JPH0311759B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a carrier-immobilized protein complex in which a protein is immobilized on a polyamide, and a method for producing the same. Support-immobilized proteins have traditionally been used in the production of biocatalysts, chromatographic materials, test strips, and the like. For the production of test strips, the carrier is particularly suitable in the form of a film or membrane.

As is known, test strips are used to semi-quantitatively or quantitatively measure certain compounds, such as glucose, albumin, bilirubin, urobilinogen, nitrous acid, ketone bodies, etc., in a simple and rapid manner in a liquid. It is often used to measure
Generally, the liquid is a biological liquid, e.g.
There is urine, blood, serum, plasma or alcoholic beverages as well as beverages or sewage.

A large number of carrier materials are known in the art for use in the manufacture of test specimens.

For example, enzymes and other reagents may be introduced into absorbent papers by impregnation, swellable natural or specially prepared polymers or latexes may be used, and complex multilayer assemblies may be used to absorb sensitive enzymes. It may also be used to protect against the harmful effects of other chemicals present in the layer.

In addition, conventionally known carrier materials are porous themselves or made into an "open-pore" form by adding fillers such as gypsum, diatomaceous earth, silica gel, etc. Microporous membranes composed of various polymers, such as polyethylene, polyvinyl chloride or polymeric compounds or mixtures thereof, may also be mentioned as absorbent carrier materials for enzymes.

As conventional carrier materials, carrier materials with so-called distribution layers whose purpose is to distribute the test liquid on the reaction surface as uniformly as possible can also be added as some examples of use. These distribution layers usually consist of pretreated glass or polymer particles of a certain size, which are then adhered to the surface with a binder. The cavity necessary for the absorption and distribution of the test liquid is thus formed. Distribution layers consisting of insoluble fibers or non-woven fabrics and thus achieving the same purpose can also be mentioned instead of those consisting of particles.

On the other hand, a particular membrane is described in European Patent Application No. 8330517.6 which is made of polyamide (nylon 66) which is rendered hydrophilic and which has functional groups, such as carboxyl groups or amino groups, on its surface.

The use of these membranes for enzyme immobilization after prior chemical modification is also described in this document. Ciba Chrome Bull (registered trademark,
One of the modifications is to fix Cibachrom Blue) on the membrane. It is known from the literature that this dye can bind NAD-dependent enzymes in a very specific manner. In Example 5 of this European patent application, it is demonstrated using lactate dehydrogenase that this fixation occurs solely with this dye.

The second method described in this European patent application is to covalently immobilize alkaline phosphatase to the membrane by using glutaraldehyde.

Surprisingly, the present inventor discovered that polyamides having a specific functional group and a specific protein exhibit excellent bonding properties without the need for prior chemical modification as described above. reached.

That is, the carrier-immobilized protein complex of the present invention is characterized by comprising a carrier-immobilized protein in which peroxidase and glucose oxidase are bound to a polyamide carrier having carboxyl groups and amino groups, and luminol.

In the first method for producing the carrier-immobilized protein complex of the present invention, a polyamide carrier having carboxyl groups and amino groups is impregnated with a solution containing peroxidase and glucose oxidase, and then dried. It is characterized by being applied and then dried.

The second production method is characterized in that a polyamide carrier having carboxyl groups and amino groups is impregnated with a solution containing peroxidase, glucose oxidase, and luminol, and then dried.

The present invention will be explained in detail below.

In the present invention, peroxidase and glucose oxidase proteins (hereinafter sometimes simply referred to as proteins) are directly bonded to a polyamide carrier having carboxyl and amino functional groups (hereinafter sometimes simply referred to as carrier). .

The carrier is preferably hydrophilic so that the proteins can be easily introduced into the carrier.

The carrier used in the present invention is preferably porous because it has a large surface and facilitates, for example, diffusion of the substrate.

The starting polymer used for the carrier according to the invention is insoluble in alcohol and CH ₂
Mention may be made of polyamides in which the ratio of to NHCO groups is in the range from 5:1 to 7:1. Polyhexamethylene sebacamide (nylon 610), poly ε-caprolactam (nylon 6) and polyhexamethylene adipic acid amide (nylon 66) having a molecular weight of 30,000 or more are preferred.

The method for making it hydrophilic and the introduction of functional groups are as follows: A non-solvent (e.g., water) is added under control to a mixed solution of polyamide and a polymer containing functional groups (MW>10,000) in formic acid. This is done by flocculating a mixed solution.

This solution is then applied to the substrate, followed by final precipitation by immersing the substrate in a precipitation bath consisting of a fixed amount of solvent and non-solvent. The membrane thus prepared is washed with water and then dried.

Carboxyl and amino groups or their derivatives may also affect protein immobilization. This makes it possible to provide a method for selectively immobilizing certain proteins.

The functional groups can also be present in "activated" form. An “activated” group is to be understood as meaning a group that is “activated” in a chemical sense by the introduction of a substituent, for example halogen, phenyl, carboxyl or sulfonyl, in other words: , the substituents have the effect of making the molecule more aggressive, which can also be seen by increasing the acidity of the methylene or carboxyl H atom located in the α-position with respect to the central atom. Introduction of a substituent into the α-position can be carried out by methods known in organic chemistry.

It has also been found that "activation" of the functional groups influences the immobilization properties of the support, in particular the selectivity of immobilization. For the preparation of test strips, the carrier is preferably used in the form of a film or membrane. The proteins immobilized on the carrier are peroxidase and glucose oxidase. The protein is preferably applied to the carrier in solution. Particularly preferred are impregnating methods, for example immersing the carrier in a protein solution or coating the carrier with a protein solution. Coating is particularly suitable for mechanically processing film or membranous carriers. The protein itself firmly adheres to the carrier and can no longer be removed by washing. If the protein is firmly immobilized, multiple coatings can be performed without washing away the protein located on the carrier. Furthermore, if proteins are firmly fixed to a carrier, these proteins can be used as biocatalysts. Multilayer assemblies can also be used, especially for test specimens. As can be seen from the examples, it is possible to prepare specimens in which various conditions tailored to specific reactions are effective at various layers. This method therefore uses complex and expensive multilayer systems containing separate layers and/or substances that change the PH of the layers to the desired one, as described in U.S. Pat. No. 4,231,754. The carrier-immobilized protein complex using luminol according to the present invention can be produced without using luminol.

The obtained carrier-immobilized protein complex can be used, for example, as a test piece for measuring glucose.

An important advantage of carriers, particularly as films or membranes, for preparing test specimens is tensile strength. Furthermore, the carrier exhibits little swelling and has a substantially uniform surface, which facilitates evaluation, especially when using a reflectance photometer.

Examples Next, Comparative Examples 1 to 2 and Examples 1 to 3 are shown,
The present invention will be explained more specifically.

Comparative Example 1 Comparative Examples 1 and 2 describe multilayer glucose test strips prepared by a known method.

The following coating solution was applied to a carrier made of polyester film with the aid of suitable applicators and dryers, such as those used in the production of single-layer and multilayer photographic materials: 0.1 M ethylenediaminetetraacetic acid in 25 ml of water. Tetrasodium {Trilon B (registered trademark, Trilon
B)} along with Luminol {manufactured by Merck}
Dissolve 0.25 g of 20% strength gelatin gel in 350 ml of water at 40°C.
The PH value of the solution obtained by melting with
20 ml) of 50% strength Trilon B solution.
I set it to 9.7. The coating thickness when wet was 170μm,
This corresponds to a gelatin content of approximately 7.0g/ ^m3 and approximately 0.07g/m3.
Corresponds to a luminol amount of m ³ .

After drying, the second layer was coated on top of the first layer; it was coated using the following coating solution:

Dissolve 0.3 g of 75% strength dodecylbenzenesulfonate (DBS paste) in 3 ml of chloroform and a 7.5% strength polycarbonate (Makrolon) solution containing chloroform; glucose oxidase (25 IU/mg) 0.123
g and 0.616 g of peroxidase (47 U/mg) were dissolved in 3.6 ml of water and dispersed in the previously prepared solution with vigorous stirring. 23 ml of a 13.2% strength dispersion of aqueous polyacrylamide in chloroform
A 7.5% strength solution of Macrolon in chloroform was combined and then mixed with the enzyme-containing dispersion.

This solution was applied to a layer of luminol-containing gelatin to a thickness of 70 μm and dried at room temperature. glucose
A sample of 30 microliters of a 250 mg solution in water (100 ml) is applied to the finished layer assembly and chemiluminescence appears according to a known reaction.

After storing the layer assembly at room temperature for about 14 days, the glucose oxidase has decayed to the extent of chemiluminescence, which is only apparent when a small amount of glucose oxidase solution is applied to the specimen before adding the glucose solution.

In this test system, glucose oxidase is not tightly immobilized on the second layer.

Furthermore, the reaction that causes chemiluminescence occurs only if the coated sample is dispensed by using filter paper or similar absorbent material.

Comparative Example 2 The procedure was similar to Comparative Example 1, but the following coating solution was used as the second enzyme-containing layer: 125 g of 20% strength gelatin gel in 100 ml of water at 40°C.
It was dissolved in 12.5 ml of an aqueous solution of glucose oxidase (1000 IU/ml) and 1.2 g of peroxidase in water (10 ml) were added to the gelatin solution, followed by 1.5 ml of a 4% strength wetting agent solution.

This solution was applied to the dry gelatin layer containing luminol at a wet coating thickness of 70 μm and then 20–25 μm thick.
Dry at °C.

No luminescence was observed in this test even when wetted with glucose solution.

Glucose oxidase is immediately inactivated here. The methods described in Comparative Examples 1 and 22 are therefore unsuitable for producing test specimens of this type.

In Examples 1 to 3, carrier-immobilized protein complexes of the present invention were prepared and evaluated as test pieces.

Example 1 A 4.5 m web of 16 cm wide was coated with the following solution to a thickness of 70 μm and dried at room temperature: Membrane a: Isotropically foamed polyamide 66 with carboxyl and amino groups on the surface. Pore diameter 0.2μm.

Dissolve 0.16 g of Luminol in 11.3 ml of 50% strength Trilon B solution and mix this mixture with 242 ml of water.
Pour in, then 2.347g glucose oxidase
A solution of 1.246 g of peroxidase in water (23.5 ml) was added. The pH value of this solution was adjusted to 9.7 with 50% strength Trilon B solution.

Upon application of 10-20 microliters of aqueous glucose solution, strong chemiluminescence was observed, which could be measured with a suitably sensitive photometer.

Instead of a glucose solution, a blood sample containing glucose was also applied to the prepared membrane, and here too luminescence appeared, but with reduced intensity.

Example 2 A piece of web as described in Example 1 was coated with two solutions: first, at a thickness of 70 μm, the following solutions were applied: 2.347 g of glucose oxidase and 1.246 g of peroxidase were dissolved in 296.4 ml of water.

After drying at room temperature, a second application was made, also at a thickness of 70 μm, using the following solution: 0.16 g of Luminol in 11.3 ml of a 50% strength Trilon B solution in water (20.6 ml). Dissolve and then add this mixture to 150 g of 20% gelatin gel in water (117.5
g) added to the solution. The pH value of this solution was adjusted to 9.7 with a Trilon B solution.

After drying, the membrane thus produced was treated with glucose solution or glucose-containing blood as described in Example 1 and the chemiluminescence obtained was then measured. Compared to Example 1, a much stronger chemiluminescence appears for both the glucose solution and the glucose-containing blood, and also for Example 1, but this luminescence increases after the membrane has been stored at room temperature for several months. Even its strength hardly diminished.

Example 3 3 ml of the coating solution used in Example 1 was applied using a doctor blade to each test piece of a membrane (similar to membrane a used in Example 1) having a length of approximately 20 cm. In each test, the amount of peroxidase was increased by 1 1/2 and decreased by 3/4, 1/2 and 1/4 of the amount used in Example 1.

Evaluation of the chemiluminescence produced by the glucose solution showed that the intensity of the chemiluminescence was significantly reduced at both higher and lower amounts of peroxidase than in Example 1.

The amount of glucose oxidase was also varied in the same way. In contrast to peroxidase, a linear relationship between enzyme amount and chemiluminescence intensity was observed for glucose oxidase within the range of amounts used.

Reference Example 1 Next, the following experiment was conducted to obtain information on the properties and strength of protein immobilization to the carrier in the carrier-immobilized protein complex of the present invention.

A piece of membrane with dimensions of 5 x 5 cm (similar to membrane a used in Example 1) was treated with 5 mg of glucose oxidase for 30 minutes.
of water (20 ml) to impregnate it. Next, remove 20 pieces of membrane to remove excess enzyme.
Washed with 500 ml of water for 3 minutes.

Each of the impregnated membrane pieces with dimensions of 1 x 1 cm was soaked at various concentrations (0.1M, 0.25M, 0.5M, 1.0M) for 15 minutes.
and 3.0 M) sodium chloride solution.

The test solution was used to determine activity, and the sodium chloride solution and membrane pieces were tested separately after removing the membrane pieces.

Test solution: Glucose 540mg Peroxidase (47IU/mg) 10mg 4-aminoantipyrine 6.2mg Sodium dichlorobenzenesulfonate
115.2 mg Water 90 ml Phosphate buffer (0.5 mol/, PH 7.0) 10 ml According to this evaluation, no enzyme activity was detected in various sodium chloride solutions, whereas it could be observed in appropriate membrane strips. Color intensity showed no significant decrease in membrane enzyme activity. This test clearly shows that the immobilization of the enzyme is not based on ionic interactions.

Obviously, many other modifications and variations of the invention described herein may be made without departing from the spirit and scope of the invention.

Claims (1)

[Scope of Claims] 1. A carrier-immobilized protein complex comprising a carrier-immobilized protein in which peroxidase and glucose oxidase are bound to a polyamide carrier having a carboxyl group and an amino group, and luminol. 2. A carrier-immobilized protein characterized in that a polyamide carrier having a carboxyl group and an amino group is impregnated with a solution containing peroxidase and glucose oxidase and then dried, and then a luminol solution is applied thereto and then dried. Method of manufacturing the composite. 3. A method for producing a carrier-immobilized protein complex, which comprises impregnating a polyamide carrier having a carboxyl group and an amino group with a solution containing peroxidase, glucose oxidase and luminol, and then drying.