Method for the determination of an analyte.

The invention relates to a method for determining an analyte and a reagent which can be used in this method.

The determination of analytes is a widespread concern, particularly in clinical diagnostics. In recent times, methods involving immunological reaction steps have been used in particular because of the great achievable accuracy and the wide applicability. Because of the advantage of easy handling, there is an increasing tendency to carry out such processes with the aid of a component of an immunological reaction bound to a solid support. For example, these methods enable the use of test strips on which an entire reaction sequence takes place simply by bringing the sample into contact with the strip.

Immunological determination methods can be divided into different classes based on the type of reaction partner involved. One of them is the class of so-called competitive displacement tests. An immobilized antigen, to which a labeled antibody is bound, is brought into contact with the sample. In the presence of the analyte, the immobilized antigen is displaced from the immune complex with the labeled antibody in an equilibrium reaction. The previously immobilized labeled antibody thus passes into the liquid phase as a complex with the analyte to be determined and can be determined via its labeling after separation of the liquid phase from the solid phase. The concentration of the analyte in the sample can be determined from this.

Such an immunoassay is described, for example, in EP-A-0173375. A labeled Fab fragment is used as the labeled antibody and is present at the beginning of the test in a complex with immobilized analyte.

A similar method is described in US-A-4436236, but an immobilized antigen is used which has a lower affinity for the labeled antibody than the analyte. Labeled antibody fragments are also preferably used in this method.

The methods of EP-A-0173375 and US-A-4436236 have the disadvantage that they have high blank values ​​for the measurement, even if there is no analyte in the sample, but the signal size is relatively small.

US-A-4277560 describes an immunoassay in which a labeled analyte is reversibly bound to a solid phase via an immobilized antibody. The labeled analyte is displaced from the solid phase by the analyte contained in the sample and can then be used as a measure of the amount of analytes to be determined. This method has the disadvantage that the accuracy of the results depends very much on the uniformity of the loading of the solid phase. Sufficient accuracy is difficult to achieve.

The object of the present invention was therefore to provide a method for determining an analyte in which the disadvantages of the methods of the prior art, in particular relatively high blank values ​​and complex measures during production, are avoided. The process should be precise and easy to automate.

The object is achieved by a method for determining an analyte in a liquid sample by bringing the sample liquid into contact with an immobilized analyte or analyte analog and a labeled antibody which has an immunological reaction both with the analyte to be determined and with the immobilized analyte or immobilized analyte analog can enter and which by binding to the immobilized analyte or the immobilized analyte analog is immobilized, characterized in that the labeled antibody per marker has at least 4 binding sites for the analyte or the analyte analog.

The invention also relates to a reagent for carrying out the method mentioned.

The analyte that can be determined in the method according to the invention is in particular an antigen or a hapten. The determination can be qualitative, that is, to determine whether the analyte is present in the sample. However, it can also be used quantitatively to determine the concentration or amount of an analyte in the sample. The liquid sample is preferably an aqueous solution, suspension or emulsion. Body fluids or fluids derived therefrom, such as blood, serum, plasma or urine, are particularly preferred.

The concentration of the analyte can be determined with the proposed method in a range from 10⁻⁹ to 10⁻⁵ mol / l, preferably from 10⁻⁸ to 10⁻⁶ mol / l. The determination of so-called highly concentrated analytes is very particularly preferred; as such, analytes in the concentration range greater than 10⁻⁸ mol / l are called. Examples of such analytes in urine are albumin or a₁ microglobulin (a₁M).

An analyte analog is a compound that is immunologically close to the analyte. The analog differs somewhat structurally from the analyte, but is recognized by an antibody against the analyte. For example, for the purposes of the present invention, analyte analogs can be used which are more strongly bound by the labeled antibody than the analyte itself. In this case the blank value, ie the measurement result without the presence of the analyte, is particularly low. An example of such an analyte / analyte analog pair is: swine albumin / human albumin, monkey albumin / human albumin.

Immobilized analytes or analyte analogs are analytes or analyte analogs bound to a solid phase. The binding can be covalent, precipitative, via specific interactions or adsorptive. Any of these types of binding can be used if it is ensured that the analyte or the analyte analog does not detach to a substantial extent from the solid phase. Methods known to the person skilled in the art are available for each of the types of binding. The type of solid phase depends on the type of binding of the analyte or analyte analog. If the analyte is covalently bound, then for example a solid phase must be used which has reactive groups. When binding via specific interactions, binding via biotin / streptavidin is preferred. For example, the solid phase is then provided with a coating containing streptavidin and the analyte bound thereto or the analog is covalently bound to biotin. A fairly firm bond is formed via streptavidin and biotin. The solid phase can be in the form of particles, papers, nonwoven materials, membranes, tissues but also cuvettes, microtiter plates etc. If the detection method is to be carried out on a test strip, absorbent nonwovens are preferred.

In the method according to the invention, labeled antibody is an antibody which can react with both the analyte to be determined and the immobilized analyte or analyte analogue. Antibodies against the analyte or the analyte analog can be produced and selected by known methods. Both polyclonal and monoclonal antibodies can be used, the monoclonal being preferred.

Any substance can be used as a label, with the aid of which the presence of the antibody can be detected quantitatively or qualitatively. A suitable label is, for example, an enzyme, a metal, a residue whose emission or absorption of light or radioactive radiation can be measured, or a residue which can be converted into such a residue by chemical or immunological reaction. A large selection of markings is available to the person skilled in the art. The condition for the label is that it must be possible to attach several binding sites of an antibody to this label. The label therefore preferably has reactive groups, for example hydroxyl, amino, mercapto or carboxyl groups, which can be linked directly or indirectly to antibodies.

Enzymes that are suitable as a label are, for example, hydrolases, such as β-galactosidase, or peroxidases, such as POD.

Metals, in particular those in the form of very finely divided particles, for example colloids, include, for example, gold. Such markings are described for example in EP-A-0258963. Fluorescent compounds are, for example, resorufins; colored compounds are, for example, phycoerythrin, colored latex particles and colored tellurium and selenium oxides (EP-A 0298368). Preferred labeling agents are enzymes and metals, particularly preferably enzymes. In contrast to the antibodies mentioned in the prior art, the labeled antibody of the method according to the invention has at least 4, preferably 6 to 12, binding sites for the analyte or the analyte analog. Such a labeled antibody is referred to below as an oligovalent labeled antibody.

To produce the labeled oligovalent antibody from antibodies and a label, it is preferred to implement a label with a plurality of antibodies which have fewer binding sites, for example 1 or 2 binding sites, such as Fab fragments or IgG.

In the processes for the production of labeled oligovalent antibodies, mixtures of labeled antibodies are also often formed which have a different number of binding sites. Mixtures of labeled antibodies can be isolated from this mixture, which are a mixture of labeled antibodies with a specific number of binding sites, for example with 4 to 7 binding sites per labeling. These mixtures can be used advantageously in the method according to the invention if they predominantly contain labeled antibodies with at least 4 binding sites per label.

For enzyme-labeled antibodies, the binding of 5 IgG or 10 Fab fragments to the enzyme is particularly advantageous.

The production of such labeled oligovalent antibodies is known and is described, for example, in Kitigawa in Enzyme Immunoassay (Eds. Ishikawa, Kuwai, Migui; Igaku Shoin Tokyo / New York (1981), pp. 81-89).

In the method according to the invention, the mixture of immobilized analyte or analyte analog and labeled antibody is preferably used as an immobilized immune complex composed of these components. Such a solid phase containing an immune complex, which contains the labeled antibody displaceable by the sample analyte, is also referred to below as the displacement matrix.

For the production, an immobilized immune complex is formed from the already immobilized analyte or analyte analog and the labeled antibody in an immunological reaction.

Another method or production of such a displacement matrix is ​​to carry out an immunological precipitation reaction between the analyte or analyte analog and an antibody against the analyte or the analog to the solid phase. It is then reacted with the labeled antibody. Such a method is described for example in EP-A-0 312 907.

If the analyte or analyte analog is to be immobilized via specific interactions, such as biotin / streptavidin, a biotinylated analyte or biotinylated analyte analog can be converted into a soluble immune complex in an immunological reaction with the labeled antibody and this then with a solid phase coated with streptavidin brought into contact. After removal of the liquid phase, a displacement matrix is ​​obtained even without washing steps, which is practically not contaminated with uncomplexed labeled antibodies or excess labeling. However, the biotinylated analyte or analyte analog can also be brought into contact with the solid phase coated with streptavidin in a first reaction.

The following quantitative ratios of the constituents have proven to be particularly advantageous: the higher the analyte concentration to be determined, the higher the amount of immobilized analyte / analyte analog should be. If the amount of labeled antibody is chosen too small in relation to the amount of analyte or analyte analog, the method is in some cases less useful.

The amount of immobilized analyte or analyte analog is preferably 1 ng - 0.1 mg / cm² matrix, particularly preferably 0.1 µg - 10 µg / cm² matrix. The amount of labeled antibody is 1 - 1000 mU / cm², particularly preferably 10-500 mU / cm² matrix.

The method according to the invention is generally carried out analogously to the principles known for competitive displacement tests, but using the displacement matrix according to the invention.

The method is particularly suitable for the determination of small sample amounts. Sample volumes from 5 µl to 1 ml are particularly suitable for testing using conventional test strips. The volumes depend on the absorbency of the matrices used. The sample volumes preferably do not exceed the suction volumes of the matrices.

At the beginning of the method according to the invention, the sample volume is brought into contact with the immobilized analyte or analyte analog and the labeled antibody and left there for a certain time. During this time, when the analyte to be determined is present in the sample, the immobilized analyte or immobilized analyte analogue is competitively displaced from the immune complex with the labeled antibody. A soluble immune complex of analyte and labeled antibody is formed. The more analyte in the sample, the more of the soluble immune complex is formed. It is therefore possible to determine the amount of the analyte from the amount of labeled antibody remaining on the solid phase or of the labeled antibody located in the liquid phase. For this purpose, the liquid phase is at least partially separated from the solid phase. Then the amount of marking in or determined in one of the two phases in the usual way. If the label is an enzyme, the phase is reacted with a substrate under conditions suitable for the enzyme reaction. The amount of substrate converted is also a measure of the amount of analyte in the sample.

By executing the method according to the invention with samples of known analyte concentration, a calibration curve is obtained, from which the concentration of the analyte in a sample, previously unknown analyte content, can be read from the measured values ​​obtained therewith.

The process can be carried out in different variations:

In one embodiment, an analyte-containing sample is pipetted into an Eppendorf cone with a test area that contains the immobilized analyte or the analyte analog and the enzyme-labeled antibody. After shaking, for example for 5 minutes, part of the solution is transferred to a cuvette which contains a chromogenic substrate for the enzyme.

The speed of color formation is measured by absorbance measurement at a wavelength at which the resulting colored product absorbs light.

In a further embodiment, the sample is added to a cuvette which has a test region described above, the mixture is incubated for a time and the liquid phase is removed from the cuvette. A washing step can then be carried out to completely remove residues of the liquid phase. A solution of a chromogenic substrate for the labeling enzyme is then added to the cuvette. The color change is also measured here. In contrast to the embodiment described above, however, the color change is smaller the more analyte was present in the sample.

A particularly preferred embodiment is a chromatography strip according to FIG. 1. The strip 1 is made up of a base film 2 on which a absorbent fleece 3, a displacement matrix 4 and a substrate layer 5 are attached to one another in an absorbent manner. The strip 1 is placed in the sample liquid such that only the absorbent nonwoven 3 comes into contact with the sample. The use of absorbent nonwovens is particularly advantageous, but not absolutely necessary. From there, the sample is sucked into the displacement matrix 4. This displacement matrix is, for example, an absorbent fleece on which the analyte or the analyte analog is immobilized and which contains the labeled antibody in the form of an immune complex with the immobilized analyte or analyte analog. Here too, the analyte to be determined displaces the immobilized analyte or the analyte analog from the immune complex with the labeled antibody. The resulting soluble immune complex flows with the sample liquid into zone 5, on which the chromogenic substrate matching the enzyme label is present impregnated. The color change is measured in the substrate zone 5. If desired, a slowly absorbing tissue can be applied between the displacement matrix 4 and the substrate zone to delay the liquid flow.

Another embodiment is a test strip 10 according to FIG. 2.

A displacement matrix 12, which contains the immobilized analyte or analyte analog and the labeled antibody, is attached to a base film 11. An at least partially transparent movable flap 14 is attached to the base film, for example via an adhesive point 13. On the side facing the displacement matrix there is a film 15 which contains the chromogenic substrate. After the incubation period, the film 15 is pressed onto the matrix 12 by lowering the flap, and the determination reaction is thereby started. A test with this test strip can be evaluated, among other things, both photometrically and, if additional reflective components are used, by reflection photometry.

The method according to the invention has the advantage that the blank value is relatively small compared to the measurement signal. It has also been found that in the competitive displacement tests known from the prior art, impurities in the labeled antibody used and non-binding impurities, for example excess marking agent, must be expected. These contaminants must be removed, for example, by washing the displacement matrix containing the immune complex of immobilized analyte or analyte analog and labeled antibody of the prior art before carrying out the test. The favorable ratio of binding sites to labeling agents in the labeled antibody of the method according to the invention largely prevents contamination by reacted labeling agents. Another advantage of the method according to the invention is that it comprises a particularly small number of steps and matrices. This eliminates time-consuming, costly and time-consuming steps.

The invention also relates to a reagent for carrying out the method according to the invention for determining an analyte in a sample, which contains an immobilized analyte or an immobilized analyte analog and a labeled antibody which contains both the analyte to be determined and the immobilized analyte or immobilized analyte analog immunological response, wherein the labeled antibody has at least four binding sites for the analyte or the analyte analogue per label.

Reference is made to the following figures:

Fig. 1 shows the longitudinal section through a chromatography strip.

Fig. 2 shows the longitudinal section through a test strip with a flap.

3 shows a calibration curve for the determination of albumin.

The following examples further illustrate the invention:

example 1

Determination of albumin

1. Production of the displacement matrix

a)

Crosslinking of human serum albumin (HSA) using disuccinidyl suberate (DSS) to poly-human serum albumin (pHSA)

1.5 g of HSA are placed in 30 ml of potassium phosphate buffer, 200 mM, pH = 8.0, and 2.5 ml of a solution of 50 mg of DSS / ml of dioxane are added within 2 hours. After the crosslinking reaction has ended, dialysis is carried out against 500 times the volume of potassium phosphate buffer, 20 mM, pH 7.2. The high molecular weight fraction (pHSA) with a molecular weight of more than 650,000 daltons is separated off on Superose 6® (Pharmacia, Freiburg, Federal Republic of Germany) via gel filtration and lyophilized after the addition of 6 mg sucrose / mg protein.

b)

Immobilization of human serum albumin

6 x 8 mm large and 0.5 mm thick non-woven pieces made of 50% polyester / 50% linters are soaked with 15 μl of a solution of 30 mg / l pHSA in 10 mM sodium phosphate buffer pH 7.5 and dried at 50 ° C. for 30 minutes .

c)

Antibody conjugates against HSA and β-galactosidase

The conjugates I to XI were made from IgG (clone 1 (I-III) or 2 (IV-VII)) or Fab (clone 3 (VIII-XI)) according to the instructions from T. Kitiwaga (Enzyme Immunoassay, ed. Ishikawa , Kawai, Migui; Igaku Shoin Tokyo / New York 1981, pp. 81-89) and each fractionated by Superose-6® chromatography.

Introduction of maleimido groups in IgG

<ul id="ul0001" list-style="none"><li>a) Add 5o µl 0.9 g / l 3-maleimidobenzoyl-N-hydroxysuccinimide ester in N, N-dimethylformamide to a solution of 1.4 mg (9.3 mmol) IgG in 0.5 ml sodium phosphate buffer (0.1 mol / l; pH 7.0).</li><li>b) Incubation at 30 ° C for 30 min. Chromatography on Sephadex G-25® (1x45 cm) with 0.1 mol / l sodium phosphate buffer pH 6.5 as eluent.</li></ul>

Conjugation with β-galactosidase

<ul id="ul0002" list-style="none"><li>a) Dissolve 1.5 mg (2.8 mmol) of β-galactosidase (lyophilized) in 1.4 ml of sodium phosphate buffer (0.1 mol / l; pH 6.5) containing 1.25 mg (8.3 mmol) of maleimido IgG. The final concentration of IgG and enzyme is 6 and 2 mmol / l, respectively.</li><li>b) Incubation 20 h at 4 ° C.</li><li>c) Separation on Sepharose 6 B® (1.5 x 45 cm column) with eluent of the following composition:<dl id="dl0001"><dt>Sodium phosphate, pH 6.5</dt><dd>10th mmol / l</dd><dt>Sodium chloride</dt><dd>0.1 mol / l</dd><dt>Magnesium chloride</dt><dd>1 mmol / l</dd><dt>Sodium azide</dt><dd>1 g / l</dd></dl></li><li>d) Reading the absorption of the eluate at 280 mm.</li><li>e) Fractionation in pools according to absorption profile</li><li>f) Determination of the β-galactosidase activity of the pools.</li></ul>

This method is also described in E. Ishikawa, J. Immunoassay 4 (3), 209-237 (1983).<tables id="tabl0001" num="0001"><img file="EP0407904B1_D0001.tif" /></tables>

d) Displacement matrix

15 μl of a solution of the conjugate (4 U / ml) from c) in 0.1 M HEPES buffer (pH 7.5) with 0.5% bovine albumin were dropped onto a fleece from b). The fleece was then dried.

e) Determination of the blank value and the measuring range

A stack of two 6 x 8 mm nonwovens from d) is mixed with 55 µl buffer solution (50 mmol / l phosphate pH 7.5)<ul id="ul0003" list-style="none"><li>A) 0 mg / l human serum albumin (HSA)</li><li>B) 100 mg / l human serum albumin (HSA)</li></ul> soaked. After 5 minutes the liquid is centrifuged off the matrix. 5 mmol / l of chlorophenol red-β-galactoside are added to the liquid and the increase in absorbance at 576 nm in a cuvette at 37 ° C. is measured using a photometer.

The blank value, ie the value that simulates a signal even without the presence of the analyte, results from the measured values ​​A).

The measured values ​​for B) correspond to the signal size for 100 mg / l analyte.

The quotient from signal size to blank value is a measure of the achievable accuracy of the test.

Table 1 shows the blank value (A), the measured value (B) and the quotient B / A for the individual conjugate mixtures I to XI:<tables id="tabl0002" num="0002"><img file="EP0407904B1_D0002.tif" /></tables>

It is clearly evident that the best accuracy can be achieved with the labeled antibodies, which have the most binding sites.

2nd Recording of calibration curves for the determination of albumin

6 x 8 mm and 0.5 mm thick non-woven pieces made of 50% polyester / 50% linters are soaked with 15 µl of a solution of 50 or 100 mg / l pHSA in 0.01 mmol / l phosphate buffer pH 7.25 and dried. Then the fleeces are each soaked with 15 μl of a solution of 4 U / ml of the labeled antibody I in HEPES buffer (100 mmol, pH 7.5) + 0.5% RSA and dried.

Both nonwovens B and C produced in this way are suitable for the determination of albumin. To record their calibration curves, they were soaked in samples containing 0 mg / l, 10 mg / l, 50 mg / l and 100 mg / l HSA. After five minutes, the liquid was separated from the fleece by centrifugation and 5 mmol / l chlorophenol red-β-galactoside (CPRG) was added to it. The increase in extinction E (mE / min) was determined photometrically as under e).

The calibration curves for the nonwovens B and C are shown in FIG. 2. Curve I shows the absorbance curve for fleece B soaked with 50 mg / l pHSA and curve II was obtained with fleece C soaked with 100 mg / l pHSA.

3rd Determination of an unknown albumine content

To determine albumin, a fleece B or C is mixed with 25 µl sample of unknown analyte content; after 5 minutes the sample liquid is removed, CPRG is added and the increase in extinction E is also measured. The calibration curve can be used to infer the albumin content from the value obtained.

Example 2

Determination of α₁-microglobulin

Production of the displacement matrix

<ul id="ul0004" list-style="none"><li>a) Production of fleece covered with thermo RSA streptavidin Thermally aggregated RSA, hereinafter referred to as Thermo-RSA, was produced in the following way: 1 g of RSA was dissolved in 100 ml of 50 mmol of potassium phosphate solution at a pH of 7.0, heated to 70 ° C. and for 4 hours with gentle stirring Temperature maintained. The solution was cooled, filtered and adjusted to a concentration of 50 mg / ml. Then was distilled against 30 times the volume. Water dialyses. Preparation of a conjugate of streptavidin with Thermo-RSA: Streptavidin obtained from Streptomyces avidinii was reacted with maleimido-hexanoyl-N-hydroxy-succinimide to obtain streptavidin bearing maleimido groups. Thermo-RSA was reacted with S-acetylmercapto-succinic anhydride and the protected SH groups were then released by adding hydroxylamine. The maleimido group-containing streptavidin was then mixed with the SH group-containing thermal RSA to form the desired conjugate. 6x8 mm and 0.5 mm thick non-woven pieces made of 50% polyester / 50% linters are soaked with 15 µl of a solution of 200 mg / l thermal RSA streptavidin in 10 mM sodium phosphate buffer pH 7.5 and 30 minutes at 50 ° C dried.</li><li>b) Production of biotinylated α₁-microglobulin analogous to biotinylation of monoclonal antibodies (according to Peters, Baumgarten, Schulze: monoclonal antibodies, production and characterization; publisher Springer 1985).</li><li>c) Conjugates from monoclonal AK directed against α₁ M and β-galactosidase. The conjugate was prepared according to the instructions of T. Kitiwaga in Enzyme Immunoassay (Eds. Ishikawa, Kuwai, Migui; Igaku Shoin Tokyo / New York (1981) pp 81-89) and with Superose ™ 6 chromatography in pools I and II fractionated. Pool I approx. 3-7 IgG / β Gal Pool II approx. 1-3 IgG / β Gal</li><li>d) Displacement matrix 15 .mu.l of a solution of 50 mg / l biotinylated .alpha.1 microglobulin in 10 mmol / l phosphate buffer pH 7.5 were dripped onto a nonwoven from a). The fleece was then dried. Then 15 μl of a solution of 4 U / ml conjugate I (fleece D) or of conjugate II (fleece E) in 0.1 mol / l HEPES buffer (pH 7.5) with 0.5% RSA were added dropwise. As in Example 1, the blank value A or the signal size B when determined with samples A 0 mg a 1 M / l B 100 mg a 1 M / l determined (see Table 2)</li></ul><tables id="tabl0003" num="0003"><img file="EP0407904B1_D0003.tif" /></tables>

Here, too, it can be seen that the blank value increases disproportionately as the number of binding sites of the labeled antibody decreases.

The calibration curve is recorded and an unknown α1 microglobulin content of a sample is determined analogously to Example 1.