DD283867A5 - PROCESS FOR DETERMINING AN IMMUNOLOGICALLY DETECTABLE SUBSTANCE AND REACTION GEFA SUITABLE FOR IT - Google Patents

Abstract

The invention relates to a method for the determination of an immunologically detectable substance and the principle of heterogeneous immunoassay using a solid phase to which one of the immunologically active reaction components is bound, wherein the solid phase used is a Reaktionsgefaesz on the inner surface streptavidin or avidin in a is bound in such amount that there are 0.1 to 2.5 mg per ml of reaction volume. A reaction vessel suitable for this purpose has opposing optically permeable wall areas and, moreover, is at least partially coated with streptavidin or avidin in the area of the inner wall provided for fluid intake, whereby the interior of the container intended for fluid intake and the streptavidin or avidin content of the coating are coordinated with one another are that 0.1 to 2.5 mg of streptavidin or avidin are present per ml of reaction volume. {Method; Reaktionsgefaesz; immunologically detectable substance; Detection methods; streptavidin; avidin}

Description

For this 5 pages drawings

Field of application of the invention

The invention relates to a method for the determination of an immunologically detectable substance according to the principle of heterogeneous immunoassay using a solid phase to which one of the immunologically active Reaktionskomnonenten is bound, a suitable reaction vessel and the use of a unit-reaction vessel.

Characteristic of the known state of the art

To determine a substance capable of binding specifically, processes based on the principle of immunoassays are frequently used. In this case, one of the members of a substance pair capable of binding to one another is reacted with the specific receptor for it, which is labeled in a manner known per se. The conjugate of these two substances can then be reacted with a receptor specific for the conjugate or one of the two parts of the conjugate. There are many variations for these immunological methods. It is advantageous if one of the receptors is bound to a solid phase. This facilitates the Ti ^- "voltage of bound and not gebunder, present neaktionspartnern To determine df.r specifically bindable substance, the amount is then measured on the solid phase bound labeled reactant or of water present in the solution labeled reactants and to the amount of to. determining reaction partner in a conventional manner in relation set.

In the immunological methods, solid tubes are usually plastic tubes or microtiter plates on whose inner surface the reaction partner is fixed, or spheres fixed on the outer surface of the reactants.

In this case, the binding of the specific reaction partner to the respective surface must be such that it does not lose the ability to specifically bind the substance which is specifically capable of binding it. For this reason, the binding of the reaction partner to the solid phase is usually adsorptive. It has therefore already been proposed to effect the fixation of the reaction particle to the solid phase via a coupling agent which mediates the binding. Care must again be taken to ensure that the binding of the reaction partner to the binding agent does not disrupt the specifically reactive region of the molecule or that the reaction partner is bound so that its reactive site faces the binding partner away from the solid phase.

Disadvantage of all these known methods is that a specific solid phase must be provided for each specific reaction. This means that for each test a different solid phase must be prepared, stored and then used for the determination, which is expensive.

Object of the invention

It is the object of the invention to provide a method and reactor suitable for many different detection methods.

Explanation of the essence of the invention

It is the object of the invention to provide a method for the determination of an immunologically detectable substance. In routine clinical diagnostics, a variety of parameters, such as hormones, tumor markers, infection, allergy, and fertility parameters are determined from a blood sample. The concentration of the parameters to be determined covers a wide range. Low concentration parameters such. B. TSH and prolactin are in the range of 10 ^"10 to 10"is' ² mol / l, while highly concentrated parameters such as T ₃ and T ₄ is about 10 ^"7 to 10" ⁸ mol / l. It would be desirable if each tube different tubes should not be used each time, but if a single reaction vessel could be used for all determinations.

This object is achieved by a method for the determination of an immunologically detectable substance according to the principle of heterogeneous immunoassay using a solid phase to which one of the immunologically active reaction components is bound, which is characterized in that the solid phase used is a reaction vessel to whose Inner surface streptavidin or avidin is bound in such an amount that per ml of reaction volume 0.1 to 2.5 \ ig are present. This amount refers to streptavidin or avidin, which is accessible to the binding partner in the context of the immunological reaction, which is in the form of a biotinylated protein or hapten.

The inventive method is applicable to all determination methods in which the receptor, the z. B. may be an antibody or an antigen to be immobilized on the solid phase, conjugated with biotin. The solid phase-fixed streptavidin or avidin has very good adhesion to all tube materials and the binding also remains stable to detergents. In the preparation of calibration curves, which are necessary for evaluation in many immunological methods, the solid-phase-bound stroptavidin or avidin according to the invention provides steep calibration curves at low blank values, which leads to an increase in accuracy. Another advantage is that batch fluctuations of the coated amount of streptavidin had only negligible effects on the measurement results. The particular advantage of the method according to the invention is that the bound amount of streptavidin or avidin is adjusted so that the foste phase can be used for all known methods according to the principle of heterogeneous immunoassay. Outside dome, there is an advantage that one can simultaneously determine several substances, in particular antibodies, but also antigens with the method according to the invention. This is explained in more detail by example 7.

In a particularly preferred embodiment, the reaction vessel used as the solid phase is also used simultaneously as a cuvette for the photometric determination of the label. This is particularly advantageous since only a single vessel is required to carry out the entire reaction. The reaction vessel is in this case equipped so that it has two opposing optically permeable Wandboreiche. In this case, the entire wall area may be coated with streptavidin or avidin.

According to the invention, a reaction vessel is used as the solid phase, to the inner surface of which streptavidin or avidin is bound. The binding of streptavidin or avidin is carried out according to methods known to the person skilled in the art (for example

EP-A-0 122 209). The streptavidin or avidin can either be bound directly to the solid phase or bound via spacer or binding agents.

In a preferred embodiment of the method according to the invention streptavidin or avidin is coupled to a soluble protein having a molecular weight above 50000000 and then the conjugate is adsorbed to a hydrophobic solid phase, such as e.g. B. in the patent application DE-A 36 40 412 is described. Preferably, a protein is used that is hydrophobized.

The hydrophobization can be carried out, for example, by application of heat, treatment with acids, denaturing agents and / or chaotropic ionone and / or by chemical coupling with a hydrophobic compound. Treatment with these agents also results in an increase in molecular weight. The increase in molecular weight can also be achieved by cross-linking with an H or polyfunctional protein reagent.

As acids, for example, acetic acid, propionic acid, lactic acid or hydrochloric acid are used. Typical concentrations are 1 to 100 mmol / l with exposure times of 10 minutes to 16 hours.

Suitable chaotropic ions are, for example, thiocyanates, iodides, fluorides, bromides, perchlorates and sulfates. Suitable denaturing agents are, for example, guanidine hydrochloride or urea. Usually, concentrations of 10 mmol / l to 6 mol / l are used here.

For derivatization with hydrophobic compounds are preferably soluble fatty acids, lipids in low or high molecular weight form and synthetic polymers such as polypropylene glycol or soluble copolymers of polystyrene used. The derivatization takes place according to the methods familiar to the person skilled in the art.

Crosslinking via bi- or polyfunctional compounds is carried out with known protein binding reagents. These are compounds that carry at least two functional groups that can be the same or different and that can react with functional groups of proteins via these functional groups. Preference is given to compounds ver consisting of an alkyl chain to which. At the end succinimide, maleimide and / or aldehyde groups are found.

The protein is crosslinked in the bi-or polyfunctional compound in a manner known per se by reacting the soluble protein and the bifunctional or polyfunctional compound.

Preferably, proteins having a molecular weight of 10,000 to 700,000 are used for the hydrophobization and / or crosslinking. Particularly preferred is bovine serum albumin, lipase or immune Y-globulin.

The protein then wild in a conventional manner, the streptavidin or avidin coupled. Suitable coupling methods are described, for example, in Ishikawa, J., Immunoassy 4 (1983), 209-327.

The resulting conjugate of streptavidin or avidin and protein is then adsorbed on the plastic surface serving as the solid phase. Before the conjugate of protein and streptavidin or avidin is adsorbed to the hydrophobic residual phase, it is also possible to pretreat the solid phase physically or chemically. For example, a Kunststoffoberflänhe be pre-swollen or activated in other known manner. The adsorptive binding to the solid phase occurs via strong and weak interactions, hydrophobic forces, dipole-dipole or ion-dipole interactions. Particularly suitable hydrophobic solid phase are reaction vessels made of support materials having a surface tension which is smaller than the surface tension of the hydrophobic soluble protein, ie are more hydrophobic than protein. Preferably, support materials having a surface tension of 40 g / cm ² are used. Particularly suitable are polystyrene, polymethacrylate, Teflon, polyamide, copolymers of styrene and acrylonitrile, glass and cellulose products.

The solid phase material prepared according to the invention is used to determine many different parameters.

In this case, vine Her with the parameters to be determined bindable substances is then conjugated with biotin, which in turn binds to the immobilized on the solid phase L'treptavdidin or avidin. In this way, the specific complexes formed by the immunoassay method can be immobilized and then determined. According to the invention, therefore, so much streptavidin or avidin is bound to the solid phase that 0.1 to 2.5 pg are available per ml reaction volume for the binding of the biotinylated, specifically bindable substance.

The reaction volume is the sum of sample volume and test reagent volume.

Preferably, the solid phase material is coated so that 1 to 2 yg of streptavidin or avidin are present for binding with biotinylated, specifically bindable substance per ml of test volume. The amount of the biotinylated specifically bindable substance in the method of the invention is preferably in the range of 10 " ^1β to 10 ~ ⁸ mol / reaction volume.

Another object of the invention is a reaction vessel with opposing optically transmissive wall areas and übi TIG, at least partially, provided in the area provided for liquid intake inner wall with streptavidin or avidin coated walls, wherein the intended for the liquid intake interior of the container and the streptavidin or Avidinehalt the coating are coordinated so that per ml reaction volume 0.1 to 2.5 pg streptavidin or avidin are present.

This reaction vessel is particularly suitable for carrying out the process according to the invention. Since it has optically transparent, opposing Wandbereichf and also has a coating with streptavidin or avidin, it can be used simultaneously for carrying out the reaction and for photometric determination. By coating with 0.1 to 2.5 μg of streptavidin or avidin per ml of reaction volume, this reaction vessel can be used to determine a wide variety of parameters according to the principle of heterogeneous immunoassay.

The reaction vessel is the solid phase. It consists of commonly used materials such as plastics, glass, etc. The optically transmissive wall regions consist of likewise known materials. Particularly preferred here are polystyrene, copolymers of polystyrene, polycarbonates, polyacrylates and polymethacrylates.

The coating with streptavidin or avidin can either be done directly or via a carrier material or a spacer.

For example, the binding of streptavidin or avidin to a soluble protein of molecular weight greater than 500,000, which is then adsorbed to the inner surface of the reaction vessel, is suitable. The invention also relates to the use of a unit-reaction vessel, which is bound from a vessel on the inner surface of streptavidin or Aviain, in such an amount that for the determination reaction per ml reaction volume 0.1 to 2.5 micrograms of streptavidin or avidin In order to determine various parameters in procedures, the principle of immunoassay is available.

According erfindungsgeinäß a method and a reaction vessel is provided in which streptavidin or avidin is fixed with good adhesion and permanently to the inner surface of a reaction vessel as a solid phase. The adhesion is so good that the addition of detergents does not lead to a detachment of the substance. The reaction vessel provided according to the invention is universally applicable and is suitable for carrying out determination methods for a very large number of parameters and therefore facilitates the performance of routine determinations.

EXAMPLES

The invention is explained below by means of figures and examples. In the drawing represent:

Fig. 1: a calibration set for a TSH determination using immobilized streptavidin in

varying amounts and biotinylated anti-TSH antibody; 2 shows a calibration cuvette for a prolactin test; FIG. 3: a calibration curve for a CEA test; and FIG. 4: a calibration curve for a T4 test FIG. 5: a calibration curve for an antiHBs determination using immobilized streptavidin in different amounts.

The TSH monoclonal antibodies used in the examples are derived from hybridoma cell lines deposited with the European Collection of Animal Cell Cultures, Porton Down, GB under the names ECACC 87 122201 and ECACC 87122202.

example 1

The solid phase adhesion of the conjugate according to the invention and hydrophobic protein and streptavidin were investigated:

Thermally aggregated RSA _1, hereinafter referred to as Thermo-RSA, was prepared in the following manner: 1 g RSA was dissolved in 100 ml BOmmol potassium phosphate solution at pH 7.0, heated to 70 ° C and held at this temperature for 4 hours with gentle stirring , The solution was cooled, filtered and adjusted to a concentration of 50 mg / ml. It was then redistilled to 30 times the volume. Water dialysed.

Preparation of Streptavidin Conjugate with Thermo-RSA: Streptavidin recovered from Streptomyces avidinii was reacted with maleimido-hexanoyl-N-hydroxy-succinimide to give streptavidin carrying maleimido groups. Thermo-RSA was reacted with S-acetylmercaptosuccinic anhydride and then the protected SH groups were released by the addition of hydroxylamine. The maleimido group-containing streptavidin was then mixed with the SH group-containing thermo-RSA to form the desired conjugate.

Polystyrene plastic tubes were then loaded with streptavidin-thcrmo-RSA conjugate. The loading of the tubes was carried out with 1 5 ml of a streptavidin-thermo-RSA solution, the molar ratio of the two components was 1.8: 1 OOug / ml) in 40mmol sodium phosphate buffer pH 7.4 to 20 ⁵ C for 18 to 24 hours.

After sucking out the Tubes 30 min at 2O ⁰ C with 1.8mi of a solution of 2% sucrose, 0.9% sodium chloride and 0.3% RSA nachbeladeri. After drying (24 hours at 2O ⁰ C and 40% relative humidity), the tubes were ready for use and durable.

Example 2

TSH one-step test.

Biotinylated monoclonal anti-TSH antibody was prepared. For this, the Ar.ti-TSH antibody, ECACC 87122201, was reacted in the usual way with biotin, wherein the coupling takes place via the amino groups. Antibody and biotin were used in the ratio 1:16. 400ng of the biotinylated antibody thus obtained was dissolved in 1 ml of buffer (40 mmol sodium phosphate, 0.5% Pluronic F68.0, 2 molar sodium tartrate / e and 0.01% phenol) and then together with 200 μl sample or a standard solution, which is a known Amount of TSH contained, and 75mU of a peroxidase-conjugated anti-TSH antibody (ECACC 87122202) incubated in a loaded according to Example 1 tube for 2 hours. Thereafter, it was washed three times with tap water and then 1 ml of ABTS substrate solution was added. After one hour the absorbance was measured at 4J5nm. The results are shown in FIG. 1 to entnehmon. It turns out that a satisfactory result with immobilized streptavidin is obtained in an amount of more than 0.1 μg / ml of test volume.

The need for streptavidin to bind the biotinylated antibody used in the assay was determined. This was

First, the amount of freely accessible biotin according to the method of Bayer, Edward A., Meir-Wi'lchek, Analytical Biochemistry 154 (1986), 367-370, determined. Starting from 1 μg of antibody which was biotinylated in the ratio of antibody to biotin 1:15 (Guesdon, IL, J. Histochem Cytochem 27 (1979) 1131-1139), it is found that 1 to 2 mol of biotin per mole of antibody for binding to Are available, ie per ug of antibody 1.5 to 3ng of biotin are freely available.

A tube loaded with 10 μg / ml streptavidin-thermo-BSA conjugate (Example 1) has a biotin binding capacity of 15 to 20 ng per ml of test volume determined with radioactive C ¹⁴ biotin.

For biotinylated immunoglobulins, the binding capacity of the streptavidin intubes is 1.7 to 2 μg / ml assay volume. The determination was carried out with! '"- labeled γ-globulin (labeled by the method of McConahey and Dixon 1966, Int. Arch. Allergy 29/185).

Example 3

Prolactin test.

Prolactin is determined in a 1-step sandwich immunoassay. For proof, a reagent having the following composition is used:

50mU / ml of a conjugate of POD and a prolactin-specific monoclonal antibody,

- 40mmol / L phosphate buffer pH: 7.0

0.5% Pluronic F65 (polyoxyethylene polypropylene)

0.2 mol / l sodium tartrate

0.01% phenol

0.2% bovine serum albumin

400ng / ml of a biotinylated monoclonal antibody to prolactin.

The two monoclonal antibodies are only required to recognize prolactin and to target various epitopes of prolactin. In a polystyrene tube coated with streptavidin-thermo-RSA conjugate as obtained according to Example 1, 1 ml of this reagent and 50 μl of sample were incubated for 30 minutes at room temperature. It was then washed three times with tap water. For the detection reaction, 1 ml of ABTS * substrate solution was added. After 30 min, the absorbance was measured photometrically at 405 nm. The results are shown in FIG. 2

 ABTS®: 2,2'-azino-di- [3-ethylbenzthiazoline sulfonate (s) 1-diammonium salt

Example 4

CEA test.

In a sample solution, CEA was determined in a 1-step sandwich immunoassay. The reagent used had the following composition:

120mU / ml of a conjugate of POD and a monoclonal antibody to CEA,

- 40mmol / IPhosphatpufforpH: 7.0 0.5% PluronicF68 0.2mol / L sodium tartrate 0.01% phenol 0.2% bovine serum albumin 500ng / ml of a biotinylated monoclonal antibody to CEA.

The two monoclonal antibodies to CEA are merely required to recognize CEA and are each directed against another epitope of CEA.

In a streptavidin-thermo-RSA-coated polystyrene tube, as obtained according to Example 1, 1 ml of the reagent and ΙΟΟμΙ sample were incubated for 2 hours at room temperature. It was then washed three times with tap water. Then, for the detection reaction, 1 ml of ABTS * substrate solution was added. After 1 hour, the absorbance at 405 nm was measured photometrically. The results are shown in FIG.

Example 5

T4 test.

T4 was determined in a competitive immunoassay using biotinylated anti-T4 antibodies. For this purpose, in a polystyrene tube coated with streptavidin-thermo-RSA conjugate, as obtained according to example 1, 500 μl of a reagent consisting of:

100mU / ml thyroxine-POD conjugate (prepared according to EP 209 155)

120mol / l barbiturate,

18.2 mmol / l phosphate buffer pH 8.6

0.04% by weight of ANS (8-anilino-1-naphthalene sulfonic acid),

0.2% by weight of bovine serum albumin

and 20μΙ sample incubated for 10min at room temperature. Subsequently, 500μΙ of a second reagent consisting of:

1 μg / ml biotinylated polyclonal antibody against T4, 120 mmol / l phosphate buffer pH: 8.6, 0.04 wt% ANS (8-anilino-1-naphthalene sulfonic acid), 0.2 wt% bovine serum albumin,

added and incubated for a further 30 min at room temperature. After washing three times with tap water, 1 ml of ABTS * substrate solution was then added and incubated at room temperature for 30 min. Subsequently, the absorbance at 405 nm was determined photometrically. The results are shown in FIG.

Examples

Anti ΗΒ antibodies are determined in a 1-step sandwich immunoassay. For proof, a reagent having the following composition is used:

60mU / ml of a conjugate of POD and HB, antigen

40mmol / L phosphate buffer, pH 7.0

200 mmol / l sodium tartrate

0.5% by weight Pluronic F68

0.01% by weight of phenol

0.2% bovine serumaluminum

0.1% by weight bovine IgG

150ng / ml biotinylated HB ₁ Ag (prepared according to Immunol., Letters 8 (1984), 273).

In a polystyrene tube coated with streptavidin-thermo-RSA conjugate according to Example 1, 1 ml of this reagent and 200 μl of sample were incubated for 4 hours at room temperature. It was then washed three times with tap water. For the detection reaction, 1 ml of ABTS substrate solution was added. After 60 minutes, the absorbance at 422 nm was measured photometrically.

The anti-HB _S test was performed using Tubes with different amounts of immobilized streptavidin. The results are shown in FIG. 5. It turns out that when streptavidin is used in amounts? Omitted, which are above the amounts used in the invention, the test is significantly less sensitive.

Example 7

Anti-HIV test

HIV antibodies are determined in a 2-step sandwich immunoassay. As proof the reagents with the following composition are used.

Reagent 1:

each 10 " ⁷ mmol / L or more biotinylated HIV antigens

40mmol / L phosphate buffer, pH 7.0

0.9% by weight of sodium chloride

10% by volume of bovine serum

The following antigens were used: genetically engineered HIV1 antigens corresponding to HIV1-gp41 (gp41-rec., Centocor ™ -p121) and HIV1-p24 (p24-rek "Contocor ™ -pg2), chemically synthesized peptides from HIV1-gp41 ( gp41-pep, Wang et al., PNAS, 83, 6159, 1986) and HIV2 gp32 (gp32-pep, Gnann, JW et al., Science, 237, 1346, 1987). These antigens have been described as labeled with biotin by Loary et al., PNAS, 80, 4045 (1983).

Reagent 2:

20mU / ml of a sheep antibody conjugate to human immunoglobulin and POD 40mmol / L phosphate buffer, pH 7.0

0.05% by weight Tween 20

0.2% bovine serum albumin

0.2% bovine IgG

In a polystyrene tube coated with streptavidin-thermo-RSA conjugate according to Example 1, 1 ml of reagent 1 and 10 μl of human serum or plasma were incubated for 1 h at room temperature. It was then washed 3 times with tap water and 1 ml of reagent 2 incubated for 1 h at room temperature. Again, it was washed 3 times with tap water and 1 ml of ABTS substrate solution was added for the detection reaction. After 60 minutes, the absorbance at 422 nm was measured photometrically.

The anti-HIV test was performed using single HIV antigens and combinations of antigens. It turned out that the test procedure in the polystyrene tube coated with streptavidin-thermo-RSA conjugate is suitable both for the determination of individual antigen-specific antibodies and for the simultaneous determination of several antibodies or antibody populations (screening test, Table 1). whether they are directed against the same virus or multiple viruses or antigens of interest.

Table 1:

Neg anti- anti- Human serum sample anti- anti- anti- HIV I HIV I anti- HIV Il HIV I HIV Il HIV antigens 52 HIV I HIV antigen amount 43 1250 812 102 " 223 75 gp41 rec. 71 786 1515 521 491 1531 263 p24-rec. 38 831 301 212 63 52 59 gp41 pep 45 41 50 295 1819 45 : .0 gp32 pep -pep 55 1402 1818 62 550 1559 266 gp41, p24 rec. 1380 1753 618 1723 1529 878 gp41, p24 rec + gp32 599

Claims (8)

1. A method for the determination of an immunologically detectable substance according to the principle of heterogeneous immunoassay using a solid phase to which one of the immunologically active reaction components is bound, characterized in that mar, used as a solid phase, a reaction vessel, on the inner surface streptavidin or avidin in is bound in such an amount that there are 0.1 to 2.5 μg per ml of reaction volume. 2. The method according to claim 1, characterized in that the reaction vessel used is a cuvette, which are coated on walls on the inner surface with streptavidin or avidin. 3. The method according to any one of the preceding claims, characterized in that coupling streptavidin or avidin to a soluble protein having a Molekularqewicht about 500,000 and then adsorbs the conjugate of streptavidin or avidin and protein to a hydrophobic solid phase. 4. The method according to any one of the preceding claims, characterized in that one simultaneously determined a plurality of substances, in particular a plurality of antibodies. 5. reaction vessel with opposing optically transmissive wall portions and otherwise at least partially in the area provided for liquid intake inner wall with streptavidin or avidin coated walls gekennzeic inet characterized in that the intended for the liquid intake of the container and the streptavidin or avidin content of the coating are coordinated so that per ml reaction volume 0.1 to 2 ^ g streptavidin or avidin are present. 6. Reaction vessel according to claim 5, characterized in that the streptavidin or avidin is bound to the Fostphase via a soluble protein having a molecular weight above 500,000. 7. Reaction vessel according to claim 5 or 6, characterized in that the reaction vessel is a cuvette. 8. Use of a unit-reaction vessel, characterized in that it is bound from a vessel on the inner surface streptavidin or avidin in such an amount that are available for the determination reaction per ml reaction volume 0.1 to 2.5pg streptavidin or avidin available , consists of determining various parameters in immunoassay procedures.