DE2857015A1 - METHOD FOR THE DETERMINATION OF ENZYMES BY DIFFUSION IN A POROUS MATRIX OR BY ELECTROPHORESIS - Google Patents

Description

rv I ι υ \ A / - Ζ - 4 Düsseldorf, August 9, 1979

Uipl.-lng. Ii. Wangemann - str «.man _n , trae _e 28

Dresdner Bank, DOs.eldorf, account 51-419 655 <G telephone 36 35 31 Postal check Konroi Cologne 1688 12

P 28 57 Ο15.2 My file no. 5836a V / Fe Hans Björne Elwing, Chalmersgatan 5 »411 35 Gothenburg / Schwiadeti.

Methods for detecting enzymes.

The invention relates to a method for detecting enzymes. This process is based on a two-phase system in which an enzyme layer is deposited on the surface of a solid phase and in which the presence and amount of an enzyme associated with the deposited on the surface of the solid phase Substance can react, is determined and established by the fact that the enzyme is from a container in a Lets liquid-saturated and immobilized phase diffuse on the aforementioned surface, and then finally the presence and the amount of the enzyme on the solid phase is determined and ascertained in a suitable manner.

Enzymatic laboratory processes play a central role in diagnostic technology in medicine, microbiology and general applied biochemistry. This is essentially due to the fact that such methods guarantee that smaller amounts of a specific reactant can be determined and ascertained within complex systems.

When diagnosing various diseases, the enzymatic

030 60 4/00 08

Elwing 5836a

laboratory methods for determining the enzyme activity in serum and in other biological fluids, also for the determination of the enzyme activity, which is indicated by'· - ' Microorganisms is caused.

These laboratory and test enzymatic procedures are generally used Affected by a number of indicator or detection systems including color changes, Zeil solutions as; Eryzothrytes or molecular degradation and the like more.

The invention is based on the object of creating a method for the detection of enzymes. It solves this problem through a process consisting of the individual steps listed below:

First, an enzyme solution-sensitive substance is called A thin layer is applied to the surface of a solid, then an immobilized one is applied Matrix on the thin layer of substance sensitive to the enzyme solution through which the enzyme passes over a given period of time can diffuse electrophoretic means, finally the matrix is removed and the investigation and determination the solution reaction area on the solid surface by adding an indicator substance or on a carried out in another suitable manner. The enzyme solution sensitive substance is shown as a thin layer in the below deposited manner on the surface of the solid body.

030804 / Ό008

Elwing 5836a

First of all, a liquid, preferably water-based, applied to the surface of the solid. Then a solution is made in which the substances already mentioned are contained are so given to the liquid that they diffuse into: this liquid, whereupon the substance is on the solid surface can be deposited, which bonds with the surface of the solid so strongly that after removal of the aqueous medium, this surface can be washed off without the coating being removed from it.

A transparent material is suitable as the solid surface, for example glass or plastics, preferably polystyrene, Polyacrylonitrile, polyolefins and their copolymers. In the last Years ago it was found that plastic surfaces adsorb macromolecules in an advantageous manner in such a way that very even and repeatable layers can be produced.

One method with which enzyme reactions can be visualized and displayed is that on the solid surface Indium particles are deposited in the vapor deposition process. The enzyme reactions are based on the indium existing layer, whereupon the reactions on the indium layer as phenomena of light propagation can be observed and examined. The main disadvantage of this method is that for the production of a uniform and reproducible indium

030604/0008

Elwing 5836a

-JB-

layer on larger areas complicated apparatuses are required. That is an obstacle to the use of such areas. In addition, it is difficult to classify a reaction as a pop.itive or a negative reaction in borderline cases because this Detection or indicator system has a flat amplitude and because the detection can only be assessed subjectively.

Another and much simpler way to make it visible and representation of enzyme reactions, on the other hand, consists in utilizing the condensation of water vapor. With this one In the process, the dried surface is exposed to water vapor, with the possibility then being given by means of the the pattern resulting from the condensation process to determine whether and to what extent a reaction has taken place. That The principle of this process was described by Langmuir in 1936. The procedure is just as delicate as that Indium layer method, but it has a steeper detection or indication amplitude. This method also enables also an objective analysis by contact copying of the condensation pattern on surfaces by exposure of Photo paper and through the development of this photo stacker.

Biological surface reactions can therefore be best dealt with through water vapor condensation on plastic surfaces make visible and represent (Vapor condensation on surface, VCS, Adams Klings, Fisher and Vroman, Journal of Immunological Methods, 3, (1973) pages 227 - 232), which because of its simple

030804/0008

Elwing 5836a

that is, the preferred method. Other methods can also be used Use, for example, the so-called ELISA method (Enzyme-Linked immunosorbent assay, J. Immun. 109: ": 129 (1972)) or also the particle adsorption process; using a barium sulfate slurry and numerous Staining process.

In the thin layer on the transparent solid surface there is a change as a result of water vapor condensation To be recognized because when a reaction has taken place, the so-called zeta potential or the surface tension against water changes. In principle, all hydrophobic surfaces have a surface tension angle from 90 ° to 170 °. Among the plastic surfaces that have these properties usually include: polystyrene, polyacrylonitrile, polyethylene and the copolymers.

The substance connected to the plastic surface is in able to react selectively with another substance, either in the parts of the substance which have reacted, or else in the parts of the substance that have not reacted, with the one in question Reaction in situ can be visualized even if it is difficult to see the reaction after it has taken place To make enzyme reaction visible directly on the layer.

03060 A / 0008

Elwing 5836a

The immobilized matrix is then applied to the surface with the antigen deposited thereon. Such immobilized matrices, which are known in laboratory technology, can contain: aqueous gels or various types of sidewall substances or fibrous substances. In the best-known method, a gel, in particular an agar gel, is used, since "_;" contains a one percent buffer solution of agar which is allowed to solidify. A basin is then formed in the matrix into which a solution containing the unknown substance is placed. The unknown substance can also be used as cellulose sheets or the like. added, the cellulose sheets are soaked to saturation with solution. The system is then left at a suitable temperature, which can be between 5 ° C and 50 ° C, so that the unknown substance can diffuse out of the basin into the system.

In comparison with the previously known methods for bringing about quantitative measurements on very small amounts of biological material, the method according to the invention has a simplicity that has not yet been achieved; associated with this are an increase in performance and a reduction in costs. Uniform and thin layers on plastic surfaces can thus be produced relatively easily, the adhesion of the layer to the plastic surfaces being independent of the concentration with which the substance that forms the layer is contained in the solution, which is in a sufficiently large amount the area was applied, and this in comparison with the adhesive forces that one

© 3 OSO-'4 / 1300 8

· ■ «- ■

Elwing 5836a

achieved in terms of glass surfaces.

The method set up by this invention will thus ^by-1 -the enzyme analysis of the substances used, for which there sensi + .I-: is the fourth. An enzyme layer is applied to the plastic surface and an immobilized matrix is applied to the enzyme layer. An enzyme-containing sample is then placed on the matrix, which can then diffuse into it. After diffusion, the matrix layer is removed, with those parts of the enzyme layer which have been affected by the enzyme being made recognizable by a solution in this area. This method is, compared to the previously known methods, very much simpler because the previously known methods work with reactions which take place in bulk and in a chromatographic system. A great deal of sensitivity is obtained due to the fact that the thin layer has a high concentration. The method provides information about the diffusion properties of the enzyme, it being possible to determine the molecular weight from the diffusion rate if the concentration and the temperature are known at the same time. With the method, the activities of the extracellular enzyme can be determined in a simple and reliable manner.

030 604/00 0

Claims (3)

η »· ι ι LJ \ A / A 4 Düsseldorf, August 9, 1979 Dipl.-Ing. H. Wangemann J str ".mann, trass. 2β Dresdner Bank, Düsseldorf, account 51-419 655 long-distance call 36 35 31 Postsdiedt account, Cologne 1688 12 Hans B.iörne Elving My file no. 5856a W / Lo P 28 57-015.2 Patent claims: 1 )) Method for detecting the enzyme activity, characterized in that a thin layer of a substance sensitive to enzyme solution is applied to a solid surface, that an immobilized matrix is applied to this thin layer, that an amount of a enzyme-containing sample is applied, which diffuses electrophoretically into the immobilized matrix and reacts with the enzyme solution-sensitive substance, whereupon the matrix is removed to determine the solution on the solid surface. 2) Method according to claim 1, characterized in that extracellular enzymes after bacterial cultivation diffuses through an immobilized matrix which has a nutrient medium for bacterial cultivation and that a solution of the substance deposited on the solid surface is determined after removal of the matrix will. 3) Method according to claim 1, characterized in that the Solid surface is made of a plastic material that belongs to the group of polystyrene, polyacrylonitrile, polyol 030604/0008 Elwing 5836a fine and their copolymers. 030604/0008 Patent attorney * λ r ..,, L, · _Α , * / 4 Düsseldorf, August 10, 1979 Uipi.-ing. π. wangemann stre _Iema nn.traß. 2β Dresdner Bank, Düsseldorf, account 51-419 655 call 36 35 31 Postsdieck account KSIn 1688 12 P 28 57 o15 2 My file no. 5836a W / Lo Hans Björne Elwing Amended claims under Article 19 PCT: 1) method for detecting enzyme activity, characterized in that that a thin layer of an enzyme solution sensitive Substance is bound to a solid surface, since3 & an immobilized matrix is applied to this thin layer, so that the immobilized matrix layer eirie ' Amount of an enzyme-containing sample in a limited area Matrix layer is applied, which diffuses electrophoretically into the immobilized matrix and dissolves with the enzyme sensitive substance reacts, whereupon the matrix is removed by determining the solution on the solid surface will. 2) Method according to claim 1, characterized in that an enzyme containing a sample is supplied to the immobilized matrix as a bacterial culture that is produced by bacterial Growth on the surface of the immobilized matrix forms extracellular enzymes, which can be passed through a nutrient medium for the immobilized matrix layer showing bacterial growth diffuses through and that a solution the substance connected to the solid surface is determined after the matrix has been removed. 030604/0008 • 5S- Elwing 5836a ti - Jfc - 3) Method according to claim 1, characterized in that the solid surface is made of a plastic material which belongs to the group of polystyrene, polyacrylonitrile, polyolefins and their copolymers. 030604 / Ό008