DD157365A5 - METHOD FOR DETERMINING A COMPONENT FROM A GROUP OF RECEPTORS AND SUBSTANCES - Google Patents

Abstract

The invention relates to methods and means for determining a component from a group consisting of specific binding receptors and substances that can be specifically bound by these receptors by measuring the activity of enzymes bound by incubation, which are characterized in that one of the After immobilization on a water insoluble carrier, incubated with the other component, then after usual washing incubated with saccharide-binding enzymes and enzyme activity determined. As saccharide-binding enzymes, those with lectin properties or lectins with enzymatic activity can be used.

Description

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, Title of the invention :

Method and means for determining a component from a group consisting of specific binding receptors and substances that can be specifically bound by these receptors

Field of application of the invention:

The invention relates to methods and means for determining a component from a group consisting of specific binding receptors and substances which can be specifically bound by these receptors, in particular methods and means for the immunological determination of antibodies, arytenes and haptens in solution via the mediating effect of an enzymatic activity.

Characteristic of the Known Technical Solutions: For the enzyme immunoassays described hitherto, coupling products of a marker enzyme and an antibody or an antigen are required (DAS 2.206.103). With the aid of these antibody-enzyme conjugates, the antigens bound via an immunosorbent are marked. The bound enzyme activity is a measure of the amount of antigen present. For competition procedures z. B.

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Coupling products are produced which compete with the antigen of the sample for binding to the immunosorbent. Here, the bound enzyme activity behaves inversely proportional to the amount of antigen present.

The need to reconcile antibody or antigen-enzyme conjugation

Having to make gate, involves a number of significant disadvantages: 'The chemical linkage of an antibody (antigen) with an enzyme, the antibody (antigen) is disturbed in its binding behavior,

d. H. the binding affinity decreases; the chemical linkage of an enzyme with an antibody (antigen) substantially reduces the enzymatic activity; the stability of the conjugates is less than the stability of the individual substances; the conjugates are very heterogeneous compounds with a broad molecular weight spectrum, whereby a reproducible production is extremely difficult; the enzymatic activity is determined with the conjugates bound to a solid phase, the diffusion of substrates and products influencing the enzymatic conversion. Aim of the invention;

The present invention has for its object to provide methods and means for the determination of antibodies, antigens and haptens, with which the described disadvantages can be avoided.

According to the invention, this object has been achieved by using, instead of a coupling product of a marker enzyme and an antibody or antigen, an enzyme or an enzymatically active lectin which, in addition to the substrate binding site, has further binding sites for the antibody or the antigen.

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Explanation of the essence of the invention;

The invention relates to a method for determining a component from a group consisting of specific binding receptors and substances that can be specifically bound by these receptors, by measuring the activity of enzymes bound by incubation, which is characterized in that one After immobilization, the components are incubated on a water-insoluble carrier with the respective other component, then after usual washing incubated with saccharide-binding enzymes and the enzyme activity is determined.

As the immobilized component, a modified component may be used whose saccharide moiety is chemically or enzymatically altered so that it no longer binds to saccharide-binding enzymes.

In a variant of the method, it is possible to incubate a component immobilized on a water-insoluble carrier simultaneously or successively with the respective other component and a coupling product of the respective other component and a saccharide-containing molecule. In a further variant, it is possible to incubate a component immobilized on a water-insoluble support with the respective other component to be determined and then, after customary washing, incubate with a further saccharide-containing component which binds to the component to be determined.

Furthermore, the invention relates to a means for carrying out this process, comprising a water-insoluble carrier, to which one of the components or a complex of the components, one of which comprises the components of a coupling product with a saccharide.

is bound, optionally a coupling product of one of the components and a saccharide-containing molecule - a saccharide-binding enzyme and substrates for determining the enzyme activity.

Surprisingly, it has been found that the process according to the invention works more accurately and faster than the conventional enzyme immunoassays. It can be dispensed with the production of antibody or antigen-enzyme conjugates, thereby disrupting the

1u immunological reaction is turned off; the enzyme does not have to be modified and can therefore be stored in a form which is preferred for the enzyme until it is used; the reagents can be produced reproducibly and standardized; since the enzyme is released from binding with the antibody (antigen) by the substrate, the enzymatic reaction proceeds in solution rather than on the tube surface; the binding of the enzyme to antibodies proceeds extremely fast (the incubation can be stopped after 15-60 minutes); the anti-competitive preparation to be made. Gen-saccharide conjugates have a much higher durability compared to antigen-enzyme conjugates, moreover, the antigen by the 'incorporation of sugar residues. only slightly modified.

In principle, all organic substances to which a binding partner exists can be quantitatively determined by the method according to the invention. The preferred embodiment is particularly simple when the substance to be determined has saccharide moieties in its molecular structure, which can be bound by a saccharide-reactive enzyme or a lectin with enzymatic activity. This is the case with antibodies, with a variety of antigens and with some haptens the V case.

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To carry out the method according to the invention, the sample solution containing the substance to be determined is incubated with the binding partner immobilized on a water-insoluble carrier, which or to which the substance to be determined specifically binds. During the incubation, an amount corresponding to the concentration of the substance binds via its binding partner to the carrier material. After removing the sample solution, the carrier material is washed to remove unspecifically adhering substances. Afterwards, this carrier material is brought into contact with an enzyme reacting with a saccharide moiety for a short time, the enzyme being bound to the substance to be determined. After washing out unbound enzyme, a solution with an enzyme substrate is added to the support material. The enzyme activity can then be measured photometrically and is directly a measure of the amount of the substance to be determined in the sample solution.

This simple method can be used when using immobilized antibodies or other receptors for the determination of saccharide-containing antigens and haptens or for immobilized antigens or haptens for the determination of their corresponding antibodies or saccharide-containing receptors.

If the binding partner immobilized on the water-insoluble support also has saccharide groups which can be recognized by the enzyme used, these may, if necessary, suitably be degraded chemically or enzymatically prior to immobilization. - '·

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If the substance to be determined does not have a suitable saccharide structure which makes it possible to bind a saccharide-reactive enzyme, a known amount of the substance to be determined can be used as an additional reagent, but has been chemically or enzymatically linked to a suitable saccharide and through this Marking of the enzyme concerned. can be known. Since one can determine this conjugate in the above manner, it is easily possible to quantitatively determine the substance to be determined by a competition method. It is possible to proceed by incubating the substance to be determined from the sample and the labeled substance simultaneously or with a time delay with a limited amount of the binding partner immobilized on the water-insoluble carrier. In this case, the substance to be determined and the labeled substance compete for binding to the common binding partner. Is the substance to be determined in a high concentration

2Q, only a small amount of the labeled substance will bind to the support material, i. H. the enzyme activity adhering to the carrier material after incubation with the saccharide-binding enzyme in this method is inversely proportional to the concentration of the substance to be determined in the sample solution.

A variant of this method consists in immobilizing the binding partner for the substance to be determined on the water-insoluble carrier and then incubating it with an excess of labeled substance. This saturates the binding partner with the labeled substance, d. H. the support material now has preformed complexes of the binding partner and the labeled substance. The test is then only in

the incubation of this carrier material with the sample solution. Depending on the concentration of the substance to be determined, the labeled substance is again displaced from the complex and the detection can then take place via the binding of the saccharide-binding enzyme.

The labeling of haptens and antigens with a suitable saccharide can be done by direct chemical or enzymatic coupling. On the other hand, the substance to be determined can also be coupled to molecules

1U, which in its molecular structure i.a. have the appropriate saccharide moiety. Such molecules may have a molecular weight of several hundred to one million or more; Preferably, the molecular weight is less than 600,000, in particular less than 300,000. Such molecules are glycoproteins, polysaccharides, glycosylated proteins or smaller molecules which, in addition to the suitable saccharide moiety, also have groups for the coupling of the substance to be determined. The conjugates resulting from the coupling then compete for binding to the immobilized binding partner as described above. The further test procedure then corresponds to that described in the competition process.

Another possibility according to the invention for the determination of substances which do not have a suitable saccharide structure

is to use an additional soluble binding partner. In this case, the sample with the substance to be determined is incubated with the immobilized binding partner, with a fraction corresponding to the concentration .30 of the substance binding via the binding partner to the carrier. After washing out the

Sample solution is added to the soluble binding partner. This soluble binding partner may correspond to the immobilized binding partner or have a different composition. The decisive factor is that it has a suitable Saccharidgruppierung and with off. reaching affinity binds to the bonded via the immobilized binding partner substance on the substrate. The binding of the soluble binding partner can then be determined again by a saccharide-binding enzyme in a known manner. The activity adhered to the carrier material is proportional to the concentration of the substance to be determined.

The prerequisite for the process variant described above is that the substance to be determined has multiple binding sites so that both the immobilized and the soluble binding partner can bind. However, if the substance to be determined has only one binding site, it is possible to chemically bind several molecules of this substance to a carrier molecule, so that conjugates are formed which can bind a plurality of binding partners. In this case, a competition procedure must first be performed, i. H. the conjugate and the substance to be determined compete for binding to the immobilized binding partner. The amount of bound conjugate depends on the concentration of the substance to be determined in the sample solution. The soluble binding partner can then bind to the bound conjugate, which in turn binds the saccharide-binding. The enzyme activity adhering to the carrier is inversely proportional to the concentration of the substance to be determined in the sample. · ..

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Enzymes with lectin properties or lectins with enzymatic activity which react with saccharides, preferably galactose oxidase, galactosidase and / or mannosidase, are suitable for carrying out the process according to the invention. Galactose oxidase from Dactylum dendroides has proven to be particularly suitable.

Plastic containers are preferably used as water-insoluble carriers, in particular test tubes made of polystyrene or polypropylene.

A determination of antigens is carried out in the simplest case so that first polystyrene tubes are coated with antibodies. The coating is carried out for about 3 to 24 hours at room temperature 'in a suitable buffer, eg. Borate buffer, phosphate buffer, carbonate buffer or "Tris buffer in a concentration of 0.01-0.5 mol / l and at pH values of 7-10 / preferably in a 0.1 M borate buffer of pH 8, 4. The tubes are then emptied and washed with a detergent-containing buffer, preferably using a phosphate buffer containing about 0.01 to 0.1% of one or more detergents such as polyoxyethylene sorbitan monooleate, polyoxyethylene ether higher alcohols, etc.

The antibody-coated tubes are then incubated with the determining antigens. This incubation is carried out for about 2 to 24 hours at room temperature in said buffers, preferably in a 0.05 to 0.2 M phosphate buffer of pH 7.5 to about 0.5 to 2% ovalbumin or a. another protein can be added. The tubes are then emptied again and washed with a detergent-containing buffer.

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Subsequent incubation with an enzyme having lectin properties or with a lectin having enzymatic activity is carried out in a 0.05 to 0.2 M phosphate buffer, preferably in a 0.1 M potassium phosphate buffer of pH 7.2. Galactose oxidase, galactosidase and mannosidase are particularly useful as saccharide-binding enzymes with lectin properties, and as lectins with enzymatic activity phytohemagglutinins from lima bean or mung bean are preferred (J.Biol.Chem. 253, 7791 (1978)). After 15 - 60.Minuten the tubes are emptied again and washed with the above-described detergent-containing buffer.

When using galactose oxidase as an enzyme, the detection reaction with galactose as a substrate. The tubes are mixed with a reaction solution, the z. B. in a 0.1 M phosphate buffer contains galactose, phenol, aminoantipyrine and peroxidase. After about one hour, the absorbance of the solution is measured at 492 nm. Other reaction solutions can be used to determine hydrogen peroxide, such as. A 0.2 M phosphate buffer containing galactose, potassium iodide, ammonium molybdate and ethylenedinitrilotetraacetic acid. The resulting color is then measured at 350 nm

 AusfUhrungsheispiele;

Example 1 .

Determination of anti-BSA antibodies

a) Preparation of BSA-coated tubes

Polystyrene tubes were adsorptively coated with bovine serum albumin (BSA). For this were. tubes with 1 ml of a 10 μg BSA solution

• filled per ml of 0.1 M borate buffer (pH 8.4) and allowed to stand at room temperature for 20 hours. Thereafter, the tubes were drained and washed twice with a phosphate buffer containing 0.05% polyoxyethylene sorbitan monooleate.

b) incubation with anti-BSA

A solution of anti-BSA (rabbit IgG) was diluted to concentrations of 1 to 10 000 ng IgG / ml by addition of 0.1 M phosphate buffer (pH 7.5) (containing 1% ovalbumin). Each 1 ml of these solutions was added to the BSA coated tubes and allowed to stand at room temperature for 18 hours. Thereafter, the tubes were emptied and washed twice with detergent-containing phosphate buffer.

c) incubation with galactose oxidase

450 units of galactose oxidase were dissolved in 2 ml of 0.1 M potassium phosphate buffer (pH 7.2) and diluted 1:30 with the same buffer before use. Each 1 ml of this solution was added to the tubes. After 15 - 60 minutes, the. Emptied tube and washed once with detergent-containing phosphate buffer.

d) detection reaction

To the tubes was added 1 ml of a reaction solution of the following composition: 100 ml of a solution of 2.35 g of phenol and 50 g of galactose per liter of 0.1 M sodium phosphate buffer (pH 7.3) were mixed with 1 ml of a 0.2 M solution Aminoantipyrine spiked. To this was added 5 ml of a solution of 1 mg / ml peroxidase. After 60 minutes

the absorbance of the solution was measured at 492 nm in the photometer. As shown in the table below, the absorbance correlates with the amount of added antibodies to BSA.

Amount of anti-BSA Absorbance at 492 nm [Ng / ml] [OD] O 0,012 1 0,035 10 0.063 100 0,080 , 1000 0,122 10000 0.268

Example 2

Determination of antigens containing sugar residues: human IgG (h-IgG)

a) Coating of tubes with antibodies against h-IgG from sheep (a-h-IgG)

Polystyrene tubes were adsorptively coated with antibodies to h-IgG (a-h IgG). For this purpose, the tubes were washed with 1 ml of a solution of. 10 μg / μg IgG per ml 0.1 M borate buffer (pH 8.4) and allowed to stand at room temperature for 20 hours. Thereafter, the tubes were emptied and washed twice with detergent-containing phosphate buffer.

b) Incubation with h-IgG

A solution of h-IgG was diluted to 1-10 ng / ml by addition of 0.1 M phosphate buffer (containing 1% ovalbumin). 1 ml each

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of these solutions was added to the a-h-IgG coated tubes and allowed to stand at room temperature for 18 hours. Thereafter, the tubes were emptied and washed twice with detergent-containing phosphate buffer.

c)

Incubation with galactose oxidase

450 units of galactose oxidase were dissolved in 2 ml of 0.1 M potassium phosphate buffer (pH 7.2) and diluted 1:50 with the same buffer before use. Each 1 ml of this solution was added to the tubes. After 15-60 minutes, the tubes were drained and washed once with detergent-containing phosphate buffer.

d) detection reaction

The tubes were mixed with 1 ml of a reaction solution analogously to Example 1 d. After 60 minutes, the absorbance of the solution at 492 nm was measured against the zero value in the photometer. The absorbance correlates with the amount of h-IgG added, such as

2ü below table shows.

Amount of h-IgG Absorbance at 492 nm [G / ml] [OD] 0 0.213 1 0,220 2 0.225 4 0.232 6 0.236 8th 0,242 10 0,271

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Example 3

Determination of antigens after labeling with sugar residues: bovine serum albumin labeled with melibiose (BSA melibiose)

u, a) Coating of tubes with antibodies against rabbit BSA (a-BSA)

Polystyrene tubes were adsorptively coated with antibodies to BSA (a-BSA). For this purpose, the tubes were filled with 1 ml of a solution of 10 yag a-BSA per ml of borate buffer (pH 8.4) and allowed to stand at room temperature for 20 hours. Thereafter, the tubes were drained and washed twice with detergent-containing phosphate buffer.

b) Preparation of BSA Melibiose Conjugates

68 mg of BSA, 100 mg of melibiose and 10 mg of sodium cyanoborohydride were dissolved in 5 ml of 0.2 M potassium phosphate buffer (pH 8.0) and allowed to stand at 37 ° C for 48 hours. The solution was chromatographed on Sephadex G-25 in 0.1 M potassium phosphate buffer (pH '7, O) to separate the excess melibiose.

C) incubation with BSA melibiose

A solution of BSA melibiose was diluted to concentrations of 0.1 - 10 ng / ml by addition of phosphate buffer (containing 1% ovalbumin). Each 1 ml of these solutions was added to the a-BSA coated tubes and allowed to stand at room temperature for 18 hours. After that, the

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Emptied tube and washed twice with detergent-containing phosphate buffer.

The incubation with galactose oxidase was carried out analogously to Example 2c

d) detection reaction

The tubes were mixed with 1 ml of a reaction solution analogously to Example 1 d. After 60 minutes, the absorbance of the solution was measured at 492 run versus zero in the photometer. The absorbance correlates with the amount of added BSA-melibiose conjugate as shown in the table below. ,

Amount of BSA metabolism [μg / ml]

0.1

1.0

10.0

Absorbance at 492 nm [OD]

0.237 0.294 0.329

Analogously, thyroxine or a conjugate of thyroxine and BSA can be labeled with melibiose. These conjugates or a conjugate of thyroxine and a γ-globulin can be quantified using anti-thyroxine coated tubes. _f

Example 4

Binding of Galactose Oxidase in a Sandwich Reaction: Determination of BSA

The coating of tubes with antibodies against rabbit BSA (a-BSA) was carried out analogously to Example 2 a.

a) Incubation with BSA

A solution of BSA was diluted to 1-10,000 ng / ml by addition of phosphate buffer (containing 1% ovalbumin). Each 1 ml of these solutions was added to the a-BSA coated tubes and allowed to stand at room temperature for 18 hours. Thereafter, the tubes were emptied and washed twice with detergent-containing phosphate buffer. ,

• J5 b) incubation with a-BSA

The tubes were incubated for 3 hours with 1 ml each of a solution of 1 ng a-BSA in 10 mM Tris buffer and then drained. It was washed twice with detergent-containing phosphate buffer.

The incubation with galactose oxidase was carried out analogously to Example 2 c.

c) detection reaction

The tubes were mixed with 1 ml of a reaction solution analogously to Example 1 d. After 60 minutes, the absorbance of the solution became 492 nm

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measured against the zero value in the photometer. The absorbance correlates with the amount of BSA added, as shown in the table below.

Amount of BSA Absorbance at 492 nm [G / ml] [QD] 1 0.396 10 0.419 100 0,465 1 000 0.493 10 000 0.543 Example 5

Binding of galactose oxidase to antibodies and modified antibodies

a) Elimination of oligosaccharides in glycoproteins

4 mg sheep anti-human IgG (a-h IgG) were dissolved in 1 ml dist. Water was dissolved and stirred with 0.2 ml of a fresh 0.1 M sodium periodate solution for 20 minutes at room temperature. Thereafter, the solution was dialyzed against 1 mM sodium acetate buffer (pH 4.4) at 4 ° C overnight. After adding 1 ml of a 0.1 M lysine solution in 0.2 M sodium carbonate buffer (pH 9.5), it was stirred for 2 hours at room temperature and then 0.1 ml of a solution of 4 mg of sodium borohydride per ml was added. After 2 hours, dialysis was carried out against borate buffer (pH 8.4).

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b) Coating of tubes with a-h-IgG and modified a-h-IgG

From the antibodies, dilutions were made at 0, Iy 1.0 and 10 μg per ml in borate buffer (pH 8.4). Polystyrene tubes were allowed to stand with 1 ml of the respective solution overnight. Thereafter, the tubes were drained and washed twice with detergent-containing phosphate buffer.

The incubation with galactose oxidase was carried out analogously to Example 2 c.

c) detection reaction

The tubes were mixed with 1 ml of a reaction solution analogously to Example 1 d. After 60 minutes, the absorbance of the solution at 492 nm was measured against the zero value in the photometer. The absorbance correlates with the amount of a-h IgG in the coating buffer as shown in the table below. Oxidized a-h IgG showed only a very low binding affinity for galactose oxidase.

a-h IgG in extinction modifier Extink Beschich at 492 nm a-h IgG in the at tung buffer coating 492 nm buffer [μg / ml]. FOD] [G / ml] [OD] 0.1 0.04 0.1 0.01 1.0 0.47 1.0 0.02 10.0 0.53 10.0 0.03

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Example 6

Determination of antigens containing sugar residues: human IgE (h-IgE)

a) Coating of tubes with antibodies to goat h-IgE (a-h-IgE)

Polystyrene tubes were adsorptively coated with a-h-IgE, which had been modified analogously to Example 5. For this purpose, the tubes were filled with 1 ml of a solution of 0.1 μg / h IgE per ml borate buffer (pH 8.4) and allowed to stand at room temperature for 20 hours. Thereafter, the tubes were emptied and washed twice with detergent-containing phosphate buffer. ,

b) Incubation with h-IgE

A solution of h-IgE was diluted to concentrations of 1-100 ng / ml by addition of phosphate buffer (containing 1% ovalbumin). Each 1 ml of these solutions was added to the a-h-IgE coated tubes and allowed to stand at room temperature for 18 hours. Thereafter, the tubes were emptied and washed twice with detergent-containing phosphate buffer.

The incubation with galactose oxidase was carried out analogously to Example 2c.

c) detection reaction. ,

The tubes were mixed with 1 ml of a reaction solution analogously to Example 1 d. After 60 minutes

The absorbance of the solution at 492 nm was measured against the zero value in the photometer. The absorbance correlates with the amount of added h-IgE, as shown in the table below.

Amount of h-IgE Absorbance at 492 nm [Ng / ml] [OD] 1 0.054 10 0,069 100 0.081 Example 7

Determination of antibodies containing sugar residues with ß-galactosidase: a-BSA - '

The coating of tubes with 'BSA and the incubation with anti-BSA was carried out analogously to Example 1 a and Ib.

a) incubation with β-galactosidase

A crystal suspension of β-galactosidase at 5 mg / ml was diluted 1: 500 with 10 mM Tris-HCl buffer containing 10 mM magnesium chloride, 10 mM mercaptoethanol and 10 mM sodium chloride. 1 ml of this enzyme solution was added to the tubes. After 2 hours, the tubes were drained and washed once with 0.1 M potassium phosphate buffer (pH 7.0).

b) detection reaction

To each tube was added 1 ml of a substrate solution composed as follows:

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15 ml of 0.3 M potassium phosphate buffer containing 3 mM magnesium chloride, 4.5 ml of 1 M mercaptoethanol, 7.5 ml of 14 mM 2-nitrophenyl-β-D-galactopyranoside in 10 mM Tris-acetate buffer containing 10 mM magnesium chloride and 18 ml of dist. Water. After 1-2 hours, the absorbance of the solution in the photometer was measured at 405 nm. The values correlate with the amount of α-BSA added, as shown in the table below.

Amount of a-BSA Absorbance at 405 nm [Ng / ml] [OD] • 1 0.14 10 0.17 100 0.22 1 000 0.25 10 000 0.33

Example 8.

Determination of chorionic gonadotropin (hCG)

a) Coating of tubes with antibodies to chorionic gonadotropin (a-hCG).

Polystyrene tubes were adsorptively coated with a-hCG. For this purpose, the tubes were filled with Ί ml of a solution of 10 μg / μg α-hCG per ml of 0.1 M carbonate buffer (pH 9 »0) and allowed to stand at room temperature for 4 hours, after which the tubes were emptied and washed twice with detergent-containing phosphate buffer. ·

b) Incubation with hCG

A solution of hCG (3000 IU / mg) was diluted to concentrations of 1 to 320 IU / ml by adding 0.1 M phosphate buffer (containing 1 % ovalbumin),

Each 1 ml of these solutions was added to the a-hCG "coated tubes and allowed to stand at room temperature for 4 hours, after which the tubes were deflated and washed twice with detergent-containing phosphate buffer.

, c) incubation with G-alactose oxidase

450 units of G-alactose oxidase were dissolved in 2 ml of 0.1 M potassium phosphate buffer (pH 7.2) and diluted 1: 100 with 0.01M 2-morpholineethanolsulfonic acid buffer (pH 5.0) before use. Each 1 ml of this solution was added to the tubes. After 5-60 minutes at 4 ⁰ C, the tubes were emptied and washed once with the same buffer. ·

d) detection reaction

The tubes were mixed with 1 ml of a reaction solution analogously to Example 1 d. After 10 minutes, the absorbance of the solution was measured at 492 nm in the photometer.

As the table below shows, the absorbance correlates with the amount of added hCG.

Amounts of hCG Absorbance at 492 nm [Ml.U./ml] [OD] 0 0,020 1.25 0.026 12.5 0,050 125.0 0,100 313.0 , , 0.174

Analogous results were obtained when instead of 2-Morpholinäthanolsulfonsäufe as Yverdünnungsuffer for galactose oxidase (step c), the following

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Buffers were used - were:

Acetate, citrate, tris, glycine, glycylglycine, N, N-bis (2-hydroxyethyl) glycine, piperazine-1,4-diethansulfonic acid.

Example 9

Determination of human IgE (h-IgE)

a) Coating of Tubes with Antibodies to Rabbit h IgE (a-h IgE)

Polystyrene tubes were adsorptively coated with a-h-IgE, for which the tubes were filled with 1 ml of a solution of 10 μg per ml of carbonate buffer (pH 8.0) and allowed to stand at room temperature for 4 hours. Thereafter, the tubes were emptied and washed twice with detergent-containing phosphate buffer.

"b) incubation with h-IgE

A solution of h-IgE was diluted to concentrations of 1-250 ng / ml by the addition of phosphate buffer (with 1 io bovine serum alburain). Each 1 ml of this solution was added to the ah-IgE "coated 20-tube and allowed to stand at room temperature for 6 hours, after which the tubes were emptied and washed twice with detergent-containing phosphate buffer.

The incubation with Galactos.eoxidas.e was carried out analogously to Example 8c at an incubation temperature of 10 ^° C.

c) detection reaction

The tubes were mixed with 1 ml of a reaction solution analogously to Example 1 d. After 10 minutes, the absorbance of the solution was measured at 492 nm against the NuIl value in the photometer. Like the following

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Table shows correlates the extinction with the amount of added h-IgE.

Amount of h-IgE [ng / ml]

Extinction "at 492 mn [OD]

10 100 250

0.276 0.396 0.755 1.458

Claims (11)

7 claims Anspruch [en] A method for determining a component from a group consisting of specific binding receptors and substances which can be specifically bound by these receptors, by measuring the activity of enzymes bound by incubation thereon, characterized in that one of the components is immobilized on a water-insoluble carrier incubated with the other component, then incubated after usual washing with saccharide-binding enzymes and determines the enzyme activity. .. ^ .-. 2. Method according to TWW 1, characterized in that are used as the saccharide-binding enzymes with lectin properties or lectins with enzymatic activity. 3. The method according to tfen "Pun (-Urt 1 and 2, characterized in that are used as the saccharide-binding enzymes galactose oxidase, galactosidase and / or mannosidase. 4. Method according to ai & n 'Pw /? V4f? 1-3, characterized in that the immobilized component used is a modified component whose saccharide moiety has been changed so that it no longer binds any saccharide-binding enzymes and / or lectins. • 10 det. 5. The method according to ' _n points' 1-4, characterized in that one immobilized on a water-insoluble carrier component simultaneously or successively with the other component and a coupling product of the other component and a saccharide-containing mole 6. The method according ö & n '7Wv * ^ 1-4, characterized .gekennzeichnet that a at a wasserunlös-' borrowed carrier immobilized component with each other, incubated component to be determined and then, after washing with üblfichem another saccharide-containing incubated to the component to be determined binding component. 7. Method according to gfett .7W # w * 1-6, characterized in that one determines the activity of the bound enzyme or lectin. 8. The method according to ßfcfi "TWjvfStt 1-6, characterized in that one determines the activity of the enzyme or lectin in the solution obtained after the last incubation. 9. Means for carrying out the process according to % m ^, tOn 1-8 / containing a water-insoluble xrager, to the one of the components or a complex of the components, of which one of the components i> consists of a coupling product with a saccharide, is bound - optionally a coupling product of one of the components and a saccharide-containing molecule - a saccharide-binding enzyme and substrates for determining the enzyme activity. 10. means according to rup ^ d .9, characterized in that the water-insoluble carrier is a plastic container. 11. Means according to the fkh * fle *> 9 and 10, thereby ge. , indicates that the water-insoluble carrier is a test tube made of polystyrene or polypropylene.