Note: Descriptions are shown in the official language in which they were submitted.
}~ 7'3/~
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
-2- ~
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.,
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. ~ .
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llOI` 7~/~
1038289
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 ~
.:
~ 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
~..... . .
~ 29 Or the free moiety of the hormone from the solution and its
~' .
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, . . . . .
... . . .
. - .,
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, - . , .
,:
1038289
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.
.
~ 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-
.
,.. : - .- , .
:: :
}IOE 7~/F 1~
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.
. .
-- - 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 usej
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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.:, ~ .:. .
. ,` :
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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
:
, r
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- 103 8 2 89
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-
,, .
:. _g_ -
' ' - ~ , : .
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:
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llo~ 7~
` ~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.
, . .
~ 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,
.~, .
, - 1 0-
- -:. -: - . - ,, . - .
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.:.- . ,,, . . . , . -
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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
.. . .
.i ~, _ 1 1 _
"'~.- `' ' ~ ' " :
'
~101~, 7'3/1? 1 1~1~
~.0382W
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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,
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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 :
:~ .
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-
: ' '
. ~ -' .
~0382~ or~ 7'3/~? ~
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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