DE2261561A1 - PROCEDURE FOR CARRYING OUT ANALYSIS BASED ON COMPETITIVE REACTIONS - Google Patents

Description

The invention relates to analyzes in which a radioactive element or a radioactive compound 2 ?; i se ... ei: divided into two phases and the proportion of activity in each phase is determined. Typical for such analyzes are Evaluations of the above measurements through competing reactions, whereby an unknown Henge of the connection, which is to be assessed, as well as a Stanäordruenge of a rauioaktivi; arkierterj Version of this compound when reacting with one Standard amount of another reagent in competition with each other step. This way of working is very widespread in medicine for the determination of hormones and other substances, where the other reagent is the antibody in immense?; or another specific lieaktionsportner in HicUtinjrnun- or ITiohthoroionsjsteraen. used viird.

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The principle of this mode of operation can be achieved by the following reactions schema can be reproduced:

0 is the connection that should definitely be grounded; C "is the radiolabelled version of this compound; R is the other reactants.

The amount R is measured in such a way that it is not sufficient to deal with

videm entire C + C to respond. Since the reaction is reversible at least to a certain extent, an equilibrium is established at which the ratio of / c 17 / ßi'J - \ - is determined by the close proximity of the unlabeled compound G. If G is changed from G - R separated and measured the activity of the two separate parts, the Heß number of the ratio can easily be calculated. The amount of unlabelled compound C can then be determined relatively or absolutely by using a standard preparation of compound G to draw up a curve.

This procedure is illustrated with examples in a journal by R.S. Yalow and S.A. Berson in IAEA-SIi-124/106, pp 455-481.

The separation of C "from G-R is generally two-step

x- ·; <performed. The first stage is either G or 0 - R caused a phase change, for example by failing from a solution, by absorption on an inert carrier or by transition to one that cannot be mixed with water Phase. The second stage is the two phases separated from one another, for example by filtration or - more commonly - by centrifugation and then Pour off any excess liquid. As soon as the two Phases are present separately in two different containers

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it is a simple matter of activity level or level to be determined in each container «,

The separation of both phases by transferring one phase from the reaction tube to another Bell container is however wasteful and also cause for inaccurate measurements or even kess errors. In particular, the entire material is rarely transferred from one container to the other. The object of the invention is therefore to avoid transfers of this known type.

This problem was solved wildly with the help of the invention Process for carrying out analyzes in which a radioactive element or a radioactive compound is distributed between two phases, which is characterized by the fact that one determines the level or level of radioactivity measures of at least one of these two phases while this phase is in contact with the other phase.

At least some of the radioactive material in one Phase must precede the radioactive material in the other phase can be separated in order to determine the level of activity of one phase. It is characteristic of the erfindungsgeraässer Procedure that the two phases themselves, not from-'each other removed v / ground, for example by transferring one phase to another vessel, but during the Kessung the degree of activity in one or in both phases stay in contact with each other.

The difficulty in measuring the level of activity in one phase while the other phase is in contact with it steiit, lies in the difficulty of the device for counting üe :: Activity in this one phase before that of the other PL.ose given Mtraiiluugei]. Jüas according to the invention

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Procedure provides two ways of overcoming this difficulty; both cages can either be independent of each other or - more commonly - applied in combination with one another will:

1) The reaction tube can be specially shaped, for example if the radioactive element «wipe one Liquid and a heavier / iest phase is distributed, for example as a centrifuge tube with a thin Capillary ata soil for the solid phase, so that this solid phase is at a noticeable distance from the bulk of the liquid Phase is present in the main body of the tube. (If the radioactive element is to be distributed between two liquid phases, a corresponding design of the tube is possible).

2) The counter can be designed or used precisely as a collimator and carefully shielded from the phase, whose activity is not measured.

The inventive method can be particularly applied to those Use cases in which the radioactive trace element involved is a soft garaaa emitter, so that a ziehalich thin lead layer can be used for shielding.

1 25
The usual I is particularly suitable because its v / cal gamma radiation is weakened to an insignificant remainder by a 3 ma layer of lead.

If a centrifuge tube with a capillary at the bottom is used as the reaction container, the degree of activity can be determined Measure the solid phase by shielding the body, i.e. the main part of the tube from the meter, whereby. only a very small error results from the addition of a very small, narrow liquid phase in the capillary tube. Of the Activity level of the liquid phase in the main body of the Reol: - tioiJGi'QhrcLeno wi ..- ύ then eaten, indeai da.o capillary tubes

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is shielded in front of the meter, with only a very small error resulting from the absence of the very small amount of liquid phase that has passed into the capillary tube.

In numerous applications, the Radioactivity is not required in either or both phases, provided that the percentage error is not is unnecessarily wide and also remains constant. Such a constant percentage error can, for example, with normal methods of iaaunological radio analysis (radio alarm assay), in which a calibration curve with reference to Establishing a standard preparation will lead to acceptably good results.

The method according to the invention will now be described in more detail with reference to the attached drawing in some exemplary embodiments explained. In the drawing show:

1 shows a perspective view of a conventional centrifuge glass with a conically tapering or pointed bottom, which contains a liquid with a solid suspended therein;

Fig. 2 is a perspective view of the centrifuge tube after centrifugation;

3 is a perspective view of a partial shielded holder; the recess for the centrifuge tube is indicated by dashed lines;

4 is a perspective view of the holder with the centrifuge tube located therein;

Fig. 5 is a perspective view of a shielded Counting device, partly in section, around the inside Tube with Ij solution and solid to show sediment.

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That in Pig. 1 and 2 shown centrifuge tubes 10 consists made of a plastic, has a closure 11 and contains a liquid phase 12 and a pesticide phase 13, of which one is radioactive or both are radioactive. In Fig. 2, the pesticide phase 13 is tapered or the top 14 of the tube 10 has been concentrated by centrifugation.

The partially shielded one shown in FIGS. 3 and 4 Holder 15 consists of a lead base 16, which sit an annular Upper part 17 made of plastic is connected. A centrifuge tube fits into the holder so that the Pointed bottom 14 and the solid material located therein is surrounded by the part of the holder made of lead.

The complete unit (holder 15 + centrifuge tubes 10) can be placed in the recess of a sodium iodide scintillation crystal be fitted, which part of an instrument ζυω let ssen the radioactivity (crystal counter). Shielded from the counter through the lead base 16 of the holder is the solid phase 13 and a small part of the liquid Phase 12, located in the top floor 14 of the centrifuge room is located. The measurement error resulting therefrom, i.e. not the entire liquid phase is visible in the meter, can generally be neglected v / ground, except there, where very precise measurements are required; he is in jeäea Case insignificant if the radioactivity levels or levels of liquids in different tubes of the same training can be compared with each other.

. The> with reference to Figures 1 to 4 described 3. 3rd; 3 ict ünzvt provided, the radioactivity of eggs virtually total liquid PLu ce Ur to measure centrifuge tube. Ue η η jeüocvi unknown samples are measured against calibrated standards,

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so the whole phase need not be eaten. It is then sufficient to ensure that the volume of liquid in the various used in the experiment Tube is the same and the radioactivity of one measure constant volume of the liquid phase. This constant volume "need not be known; it must but make up a constant proportion of the total liquid phase, such as with different, im tubes used in the same experiment.

This principle is explained with reference to I ¹ Ig. 5 explained in more detail: the counting device comprises a (not shown) sodium iodide scintillation crystal, in the recess of which the lead shielding 18, 19 has been arranged or installed. A tube 20 with a closure 21, which contains a liquid phase 22 up to the mirror (surface) 23 and a sedimented solid phase 24, fits exactly into the lead shielding. The upper and lower parts 18 and 19 of the lead shield limit the field of view of the counting device to a fixed column 25 of the liquid phase 22. Provided that the diameter of the column 25 is fixed, i.e. provided that the inside diameters of the tubes used in the experiment are all the same and provided that the surface of the liquid 22 in the top 19 of the lead shield in each tube is always at the same level, the counter measures the radioactivity of a constant proportion of the total liquid phase, as between different tubes used in the same experiment.

This procedure can be modified to that the liquid surface 23, the liquid level, is located within the fixed column 25. in the field of view of the counting device. However, this alternative will

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not preferred because the surface 23 is always yarn; have exactly the same height for different tubes used in the same experiment nut.

Errors occur when the tubes do not all have exactly the same inner diameter or when the total volume the liquid phase is not exactly the same in each tube; however, these errors are in practice does not matter unless extremely accurate measurements are sought and needed.

Example General Methods '? ·

The insulin inraonoassay kit analysis unit manufactured by The Radiochemical Center and based on the method of Haies and Rändle (Lancet, i, 200, 1963; 3iochera, J., 88, 137, 1963) was used to deny the use of the Heßsysteas explain. A generally recognized or approved serum was used as "unknown". This Serum had been scored 3 times on more than 15 occasions using the standard filtration method. The measurements ranged from 43.0 to 58.5 milliunits each era rait an overall mean of 49.7.

Procedure

The procedure was carried out exactly as outlined in the instructions for use enclosed with the analysis unit is, with the exception of those used to prepare the incubates volumes used: 300 / ul were used instead of the specified 100 yul geiJOüv.neD. As a result of this greater volu ^ cnu could use the existing shielded containers for

by doing

a noun volume of 1 era ^{5 is} created, without modification;

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Insulin stabilization was 250 * 1ΌΟ> 5Ö »25 *" 12 * 5

"null" Miksöeisheiten f / tiS) / "Gtä ^ produced, JeSe K © nzesträtioo was prepared in 3-iSchif execution, same ΫώΙ ^ ώιώθ by ZQ & j & i in

4-fold iieiegesiielliii itiäöffl each 500 ^ ΐϊϊ Serial) In eiäneni similar ESlilciieia submitted

each) Röliiülien "wurtäea 3007 ^ 1 binding reagent given the & ai3 ^: ze Te ^: rt3i" söh% »The EBUrekeü was te-rsciilöBseiii and worked for 6 hours at 4- ^Θ Θ; it was i gleiclier iö1> / added to each Eöiirciien a eil 300 ul 125-1> -ϊη3υ1ιή The Iniialii the Eöiirölien was getoisclit., the Röhrclien were verschlössen and a further 18 hours Dei ialten 4 ⁰ C ge /.

liacii "upon completion of the incubation, the tubes became 6 minutes long in a "Tinipaη 320" centrifuge toit 14 500 rpm (Η 000 g) hurled. They were then placed in the closed holder described above, and each tube was placed for 60 seconds long in a Hucleär Sn-terprises Äutö- &admä3; ähler. Type 83II counted, '

Results: Values obtained in clipped containers.

insulinyuE / Gs ö / rnin 3 measurements

"zero" 43169 44325 Φ3391 12 * 5 SG2i6 5Ö96O 49519 i .25 57H4 57108 56710 5Ö 6BI91 67349 ^ 579 j 1ΟΘ T6Ö45 76520 764Q9 \ 250 8Ö559 80705 62078

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c / rain found ^ / uE / cö ⁵ insulin mean value for in · ¹ unknown ¹ 'Sera readsimerit ⁿ Wöbekaant "

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The result obtained for the insulin concentration of an approved serum using the separate measuring system is very favorably comparable to the result obtained with the help of the usual metaphoric filtration

As can be seen from the above description, the material nature of the analysis is not, erfinuungsv / eöeir-liGii. Imnuno assessment and similar measurements or assessments based on competing reactions »v / as described above! Are Examples of suitable anal; ysenforcien »The radioactive involved Element is preferably a soft osteoarthritis, but the nature of the two phases and you ilethode, according to which the radioactive element between the piiases distributed do not play an essential role. In the same Any suitable means of advising the Degree of activity in one of the two phases or Ia both Phases are applied »

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Claims (9)

P A 3} El ·! T A H S PR Ü C H E 1.) Procedure for carrying out competitive actions based analyzes "in which a radioactive element or a radioactive compound between two ' Phases distributed is characterized by that one has the degree of radioactivity of at least one the "two phases determined, while this phase with the other phase is in contact. 2. The method according to claim 1, characterized in that the measurement on two in one single container makes present phases. 3. The method according to claim 2, characterized in that the radioactivity of the one Measure phase with a measuring device, in front of which a part the contents of the container is shielded. 4 «Method according to one of claims 1 to 3 thereby know that you can use a liquid Phase and a denser solid phase works. 5. Method according to claim 4, characterized in that g ek e η .η - draws that one corresponds to the solid phase in one shaped tubes during the measurement in aerklichera Keeps distance from the Ila-up amount of the liquid phase, 6th The method of claim 4 or ρ characterized geke η η ζ eie Ii net that is in Heßvorrichtungen - 12 - 3098287 07 22S15B1 cJerWise as a collimator and shields that one measures the radioality of virtually all of the liquid phase but not the solid phase. 7. The method nacii claim 4 or 5, characterized in that the Ileßvorrichtung in such a way as a collimator and shields that aan measures the radioactivity of only a selected area of the liquid phase. 8. Procedure according to. Claim 4 or 5, characterized in that the measuring device in such a way as a collioator and shields, that as η measures the radioactivity of the solid phase but not the liquid phase. 9. The method according to any one of claims 1 to 8, wherein man (a) in a tube provides: 1. A known volume of a liquid containing the compound to be assessed, 2. a standard version of a radiolabelled version of the compound to be evaluated and 3. a standard amount of reagent sufficient to υπ reversible with part of the compound to be assessed and with part of the radiolabelled version this compound * which gives the compound to be evaluated and its radiolabelled version sv / isclien the liquid phase and the solid phase are distributed, (b) the solid phase is located at the bottom of the tube and (c) the level of radioactivity from at least a part of the liquid phase, characterized in that when performing the 3tiue to respond _Λ ~. - λ j - 309828/0731 (c) measures the degree of adioactivity of at least a portion of the liquid phase while this liquid phase remains in contact with the solid phase in the container. ■. 309828/0731 ft Blank page