METHOD FOR THE DETERMINATION OF ENZYMES BY DIFFUSION IN A POROUS MATRIX OR BY ELECTROPHORESIS

Abstract: Method for the determination of enzymatic activity in which a sample is added to a basin in or a defined surface of a liquid-saturated porous matrix covering a thin layer of a substance sensitive to enzymatic lysis deposited on the solid surface of a carrier. The sample is permitted to diffuse or migrate electrophoretically in the matrix and react with the substance sensitive to enzymatic lysis. The matrix is then removed whereon lysis can be indicated on the solid surface. The enzym-containing sample can be a bacteria colony on the matrix. Plastic material is preferred for the solid surface.

Description: An enzymatic indicator system The present invention relates to an enzymatic indicator sys¬ tem based on a two-phase system, in which an enzyme substrate is bound to the surface of a solid phase and in which the presen¬ ce and quantity of an enzyme capable of reacting with the compound bound to said surface is determined by permitting the enzyme to diffuse from a basin in a liquid-saturated immobilized phase lo¬ cated on said surface. The presence and quantity of enzyme is then indicated on the solid phase in a suitable manner. Enzymatic types of testing methods have a central position within the diagnostic techniques of medicine, microbiology and applied biochemistry in general. This is predominantly due to the fact that such methods afford the possibility of determining minute quantities of the specific reactants in question in complex systems. Enzymatic test methods have been used in diagnosing vari¬ ous diseases by determining enzyme activity in . serum, and other biological fluids, as well as enzyme activity generated by microorganisms. These enzymatic tests are generally effected by . various indicator systems involving colour changes, lysis of cells as erythrocytes, or degradation of molecules or the like. The method according to the invention involves depositing a substrate on a solid surface as a thin layer of a substance sensitive to enzymatic lysis, whereafter there is placed on the thin layer an immobilized matrix in which the enzyme to be de¬ termined is permitted to diffuse or electrophoretically mig¬ rate during a measured period of time, whereafter the matrix is removed and the lysis reaction surface area on the solid surface is determined by adding an indicator substance, or in some other suitable way. The thin layer of said substance is suitably applied to the solid surface in the following manner: A liquid medium, suitably based on water, is first dispense^ to the solid surface,, A solution containing said substances is then added to the liquid medium to diffuse therein, where¬ after the substance is permitted to deposit itself onto the. so lid surface, becoming bound to the solid surface with a force such magnitude that said surface, subsequent to the aqueous me dium having been removed, can be washed without the layer bein removed therefrom. The solid surface is suitably a transparent material, suc as glass or a plastics material, e.g. polystyrene, polyacryl- nitrile, polyolefines and copolymers thereof. It has been foun in recent years, that plastics surfaces advantageously adsorb macro-molecules to form very uniform and reproducable layers β A technique for.visualizing enzyme reactions is one in wh a thin layer of indium particles is vapor-deposited on the sol surface. The enzyme reactions are carried out on the indium layer, whereafter the reactions can be observed as a light-pro pagation pehno ena on the indium surface. The most serious dis advantages with this technique, appear to be the requirement of advanced apparatus for producing a uniform and reproducablelay of indium on large surfaces. This restricts the rational use of such surfaces. Furthermore, it is difficult to classify in a reaction as a positive or a negative one in borderline cases since this indicator system has a flat amplitude and the indic tion can only be judged subjectively. Another]^,much simpler technique for visualizing enzymatic reactions on solid surfaces is one employin the condensation of water vapor. This technique involves exposing the dried sur face to vapor, whereupon it is possible to determine whether a reaction has taken place and the extent of any such reaction from the pattern formed by the condensation. The principles of this technique were described by Langmuir in 1936. This method is as sensitive as the method employing an indium layer, but OMPI - - has a steeper indication-amplitude. Moreover, it permits ~±he objec¬ tive analysis by contact-copying of the condensation pattsrn on the surfaces by irradiation of photographic paper and development thereof. 5 The indication of biological surface reactions is tfeωs best effected with .vapour condensation on the plastics surface (Vapour condensation on surface, VCS, see Adams, Klings, Fisher and Vroman, Journal of Immυnological Methods, 3, (1973) pages 227-232), which, because of its simplicity, is the preferred method. Other 10 known methods can also be used, such as the so-called ELXSA- method (Enzyme-linked immunosorbent assay, J. Immun β 109 129 (1972), or particle adsorption technique, using a slurry of barium sulphate for example, and various colouring techniques,, The reason why it is possible to observe a change in the thin 15 layer on the solid transparent surface as a result of vapour con¬ densation is due to the fact that the so-called Zeta-potential or surface tension against water is changed on the surface when a reaction has taken place. In principle, all hydrophobic surfaces have a surface-tension angle of from 90 to 170 „ Those plastics sur- 20 faces which normally have such properties include polystyrene, poly- acrylnitril, polyethylene and copolymers thereof,, The substance bound to the plastics surface is able to react selectively with a further substance, either on reacted parts or on unreacted parts of the substance, and the reaction in question can 25 be shown visually iri situ even when it is difficult to make the reaction directly visible on the substrate after the en__y.me reac¬ tion. An immobilized matrix through which the unknown substance shall diffuse is then applied to the surface having the antigen 30 thereon. Such immobilized matrices are well known within the tech¬ nique of analysis, and may comprise aqueous gels or various types of sediment or fibrous substances. The most conventional method is π--.. one in which α gel is used, in particular an agar gel, suitab comprising a buffered lyt-solution of agar which is permitted solidify. A basin is then formed in the matrix, there being s lied to the basin a solution containing the unknown substance unknown substance may also be supplied in cellulose plates or like, which have been saturated with the solution. The syste is left at a suitable temperature of between 5 and 50 C, for unknown substance to diffuse from the basin. Compared with previously known methods of obtaining quan tative measurements of minute quantities of biological materi the novel method exhibits a simplicity which has not previous been achieved, and therewith a subsequent increase in capacit and decrease of costs. Thus, it is relatively simple to obtai uniform, thin component layers on plastics surfaces, the adhe of the layer to the plastics surface being independent of the 1 concentration of the substance forming the layer in the solut applied to the surface in a sufficient quantity, as compared the adhesion forces obtained in respect of glass surfaces. The method according to the invention is thus used for e zymatic analysis of substances sensitive thereto, the plastic surface having applied to it a thin layer of an enzyme subst and an immobilized matrix over said substrate, whereafter an enzyme-containing sample quantity is applied to the matrix an permitted to diffuse therein; whereafter the matrix is remov and those parts of the enzyme substrate which have been affec by the enzyme are indicated by lysis being obtained in this r gion. The method is much simpler to carry out than known meth the latter employing reactions in a mass and chromatographic systems. A high-degree of sensitivity is achieved,which can b subscribed to the fact that a high substrate concentration is tained in the thin layer. The method provides information con cerning the diffusion properties of the enzyme, it being pos IjUR OM - to determine the molecular weight from the diffusion rate, if the concentration and temperature are known at the same time. The method enables the extra-cellular enzyme activity of bac¬ teria to be examined in a simple and reliable manner. gϋREA } ' #8k QMPI

Claims: CLAIMS:- 1„ A method of determining enzymatic activity, characterized that there is deposited on a solid surface a thin layer of a substance which is sensitive to enzymatic lysis, whereafter an immobilized matrix is applied to the thin layer and in that th is supplied to . the immobilized matrix a quantity of an enzyme- containing sample, which is permitted to diffuse or migrate el • trophoretically in the matrix and react with the substance sen sitive to enzymatic lysis, whereafter the matrix is removed fo allowing the lysis to be indicated on the solid surface. 2. A method according to claim 1, characterized in that extr cellular enzymes formed upon bacterial growth are permitted to diffuse through an immobilized matrix comprising a nutrient fo bacterial growth, and in that a lysis of the substance deposit on the solid surface is allowed to be indicated subsequent to removing the matrix. 3. A method according to claim 1, characterized in that the solid surface constitutes a plastics material selected from th group polystyrene, polyacrylnitrile, polyolefines and c polyme thereof. OMPI _ WIPO

Inventors: ELWING HANS BJOERNE
Applicants: ELWING HANS BJOERNE
Application year: 1978
Main classification: G01N33
IPC codes: C12Q1, G01N27, G01N33