~ .
P~ methylol or ester groups or by cocondensation.
- As ion exchangers~ there may be admixed anion and cation !' exchangers having medium particle sizes from 0.04 to 1.0 mm -~ ~ diameter, preferably from o.o8 to 0.2 mm diameter. The -, exchange capacity is 0.3 to 3 Yal/l, preferably 0.5 to 1.5 ~ Val/l, Generally, the exchan6e resins are cross-linked, the -~:
eross-linking portion ranging from 1 to 12 ~o, preferably from 4 to 10 ~. Suitable ion exchangers in powder form available 29 in commerce are Amberlite (R) CG 400, Amberlite(R)IRA 402, .~, .
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~T0~ 73/l` 14ll 1W82~39 Ambcrlitc(R)200 nnd Dowex(l~) (registered trade marks).
The mi~tures of thermoplasts and ion exchangcr, which Advantageously contain from 10 to 60 % by wei~ht, prefcrably from 20 to 40 % by weight of exchange resin, are homogenized on rollers, calanders, kneaders or extruders, preferably double screw extruders, at a temperature ranging from 100C
to 200C, preferably from 130C to 170C.
The adsorption vials can be manufactured on extruders or on injection molding machines.
In the extrusion procedure, the homogenized mixture is preferably carried through a tubular die at a temperature ` ranging from 130C to 170 C and synchronously talcen off over a gaging tank under a negative gage pressure of from 1 to 15 m - water column, preferably from 2 to 10 m water column. The- t5 endless tube thus obtained is cut to the length corresponding to that of the adsorption vials and the outer surface is turned - off at the extremities to guarantee a tight closure between the adsorption vial and the measuring vial and the cap.
In the injection molding, the mixt~re is worked within the same temperature range. The tube thus obtained has the dimensions necessary for the tightness of the device of the invention. Afterwards1 the inner surface of the shaped article can be bored open in this case in order to improve the adsorptive properties.
, - 25 The process and device of the invention allow a simpler ,.~ , .
and safer determination of the hormones, especially with ~-~ respect to the separation of the free moiety of the hormone .' '~ ~ .
-~ from the solution. Complicated steps, such as pipetting, - 29 centrifugin~ and washing are no longer necessary. The values .. . .

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obtained are very ~cll reproducible.
Surprisingly,it was ~ound that the adsorption vial Or thermoplastic material and ion oxchan~er did not show any swelling processes ~hich could make burst the measuring vial ; 5 being tightly connected.
Examples of manufacture (adsorption vial) E X A M P L E 1:
Low pressure polyethylene having 30 ~ of Amberlite(R)CG
400 I (registered trade mark), chloride form, was dried ever 10 night under reduced pressure and granulated on a double screw extruder after mixing through. The granules were dried~ and an extruder was used to produce an endless tube. From the tube produced according to that process pipes 6 cm long were cut and used for adsorption.
15 E X A M P L E 2s High pressure polyethylene having 35 ~ Amberlite( )CG
~ 400 (registered trade mark) in chloride form were dried for ``~ 24 hours under reduced pressure, thoroughly mixed and granu-lated on a double screw extruder~ The dried granules were -~s 20 used for the production of an endless tube from which pipes 5 cm long were cut and used for adsorption.
E X A M P L E 3:
Granules of high pressure polyethylene having 35 ~
~--` Amberlite CG ~00 I (registered trade mark) in chloride form ~~ 25 wereused for the production of injection-molded pipes 5 cm long.
~ - Example of application: determination of the T~G capacitY
t a) Preparation of the L-triiodothyronine-I125 solution.
A corresponding amount of L-triiodothyronine-I1 S ~ ~ ) 29 haYing the specific acti~ity 100 m~i/mg T3 was added to a .
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solution of 1 r tris(hydroxyllletllyl)-aminol~ethalle tho pll of - which was adjustod to 7.4 with conc.h~drocllloric acid, 90 that the concentration of radioactivity wa9 about 0.7 ~Ci/ml.
b) Standard serum Serum having normal TBG binding capacity obtained from the blood of human beings having a thyroid gland with normal function.
c) Determination of the TBG capacity.
About 5 ml of blood were taken from the patient the thyroid gland function of whom was to be examined. From the serum obtained therefrom after coagulation and centrifuging ` ` of the blood corpuscles 0.2 ml were pipetted into a measuring :~4 vial made of polypropylene which contained 3,3 ml of the T
-solution, the adsorption vial manufactured according to ex-amples 1 to 3 was put thereon, the device was closed and the whole was allowed to stand for about 10 minutes. The corres-. ~ .
ponding procedure was applied to the standard serum, During that period of time the total radioactivity of the liquid ~-~ was measured in a well type scintillation counter or in an automatic gamma sample changer. Thereafter, the device was fixed on a rotator and turned for an hour with 13 revolutions per minute. The device was set up, the liquid in the adsorption ~ial was allowed to flow downwards and the activity remaining ~i in the liquid phase was measured. The measurement was per-- 25 formed by calculating the quotient G = residual activity :
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initial activity as to the patient and the standard serum and the thyroxine binding capacity in percentages of the patient serum according to the equation TBK f~ = G(patient s~um) . 100 ~ G(standard 5 eru~) ; -13-: ' ' . ~ -' .

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Generally, the T~K value rises as comparad to the normal serum in the prescnce of n hypothyrcosis and falls in the prescnce of a hyperthyreosis.
E X A M P L E 5:
Determination of the thvroxine concentration in the serum 0.5 ml each of the serum to be examined and t~e control eerum wlth a known amount of not labelled T4 were introduced ~- each time in a vial capable of being centrifuged of each 1.0 ml and mixed on a whirl mixer for 30 seconds. The mixture was allowed to dwell for 10 minutes and the denaturated proteins were centrifuged at 2500 rpm. The extraction yield was 72 ` ~ of the T4 initially present.
; The thyroxirecontent was determined by pipetting each 0.3 ml of the alcoholic extract in measuring vials which ~j 15 contain 3.3 ml of a TBG solution prepared according to kno~m `~ methods of column chromatography and 0.02 ~uCi/ml of thyroxine ~ labelled with 125I. To draw the standard curve each 0,3 ml of a standard solution of 5 and 20 JUg of thyroxine/100 ml were ' .-'1 - .
~ treated in the same manner. The adsorption vials were put - .s;
on the measuring vials, the devices were closed and the total l~ radioactivity of the solutions was measured after thorough ~ mixing. To adsorb the free T4- 25I-activity the devices were ~ . .
turned headover on a rotator at 13 rpm for 60 minutes at room temperature. The devices ~ere set upright so that the solution i l 25 flowed from the adsorption vials entirely into the measuring , ~,-, , .
vials~ where the res~dual radioactivity ~hich had remained in the solution ~as measured. Then, the quotient :...... .
G = residual ra~ioactivity . 100 -~ total radioactivity ;. ~,.. ~
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103~2~39 was calcul~tcd for eaCII sample. Tho ~alues obtained for the standarcl salllples were insert;ed into a dia~ram ag~inst the amounts of thyroxinc contained in the standard solutions and the two points were ~oined with a straight line, thus ~i~ing the standard curve. The unknown content of the sera to be examined would nowbe evaluated by means of the quotient G of the standard curve. The thyroxine content of the serum results from the value read off and the extraction yield.
The TBG solution mentioned abo~e was prepared from a , .
fraction containing TBG which was obtained upon purifying the ; serum by column chromatography (so-called "cast II" (~bguB II) according to K. Heide and H, Haupt, Behringwerke Mitteilungen , vol. 43, 1964, 161). In this process the corresponding -, fraction is, at first, dialysed against water and then diluted ~ 15 with 0.1 M tris-(hydroxymethyl)-aminomethane/HCl buffer to give .,~d ~ a total protein content of 0.022 g/100 ml. The solution was ~ stabilized and conser~ed by adding 0.02 ~ by weight of NaN3.

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Claims (14)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for determining the concentration of a hormone or a protein in a liquid, which comprises mixing, in any order, the following components: (i) a sample of the liquid containing the hormone or protein, (ii) where appropriate, a known amount of a complementary substance capable of forming a complex with the hormone or protein, and (iii) a known amount of the hormone, protein or complementary substance which has been labelled with a radioactive nuclide, measuring the radioactivity of the re-sulting liquid, contacting that liquid with the inner surface of an adsorption vessel, said inner surface being capable of specifically adsorbing the hormone or protein and comprising a thermoplastic material containing from 10-60% by weight of an ion exchanger, removing the liquid from the adsorption vessel and measuring the radioactivity of the removed liquid.

2. A process as claimed in claim 1, wherein the components (i), (iii) and, where appropriate, (ii), are mixed and the radioactivity of the resulting liquid is measured in a measuring vessel, and, after the liquid has been contacted with the inner surface of the adsorption vessel, the liquid is removed to the same or another measuring vessel.

3. A process as claimed in claim 2, wherein the adsorption vessel is attached to the measuring vessel before or after the radioactivity of the liquid in the measuring vessel is measured.

4. A process as claimed in claim 3, wherein the adsorption vessel is attached directly to the measuring vessel and the liquid is caused to return to the same measuring vessel after its contact with the inner surface of the adsorption vessel.

5. A process as claimed in claim 1, claim 2 or claim 3 in which the liquid is brought into contact with the inner surface of the adsorption vessel several times by turning the attached measuring and adsorption vessels around an axis 90° to the cylindrical axis.

6. A process as claimed in claim 1, claim 2 or claim 3 in which the concentration of hormones in the blood is determined.

7. A process as claimed in claim 1, claim 2 or claim 3 in which the TBG capacity of the blood serum is indirectly determined, the radioactively labelled indicator used being L-triiodothyronine labelled with 125I or 131I.

8. A process as claimed in claim 1, claim 2 or claim 3 in which the concentration of thyroxine in the blood is determined, the radioactively labelled indicator used being thyroxine labelled with 125I or 131I.

9. A device, for use in determining the concentration of a hormone or protein in a liquid using a radioactively labelled compound, said device consisting of an adsorption vessel de-tachably connected to a measuring vessel, the measuring vessel being closed at the bottom and capable of being fed with the liquid to be examined, a solution of the hormone labelled with radio-activity and the antibody or the hormone-binding protein, and the adsorption vessel is capable of being detachably connected to the measuring vessel at the lower extremity of the adsorption vessel and capable of being closed at its upper extremity, and said adsorption vessel having an inner layer, consisting of a thermoplast containing 10-60% of ion exchanger, which has adsorptive properties in relation to the hormone to be determined.

10. A device as claimed in claim 9 in which the adsorption vessel is formed from a thermoplast containing 10 to 60% of ion exchanger.

11. A device as claimed in claim 9 in which the adsorption vessel is formed from high pressure polyethylene containing from 20 to 40% by weight of an ion exchanger.

12. A device as claimed in claim 9, claim 10 or claim 11, in which the ion exchanger used therein has a medium particle size of 0.04 to 1 mm diameter, an exchanger capacity of 0.3 Val/1 and a cross-linked moiety ranging from 1 to 12%.

13. A device as claimed in claim 9, claim 10 or claim 11 in which the ion exchanger used therein has a medium particle size of 0.08 to 0.2 mm diameter, an ion exchanger capacity of 0.5 to 1.5 Val/1 and a cross-linked moiety of 4 to 10%.

14. A device as claimed in claim 9, claim 10 or claim 11, in which an anion exchanger is used carrying quaternary amine groups capable of being ionised.