DD245727A5 - A METHOD FOR DETECTING AND / OR MEASURING CLINICAL PARAMETERS IN THE IMMUNIZATION PROCESS OF ENZYMES - Google Patents

Abstract

The invention relates to a method for determining clinical parameters in biological fluids for use in medical diagnostics. The object of the invention is to provide an improved method based on an enzyme immuno-reaction which combines rapid and rapid performance with high sensitivity and specificity. In accordance with the present invention, the biological fluid is sequentially contacted with (a) enzyme-labeled antibodies or antigens reversibly adsorbed on a solid support; (b) antibodies, antigens, anti-antibodies, or antigen-antibody complexes that are supported on a solid support in (c) one or more chromogen substrates capable of proving a detection reaction with said antibody or antigen associated enzymes Fig. 1

Description

Field of application of the invention

The invention relates to a method for determining clinical parameters in biological fluids by enzyme processes. More particularly, the invention relates to a diagnostic method and apparatus for the qualitative and / or quantitative determination of clinical parameters by an enzyme immunoprocess using affinity chromatography. The invention is particularly useful for the analysis of biological substances in biological fluids such as ureas, plasma, blood, exudates, tissue extracts, etc.

Characteristic of the known technical solutions

The immunoenzyme diagnostic methods have been known for a long time. They are based on the reaction between an antibody and its antigen, one of which is associated with an enzyme capable of producing the detection reaction (of chromogen or otherwise) when contacted with a suitable substrate. Thereafter, the enzyme-labeled antigen-antibody complex, by contact with the detection substrate, results in a reaction that can be used for the qualitative or quantitative determination of the antigen or antibody of interest.

All of the most commonly used enzyme immunoengines in diagnostics have a number of deficiencies attributable essentially to the complicated and delicate as well as the long incubation times which require repeated washing of the reactive system.

The execution of these techniques is therefore limited to qualified laboratories, whereas a more general application, for example in ambulatory or even domestic practice, has not been possible up to now. In fact, while conventional methods do not pose any particular difficulty for professionals, handling and reading the test results involve problems that are difficult, if not impossible, to overcome by inexperienced forces.

For example, there is a method known by the abbreviation "ELISA" (enzyme-linked immunosorbent sarialysis, Immunochem., 8, 871-874 [1971]) which requires, in addition to long incubation and reaction times as well as repeated washes, also skilled handling and manipulation.

The "ΕΜΓΓ" method, on the other hand, has the limitation that it does not allow the determination of high molecular weight molecules.

Finally, a method known in the art as the "FIA" method requires the use of specialized gauges.

For a detailed description of the above diagnostic methods, reference is also made to "Enzyme Immunoassay", E. Ishikawa, T. Kawai, K. Miyai, Igaku-Shoin-Tokyo-New York (1981).

Object of the invention

The object of the invention is to provide an improved diagnostic method based on an enzyme immuno-reaction which combines easy and rapid execution with responsiveness and specificity peculiar to these immunoenzymatic methods.

Explanation of the essence of the invention

The invention has for its object to provide a device that allows the execution of the method according to the invention in the desired manner.

The invention overcomes the problems encountered in the prior art techniques by providing a method and apparatus for determining, even on a quantitative basis, clinical parameters of any kind without recourse to particular manipulations.

According to the invention, a biological fluid containing the parameter (s) to be determined is flowed upwards or downwards through a conventionally formed support which consists of

(a) a first or intermediate zone on which substances can be adsorbed which can prevent interference with the sample;

(b) a zone previously precoated with one or more antigens or enzyme-labeled antibodies raised relative to one or more epitopes (NR Jerne, Angew Chem Chem Int Ed Engl., 24, 1985, 810-816), which are specific for the clinical parameters to be determined were adsorbed in a reversible manner;

(c) another zone adjacent to the previous zone on which the antibodies or antigens forming the clinical parameters to be determined are immobilized by known methods in an amount equal to or greater than that of the enzyme-labeled antibodies or antigens is. Antibodies may also be used in this zone as compared to one or more epitopes other than the epitope (s) recognized by zone (b) antibodies (sandwich analysis). In this case, the chromogenic substrates (the substrate) for the enzyme-labeled antibodies of zone (b) should be bound to zone (c). Alternatively, the chromogenic substrate (s) could be reversibly adsorbed on a subjacent zone. As an alternative, this zone (c) may have an anti-antibody immobilized thereon and which is specific for an antigen-antibody complex, in which case the zone (b) is again subdivided into two sections, e.g. first portion, the enzyme-labeled antigen is adsorbed, and a second portion carries the corresponding antibody, or vice versa.

(d) a zone on which a chromogenic substrate is adsorbed for the antigenic enzyme or antibody-associated enzyme present in the zone (b) or one or more carriers capable of flowing biological fluids (sandwich analysis).

The biological fluid flowing up or down through the overlay by gravity, capillary action, chromatography, or diffusion is caused to first contact zone (b), the antigen or antibody that may be present react with the complementary ones enzyme-labeled antibodies or antigens.

The complex or system thus formed chromatographically (or precipitates) through the overlay to pass through zone (c) where the excess enzyme-labeled antibody or antigen, if present, binds to the complementary immobilized antigen or antibody become. In the sandwich analysis, the specific complex is bound, resulting in a "sandwich" arrangement.

After passing through the intermediate zone, where all interfering substances can be removed (for example, by adsorption, chemical bonding, decomposition, etc.), the enzyme-labeled antigen-antibody complex reaches the chromogenic substrate to produce a color (positive test) that is appropriate for both quantitative as well as for the qualitative determination, for example by comparison with a color scale. On the other hand, if no clinical parameter is present in the biological fluid (negative test), the enzyme-labeled antigen or antibody in one case is completely fixed on the zone (c) by 1 mm unochemical reaction, preventing it from reaching the chromogen substrate; or in the other case (sandwich analysis) the enzyme-labeled antibody is not fixed to the zone (c) and the color formed during passage of the antibody-associated enzyme is washed out of the zone by the flow of biological fluid (negative test).

In other words, the system is based on a struggle for existence between the antigen or antibody present in the biological fluid and the immobilized antigen or antibody which, in the event of a negative reaction in the first case, functions as a barrier to the enzyme-labeled antibody or antigen to prevent its migration to the chromogen substrate-containing zone, or in the second case as a non-reactive material which is unable to bind enzyme-labeled antibodies.

The device according to the invention may be in the form of a strip, tape, film, tube, specimen, piston, pad, etc. The various constituents may either be adsorbed or immobilized directly on the material forming the device, or a suitable solid support substance may be used which is present in the device or on the surface thereof in the form of a gel, powder, granules, small globules , Balls etc. applied

The materials of the device and the solid supports or carriers in the various reaction zones may be the same or different. Examples of suitable materials and carriers are glass, silica derivatives, paper or cellulose derivatives, metals, polymers such as polyvinyl chloride, polystyrene, polybutadiene, nylon, polyacrylamides, methacrylates, etc., polysaccharides or polyols, starch, stabilized human or animal red blood cells, organic substances such as BaSO ₄ , TiO ₂ , kaolin, diatomaceous earth, etc. The device is preferably made of an opaque material, assuming the display area is transparent.

The immobilizing binding of the antigen or antibody to said materials can be by physical or chemical processes (ester or amide bond, etc.) after

U.S. Patent No. 4,003,988 and following references: B.K. VanWeemen and A. H.W.Schuurs, Febs Letters-Bd. 15, No.3, June 1971, p.232-235; P. Leinikki and Suvi Passila, J. din. Path., 1976, 29, pp. 1116-1120; B.R.Brodeur, F.E. Ashton and

B. Diena, The Journal of Medical Microbiology, Vol. 15, No. 1,1981, pp. 1-9; A. Full 1, D.E. Bidwell 2, A. Barlett 2, D.G. Fleck 3,

M. Perkins 3 and B. OIadehin 3, J. Clin. Path., 1976, 29, 150-153; Howard, H.Wetall, Immobilized Enzymes, Antigens, Antibodies, and Peptides (Immobilized Enzymes, Antigens, Antibodies and Peptides), Vol. 1.

The adsorption binding of the non-immobilized components, on the other hand, can be achieved by known techniques, a matter well known from affinity chromatography.

Both immobilized and enzyme-labeled antibodies useful in the invention may be polyclonal or monoclonal in toto or in fragments, such as Fab 'and F) ab') 2, possibly in admixture with each other, and may be specific to be one or more active sites of the antigen.

Enzyme labeling of the antigens or antibodies may be by known methods, such as those mentioned in the above text "Enzyme Immunoassay", any enzyme which can induce a chromogen reaction to a substrate, such as peroxidase, alkaline phosphatase , / 3-galactosidase or the like These enzymes can be associated, for example, with glutaraldehyde, dimaleimide and their esters by the methods described on pages 54 to 113 of the above-mentioned text "Enzyme Immunoassay".

Both antibodies and antigens can be applied to the substrate without restriction on the amount, the amount depending on the type of analysis in question may vary at least between 1 and 10 mg / cm ² and it is only to be considered that the enzyme-labeled component is at least is present in stoichiometrically equal amounts.

As already stated, a quantitative determination is possible by determining the level of the developed color either in an arbitrary manner or by means of a suitable display device.

Finally, the first or intermediate separation zone may simply consist of a material which can serve as a chromatographic support on which such substances capable of being adsorbed can be adsorbed. Remove compounds that may interfere with the identification reaction. It may for example consist of ion exchange resins for masking heavy metals; immobilized enzymes for degradation of urea or other metabolites; Antiprotein antibodies to remove albumin; Antibodies that are specific for molecules with a similarity to the antigen to be determined, etc.

With the method and apparatus described above, the invention provides a means by which it is possible to determine in a simple, rapid and accurate manner a large number of different clinical parameters, for example peptide hormones such as HCG, LH, steroid hormones such as progesterone, estrone glucuronide, pregnane diol glucuronide or other parameters of general clinical interest such as streptolysin, immunoglobulins, viruses such as herpesviruses, HTLV-3 viruses, etc.

In addition, several enzyme-labeled antigens and / or antibodies can be accommodated on the same support together with the corresponding immobilized antibody / antigens, whereby it is possible to obtain, by the presence of a corresponding number of chromogen substrates, which are preferably selected to be different by reaction with each other enzyme-labeled complexes develop different colors, in different zones at the same time to determine a number of clinical parameters.

Ausführungsbeispiei

The invention is explained in more detail below with reference to some examples.

In the accompanying drawing figures, the symbol -vp> denotes the antigen; denotes the symbol

\ 0 the immobilized antigen; , the symbol - ^ J> C., the enzyme-associated antigen,

while the symbols > -,> 0,} T "in the same way the antibody,

denote the immobilized antibody or the enzyme-associated antibody. Finally, the symbol denotes

Odas chromosome substrate, Q 0 denotes the immobilized anti-antibody, and

the arrow indicates the direction of the incoming liquid.

In the drawings show:

Fig. 1 shows schematically an arrangement which, according to an embodiment of the invention, allows to determine an antigen and which consists of a support (strip, tube or other element) with a first zone containing the enzyme-labeled antibody specific for the antigen to be determined is a second zone adjacent to the first containing the immobilized antigen and followed by a separation zone and finally a zone containing the chromogen substrate. If a pipe is used as a support, the pipe ends can be closed by porous membranes.

Fig. 2: the case that an antibody is to be determined. In this embodiment, unlike the previous case, the biological fluid is caused to pass in the downward direction indicated by the arrow to successively first contact the enzyme-labeled antigen, then the immobilized antibody, and finally the chromogen substrate, which instead be adsorbed on the solid support, which can be added to the emerging from the lower end of the device liquid.

FIG. 3: for the case shown in FIG. 1, the different migration steps under the conditions of a positive (+) and a negative (-) course of the test (presence or absence of the relevant antigen).

Fig. 4 shows another embodiment similar to that shown in Fig. 1, differing only in that the immobilized antigen is pre-glutinated with the enzyme-labeled antibody; any antigen present in the biological fluid is thus in competition with the immobilized antigen to release the labeled antibody, allowing it to migrate into the chromogen substrate.

Fig. 5: another variant of the method for the determination of antigens. In this case, the pad consists of a first zone containing an enzyme-labeled antigen, a second zone to which a corresponding antibody has been adsorbed, and finally a zone containing an immobilized anti-antibody (for example, Sepharose protein A or anti-IgC, bonded to PVC), which in turn is followed by the chromogen substrate; also in this case, it is only the fight with the free antigen present in the biological fluid which causes the enzyme-labeled antigen to reach the substrate while the antibody-antigen complex remains trapped by the anti-antibody. I

Fig. 6a and 6b: another type of device, seen in cross section and in plan view, respectively. The device consists of a support strip 2 which is provided with holes 1 for the passage of a liquid, and which has an impermeable transparent coating 3, which makes it possible to visually check the chromogen-containing layer 4. In this case, the reading for qualitative determination can be made visually or by means of a deflection meter for quantitative determination.

The embodiments shown are only intended to illustrate the principle of the invention without limiting it. For example, the size of the individual zones may vary in each case, usually ranging between a few millimeters and a few centimeters. In addition, certain details have not been shown that could allow for easier application of the invention, such as carrying handles, biological fluid aspirator, markers indicating the side on which the biological fluid is to be introduced, "color gamuts displayed on one side of the biological fluid The devices according to the invention may also be a symmetrical device with an enzyme-labeled component arranged at both ends, to which the above-mentioned follow-on zones then adjoin, this measure serving to introduce a wrong introduction of the liquid In addition, for domestic use packaged compact units can be created which have the requisite degree of sterile, stable, coordinated properties, etc.

From the foregoing it will be seen that the method and apparatus of the invention are versatile and practical in application and can be applied in a wide range of clinical analysis without the need for specialized equipment or skills. In addition, they make it possible to determine analysis results in a short time, usually in the span of minutes.

The invention will now be explained in more detail by the following example, which is not intended to limit the spirit and scope of the invention in any way.

example

a. Preparation of PVC immobilized on PVC

100 g PVC, particle size 200μ, are treated in a solution of HCG at 50 units / ml in phosphate buffer at pH 7.2 at 37 ^° C. overnight. The coated polymer is washed five times with the same buffer and dried at 37 ^° C. for 10 hours.

b. Preparation of peroxidase-labeled anti-HCG

Rabbit anti-HCG (10 ml) is precipitated three times with 18% anhydrous sodium sulfate and the product is recovered from each 10 ml of distilled water after each precipitation. Then, the product overnight at +4 ⁰ C, using a phosphate buffer at a pH of 7,2,1OmMoI is diaiysiert.

The dialyzed solution is treated with 100 mg of peroxydase RZ 3.00 and 25 ml of 25% glutaraldehyde at +4 ⁰ C for 48 hours. The solution is eluted on Sephadex G 150, which is balanced with sodium chloride, 50mMoI. The eluted anti-HCG peroxidase complex can be stored at -2O ⁰ C.

c. Preparation of labeled antibodies adsorbed on the support

Glass, 50g, is treated with anti-HCG peroxidase, 5ml, diluted 1: 500 in phosphate buffer, 10mMoI, pH 7.2, and the product is dried overnight at 37 ° C.

d. Preparation of the chromogen adsorbed on the support

50g of glass are treated with 50mg of tetramethylbenzidine dissolved in 5ml of DMSO water.

e. column packing

A glass column (3.5 mm inner diameter, 15 cm height) is filled from bottom to top with the following materials: adsorbed anti-HCG peroxidase, immobilized HCG, adsorbed chromogen prepared in the above manner.

The glass column is closed at both ends with cotton or another suitable porous plug.

f. Carrying out the analysis

The column described above is contacted with the urea to be analyzed, which rises by capillary action in the column. When HCG is present in urea, it is bound to the anti-HCG peroxidase contained in the first column section; on further increase, it does not cause any further agglutination reaction with the immobilized HCG contained in the second column section so that it continues its way to the last column section in which is the chromogen substrate which develops a blue color upon reaction with the enzyme ; in a sandwich analysis, the complex is specifically bound to zone (c) by immunochemical reaction, and the blue color is evoked by the reaction with the enzyme and chromogen substrate, the blue color becoming visible in zone (c). On the other hand, if no HCG is contained in the urea, the free HCG peroxidase complex in the first column section reaches the column zone in which the immobilized HCG is located, and is bound thereto by immunochemical reaction, so that it can not reach the chromogen substrate. So no color is formed; in the sandwich analysis, the anti-HCG peroxidase in the zone (c) is not bound and washed out of this zone, also in this way no color is developed

 The process described above can also be used in descending liquid systems.

Claims (22)

-1- 245 72: Invention claim: 1. A method for determining clinical parameters in biological fluids by the enzyme immuno process, characterized in that a biological fluid is brought into contact with successively a. enzyme-labeled antibodies or antigens reversibly adsorbed on a solid support; b. Antibodies, antigens, anti-antibodies or antigen-antibody complexes immobilized on a solid support in an amount at least stoichiometrically equal to the enzyme-labeled antibodies or antigens; and c. one or more chromogen substrates capable of proving a detection reaction with the enzymes associated with the antibodies or antigens. 2. Method according to item 1, characterized in that the biological fluid, prior to contact with the chromogen substrate, flows through a chromatographic material on which substances may be adsorbed which can remove compounds which may interfere with the analysis in question. 3. The method according to item 1 or 2 in the application for the determination of antigens, characterized in that the biological fluid is brought into contact with successively a. the complementary enzyme-labeled antibody adsorbed on a solid support in a reversible manner; b. the same antigen immobilized on a solid support in an amount at least stoichiometrically equal to that of the enzyme-labeled antibody; c. a chromogen substrate. 4. The method according to item 1 or 2, used for the determination of an antigen, characterized in that the biological fluid is brought into contact with a. the complementary, enzyme-labeled antibody reversibly adsorbed on a solid support; b. one or more immobilized antibodies and a chromogen substrate which are reversibly or immobilized adsorbed on a solid support; c. one or more documents that can allow the flow of biological fluids. 5. The method according to item 1 or 2 in the application for the determination of an antibody, characterized in that the biological fluid is brought into contact with successively a. the complementary enzyme-labeled antigen reversibly adsorbed on a solid support; b. the same antibody immobilized on a solid support in an amount stoichiometrically at least equal to the enzyme-labeled antigen, and c. a chromogen substrate. 6. The method according to item 3, characterized in that the enzyme-labeled antibody and the immobilized antigen are agglutinated on the same solid support. 7. The method according to item 1 or 2 in the application for the determination of an antigen, characterized in that the biological fluid is brought into contact with successively a. the same enzyme-labeled antigen which is reversibly adsorbed on a solid support; b. the complementary antibody reversibly adsorbed on a solid support; c. an anti-antibody specific for the antigen-antibody complex immobilized on a solid support, and d. a chromogen substrate. 8. The method according to item 1 in the application for the determination of various clinical parameters in a single operation, characterized in that on the same solid support, the corresponding enzyme-labeled antibodies or antigens and the corresponding immobilized antibodies or antigens are applied and that for each enzyme used specific chromogen substrates are on individual bases. 9. Method according to one of the preceding points, characterized in that the supports, which may be the same or different for the different components, consist of glass or silicon dioxide derivatives, paper or cellulose derivatives, polymers, polysaccharides or polyols, kaolin, T1O2, BaSO ₄ , Diatomaceous earth, metals or stabilized red blood cells can exist. 10. The method according to any one of the items 1,2,3,6 to 9, characterized in that it is used for the determination of peptide hormones (HCG, LH), steroid hormones, proteins, streptolysin or viruses. 11. The method according to any one of the items 1,2,5,8 or 9, characterized in that it is used for the determination of immunoglobulins and other antibodies of diagnostic interest. Apparatus for determining clinical parameters of the kind that can be transported by the ascending or descending biological fluids having the shape of a tube, specimen, strip, pillow, piston, film, characterized in that the device consists of a. a first or separation intermediate zone on which the substances capable of removing compounds capable of interfering with the analysis in question can be adsorbed; b. a zone on which one or more enzyme-labeled antibodies or antigens have previously been adsorbed which are specific for the clinical parameters to be determined; c. a further zone adjacent to the preceding zone to which the antibodies, antigens, anti-antibodies or antibody-antigen complexes which constitute the parameters to be determined have been previously immobilized by known amounts in an amount at least equal to that of the enzyme-labeled one Antibody or antigens is, and d. a zone on which chromogen substrates have been adsorbed for use by the antigen or antibody-associated enzymes. Apparatus for determining clinical parameters of the type that can be transported by ascending or descending biological fluids having the shape of a tube, specimen, strip, pillow, piston, film, characterized in that the device consists of a. a first or intermediate separation zone to which the substances have been adsorbed, which may remove those compounds which may affect the analysis in question; b. a zone previously adsorbed with one or more enzyme-labeled antibodies (raised relative to one or more epitopes) specific for the clinical parameters to be determined; c. a zone on which one or more antibodies (drawn in comparison to one or more epitopes other than the epitopes recognized by the enzyme-associated antibodies of zone b) have been immobilized and chromogen substrate (s) is reversibly adsorbed or immobilized ( are); d. a zone with one or more overlay materials that allow the flow of biological fluids. 14. Device according to item 12 or 13, characterized in that it consists of one or more materials selected from glass, metals, synthetic or natural polymaterials or paper coated in the form of a gel, of powders, granules, microspheres, Suitable pad materials can be applied, which may be the same or different for the individual zones and are selected from starch, diatomaceous earth, kaolin, BaSO ₄ or TiO ₂ . 15. The device according to item 12 or 14, characterized in that the first or intermediate zone (a) ion exchange resins, enzymes such as urease or protease, oxidizing or reducing substances, antibodies or antigens as the substances that can remove interfering substances. 16. Device according to one of the items 12 to 15 in the application for the determination of an antigen, characterized in that it consists of a. a first zone having a complementary, enzyme-labeled antibody reversibly adsorbed thereon; b. a zone adjacent to the first zone on which the same antigen is immobilized in an amount at least equal to that of the enzyme-labeled antibody; c. a subsequent zone which effects chromatographic separation and, as an option, interference removal: and d. a final end zone where the chromogen substrate is for the enzyme. 17. Device according to one of the points 12 to 15, characterized in that for the determination of antibodies, the device consists of a. a first zone to which the complementary enzyme-labeled antigen is reversibly adsorbed; b. a zone adjacent to the first zone on which the same antibody is immobilized in an amount at least equal to that of the enzyme-labeled antigen; c. a separation zone and - ~ .. d. a chromogen detection zone. 18. Device according to items 12 to 15, characterized in that for the determination of an antigen, the device has a first zone, on which the applied to the pad antibody is preagglutinated with the enzyme-labeled antigen, a separation zone and a chromogen detection zone. 19. Device according to items 12 to 15, characterized in that for the determination of an antigen, the device consists of a first zone, which is provided with an enzyme-labeled antigen, a second zone, on which the antibody is reversibly adsorbed, a zone with the immobilized anti-antibody, and finally the separation and chromogen detection zone. 20. Device according to one of the items 12 to 19, characterized in that for the simultaneous determination of different clinical parameters, each of the individual zones (a) and (b) each contains a corresponding number of enzyme-labeled antibodies or antigens and immobilized antigens or antibodies and various chromogen detection zones available. 21. Device according to one of the items 12 to 20, characterized in that the device is suitable for the determination of HCG, LH or other peptide hormones, progesterone, Estromglukuronid, Prägnandiolglukuronid or other steroid hormones, streptolysin, immunoglobulins, viruses. Device according to any one of items 12 to 21, characterized in that the whole apparatus, with the exception of the display zone, which is made of transparent (transparent) material (s), is of opaque material (s). For this 4 pages drawings