DK172581B1 - A method for determining an immunologically detectable substance and a suitable reaction vessel - Google Patents

Abstract

To determine an immunologically detectable substance on the principle of the heterogeneous immunoassay using a solid phase to which one of the immunologically active reactants is bound, the solid phase used is a reaction vessel to whose inner surface streptavidin or avidin is bound in an amount such that 0.1 to 2.5 mu g are present per ml of reaction volume. A reaction vessel suitable for this purpose has opposite optically transparent wall regions and, moreover, is coated, at least partially in the region of the inner wall provided to receive liquid, with streptavidin or avidin, where the interior of the container intended to receive liquid, and the streptavidin or avidin content of the coating are matched with one another so that 0.1 to 2.5 mu g of streptavidin or avidin are present per ml of reaction volume. <IMAGE>

Description

in DK PR 172581 B1

The invention relates to a method for determining 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 attached, a suitable reaction container and the use of a unitary reaction vessel.

From FR Publication No. 2,601,455, a solid biological reaction vessel is known for binding a reagent via a biotin-avidin complex, with biotin being bound to the reagent and avidin being bound to the solid support. There is no indication of the amount of avidin that binds to the solid phase.

In determining a specific binding agent, methods 10 are often employed according to the principle of immunoassay. Thus, one of the partners of a specific with each other the binding substance pair is reacted with that of the specific receptor which is marked in a manner known per se. The conjugate of these two substances may then also be reacted with a receptor specific to the conjugate or one of the two portions of the conjugate. There are many variations to this immunological approach. It is advantageous if one of the receptors is bound to a solid phase. This facilitates the separation of bound and non-bound reaction partners. In order to determine the specific binding agent, the amount of labeled reaction-bound or solution-labeled reaction partner is then measured and, in a manner known per se, relative to the amount of the reaction partner to be determined.

As the solid phase, the immunological methods usually employ small plastic tubes or microtiter plates on which the inner surface of the reaction partner is fixed, or beads on whose outer surface the reaction partner is fixed.

Hereby, the binding of the specific reaction partner to the surface in question must be such that it does not lose the ability to specifically bind it to the specific binding agent. For this reason, the reaction of the reaction partner to the solid phase is mostly adsorptive. Therefore, it has already been proposed to effect the fixation of the reaction partner to the solid phase via a coupling agent which mediates the bond. Here again care must be taken to ensure that the binding of the reaction partner to the binding agent does not destroy the specific reacting region of the molecule or that the reaction partner is bound so that its reactive site is away from the solid phase and towards the binding partner.

A disadvantage of all these known methods is that a specific solid phase must be provided for each specific reaction. This means that for each sample, another solid phase must be prepared, stored and then determined, which is labor intensive.

It is therefore an object of the invention to provide a method and a reaction vessel suitable for many different detection methods In the clinical routine diagnosis, a blood test is determined in each case many different parameters such as hormones, tumor markers, infection parameters, allergy parameters and fertility parameters. The concentration of the parameter to be determined encompasses a wide range. TSH (thyroid stimulating hormone) and prolactin are in the range of 1'10-10'12 mol / liter, while high concentration parameters such as Tj and T4 are at about 10'T-10 '* mol / liter . It would be desirable if the 15 individual regulations did not use different pipes each time, but if all the provisions could be used a single reaction vessel.

This task is solved by a method for determining an immunologically detectable substance according to the principle of heterogeneous immune determination using a solid phase to which is attached one of the immunologically active reaction components, which is characterized by the use of a reaction vessel as a solid phase, on the inner surface of which is bound strep-tavidin or avidin in such an amount that ml of reaction volume is 0.1 - 2.5 µg.

This quantification refers to streptavidin or avidin, which is available to the binding partner in the context of the immunological reaction that is in the form of a biotinylated protein or hapten.

The method of the invention is applicable to all assay methods in which the receptor, e.g. may be an antibody or an antigen to be immobilized on the solid phase is conjugated with biotin. The streptavidin or fixed avidin fixed in accordance with the invention according to the invention exhibits good adhesion to all pipe and glass materials and the bond remains stable to detergents. In preparing adjustment curves which are necessary for assessment by many immunological methods, the solid phase bound streptavidin or avidin provides steep adjustment curves at low voids, leading to an increase in accuracy. A further advantage is that charge fluctuations in the amount of coated streptavidin had only an insignificant influence on the measurement results. The particular advantage of the method according to the invention lies in the fact that the bound amount of streptavidin or avidin is adjusted so that the solid phase can be applied to all known methods according to the principle of heterogeneous immune determination. In addition, an advantage lies in the fact that by the method according to the invention, several substances, especially antibodies, but also antigens can be determined simultaneously. This is further illustrated by Example 7.

In a particularly preferred embodiment, the reaction vessel used as a solid phase is simultaneously used as a cuvette for photometric determination of the marker. This is particularly advantageous since only a single vessel is required to perform the overall reaction.

In this case, the reaction vessel is designed to have two mutually optically permeable wall regions. Thereby the total wall area can be coated with streptavidin or avidin.

According to the invention, a reaction vessel is used as a solid phase on the inner surface of which is streptavidin or avidin bound. The binding of the streptavidin or avidin occurs in the manner well known to those skilled in the art (e.g., EP-A 0.122.209). The streptavidin or avidin can either be directly bonded to the solid phase or be bonded via an intermediate or through substances which mediate the binding.

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 ca. 500,000, and this conjugate 25 is then adsorbed on a hydrophobic solid phase, as described e.g. in patent application DE-A 36.40 412. Preferably, a protein which is hydrophobized is used.

4 DK PR 172581 B1

The hydrophobization can be effected, for example, by the use of heat, treatment with acids, denaturing agents and / or chaotropic ions and / or by chemical coupling with a hydrophobic compound.

Treatment with these agents also leads to an increase in molecular weight. The increase in molecular weight can also be achieved by cross-linking with a bifunctional or polyfunctional protein reagent.

As acids, e.g. acetic acid, propionic acid, lactic acid or hydrochloric acid. Usual concentrations are I - 10O mmol / liter at impact times of 10 minutes to 16 hours.

Suitable chaotropic ions are e.g. thiocyanates, iodides, fluorides, bromides, perchlorates and sulfates Suitable denaturing agents are e.g. guanidine hydrochloride or urea. Usually 10 concentrates of 10 mmol / liter - 6 mol / liter are used here.

For derivatization with hydrophobic compounds, soluble fatty acids, low molecular or high molecular weight lipoids and synthetic polymer polymers such as polypropylene glycol or soluble copolymers of polystyrene are preferably used. Derivatization occurs in ways well known to those skilled in the art.

The crosslinking via bifunctional or polyfunctional compounds is carried out with protein binding reagents known per se. These are compounds that carry at least two functional groups that may be the same or different and that can react via these functional groups with functional groups in proteins. Preferably, compounds consisting of an alkyl chain at the ends of which are succinimide, maleimide and / or aldehyde groups are used.

The protein is cross-linked in a manner known per se with the bifunctional or polyfunctional compound, with the soluble protein and the bi- or polyfunctional compound being reacted.

Preferably, for the hydrophobization and / or crosslinking, proteins having a molecular weight of 10,000 - 700,000 are used. Particular preference is given to bovine serum albumin, lipase or immune-γ-globin.

5 DK PR 172581 B1

To the protein is then coupled in a manner known per se, the streptavidin or avidin. Suitable coupling methods are e.g. described in Ishikawa, J. Immunoassay 4 (1983), 209 - 327.

The resulting conjugate of streptavidin or avidin and protein is then adsorptively adsorbed onto the solid-phase plastic surface. Before the conjugate of protein and streptavidin or avidin is adsorbed onto the hydrophobic solid phase, it is also possible to pre-treat the solid phase physically or chemically. Thus, e.g. a plastic surface is pre-swelled or activated in other ways known per se. The adsorptive bond to the solid phase takes place via strong and weak interactions, hydrophobic forces, dipole-dipole or ion-dipole reactions.

Particularly suitable as hydrophobic solid phase, reaction vessels are carrier materials having a surface tension less than the surface tension of the hydrophobic soluble protein, i.e.

more hydrophobic than protein. Carrier materials having a surface tension of 40 erg / cm 2 are preferably used. Particularly suitable are polystyrene, polymethacrylate, teflon, polyamide, copolymers of styrene and acrylonitrile, glass and cellulose products.

The solid phase material of the invention is used to determine many different parameters. Hereby, one of the substances which is capable of binding with the parameter to be determined is conjugated with biotin, which in turn binds to the solid phase streptavidin 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 µg are bonded to the binding of the biotinylated, specifically binding agent.

The reaction volume is hereby referred to as the sum of sample volume and sample reagent volume.

Preferably, the solid phase material is coated so that 1 to 2 µg of streptavidin or avidin is bonded to the bond with biotinylated, specifically binding agent per ml. ml sample volume.

The amount of the biotinylated, specifically binding agent, in the process of the invention is preferably in the range of from 10 to 16 to 10 to 1 mole per day. reaction volume.

6 DK PR 172581 B1

The invention further relates to a reaction vessel having opposite, optically through permeable wall areas and otherwise, at least in part, within the area of the liquid-absorbed inner wall, with streptavidin or avidin-coated walls, the liquid compartment interior being provided. and the streptavidin or avidin content of the coating is matched to each other so that In the reaction volume, 0.1-2.5 µg of streptavidin or avidin are found.

This reaction vessel is particularly suitable for carrying out the process of the invention.

Since it has optically permeable, opposite wall regions and additionally has a coating with streptavidin or avidin, it can be used simultaneously for carrying out the reaction and for photometric determination. By virtue of the coating with 0.1 - 2.5 µg of streptavidin or avidin per ml. In reaction volume, this reaction vessel can be used to determine the most diverse parameters according to the principle of heterogeneous immune determination.

The reaction vessel forms the solid phase. It consists of the commonly used materials such as plastics, glass, etc. The optically permeable wall areas consist of also 15 known materials. Herpolystyrol, copolymers of polystyrol, polycarbonates, polyacrylates and polymethacrylates are particularly preferred.

The streptavidin or avidin coating can be done either directly or via a carrier or intermediate. Suitable for example. the binding of streptavidin or avidin to a soluble protein with a molecular weight above 500,000, which is then adsorbed on the inner wall of the reaction vessel.

The invention also relates to the use of a unit reaction vessel consisting of a container on the inner surface of which is streptavidin or avidin bound in an amount such that the 0.1 to 2.5 µg of streptavidin or avidin are used to determine various parameters by methods according to the principle of immunoassay 25.

7 DK PR 172581 B1

According to the invention, there is provided a method and a reaction vessel by which streptavidin or avidin is well adhered and permanently fixed to the inner surface of a reaction vessel as a solid phase. The swelling is so good that not even the addition of detergents leads to a release of the substance. The reaction vessel of the invention is universally applicable and suitable for carrying out assay methods for many parameters, thereby facilitating the execution of routine determinations.

The invention is illustrated below with drawings and examples. In the drawing, Figure 1 shows a series of adjustment curves for a TSH assay using immobilized streptavidin in various amounts and biotinylated anti-TSH antibody, Figure 2 an adjustment curve for a prolactin sample, Figure 3 an adjustment curve for a CEA sample, Figure 4 an adjustment curve for a T4 sample, and Figure 5 a plurality of adjustment curves for an anti-HB.-B assay using immobilized streptavidin in various amounts.

15 Examples employ monoclonal antibodies against TSH strains from hybridoma cell lines deposited in the European Collection of Animal Cell Cultures, Porton Down, GB, under the designations ECACC 87.122201 and ECACC 87.122202.

EXAMPLE 1.

The solid phase adhesion of the conjugate of the invention and hydrophobic protein and streptavidin were investigated: 8 DK PR 172581 B1

Thermally aggregated RSA, hereinafter referred to as thermo-RSA, was prepared as follows: 1 g of RSA was dissolved in 100 ml of 50 mmol potassium phosphate solution at a pH of 7.0, heated to 70 ° C and kept for 4 hours under slight stirring at this temperature. The solution was cooled, filtered and adjusted to a concentration of 50 mg / ml. Then, 5 were dialyzed against a volume 30 times the volume of redistilled water.

Preparation of a Conjugate of Streptavidin with Thermo-RSA: Streptavidin, extracted by Streptomyces avidinii, was reacted with maleimido-hexanoyl-N-hydroxysuccinimide to give a streptavidin bearing maleimido groups. Thermo-RSA was reacted with S-acetylmercaptoric anhydride and then the protected SH groups were released by the addition of hydroxylamine. The streptavidin containing maleimido groups were then mixed with the SH group-containing thermo-RSA to form the desired conjugate.

Polystyrene plastic glass was then charged with the streptavidin thermo-RS A. conjugate. The glass was charged with 1.5 ml of a streptavidin thermo-RSA solution, the molar ratio of the two components being 1.8: 1 ( 10 µg / ml), in 40 mmol sodium phosphate buffer, pH 7.4, at 20 ° C for 18-24 hours.

After extraction of the glass, it was left for 30 minutes at 20 ° C with 1.8 ml of a solution of 2% sucrose, 0.9% sodium chloride and 0.3% RSA. After drying (24 hours at 20 ° C and 40% relative humidity), the glasses were ready for use and durable.

EXAMPLE 2.

20 ---------------- Single Step TSH Trial.

Biotinylated anti-TSH monoclonal antibodies were prepared. For this purpose, the anti-TSH antibody, ECACC 87.122201, was reacted in the usual way with biotin, with the coupling taking place over the amino groups. Antibody and biotin were used at a ratio of 1: 16,400 ng of the 25 biotinylated antibody thus obtained was dissolved in 1 ml buffer (40 mmol sodium phosphate), 0.5% Pluronic F68, 0.2 mol sodium tartrate per ml. and then incubated together with a 200 µl sample or standard solution containing a known amount of TSH and 75 mU of a peroxidase conjugated anti-TSH antibody (ECACC 87.122202) in a according to Example 1 coated glass for 2 hours. Then three times with wash water 5 was washed and then 1 ml of ABTS substrate solution was added.

After one hour, the extinction was measured at 405 nm. The results are shown in Figure 1. It is found that a satisfactory result is obtained with immobilized streptavidin in an amount greater than 0.1 pg / ml of sample volume.

The need for streptavidin was found to bind the biotinylated antibody used in the sample. For this purpose, the amount of freely available biotin was first determined by the method of Bayer, Edward A., Meir-Wilchek, Analytical Biochemistry 154 (1986), 367-370.

Starting from 1 µg of antibody that was biotinylated in the antibody to biotin ratio 1:15 (Guesdon, I.L., J.Histochem.Cytochem. 27 (1979), 1131-1393), it is obtained that 1-2 moles of biotin per moles of antibody are available for binding, i.e. that per. pg antibody is 1.5 - 3 ng of biotin freely available.

With 10 µg / mIstreptavidin thermo-RSA conjugate charged glass (Example 1), a biotin binding capacity of 15-20 ng ml of sample volume determined with radioactive Cu biotin.

For biotinylated immune-γ-globulins, the binding capacity of the streptavid glass is 1.7-2 pg / ml sample volume. The assay was performed with ln5-labeled γ-globulin (labeled according to the method of McConahey & Dixon (1966), Int.Arch.Allergy 29/185).

EXAMPLE 3.

Prolactinprøve.

Prolactin is determined by a one step sandwich immunoassay. For the detection, a reagent of the following composition is used: DK PR 172581 B1 io 50 mU / ml of a POD conjugate and a prolactin-specific monoclonal antibody, 40 mmol / l phosphate buffer, pH 7.0, 0.5% PluronicF65 (polyoxyethylene polypropylene), 0 , 2 mol / l sodium tartrate, 0.01% phenol, 0.2% bovine serum albumin, 400 ng / ml of a biotinylated monoclonal antibody to prolactin.

For the two monoclonal antibodies, only the requirement is that they recognize prolactin and target different epitopes of prolactin. In a polystyrene coated glass streptavidin thermo-RSA conjugate prepared according to Example 1, 1 ml of this reagent and 50 µl of sample were incubated for 30 minutes at room temperature. Then it was washed three times with tap water. To the detection reaction was added 1 ml of ABTS® substrate solution. After 30 minutes, the extinction was measured photometrically at 405 nm. The results are shown in Figure 2.

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

EXAMPLE 4.

CEA test.

In a sample solution, CEA was determined by one-step sandwich immunoassay. The reagent used had the following composition: 20 120 mU / ml of a POD conjugate and a monoclonal antibody, 40 mmol / l phosphate buffer, pH 7.0, 0.5% PluronicF68, 0.2 mol / l sodium tartrate, 0.01 % phenol, 11 DK PR 172581 B1 0.2% bovine serum albumin, 500 ng / ml of a biotinylated monoclonal antibody to CEA.

For the two monoclonal antibodies against CEA, only the requirements are that they must recognize CEA and are directed to another epitope of CEA.

In a polystyrene-coated streptavidin-thermo-RS coated glass, prepared as in Example 1, 1 ml of the reagent and 100 μΐ sample were incubated for 2 hours at room temperature. Thereafter, three times were washed with tap water. Then, 1 ml of ABTS® substrate solution was added to the detection reaction. After one hour, the extinction was measured at 405 nm photometrically.

The results are shown in Figure 3.

EXAMPLE 5.

T, skill test.

T4 was determined by a competitive immunoassay using biotinylated anti-T4 antibodies. To this end, in a polystyrene-coated polystyrene glass coated with streptavidin thermo-RSA conjugate, 500 μΐ of a reagent consisting of 100 mU / ml of thyroxine-POD conjugate (prepared according to EP 209,155) was incubated. , 120 mmol / l barbiturate, 18.2 mmol / l phosphate buffer, pH 8.6, 0.04 wt% ANS (8-anilino-1-naphthalene sulfonic acid), 0.2 wt% bovine serum albumin and 20 µl sample 10 minutes at room temperature. Then, 500 µl of another reagent consisting of 12 µg PR 172581 B1 was added 1 µg / ml biotinylated polyclonal antibody to T4, 120 mmol / 1 phosphate buffer pH 8.6, 0.04 wt% ANS (8-aniino-1 -naphthalene sulfonic acid), 0.2% by weight of bovine serum albumin 5 and further incubated for 30 minutes at room temperature. After washing the trays with tap water, 1 ml of ABTS® substrate solution was added and incubated for 30 minutes at room temperature.

Then, the extinction was determined photometrically at 405 nm. The results are shown in Figure 4.

EXAMPLE 6.

10 -----------------

Anti-HBj sample HB1 antibodies are determined by one-step sandwich immunoassay. For the detection, a reagent of the following composition is used: 60 mU / ml of a conjugate of POD and HB, antigen, 40 mmol / l phosphate buffer, pH 7.0, 200 mmol / l sodium tartrate, 0.5 wt% Pluronic F68, 0.01 wt% phenol, 0.2% bovine serum albumin, 0.1 wt% bovine IgG, 150 ng / ml biotinylated HB, Ag (prepared according to Immunol.Letters 8 (1984) 273).

With streptavidin thermo-RSA conjugate-coated polystyrene glass of Example 1, 1 ml of this reagent and 200 μΐ sample were incubated for 4 hours at room temperature. Thereafter, three times were washed with tap water. To the detection reaction, 1 ml of 25 ABTS substrate solution was added. After 60 minutes, the extinction was measured photometrically at 422 nm.

13 DK PR 172581 B1

The anti-HB1 test was performed using glass with various amounts of immobilized streptavidin. The results are shown in Figure 5. This shows that when streptavidin is used in amounts that exceed the amounts used according to the invention, the sample becomes clearly more insensitive.

EXAMPLE 7.

Anti-HIV test.

HIV antibodies are determined by a two-step sandwich immunoassay For the detection, the reagents of the following composition are used: 10 Reagent 1: 10'7 mol / liter of one or more biotinylated HIV antigens, 40 mmol / l phosphate buffer, pH 7.0, 0.9% by weight boiling salt, 10% by volume bovine serum.

The antigens used were the following: Genetically engineered HIV1 antigens corresponding to 1 HTV1-gp41 (gp41 rec., Centocor®-p 121) and HIV1-p24 (p24 rec., Centocor®-gp2), chemically synthesized peptides of HIV1 -gp41 (gp41-pep. Wang et al., PNAS 83, 6159 (1986)) and HLV2-gp32 (gp32-pep., JWGnann et al., Science 237, 1346 (1987)). These antigens were labeled with biotin as described by Leary et al., PNAS 80, 4045 (1983).

Reagent 2: 20 mU / ml of a sheep antibody conjugate against human immunoglobulin and POD, 40 mmol / l phosphate buffer, pH 7.0, T4 0.05 wt% Tween 20, 0.2% bovine serum amburain, 0.2% bovine IgG.

In a streptavidin thermo-RSA conjugate coated polystyrene tube of Example 1, 5 L ml of the reagent 1 and 10 μL of human serum or plasma were incubated for one hour at room temperature. Then, three times wash with tap water and 1 ml of reagent 2 incubated for one hour at room temperature. Again, three times was washed with tap water and 1 ml of ABTS substrate solution was added to the detection reaction. After 60 minutes, the extinction was measured photometrically at 422 nm.

The anti-HTV test was performed using single HIV antigens and antigen combinations. Thus, it was found that the test in polystyrene glass coated with Streptavidin thermoRS A conjugate is suitable both for the determination of single antigen-specific antigens and for simultaneous assay. of several antibodies or antibody populations (screening test, Table 1), regardless of whether they are directed against the same virus or multiple viruses or interesting antigens.

Claims (8)

A method for determining an immunologically detectable substance according to the principle of heterogeneous immunoassay using a solid phase to which is attached one of the immunologically active reaction components, characterized in that as a solid phase a reaction vessel is used on its inner surface which is bound streptavidin or avidin in such amount that 0.1 - 2.5 pg. Process according to claim 1, characterized in that a cuvette whose walls on the inner surface are coated with streptavidin or avidin is used as reaction vessel 25. 16 DK PR 172581 B1 A method according to any one of the preceding claims, characterized in that streptavidin or avidin is coupled to a soluble protein having a molecular weight above approx. 500,000 and then adsorb the streptavidin or avidin and protein conjugate on a hydrophobic solid phase. Method according to the preceding claims, characterized in that at the same time several substances are determined, in particular several antibodies. A reaction vessel for use in the method according to claims 1 to 4, with opposite, optically permeable wall regions and, at least in part, in the area of inner wall intended for fluid uptake, with streptavidin or avidin coated walls, the streptavidin or avidin content of the coating is matched to each other so that There are 0.1 to 2.5 µg of streptavidin or avidin in the reaction volume. 5 Anti-Anti-Anti-Anti-Anti-Anti-HIV Antigens Neg. HIVI HIVI HIVI HIV II HIVI HIVII HIV antigen amount of gp41 rec. 52 I250 812 521 102 223 75 10 p24 rack. 43 786 1515 212 491 1531 263 gp41-pep. 71 831 301 295 63 52 59 gp32 pep. 38 41 50 62 1819 45 810 gp41, p24 rack. 45 1402 1818 618 550 1550 266 gp41 p24 stretch + 15 gp 32 pep. 55 1380 1753 599 1723 1529 878 Patent Claims. A reaction vessel according to claim 5, characterized in that the streptavidin or avidin is bound via a soluble protein with a molecular weight above 500,000 to the solid phase. Reaction vessel according to claim 5 or 6, characterized in that the reaction vessel is a cuvette. Use of a unitary reaction vessel consisting of a single vessel, if internal surface bound to streptavidin or avidin in an amount such that the determination reaction per 0, 1 - 2.5 µg of streptavidin or avidin are available for the determination of various parameters by methods according to the principle of immunoassay. 20