Method for the immunological determination of ligands

The invention relates to an immunological method for the determination of a ligand and a suitable therefor reagent composition.

In body fluids and tissue, the substances are found, which are capable of binding to a specific binding partner and serve as parameters for specific diseases or the health of the human body. For this purpose, on the one hand include immunologically active proteins, for binding sites on their surface, such as tumor markers, hormones or viral proteins, and on the other hand DNA fragments. Since these substances referred to as "ligand" designated often occur only in very small quantities, are used for their detection method according to the immunoassay principle by which these substances can be determined very specifically and accurately. The known immunological method of determination can be divided into homogeneous and heterogeneous methods. In the heterogeneous method, a solid phase reaction is always involved in order to immobilize complexes which contain the substance to be detected and a labeled component and thereby separated from unbound components. In the homogeneous method is no separation of bound label and unbound label so that a differentiation between bound and unbound label must be carried out by other methods.

The heterogeneous immunoassays are based essentially on two variants, namely competitive and sandwich assays. In these variants of the process is generally at least two receptors are used which are connected to the ligand to be detected and capable of binding one of which carries a label and the other is bound to a solid phase or mediates binding to a solid phase. A number of variants are known for this purpose using other receptors. In determining now go an all complexes, which are bound to the solid phase and having a label.

In order to perform competitive heterogeneous immunoassays, there are essentially two types, either an antibody to the ligand is immobilized, or to the ligand analogue is immobilized substance. In the first variant, a sample solution containing the ligand and a conjugate of ligand and a label with the immobilized antibody is incubated. Thereby competing ligand and labeled substance binding to the antibody. is present The more ligand in the solution, the less labeled substance can be bound. After the separation of solid and liquid phase can then be determined in one of the two phases the mark. The amount of bound labeled substance is an indirect measure of the amount of substance to be determined, ie, of ligand.

In the second variant, the sample solution containing the ligand, is incubated with a specific antibody for it as well as the immobilized substance analogs. Here compete immobilized ligand and ligand present in the solution binding to the antibody. The more ligand is present in the solution, the less antibody is bound by binding to the immobilized ligand analogue on the solid phase. Again, the amount of bound label is determined again after separation of the solid phase from the liquid phase, which is indirectly proportional to the amount of ligand in the sample solution again.

In the variants of the sandwich immunoassay, an antibody to the ligand is immobilized, and the sample solution is incubated in the presence of this immobilized antibody to a further antibody which is bindable with the ligand and is labeled. The receptors are thereby added in excess, so that all in the sample molecules to be detected are bound to the solid phase and bind with a labeled receptor.

A disadvantage of this method is that for each substance to be determined (ligand) several specially adapted receptors must be provided. So often marked specific antibodies are required, which are different from ligand to ligand. The covalent binding of marker groups to antibodies, which usually engages in several places of the antibody molecule, can cause undesirable changes in the binding properties of the modified antibody.

EP-A-0344578 discloses a process wherein an avidin-coated solid phase for binding of biotinylated ligand-specific antibodies and enzyme-labeled antibodies for the detection ligandspezifische be used.

US-A-4,228,237 describes methods for the immunological determination, using the enzyme-labeled avidin and biotinylated antibodies specific for the detection and a solid phase with specific antibodies.

Another problem occurs in the detection of proteins or DNA or RNA fragments which have more than one binding site.

Here, each of a plurality or multiplicity of receptors bind with the resulting inaccuracy caused by use of standards is to compensate yet. This imprecision is still potentiated when all receptors used themselves are bi- or polyvalent again.

It was therefore an object of the invention to provide a method, can be detected with the ligand with high accuracy, good reproducibility and high specificity and in which also universal, selected components and universally suitable solid phase materials can be used. In particular, only one specially adapted receptor should be required.

This object is achieved by a method for the immunological determination of a ligand, which is characterized in that with the ligand to be determined <ul><li>a) at least 2 molecules of a P1 specific bindable substance P2 of which at least one of these molecules carries a label and</li><li>b) a reagent comprising receptors R that specifically bind the ligand, each receptor of a single molecule of a P2 monovalent bindable binding partner P1 and the only specific binding molecule R₁ exists for the ligand to be determined and wherein P1 in all receptors R is the same and R₁ may be the same in all the receptors, and then converting the label is determined.</li></ul>

In accordance with feature a) labeled P2 in a known amount is preferably in excess with respect to the possible amount used of ligand, and this excess, if everything P2 is selected, is based on twice the stoichiometric amount of ligand, although unlabeled P2 is used, the simple stoichiometric amount is based ligand. In the latter embodiment also unlabeled P2 in the simple excess is present.

The inventive method is suitable for the determination of all in body fluids or tissue extracts to be detected, capable of a specific binding ligand with low concentrated substances are also well documented as highly concentrated. The sensitivity and accuracy of the method is improved over the previously known methods. The invention provides the ability to quickly and reliably perform with simple reagents provisions.

The process is particularly suitable for the determination of ligands with multiple binding sites, and DNA or RNA molecules. With specific binding site is a binding site thereby understood that can specifically bind with another substance. Examples of these are antigenic determinants specific binding sites on proteins or a specific nucleic acid sequence to DNA or RNA.

An important feature is that each receptor R one partner P1 of the specific binding pair P1 / P2 contains and these partners P1 towards P2 is monovalent, so that only a single molecule P2 can be bound per molecule receptor R. As component R₁ are substances suitable which are capable of specific binding to the ligand. This can, for example, macromolecules such as antibodies, antibody fragments, and specifically bindable binding proteins, haptens, epitopes, or, in the case of DNA or RNA detection, DNA probes. Preferably, the component R₁ only one binding site on the ligand. In the case of a protein detection antibody fragments are preferred, which have only one paratope or substances with which the protein specifically binds, for example, T4 for the detection of T4 binding protein. For the detection of DNA or RNA is used as component R₁ for example, a probe used is capable of hybridizing with a sequence of the DNA or RNA. Particularly preferred as component R₁ in the receptor R a Fab 'fragment of a bindable with the ligand antibody used. R₁ may be a single substance or a mixture depending on the ligand.

The component P1 of the receptor R is a partner of a specifically binding pair. Specifically, each binding pairs are known. Suitable binding pairs are in particular biotin-streptavidin or avidin; Hapten-antibody antigen-antibody, Concavalin antibody; Sugar-lectin; Hapten-binding protein, eg, thyroxine binding globulin and thyroxine or oligopeptide-antibody.

Particular preference is used as the binding pair biotin and streptavidin or avidin so that receptor R₁ more preferably a single molecule contains biotin as partner P1.

The preparation of conjugates is provided using the known to the expert methods (eg analogous to Eur. J. Biochem. 131 (1980) 333-338).

In the case of a heterogeneous method guide P2 conveyed in non-labeled form, the binding to the solid phase.

According to the invention it is achieved to provide a universal test available as solid phase and label conjugate can remain the same for all the provisions and only a single receptor must be matched to the respective ligand to be detected. Since the binding of R, ligand and labeled P2 takes place in homogeneous phase, it is compared to the binding of R to the solid phase P2, which proceeds in the heterogeneous phase, is preferred. The bound to the solid phase unlabeled P2 of the specific binding system is therefore used in excess.

receptor are preferred as R preferably conjugates used consisting of an antibody or antibody fragment R₁ and biotin as P1 exist.

The preparation of the conjugates R is carried out in known manner by reacting the component R₁Such as an antibody or antibody derivative with the partner P1 in a stoichiometric ratio of 1: 1. Preferably, the coupling via a free SH group or an amino group is carried out. The case caused statistically-the products with two or more components P1 can be separated by gel chromatography method. Conjugates R are particularly preferably used for the novel process, consisting of a Fab 'fragment and biotin. To produce such conjugates, for example, a bindable with the ligand to be detected antibodies with pepsin is treated, the resulting F (ab ')₂exposed fragment reducing conditions and subsequently reacted with the component P1, wherein the P1 component binds either via an in component P1 existing functional group or via a spacer, optionally after activation of the binding sites to a free SH-group or amino group of the Fab 'fragment , Products that contain one or more partners P1, can be separated, for example by a process described in EP-A 0,131,343, the difference in charge of related proteins is used for separation. Upon binding of the partner via the amino functions of a protein single or multiple bond leading to conjugates with different charge, which allows their separation.

As a solid phase test tubes or microtiter plates are especially suitable polystyrene and like plastics which are adsorptively or covalently coated on the inner surface with P2. Also suitable are particulate substances such as molecular sieve materials, glass beads, plastic tubes and the like, and porous layered support such as paper. The binding of the partner P 2 is carried out in a conventional manner.

At least a portion of P2, P2 at all homogeneous test procedure is used in labeled form. As mark in this case are the usual markers of immunological tests, such as an enzyme, a fluorescent, chemiluminescent or radioactive substance. Methods for labeling are known in the art, for example from Clin.Chim. Acta<u>81</u> (1977) 1-40 and require no further explanation here. The label may be determined in a conventional manner.

If the invention is used with a homogeneous process management, namely, so everything is P2 used without using a solid phase in a labeled form. The complexes thus formed from ligand, at least two molecules of one of R and R in the complex number corresponding to number of molecules marked P2 is then detected according to the methods known in the art for the measurement of a mark with a homogeneous process procedure. Suitable detection methods are described for example in EP-B 0084 807th

Both heterogeneous and homogeneous-guide of the inventive method, a conjugate K may additionally be used, which contains the ligand or a ligand analog with a binding partner P1 which is identical to the binding partner P1 in the receptor R. In this procedure competing ligand in the sample solution with ligand or ligand analog in the added conjugate K to the component R₁ in the receptor R, so that there is a competitive method. This embodiment can be applied to both the solid phase method and the process in homogeneous phase. For the preparation of the conjugate K, the considerations for the preparation of the receptor R in the same manner. The ligand analog while a substance is considered, the R in terms of their binding ability with component₁ in the receptor R has similar properties to the ligand itself, so that in fact there is a competition for the binding site among the employed stoichiometric conditions.

The process may be carried out in one or more stages. The evaluation is carried out in a conventional manner. Since each of the receptors and the substance to be determined can react only with the specifically intended for it reactants, it is possible to incubate all of the receptors and the sample together and carry out the process in one stage. This is particularly advantageous in carrying out the process in an automated analyzer. In the event that the process is carried out heterogeneously stage, preferably, the substance P2 carries a label used in a substoichiometric amount relative to the receptor R.

The implementation of all process variants is preferably carried out in a buffered solution. Buffer systems for this method are known per se. Particularly suitable for this purpose are GOOD-buffers and phosphate buffers.

To carry out the process in a heterogeneous phase, the sample solution with receptors R, labeled P2, and optionally in the presence of K a with P2 unmarked coated solid phase is incubated either simultaneously or sequentially. In this example, bind two receptors R to the ligand on the components R₁, About P1 partner of a receptor labeled R P2 is then bound, and on the other receptor R binding pair is effected by the P1 / P2 unlabeled binding to the solid phase. In the evaluation of all the complexes display, which are bound to the solid phase and carry a label.

According to the invention, a method is provided which is carried out quickly and easily and is very sensitive even with the use of polyclonal antibodies.

Another object of the present invention is a reagent for the immunological determination of a ligand, which is characterized in that it<ul><li>a) at least 2 molecules of a specifically bindable substance for P1 P2, of which at least one of these molecules carries a label and at least one other molecule P2 is bound to a solid phase or all P2 carries a label and no solid phase is present.</li><li>b) a reagent comprising receptors R that specifically bind the ligand, each receptor of a single molecule of a P2 monovalent bindable binding partner P1 and the only specific binding molecule R₁ exists for the ligand to be determined and wherein P1 in all receptors R is the same and R₁ may be the same in all receptors contain.</li></ul> Preferably, the reagent containing the substance P2 in labeled form and as a solid phase-bound form in united assembly (eg layer of the test carrier or Device magm. Particle solution).

In a preferred embodiment, this reagent contains unlabeled, bound to a solid phase or capable of binding to a solid phase P2. According to another preferred embodiment, the reagent comprises a conjugate zusätzllich K of ligand or ligand analog and a component P1 as defined above.

This reagent is suitable for determining a plurality of parameters in body fluids and tissue extracts.

In a preferred embodiment, the reagent in addition contains buffer substances. Particularly preferably, it contains phosphate buffer or Good buffer.<dl id="dl0001"><dt>Fig. 1</dt><dd>shows a diagram in which the results are plotted for the determination of thyroxine binding index.</dd><dt>FIG. 2</dt><dd>shows a diagram in which the results are plotted for the determination of anti-T4 antibody.</dd></dl>

The invention is illustrated by the figure and the examples.

example 1

Determination of AFP (α-feto protein)

<ul><li>a) Preparation of a conjugate of biotin and Fab 'fragments of anti-AFP antibody (anti-AFP Fab'-biotin) Polyclonal antibodies against AFP are immunosorptively on cleaned and prepared therefrom Fab 'fragments. These are according to Analyt. 161 (1987) 262-271 or Analyt. 149 (1985) 529-536 coupled to biotin.</li><li>b) Test procedure<ul><li>Buffer A:</li><li>120 mmol / l Natriumbarbiturat</li><li>18.2 mmol / l phosphate buffer, pH 8.6</li><li>1.27 mmol / l 8-anilino-1-naphthalene sulfonic acid</li><li>0.2 wt .-% bovine serum albumin</li><li>(Final concentration in the test)</li></ul></li></ul>

480 ul buffer A and 20 ul anti-AFP-Fab 'biotin (final concentration in the assay: 4 ug / ml) to be increased in a coated with streptavidin polystyrene vessel (produced according to EP-A 0 269 092), together with 50 ul of sample (human serum added with AFP) and incubated at 25 ° C for 30 minutes. Then 480 ul buffer A and 20 microliters of a solution of streptavidin-POD conjugate (50 mU / test) are added and incubated at 25 ° C for 30 minutes. It is washed and 1 ml ABTS® solution (9.1 mmol / l ABTS®, 2,2'-azino-di [3-ethylbenzthiazoline-sulfonic acid (6)] - di-ammonium salt, 100 mmol / l phosphate citrate buffer, pH 4.4, 3.2 mmol / l sodium perborate) is added, incubated for 30 minutes at 25 ° C and the optical density at 422 nm as a measure for AFP content.

example 2

T-Uptake Test

The determination is performed in such a manner that T4 is added to the sample in order to saturate excess TBG. Then the T4 unbound is measured. Thus a the thyroxine binding index (TBI) is obtained directly proportional calibration curve.

Sample: standards are used as samples containing amounts of TBG and T4 as defined in human serum. For Sample 1 a TBI of 0.44 and sample 2 gives a TBI of 1.44 (see. Instructions to Enzymun® test® TBK from Boehringer Mannheim GmbH, Order no. 249 416).

reagent 1

<ul><li>100 U / l conjugate of streptavidin and POD</li><li>36 pmol / l T4</li><li>120 mmol / l Natriumbarbiturat</li><li>18.2 mmol / l phosphate buffer, pH 8.6</li><li>0.2 wt .-% bovine serum albumin</li></ul>

reagent 2

<ul><li>0.2 nmol / l conjugate of T4 and biotin</li><li>0.15 mg / l Anti-T₄-Fab'-Biotin (prepared as in Example 1) of polyclonal antibodies to T4</li><li>120 mmol / l Natriumbarbiturat</li><li>18.2 mmol / l phosphate buffer, pH 8.6</li><li>0.2 wt .-% bovine serum albumin</li></ul>

50 ul of sample are mixed with 500 .mu.l reagent 1 added to a substrate coated with streptavidin polystyrene vessel and incubated at 25 ° C for 30 minutes. There are 500 .mu.l reagent 2 was added and incubated at 25 ° C for 30 minutes. Then (see Example 1.) Is washed with water and 1 ml ABTS.RTM solution was added, incubated for 30 min at 25 ° C and the optical density at 422 nm as a measure for the thyroxine binding. The results are shown in FIG. 1.

example 3

Determination of an antibody against T4

As a sample standard solutions of polyclonal antibodies against T4, with the concentrations of 0, 0.5 and 1.0 mg / l are used.

reagent:<ul><li>10 mmol / l T4-Biotin conjugate</li><li>50 U / l streptavidin-peroxidase conjugate</li><li>120 mmol / l Nariumbarbiturat</li><li>18.2 mmol / l phosphate buffer, pH 8.6</li><li>1.27 mmol / l 8-anilino-1-naphthalene sulfonic acid</li><li>0.2 wt .-% bovine serum albumin</li></ul>

50 ul of sample and reagent 1 ml are incubated in a streptavidin-coated polystyrene vessel at 25 ° C for 30 minutes. It is then washed and 1 ml ABTS® solution (cf. Example 1) was added, incubated for 30 minutes at 25 ° C and the optical density at 422 nm as a measure of the level of antibodies directed against T4. The results are depicted in FIG. 2

example 4

Preparation of IgG-Biotin (1: 1)

<ul><li>1) 50 mg of a monoclonal antibody to TSH (ECACC 87122202) is reacted with 2-fold molar excess of D-biotinyl-ξ-amidocaproic acid N-hydroxy succinimide ester according to. JACS 100 (1978), 3585-3590 implemented. Is obtained as the main product monobiotinylated IgG's and höherbiotinylierte by-products are separated off.</li><li>2) The removal is effected by means of DIP chromatography (delta Isoelectric-<u>P</u>oint) for example, is described in EP-A 0,131,343.</li></ul>

The separation of the mixture was performed on a Mono-S cation exchange column (Pharmacia).

The mixture was dissolved in 1 mmol / l potassium pyrophosphate buffer, pH abandoned 6.9 (buffer A). The bound components were eluted by applying a linear gradient with 20 mmol / l Kaliumpyrophosphatpuffer / 200 mmol / l NaCl, pH 6.9 (buffer B). pure monobiotinylyierte IgG fraction is obtained in high yield, in addition to pools with higher degree of biotinylation.

example 5

Preparation of Fab-Biotin (1: 1)

<ul><li>1) Monoclonal antibodies against TSH (ECACC 87122202) will be distributed acc. A. Johnstone, R. Thorpe; Immunochemistry in Practice, Blackwell Scientific Publications (1982), 52 -53, split into Fab. Fab 50 mg are reacted as described in Example 4. FIG.</li><li>2) The separation is effected here under modified conditions:<ul><li>Buffer A: 50 mmol / l MES pH 5.6</li><li>Buffer B: 50 mmol / l MES / 200 mmol / l NaCl pH 5.6</li></ul></li></ul> Under these conditions, Fab-biotin are obtained pure at high yields.

Abbreviations

Biotin X-OSu:

N-Biotinoyl-aminocaproic ξ--hydroxysuccinimide

MES:

2- (N-morpholino) ethanesulfonic acid<ul><li><u>KPP:</u> potassium pyrophosphate</li><li><u>RSA:</u> Bovine serum albumin</li><li><u>POD:</u> peroxidase</li></ul>

example 6

TSH test

The test is carried out with a mixture of the two monoclonal antibodies against TSH (ECACC 87122201 and ECACC 87122202) is gem. Example 5 was prepared as a Fab-biotin.

Step 1:

3 ug Fab-biotin mixture in 1 ml of 50 mmol / l phosphate buffer pH 7.5, 0.1% BSA are mixed with 200 ul of sample (human serum spiked with TSH) for 2 hours in a tube coated with streptavidin (Example 1, see AFP) incubated. Thereafter, unbound material is washed out.

Step 2:

200 mU streptavidin-peroxidase conjugate in lml buffer (see above) is incubated for 1 hour and then washed unbound material.

Step 3:

1 ml Substratlsg (Example 1, see AFP) is incubated for 1 hour and the optical density at 422 nm as a measure of the TSH content.