Method for the determination of an analyte.

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

The determination of analytes is a widespread concern in particular in clinical diagnostics. More recently, in particular applied to because of the sizes achievable accuracy and the broad applicability methods include the immunological reaction steps. Because of the advantage of ease of handling, it is more and more over to carry out such methods with the aid of a bound to a solid carrier component of an immunological reaction. For example, these methods enable the use of test strips on which runs an entire reaction sequence simply by contacting the sample with the strip.

Immunological determination method can be divided into different classes because of the nature of the reactants involved. One is the class of drugs called competitive displacement tests. In this case, an immobilized antigen to which a labeled antibody is bound, is brought into contact with the sample. The immobilized antigen is displaced in the presence of the analyte in an equilibrium reaction from the immune complex with the labeled antibody. The former immobilized labeled antibody is thus a complex with the analyte to be determined in the liquid phase and can be determined after separating the liquid phase from the solid phase via its label. From this the concentration of the analyte in the sample determined.

Such an immunoassay is for example described in EP-A-0,173,375th As a labeled antibody, a labeled Fab fragment is used, which is present at the beginning of the test in complex with immobilized analyte.

In US-A-4436236 a similar process is described, however, it is used an immobilized antigen which has a lower affinity for the labeled antibody than the analyte. Also in this process preferably labeled antibody fragments are used.

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

In US-A-4277560 is described an immunoassay wherein a labeled analyte is bound via an immobilized antibody reversibly to a solid phase. The labeled analyte is displaced by the analyte contained in the sample from the solid phase and can be used as a measure of the amount of analyte to be determined thereafter. This method has the disadvantage that the accuracy of the results is highly dependent on the uniformity of the loading of the solid phase. Sufficient accuracy is difficult to achieve.

Object of the present invention was therefore to provide a method for determining an analyte, in which the disadvantages of the processes of the prior art, in particular relatively high blank values ​​and expensive measures in the manufacture can be avoided. The method should be accurate and easy to automate.

The object is achieved by a method for determining an analyte in a liquid sample by contacting the sample liquid with an immobilized analyte or analyte analogue and a labeled antibody with both the analyte to be determined, but also with the immobilized analyte or immobilized analyte analogue, an immunological reaction may enter into and immobilized by binding to the immobilized analyte or immobilized analyte, characterized in that the labeled antibody per marker has at least 4 binding sites for the analyte or the analyte analogs.

Likewise provided by the invention is a reagent for carrying out the said method.

The analyte in the AV invention can be determined Ahren, in particular an antigen or a hapten. The determination can be qualitative, ie to determine whether the analyte is present in the sample. However, they 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. Particularly preferred are body fluids or fluids derived therefrom, such as blood, serum, plasma or urine.

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

Under an analyte analogue is analyte immunologically related compound to understand. The analog differs although structurally somewhat from the analyte, but is recognized by an antibody against the analyte. For example, such analyte analogs can be used, which are tied high by the labeled antibodies as the analyte itself in terms of the present invention. In this case, the blank value, so the measurement result without the presence of analyte is particularly low. Example of such an analyte / Analytanalogonpaar is: porcine albumin / human albumin, albumin monkey / human albumin.

Immobilized analytes or analyte analogues are bound to a solid phase analyte or analyte analogues. The binding may be covalent, präzipitativ, specific interactions or adsorptive. Each of these types of bonding can be used if it is ensured that the analyte or the analyte does not separate to a substantial extent from the solid phase. For each of the types of binding are known in the art methods. The nature of the solid phase depends on the nature of the binding of the analyte or analyte analogue. Is the analyte covalently bound, such as a solid phase has to be used which has reactive groups. Upon binding of specific interactions, the binding via biotin / streptavidin is preferred. For example, then the solid phase is provided coating containing a streptavidin and the analyte bound thereto, or the analog of biotin covalently bound. It forms a rather tight binding on streptavidin and biotin. The solid phase may also be present cuvettes, microtiter plates, etc. in the form of particles, paper, nonwoven materials, membranes, tissues, however. If the detection methods are carried out on a test strip, as absorbent non-woven fabrics are preferred.

Labeled antibody is in the process of the invention, an antibody which can undergo an immunological reaction with both the analyte to be determined and the immobilized analyte or analyte analogue. Antibody for the analyte or the analyte-analog can be prepared and selected by known procedures. can be used both polyclonal and monoclonal antibodies, wherein said monoclonal is preferred.

When marking can serve any substance, the presence of the antibody can be detected quantitatively or qualitatively using those. A suitable label is for example an enzyme, a metal, a residue whose emission or absorption can be measured by light or radioactive ray, or a radical which can be converted by chemical or immunological response in such a radical. The skilled worker is a size range of markers available. A condition of the label is that it must be possible to attach to this marking multiple binding sites of an antibody. Therefore, the mark preferably has reactive groups, such as hydroxyl, amino, mercapto or carboxyl groups which can be linked to antibodies directly or indirectly.

Enzymes which are suitable as a marker, are recordable as hydrolases, such as beta-galactosidase, or peroxidases such as POD.

Metals, particularly those most finely distributed in the form of particles, such as colloids, for example, include gold. Such labels are described for example in EP-A-0,258,963th Fluorescent compounds include resorufins; colored compounds are, for example, phycoerythrin, dyed latex particles and dyed tellurium and selenium oxides (EP-A 0,298,368). Preferred labeling agents are enzymes, and metals, particularly preferably enzymes. The labeled antibody of the present process, in contrast to the antibodies mentioned in the prior art, at least 4, preferably 6 to 12 binding sites for the analyte or the analyte analogue on. Such a labeled antibody is hereinafter referred to as oligovalent labeled antibody.

For the preparation of the labeled antibody from oligovalent antibodies and a label is preferably a marker with several antibodies that have less binding sites, for example 1 or 2 binding sites, such as Fab fragments or IgG reacted.

In the methods for preparing labeled antibodies oligovalent often arise mixtures of labeled antibodies that have a different size number of binding sites. From this mixture, mixtures of labeled antibodies can be isolated, which represent a mixture of labeled antibodies with a specific binding site number, for example, 4 to 7 binding sites per label. These mixtures can be used in the method of the invention advantageous if they contain predominantly labeled antibody with at least 4 binding sites per label.

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

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

Preferably, in the process of the invention the mixture of immobilized analyte or analyte analogue and labeled antibody is used as an immobilized immune complex of these constituents. Such an immune complex containing solid phase containing the labeled antibody displaced by the sample analyte is also hereinafter referred to as displacement matrix.

For preparing an immobilized immune complex is formed from the already immobilized analyte or analyte analogue and the labeled antibody in an immunological reaction.

Another method or preparing such a displacement matrix is ​​the implementation of an immunological precipitation reaction between the analyte or analyte analogue and an antibody against the analyte or the analogue on the solid phase. Is then reacted with the labeled antibody. Such a method is described for example in EP-A-0312907.

If the immobilization of the analyte or the analyte analogue of specific interactions such as biotin / streptavidin is to take place, a biotinylated analyte or biotinylated analyte in an immunological reaction with the labeled antibody can be converted to a soluble immune complex, and then with a streptavidin-coated solid phase contacted. After separation of the liquid phase even without washing steps a displacement matrix, which is practically not contaminated with non-complexed labeled antibodies or excess label. But it can also be brought into a first reaction the biotinylated analyte or analyte to the streptavidin-coated solid phase in contact.

Be advantageous in particular the following proportions of the ingredients have been found, the higher the analyte to be determined, the higher the amount of immobilized analyte / analyte should be. If the amount of labeled antibody chosen in relation to the amount of analyte or analyte analogue is too low, then the process becomes worse in some cases useful.

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

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

The process is particularly suitable for the determination of small Probenmengen.Geeignet to study by conventional test strips are particularly sample volumes of 5 .mu.l to 1 ml. The volumes depend on the absorbency of the matrices used. Preferably, the sample volumes do not exceed the suction volumes of the matrices.

At the beginning of the method according to the invention the sample volume with the immobilized analyte or analyte analogue and the labeled antibody is brought into contact and held there for a certain time. During this time found in the presence of analyte to be determined in the sample, the competitive displacement of the immobilized analyte or immobilized analyte analogue from the immune complex with the labeled antibody instead. It forms a soluble immune complex of analyte and labeled antibody. The more analyte is present in the sample, the more soluble immunocomplex is formed. Therefore, it is possible to determine the amount of analyte from the amount of remaining on the solid phase or the labeled antibody contained in the liquid phase labeled antibody. For this purpose, the liquid phase from the solid phase is at least partially separated. Then the amount of label in or on one of the two phases is determined in the usual manner. Where the label is an enzyme, the phase is reacted under conditions suitable for the enzyme reaction with a substrate. The amount of reacted substrate is also a measure of the amount of analyte in the sample.

By carrying out the method according to the invention with samples of known analyte concentration to obtain a calibration curve can be read from the concentration of the analyte not previously known analyte content in a sample from the measured values ​​thus obtained.

The process may be carried out in different versions:

In one embodiment, analyte-containing sample is placed in an Eppendorf tube with a test zone containing the immobilized analyte or the analyte and the enzyme-labeled antibody, a pipette. After shaking, for example for 5 min., A portion of the solution is transferred to a cuvette that contains a chromogenic substrate for the enzyme.

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

In a further embodiment, a cuvette having a test zone described above, mixed with the sample, the mixture incubated for a time and remove the liquid phase from the cuvette. It may then be connected for the complete removal of residues of the liquid phase, a wash step. Subsequently, a solution of a chromogenic substrate for the labeling enzyme in the cuvette will be given. Again, the color change is measured. In contrast to the above-described embodiment, however, the color change is the smaller, the more analyte present in the sample.

A particularly preferred embodiment is a chromatographic strip according to Figure 1. The strip 1 is constructed of a base film 2, mounted on the breather a 3, a 4 and a displacement matrix Substratylies 5 interconnected absorbent. The strip 1 is placed into the sample liquid that only breather 3 comes from the webs with the sample into contact. The use of breathers is particularly advantageous, but not absolutely necessary. The sample is drawn from there into the displacement matrix. 4 This displacement matrix is, for example, an absorbent nonwoven, on which the analyte or analyte analogs are immobilized and containing the labeled antibody in the form of an immune complex with the immobilized analyte or analyte analogue. The analyte to be determined here also displaces the immobilized analyte or the analyte analogue from the immune complex with the labeled antibody. Characterized the resulting soluble immune complex with the sample liquid flows in the zone 5, on which is present impregnated on the right of the enzyme label chromogenic substrate. In the body region 5, the color change is measured. If desired, can be attached to the delay of the liquid stream between displacement matrix 4 and substrate zone slowly sucking tissue.

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

On a base film 11 a displacement matrix 12 is attached, containing the immobilized analyte or analyte and the labeled antibody. At the base film at least partially transparent movable flap 14 is for example mounted on a splice. 13 On the displacement matrix facing side is a film 15 containing the chromogenic substrate. After the incubation period of the film 15 is pressed by lowering the flap on the matrix 12 and thereby started the determination reaction. A test with this test strip can both photometric and with the additional use of reflective elements are by reflection among others.

The inventive method has the advantage that the blank value compared to the measurement signal is relatively small. It has also been found that must be counted in the known from the prior art competitive displacement tests with impurities of the labeled antibody used with non-binding contaminants, such as excess labeling agent. These impurities must be removed for example by washing the containing the immune complex of immobilized analyte or analyte and labeled antibody of the prior art Verdränungsmatrix before performing the test. The favorable ratio of binding sites to marker-labeled antibody in the inventive process contamination by unreacted labeling agent is largely avoided. Another advantage of the invention is that it comprises more steps and matrices. This eliminates expensive and cost as well as time-consuming steps.

Also subject of the invention is a reagent for performing the method according to the invention for determining an analyte in a sample, the immobilized analyte or an immobilized analyte analogue and a labeled antibody having a both the analyte to be determined and also with the immobilized analyte or immobilized analyte analogue, can undergo immunological reaction, wherein the labeled antibody per mark has at least four binding sites for the analyte or the analyte.

Reference is made to the following figures:<ul><li>Fig. 1 shows the longitudinal section through a chromatography strip.</li><li>Fig. 2 shows the longitudinal section through a test strip with the flap. </li><li>Fig. 3 shows a calibration curve for the determination of albumin.</li></ul>

The following examples further illustrate the invention:

example 1

Determination of albumin

1. Preparation of the displacement matrix

a) cross-linking of human serum albumin (HSA) by Disuccinidylsuberat (DSS) to poly-human serum albumin (PHSA)

1.5 g HSA are introduced into 30 ml of potassium phosphate buffer 200 mM, pH = 8.0 and added over 2 hours with 2.5 ml of a solution of 50 mg DSS / ml dioxane. After expiration of the crosslinking reaction of potassium phosphate buffer, 20 mM, pH 7.2 is dialyzed against a 500 fold volume. The high molecular weight fraction (PHSA) having a molecular weight greater than 650,000 Dalton is on Superose 6^R (Pharmacia, Freiburg, Federal Republic of Germany) gel filtration and, after addition of 6 mg sucrose / mg protein lyophilized.

b) Immobilization of human serum albumin

6 x 8 mm wide and 0.5 mm thick pieces of fleece of 50% polyester / 50% linters are impregnated with 15 .mu.l of a solution of 30 mg / l PHSA in 10 mM sodium phosphate buffer pH 7.5 and 30 minutes at 50 ° C dried.

c) conjugates of antibodies to HSA and ß-galactosidase

The conjugates I to XI were obtained from IgG (clone 1 (I-III) or 2 (IV-VII)) or Fab (clone 3 (VIII-XI)) by the method of T. Kitiwaga (Enzyme Immunoassay, eds. Ishikawa , Kawai, Migui; Igaku Shoin Tokyo / New York 1981, pp 81-89) and made respectively by Superose -6^RChromatography fractionated.

Introduction of maleimido groups in IgG

<ul><li>a) addition of 5o .mu.l 0.9 g / l of 3-maleimidobenzoyl-N-hydroxysuccinimide ester in N, N-dimethylformamide to a solution of 1.4 mg (9.3 mmol) of 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 ß-galactosidase

a) dissolving 1.5 mg (2.8 mmol) ß-galactosidase (lyophilized) in 1.4 ml of sodium phosphate buffer (0.1 mol / l; pH 6.5) containing 25.1 mg (8.3 mmol) maleimido IgG. Final concentration of IgG and enzyme is 6 and 2 mmol / l. b) incubating 20 h at 4 ° C. c) separation on Sepharose 6 B (1.5 x 45 cm column) with eluent the following composition: <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Sodium phosphate, pH 6.5</entry><entry namest="col2" nameend="col2" align="char" char=".">10 mmol / l</entry></row><row><entry namest="col1" nameend="col1" align="left">sodium chloride</entry><entry namest="col2" nameend="col2" align="char" char=".">0.1 mol / l</entry></row><row><entry namest="col1" nameend="col1" align="left">magnesium chloride</entry><entry namest="col2" nameend="col2" align="char" char=".">1 mmol / l</entry></row><row><entry namest="col1" nameend="col1" align="left">sodium azide</entry><entry namest="col2" nameend="col2" align="char" char=".">1 g / l</entry></row></tbody></tgroup></table></tables> d) reading the absorbance of the eluate at 280 mm. e) fractionating in pools after absorption profile f) determination of the ß-galactosidase activity of the pools.

This method is also described in E. Ishikawa, J. Immunoassay 4 (3), 209-237 (1983). <tables id="tabl0002" num="0002"><table frame="all"><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">I</entry><entry namest="col2" nameend="col2" align="left">about 4 - 7 IgG per molecule β-galactosidase</entry></row><row><entry namest="col1" nameend="col1" align="left">II</entry><entry namest="col2" nameend="col2" align="left">2-5 IgG per molecule β-galactosidase</entry></row><row><entry namest="col1" nameend="col1" align="left">III</entry><entry namest="col2" nameend="col2" align="left">1-2 IgG per molecule β-galactosidase</entry></row><row><entry namest="col1" nameend="col1" align="left">IV</entry><entry namest="col2" nameend="col2" align="left">4-7 IgG per molecule β-galactosidase</entry></row><row><entry namest="col1" nameend="col1" align="left">V</entry><entry namest="col2" nameend="col2" align="left">3-5 IgG per molecule β-galactosidase</entry></row><row><entry namest="col1" nameend="col1" align="left">VI</entry><entry namest="col2" nameend="col2" align="left">2-3 IgG per molecule β-galactosidase</entry></row><row><entry namest="col1" nameend="col1" align="left">VII</entry><entry namest="col2" nameend="col2" align="left">1-2 IgG per molecule β-galactosidase</entry></row><row><entry namest="col1" nameend="col1" align="left">VIII</entry><entry namest="col2" nameend="col2" align="left">8-14 Fabfragmente per molecule β-galactosidase</entry></row><row><entry namest="col1" nameend="col1" align="left">IX</entry><entry namest="col2" nameend="col2" align="left">6 - 10 Fabfragmente per molecule β-galactosidase</entry></row><row><entry namest="col1" nameend="col1" align="left">X</entry><entry namest="col2" nameend="col2" align="left">4-8 Fabfragmente per molecule β-galactosidase</entry></row><row><entry namest="col1" nameend="col1" align="left">XI</entry><entry namest="col2" nameend="col2" align="left">1-4 Fabfragmente per molecule β-galactosidase</entry></row></tbody></tgroup></table></tables>

d) displacement matrix

It 15 .mu.l of a solution of the conjugate (4 U / ml) of c) in 0.1 M HEPES buffer (pH 7.5) was spotted with 0.5% bovine albumin to a web of b). Subsequently, the web was dried.

e) Determination of the blank value and the measurement range

A stack of two 6 x 8 mm sizes webs of d) with 55 .mu.l of buffer solution (50 mmol / l phosphate pH 7.5) with<ul><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 from the matrix. to the liquid, add 5 mmol / L chlorophenol red ß-galactoside and measures the increase in absorbance at 576 nm in a cuvette at 37 ° C by means of a photometer.

From the measured values ​​A) results in the blank value, ie the value which simulates a signal even without the presence of the analyte.

The measured values ​​for B) in accordance with the signal size of 100 mg / l analyte.

The ratio of signal amplitude to blank value is a measure of the achievable accuracy of the test.

Table 1 gives the blank value (A), the measured value (B) and the ratio B / A for each Konjugatmischungen I to XI again: <tables id="tabl0003" num="0003"><table frame="all"><tgroup cols="4" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">conjugate</entry><entry namest="col2" nameend="col2" align="center">Blank (A) [mU / min]</entry><entry namest="col3" nameend="col3" align="center">Measured value (B) [mU / min]</entry><entry namest="col4" nameend="col4" align="center">B / A</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">I</entry><entry namest="col2" nameend="col2" align="right">55</entry><entry namest="col3" nameend="col3" align="right">1190</entry><entry namest="col4" nameend="col4" align="char" char=".">21.6</entry></row><row><entry namest="col1" nameend="col1" align="left">II</entry><entry namest="col2" nameend="col2" align="right">175</entry><entry namest="col3" nameend="col3" align="right">1900</entry><entry namest="col4" nameend="col4" align="char" char=".">10.8</entry></row><row><entry namest="col1" nameend="col1" align="left">III</entry><entry namest="col2" nameend="col2" align="right">520</entry><entry namest="col3" nameend="col3" align="right">2370</entry><entry namest="col4" nameend="col4" align="char" char=".">4.5</entry></row><row><entry namest="col1" nameend="col1" align="left">IV</entry><entry namest="col2" nameend="col2" align="right">70</entry><entry namest="col3" nameend="col3" align="right">940</entry><entry namest="col4" nameend="col4" align="char" char=".">13.4</entry></row><row><entry namest="col1" nameend="col1" align="left">V</entry><entry namest="col2" nameend="col2" align="right">70</entry><entry namest="col3" nameend="col3" align="right">1385</entry><entry namest="col4" nameend="col4" align="char" char=".">19.7</entry></row><row><entry namest="col1" nameend="col1" align="left">VI</entry><entry namest="col2" nameend="col2" align="right">385</entry><entry namest="col3" nameend="col3" align="right">2175</entry><entry namest="col4" nameend="col4" align="char" char=".">5.6</entry></row><row><entry namest="col1" nameend="col1" align="left">VII</entry><entry namest="col2" nameend="col2" align="right">1330</entry><entry namest="col3" nameend="col3" align="right">2300</entry><entry namest="col4" nameend="col4" align="char" char=".">1.7</entry></row><row><entry namest="col1" nameend="col1" align="left">VIII</entry><entry namest="col2" nameend="col2" align="right">80</entry><entry namest="col3" nameend="col3" align="right">580</entry><entry namest="col4" nameend="col4" align="char" char=".">7.2</entry></row><row><entry namest="col1" nameend="col1" align="left">XI</entry><entry namest="col2" nameend="col2" align="right">106</entry><entry namest="col3" nameend="col3" align="right">660</entry><entry namest="col4" nameend="col4" align="char" char=".">6.2</entry></row><row><entry namest="col1" nameend="col1" align="left">X</entry><entry namest="col2" nameend="col2" align="right">180</entry><entry namest="col3" nameend="col3" align="right">1000</entry><entry namest="col4" nameend="col4" align="char" char=".">5.5</entry></row><row><entry namest="col1" nameend="col1" align="left">XI</entry><entry namest="col2" nameend="col2" align="right">310</entry><entry namest="col3" nameend="col3" align="right">1060</entry><entry namest="col4" nameend="col4" align="char" char=".">3.4</entry></row></tbody></tgroup></table></tables>

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

2. Recording of calibration curves for the determination of albumin

6 x 8 mm wide and 0.5 mm thick pieces of fleece of 50% polyester / 50% linters are mixed with 15 microliters of a solution of 50 or 100 mg / l PHSA at 0.01 mmol / l phosphate buffer pH 7.25 and soaked dried. After the webs are soaked in 15 microliters of a solution of 4 U / ml of the labeled antibody I in HEPES buffer (100 mM, pH 7.5) + 0.5% BSA and dried.

Both webs B and C thus produced are suitable for albumin determination. To accommodate their calibration curves were with samples containing 0 mg / l, 10 mg / l, 50 mg / l or 100 mg / l HSA, soaked. After five minutes, the liquid was separated from the web by centrifugation and their 5 mmol / L chlorophenol red ß-galactoside (CPRG) added. The extinction E (mE / min) was determined photometrically as described under e).

The calibration curves for the webs B and C are shown in Fig. 2. Curve I gives the extinction for the 50 mg / l PHSA soaked fleece B again and curve II was added to the soaked with 100 mg / l PHSA fleece C.

3. Determination of unknown albumin content

For the determination of albumin, a nonwoven B or C is treated with 25 ul of sample unknown analyte content; after 5 minutes, the sample liquid is removed, CPRG added and also the extinction E measured. From the value obtained can be concluded by means of the calibration curve on the albumin content.

example 2

Determination of α₁-microglobulin

Manufacture of displacement matrix

a) Preparation of coated with thermal-BSA streptavidin nonwoven

Thermally aggregated BSA, referred to below as thermo-BSA was prepared in the following manner: 1 g of BSA was dissolved in 100 ml of 50 mM potassium phosphate solution at pH 7.0, heated to 70 ° C and 4 hours with gentle stirring at this temperature maintained. The solution was cooled, filtered and adjusted to a concentration of 50 mg / ml. Then, double-distilled to a 30-fold volume. Water dialyzed.

Preparation of a conjugate of streptavidin with thermo-BSA:

From Streptomyces avidinii streptavidin obtained was reacted with maleimido-hexanoyl-N-hydroxy-succinimide, to obtain in this way a maleimido carrying streptavidin. Thermo-BSA was reacted with S-acetylmercaptosuccinic and then the protected SH groups released by adding hydroxylamine. The maleimido-containing streptavidin was then the containing SH groups thermo-BSA are mixed to form the desired conjugate.

6x8 mm wide and 0.5 mm thick pieces of fleece of 50% polyester / 50% linters are impregnated with 15 .mu.l of a solution of 200 mg / l Thermo-BSA-streptavidin in 10 mM sodium phosphate buffer pH 7.5 and at 50 ° C for 30 minutes dried.

b) Preparation of biotinylated α₁ -microglobulin analogous provision Biotinylation of monoclonal antibodies (according to Peters, Baumgarten, Schulze. Monoclonal antibodies, production and Charkaterisierung; Verl Springer 1985).

c) conjugates of monoclonal AK directed against α₁ (M and ß-galactosidase.

The conjugate was by the method of T. Kitiwaga in Enzyme Immunoassay (Eds Ishikawa, Kuwai, Migui; Igaku Shoin Tokyo / New York (1981) pp 81-89.) Produced and Superose ™ 6 chromatography in the pools I and II fractionated. Pool I ca. 3-7 IgG / ß Gal Pool II ca. 1-3 IgG / ß Gal

d) displacement matrix

It 15 .mu.l of a solution of 50 mg / l biotinylated α₁-microglobulin in 10 mmol / l phosphate buffer pH 7.5 was dropped onto a web of a). Subsequently, the web was dried. Thereafter, 15 ul of a solution of 4 U / ml of conjugate I (Fabric D) or conjugate II (non-woven E) in 0.1 mol / l HEPES buffer (pH 7.5) with 0.5% BSA was dropped.

As in Example 1 the blank value A or the signal B were size when measured using samples A 0 mg a₁M / l B 100 mg a₁M / l (see Table 2) <tables id="tabl0004" num="0004"><table frame="all"><tgroup cols="4" colsep="0" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">conjugate</entry><entry namest="col2" nameend="col2" align="center">Blank (A) [mU / min]</entry><entry namest="col3" nameend="col3" align="center">Measured value (B) [mU / min]</entry><entry namest="col4" nameend="col4" align="center">B / A</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">I</entry><entry namest="col2" nameend="col2" align="right">175</entry><entry namest="col3" nameend="col3" align="right">803</entry><entry namest="col4" nameend="col4" align="char" char=".">4.6</entry></row><row><entry namest="col1" nameend="col1" align="left">II</entry><entry namest="col2" nameend="col2" align="right">327</entry><entry namest="col3" nameend="col3" align="right">960</entry><entry namest="col4" nameend="col4" align="char" char=".">2.9</entry></row></tbody></tgroup></table></tables>

This also shows that the blank with decreasing number of binding sites of the labeled antibody increases disproportionately.

The inclusion of the calibration curve, and the determination of an unknown α1 Mikroglobulingehalts a sample carried out analogously to example 1.