WO1989004487A1

WO1989004487A1 - Immunological separation and detection process for multiple determinations

Info

Publication number: WO1989004487A1
Application number: PCT/EP1988/001016
Authority: WO
Inventors: Armin Gilak
Original assignee: Armin Gilak
Priority date: 1987-11-13
Filing date: 1988-11-10
Publication date: 1989-05-18
Also published as: EP0386074A1

Abstract

In a simultaneous IRA/IRMA process carried out on three or possibly two antibodies using a chromatographic separation column, at least two determinations of immunologically detectable substances can be carried out rapidly without time consuming separation techniques.

Description

Immunological separation and detection procedure for multiple determinations

The invention relates to an immunological separation and detection method for the determination of at least two antigens in a liquid using a tube coated on the inside with antibodies, a sphere coated with antibodies and only one type of labeling.

With the large number of parameters to be determined for diagnostic or therapeutic purposes, there is a considerable need to carry out such determinations quickly and safely for laboratory personnel, preferably in parallel.

For these purposes, immunological examination methods have proven to be particularly suitable.

For example, DE-C2 29 43 648 suggests using different solid phases coated with antibodies for the detection of different ligands in a liquid sample, which solid phases can be easily separated from one another. After the incubation of these solid phases with the sample solutions, the radioactive supernatant must be removed and then the solid phases separated.

A simplified separation method for immunological determinations is known from DE-C2 32 17 032. According to this method, an antigen / antibody complex is separated from the supernatant reagent solution by a dry chromatographic column. However, this method is only suitable for determining an unknown substance.

DE-C2 33 22 373 proposes to mark carrier particles with a fluorescent marking substance and then to load them with antigens and / or antibodies. The distinction between the individual particles can be made either by using different fluorescent markers or by connecting the particles with different concentrations or with several markers. This method has disadvantages with regard to the different marking substances and the associated various settings on the detection device.

General procedures for performing a radioimmunoessay or a so-called immunoradiometric test (IRMA) are set out in Chemiker-Zeitung, 103, (1979) No. 2, pp. 53-68.

The aforementioned prior art has the disadvantages that either only one substance can be detected or the separation of the substances to be determined requires a comparatively high outlay.

Accordingly, the object of the invention is to provide a method which enables simple and rapid separation of immunologically detectable substances and which, in the case of the use of radioactive marking agents, permits a contamination-proof procedure.

This object is achieved in that a first antigen to be determined by the IRMA technique is bound to the inside wall of the reaction vessel and a second antigen according to a conventional solid-phase RIA to a ball, the supernatant liquid, which, in addition to excess reagents, also contains a third contains immune complex to be determined, separated from the solid-phase complexes by a dry chromatography column and thereby the third complex to be determined migrates into the upper part of the column, then removes the chromatographic column for measurement and places it in a suitable measuring device, closes the tube, turns it upside down and the immune complex in the lower part and on the ball in the Closure part measures one after the other.

According to a preferred embodiment, the method according to the invention can also be used for the determination of only two antigens either by omitting the coated sphere and detecting a second immune complex in the chromatography column or by using a sphere coated with antibodies and the chromatography column only for separation or Absorbing excess reagents used.

The labeling of the antibodies or antigens is preferably carried out using radioisotopes, but depending on the analytical problem or laboratory situation which is treating _Z u, it can advantageously also be done by fluorescence and

Luminance labeling or by enzymatic methods. Iodine should be mentioned as the preferred radioisotope; fluorescein isothiocyanate could be used in a suitable manner for the fluorescence labeling. In case of a

Enzyme labeling would use a permeable chromatography column and the determination would be made photometrically.

Depending on the analytical problem, polyclonal or monoclonal antibodies will preferably be used. In a preferred manner, both types of antibody can also be used next to one another or microfine silica or proteins can be used to bind antigens. The biological materials can advantageously be coupled to the solid phase using the carbodiimide process.

The device from DE-C2 is particularly suitable for carrying out the method according to the invention

33 47 464 in connection. with the chromatography column from DE-C2 37 17 032. If necessary, a ring is provided in the upper inner part of the reaction vessel, the inner diameter of which is preferably 1 to 2% smaller than the diameter of the ball, thus the ball is measured either by adhesion in the cap of the device or fixed to the ring of the reaction vessel. It remains in this position even after the reaction vessel has been turned several times.

Magnetic means can advantageously be used to separate the two solid-phase immune complexes. For example, a permanent magnet in the closure flap is suitable for fixing a ball containing a metallic substance.

The reagents and device parts required to carry out the method according to the invention are compiled in a test combination. This test combination contains the device described above, for example the lower part of the reaction vessel and the sphere being provided with biological materials, for example antigens, antibodies and other proteins. This test combination also contains a cap, a dry chromatographic column, other labeled and unlabelled antibodies, as well as labeled and unlabelled antigens and 0.9% saline.

The advantages achieved by the invention are that the reaction, separation and detection can be carried out in one device without the need for centrifuging or decanting steps need to be carried out without any risk of contamination of the laboratory or laboratory personnel by splashing reagent solution. It is particularly suitable for immunologically detectable substances that are sensibly determined side by side.

The implementation of the method according to the invention is explained in more detail below.

One way of carrying out the method according to the invention is to use a reaction and separation device which has a ring 4 in the upper part of a tube 1 near the tube opening. The difference between the outer and inner ring radius is about 1/10 of the outer diameter of the ring, between the bottom of the tube 1 and the ring 4 there is a ball 3, the diameter of which is slightly larger than the inner diameter of the ring 4. Depending on the measurement to be carried out, the lower part of the inner tube wall 2 and / or the ball 3 are coated with biological material. The device contains a removable chromatography column 6 between the ball 3 and a sealing cap 5.

Antibodies 1 and l1 react below with antigen A, antibody 2 with antigen B and antibody 3 with antigen C.

The lower part of an inner tube wall is coated with unlabeled antibody 1 directed against antigen A. One sphere is directed against antigen B.

Antibody 2 coated, to carry out the test, a mixture of antibody 3, ¹²⁵ I-labeled antibody 1 'is added to the antigen A, B and C standard solutions and, if appropriate, diluted patient sera, which have been pipetted beforehand into a sample tube coated with unlabeled antibody 1. and also labeled antigen B and Antigen C added, for volume compensation and to increase the specificity of the detection reaction, further additives may be used. The test tubes are closed with a cap, shaken thoroughly with a vibration mixer and then incubated for about 1 hour at room temperature.

Since there is an excess of antibody 1, after the incubation all antigen A molecules are bound in a sandwich-like manner between the antibodies 1 immobilized on the vessel wall and the radioactively labeled antibodies 1 '. At the same time, unlabeled antigen C and labeled antigen C as well as unlabelled antigen B bound to the sphere and labeled antigen B compete for the binding sites on their respective antibodies.

After the approximately one-hour incubation, a chromatography column is placed in the tube on the ball and the solution of the sample tube is sucked up by capillary action. It is separated from the antigen A sandwich complex located on the tube wall and the antigen B antibody complex present on the sphere. By adding 0.5 ml of physiological saline, the solid-phase immune complexes are washed and, moreover, material adsorbed on the vessel wall is dissolved and also sucked into the column.

Chromatography separates reaction products and reactants according to their molecular weight. Antibodies and immune complexes migrate to the upper part of the chromatography column due to their higher molecular weight, whereas low-molecular-weight reactants remain in the lower part of the column, including the labeled antigens. The antigen

C-antibody complex will therefore be found in the upper part of the column. For the antigen C measurement, the entire device is placed in the measuring vessel of a gamma counter and the pulse rate is determined. The antigen B measurement is carried out without a column, the tube being turned over, that is to say with the opening facing downward, into the measuring vessel. The sphere with the antigen B antibody complex to be measured is now in the cap. To determine antigen A, the tube is turned over again, that is to say brought into the normal position, the hollow sphere remaining in the cap in a slightly wetted state and not falling back onto the bottom of the tube. In this position, the tube is placed in the measuring cell of the gamma counter for measuring the wall-mounted antigen A solid-phase immune complex.

The method according to the invention can, however, also be carried out with a ring on the upper inside of the tube, in which case the ball does not adhere to the cap, but rather to the ring by means of adhesive force, and likewise does not fall back onto the bottom of the tube when the tube is in the normal, normal position.

The invention is illustrated by the following examples.

example 1

To determine unknown amounts of TSH (Thyrotropic Hormone) and FT. (free triiodothyronine) and FT. (free thyroxine) is added to 100 µl serum samples and FT ₃ , FT ₄ and TSH standard solutions in a coated with mouse anti-TSH antibodies Rctcchen, which contains a hollow sphere coated with T ₄ antibodies, a mixture of 200 / ul of 1 ²⁵ j labeled monoclonal TSH antibodies and ¹²⁵ j labeled T ₃ and T ₄ and corresponding highly specific polyclonal T ₃ antibodies was added. The reaction mixture is mixed on a vibratory mixer and incubated for about one hour at 37 ° C or overnight at room temperature.

Now a chromatography column is introduced into the reaction vessel and the liquid phase is thereby sucked up and separated according to its molecular weight in such a way that the high molecular weight solution components migrate into the upper part of the column. After the tube wall and thus the solid-phase immune complexes of both the hollow sphere and the tube wall have been washed with 0.5 ml of physiological saline and this solution has also been sucked up through the chromatography column, the tube is held upright with the hollow sphere and column in the measuring cell

Gamma counters placed for FT ₃ measurement and the pulse rate measured.

After removing the chromatography column and turning the almost dry tube upside down, it is brought into the measuring cell with the opening downward, with the hollow sphere resting on the ring, to determine the FT ₄ pulse rate. The tube is then turned back into the normal position, with the tube opening facing upwards, the ball being held in place by the ring in the upper part of the tube, and placed in the measuring cell of the gamma counter to determine the TSH pulse rate.

The measurement results of FT ₃ , FT ₄ and TSH were evaluated using the available measurement protocols and calibration curve from 5 standards. To create the standard calibration curve, the measured percentage binding was plotted against the corresponding standard concentration according to the spline approximation standard curve. Measurement for example (1)

Protocol: FT3-RIA

Standards cpm 1 cσm 2 cpm x (pmol FT3 / 1)

S0 34230 34501 34365 0.00 S1 35541 35303 35422 2.20 S2 38328 38720 38524 4.40 S3 43310 43219 43264 8.80 S4 50701 50501 50601 17.60 S5 61420 61880 61650 35.20

Control 1 36989 36798 36893 3.40 Control 2 46322 46450 46386 12.50

Protocol: FT4-RIA

Standards cpm 1 cpm 2 cpm x (pmol FT4 / 1)

S0 12363 12280 12321 0.00

S1 11500 11620 11560 3.20

S2 9435 9530 9482 6.40

S3 7004 7100 7052 12.80

S4 4910 4790 4850 25.60

S5 2783 2801 2792 51.20

Control 1 5901 5699 5650 19.4

Control 2 3288 3201 3244 43.1

Protocol: TSH-IRMA

Standards cpm 1 cpm 2 cpm x (mU TSH / ml)

So 252 263 257 0.00

S1 990 1005 997 1.50

S2 2271 2199 2235 5.00

S3 4171 1161 1166 10.00

S4 9831 9888 9859 25.00

S5 13270 18360 1S315 50.00

Control 1 755 770 762 0.90

Control 2 8148 8190 8169 20.90

Example 2

To determine the total T3 and total T4 and the TSH in the serum, 100 μl of serum samples and standard solutions from total T3, total T4 and TSH are pipetted as in Example 1 into reaction tubes with the appropriate antibodies and 500 μ.1 of a liquid Mixture of corresponding tracers and T3 antibodies with the addition of 0.03 M thiomersal as deblocking agent for the complete release of T3 and T4 from their carrier proteins such as TBG. The procedure is then as described in Example 1. The values obtained are listed in the following measurement reports and curves.

Measurement for example (2)

Protocol: TT3-RIA

Standards cpm 1 cpm 2 cpm x (nmol TT3 / ml)

S0 41411 41112 41261 0.00

S1 42461 42220 42340 0.76

S2 46801 46422 46611 1.53

S3 51961 51798 51879 3.07

S4 61433 61433 61317 6.15

S5 74533 74333 74433 12.30

Control I 44255 43998 44126 1.13

Control II 55693 55100 55396 3.89

Protocol: TT4-RIA

Standards cpm 1 cpm 2 cpm x (nmol TT4 / ml)

S0 14835 14750 14792 0.00

S1 13944 13855 13899 25.74

S2 11322 11299 11310 64.35

S3 8523 8460 8491 102.96

S4 5868 5766 5817 157.44

S5 3372 3402 3387 257.40

Control I 6828 6730 6779 138.99

Control II 4066 3999 4032 217.50

Protocol: TSH-IRMA

Standards cpm 1 cpm 2 cpm x (mU TSF / ml)

S0 210 215 212 0.00

S1 990 960 975 1.50

S2 2381 2299 2340 5.00

S3 4330 4420 4375 .10.00

S4 10982 10882 10932 25.00

S5 16840 16799 16S19 50.00

Control I 822 7S9 805 1.10

Control II 8433 8400 S416 19.40

Claims

1. Immunological separation and detection method for the determination of at least two antigens in a liquid using a tube coated on the inside with antibodies, characterized in that

a first antigen to be determined using the IRMA technique on the inner wall of the tube, a second

Binds antigen to a ball using a conventional solid phase RIA,

the supernatant liquid, which, in addition to excess reagents, also contains a third immune complex to be determined, is separated from the solid-phase complexes by a dry chromatography column and thereby allows the third immune complex to be determined to migrate into the upper part of the column.

for the measurement, insert the chromatographic column into a suitable measuring device, close the tube, turn it upside down and carry out the measurement separately in the lower part of the tube and in the closure part in a measuring device.

2. Immunological separation and detection method according to claim 1, characterized in that one works to determine two antigens with the coated tube and the chromatographic column.

3. Immunological separation and detection procedure according to

Claim 1 or 2, characterized in that one

Antibodies or antigens labeled with a radioisotope.

4. Immunological separation and detection procedure according to

Claim 3, characterized in that iodine is used as the radioisotope ¹²⁵ .

5. Immunological separation and detection procedure according to

Claims 1 to 4, characterized in that monoclonal antibodies are used.

6. Immunological separation and detection method according to claim 1 and 2, characterized in that

Antibodies or antigens are fluorescently labeled.

7. Immunological separation and detection method according to claim 1 and 2, characterized in that antibodies or antigens are luminescent-labeled.

8. Immunological separation and detection method according to claim 1 and 2, characterized in that one labels antibodies or antigens enzymatically.

9. Separation and detection method according to claim 1, characterized in that the ball is fixed for measurement on a ring arranged in the upper inner part of the tube.

10. Separation and detection method according to claim 1, characterized in that the ball is separated by magnetic means from the solid phase complex present at the bottom of the tube.

11. Reaction and separation device for performing the method according to claim 1, characterized in that a tube (1) in the upper part near the opening has a ring (4), the difference between the outer ring and inner ring radius about 1/10 of the outer diameter of the ring and a hollow ball (3) is provided between the tube bottom and the ring (4), the diameter of which is slightly larger than the inside diameter of the ring (4).

12. Reaction and separation device according to claim 11, characterized in that the ball diameter is 1 to 2% larger than the inner diameter of the ring.

13. Reaction and separation device according to claim 11 and 12, characterized in that biological materials are bound to the inner tube wall and / or the ball.

14..Test combination, comprising a device according to

Claims 11 to 13 for carrying out the method according to one of the preceding claims from tubes and spheres coated with antibodies, a closure part and a chromatographic column, the tube optionally containing a ring arranged in the upper inner part, the inside diameter of which is 1 to 2% smaller than that Ball diameter is, as well as labeled and unlabelled antibodies or antigens and 0.9% saline.