DE2261561C3 - Methods for Carrying Out Competitive Response Analysis - Google Patents

Description

The invention relates to a method for performing competitive reactions Analyzes according to the preamble of the claim.

Such a method is from US Pat. No. 3,615,222 known.

Typical of such analyzes are determinations by competitive reactions, with one unknown Amount of the compound to be determined and a standard amount of a radiolabelled form this compound competes in the reaction for a standard amount of another reagent step with each other. This working method is very widespread in medicine for the determination of Hormones and other substances, being the other reagent of antibodies in immune systems or another specific reactant used in nonimmune or nonhormone systems will.

C is the compound to be determined; C * is the radiolabelled form of this compound; R is the other reactant.

ίο The amount R is measured in such a way that it is not sufficient to react with all of the C + C *. Since the reaction is reversible, at least to a certain extent, an equilibrium is established at which the ratio of

[C * KC ^# ] + [C * -R]

is determined by the amount of unlabeled compound C present. If C * is separated from C * - R If the activity of the two separate parts is measured, the relationship can be determined easily calculate. The amount of unlabelled compound C can then be determined relatively or absolutely by using a standard preparation of compound C to establish a calibration curve.

This working method is illustrated with examples in a journal from RA. Yalow and SA. Berson in IAEA-SM-124/106, pages 455-481.
The separation of C * and C * - R is generally carried out in two stages. In the first stage, either C * or C * -R is caused to change phase, for example by precipitation from solution, by absorption on an inert support or by transition to a phase which is immiscible with water. In the second stage, the two phases are separated from one another, for example by filtration or - more commonly - by centrifuging and then pouring off the supernatant liquid. Once the two phases are separated in two different containers, it is easy to determine the activity in each container.

The separation of the two phases by passing one phase from the reaction tube into one However, other containers are time-consuming and also give rise to inaccurate measurements or even measurement errors. In particular, all of the material from "" one container is seldom transferred to the other.

It is an object of the present invention to provide a method for performing competitive reaction to develop based analyzes that are faster and easier to carry out than the known method and that still works with sufficient accuracy.

This object is achieved by the method characterized in the patent claim.

The subject matter of the claim makes the difficulty in measuring the activity in a phase while this is in contact with the other phase, which is the device for counting the

bo activity in this one phase from that of the other Phase shielding emitted radiation overcome.

The method according to the invention can be applied particularly to those cases in which the involved

hi radioactive marker is a soft y-emitter, so that a fairly thin layer of lead can be used for shielding. The common ¹²⁵ J is particularly suitable because of its soft

y-Strahhmg already through a 3 mm thick lead layer is weakened except for an insignificant remainder.

When the radioactive element is distributed between a liquid and a heavier solid phase and a centrifuge tube as the reaction vessel is used with a capillary at the bottom, the activity of the solid phase, which is in the Capillary at a considerable distance from the liquid phase in the main part of the tube measure by shielding the main part of the tube from the meter, leaving only one very small error due to the addition of a very small amount of liquid phase in the capillary tube The activity of the liquid phase in the main part of the reaction tube is then measured by the Capillary tube is shielded from the meter, with only a very small error from the In the absence of the very small amount of liquid phase that has passed into the capillary tube If the radioactive element is to be distributed between two liquid phases, a corresponding one is required Formation of the tube possible.

In many applications, there is a very precise measurement of radioactivity in one or both Phases not required, provided that the percentage error is not too wide and also constant In spite of such a constant percentage error, for example, in the usual radioimmunoassay, in which a calibration curve with reference to a Standard preparation is established, acceptably good results are obtained.

The method according to the invention will now be described in some exemplary embodiments with reference to the drawing explained in more detail in the drawing shows

F i g. 1 is a perspective view of a conventional centrifuge tube having a liquid therein contains suspended solid,

F i g. 2 is a perspective view of the centrifuge tube after centrifugation;

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

F i g. 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, to show the to show tubes with solution and solid sediment arranged in it.

The centrifuge tube 10 shown in Fig. 1 and 2 is made of plastic, has a closure 11 and contains a liquid phase 12 and a solid phase 13, one or both of which are radioactive. In Fig. 2 is the solid phase 13 concentrated in the conical bed 14 of the tube 10 by centrifugation been.

The partially shielded holder 15 shown in Figures 3 and 4 consists of a lead base 16, which is connected to an annular upper part 17 made of plastic. A centrifuge tube thus fits into the Holder in that the bottom · .η 14 and that in it Solid material located is surrounded by the part of the holder made of lead.

The complete unit (holder 15+ centrifuge tube 10) can be fitted into the recess of a sodium iodide scintillation crystal, which part of an instrument for measuring radioactivity (crystal counter). Shielded from the meter by the Lead base 16 of the holder 15 is the solid phase 13 and a small part of the liquid phase 12, which is in the Pointed bottom 14 of the centrifuge tube 10 is located. The measurement error, which results from the fact that not the entire liquid phase is visible in the meter can generally be neglected with the exception of there where very precise measurements are required; it is insignificant in any case if the radioactivity of Liquids in different tubes of the same design are compared with one another.

With reference to FIG. 1 to 4 described system is provided to the radioactivity of practically measure the total liquid phase in the centrifuge tube. However, if unknown samples against calibrated standards are measured, the entire phase does not have to be measured. Then it is enough ensure that the volume of liquid in the various tubes used in the experiment is the same and measure the radioactivity of a constant volume of the liquid phase. This constant volume does not need to be known; however, it must be a constant proportion of the total make up the liquid phase as, for example, different ones used in the same experiment Tube.

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

This method can be modified so that the liquid surface 23 is within the fixed column 25 is in the field of view of the counting device. However, this alternative is not preferred, because the surface 23 always has exactly the same height with different in the same experiment used tubes must have.

Errors occur when the tubes do not all have exactly the same inner diameter or when the total volume of the liquid phase is not exactly the same in each tube; these errors are, however, of no importance in practice, unless extremely precise measurements are sought and are needed.

example General methods

C ^; An insulin immunoassay analysis unit based on the method of Haies and Rändle (Lancet, i, 200, 1963; Biochem. J., 88, 137, 1963) was used to explain the use of the measuring system. A defined serum was used as "unknown". This serum was 3 fold on more than 15 occasions

evaluated using the usual filtration method. The measured numbers ranged from 43.0 to 58.5 milliunits per cm ³ with an overall mean of 49.7.

Procedure

The method was carried out exactly as it is set out in the instructions for use enclosed with the analysis unit, with the exception of the volumes used to prepare the incubates: 300 μΐ were used instead of the 100 μ indicated. As a result of this larger volume, the existing shielded containers, which are designed for a nominal volume of 1 cm ³ , could be used without modification 15 68. 67

Insulin standards of 250, 100, 50, 25, 12.5 68 974 and “zero” microunits (μΕ) / αη ³ were prepared. Each 68 185 concentration was prepared in triplicate, «\ _c = counting impulses

placing equal volumes of 300 μΐ in centrifuge tubes were given. The "unknown" sera were produced 4 times by adding 300 μΐ serum in each case tubes of the same type were presented.

300 μl of binding reagent were added to each tube and the whole thing mixed. The tubes were sealed and ^{kept at 4 °} C. for 6 hours; an equal amount of 300 μΐ of 125 l insulin was then added to each tube. The contents of the tubes were mixed, the tubes capped and held at 4 ° C for an additional 18 hours.

When the incubation was complete, the tubes were centrifuged at 14,500 rpm for 6 minutes (14,000 g). They were then placed in the shielded holders described above and each tube counted in a y-counter for 60 seconds

Results: Counts received in shielded containers.

insulin c *) / min 3 measurements 43 391 μΕ / cm ³ 49 919 "zero" 43 169 44 325 56710 12.5 50 286 50 960 67 579 25th 57 114 57 108 76 409 50 68 191 67 349 82 078 100 76 045 76 520 Mean for 250 80 559 80 705 "unknown" c *) / min found in μΕ / cm ³ insulin "Unknown" sera certainly

52.5
50.5
54.0
52.0

The result obtained for the insulin concentration of a defined serum using the shielded measuring system, is very exactly comparable to the result obtained with the help of the usual membrane filtration is obtained.

As can be seen from the above description, the type of analysis is not essential to the invention. Immunoassays and similar determinations due to competing reactions as described above are examples for suitable forms of analysis. The radioactive element involved is preferably a soft y-emitter, but the nature of the two phases and the method by which the radioactive element distributed between the phases do not play an essential role. In the same way, everyone can suitable means for determining the degree of activity in one of the two phases or in both phases can be applied.

1 sheet of drawings

Claims (1)

Claim: Method for Carrying Out Competitive Reactions based analyzes in which the following is introduced into a tube: 1. a known Volume of a liquid containing the compound, the proportion of which is to be determined; 2nd one Standard amount of a radioactively labeled form of the compound to be determined and 3. one Standard amount of a reagent which is sufficient to be reversible with a part of the to be determined Compound and with part of the radioactively labeled form of this compound with formation another liquid or solid phase to react, creating the compound to be determined and their radioactively labeled form between the liquid phase and the other liquid or solid phase are distributed, in which the other liquid or solid phase at the bottom of the tube is localized and the level of radioactivity of at least part of the liquid phase or the other liquid or solid phases is measured with the aid of a measuring device and for measurement of the unknown part of the connection against calibrated standard connections, the geometric Dimensions of the tube are kept constant for all measurements, characterized in that that one can measure the level of radioactivity on at least part of the liquid Phase or the other liquid or solid phase carries out, while this phase (measuring phase) with the other phase is in contact, and that the shape of the tube is formed so that the Measuring phase has a considerable distance from the main volume of the other phase, and / or a shield is formed so that it is in the area of the other phase as well at least in a sub-area of the measuring phase immediately following the phase boundary shields radiation emerging from the tube from the measuring device, which also collimates in this way is that it only absorbs radiation coming out of the measuring phase. The principle of this procedure can be represented by the following reaction scheme: