FI85194C - Determination of clinical parameters using an enzymatic immunoprocess - Google Patents

Description

85194

Determination of clinical parameters by enzymatic immunoprocessing

Improvement of clinical parameters with 5 enzymatic immunoprocesses 10

The present invention relates to a diagnostic method and a device for the qualitative and / or quantitative determination of clinical parameters by an enzymatic immunoprocess using affinity chromatography. The invention relates in particular to a method according to claim 1 and to a device according to claim 10.

The invention is particularly useful for analyzing biological agents in biological fluids such as urine, plasma, blood, inflammatory fluid, tissue extracts, etc.

20

Immunoenzymatic diagnostic techniques have long been known in the art. They are based on the reaction between an antibody and its antigen, either the antibody or the antigen being conjugated to an enzyme that produces an expressive reaction (chromogenic or similar in nature) when contacted with a suitable substrate. This enzyme-labeled complex between the antigen and the antibody then, upon contact with the detecting substrate, causes a reaction that results in the antigen or antibody of interest. . The substance can be determined qualitatively or quantitatively.

All of the enzymatic immunoassays currently used in diagnostics have the disadvantage of certain shortcomings, which are essentially due to the complex and sensitive procedures involved in these techniques and the long incubation times between which the reaction system must be washed repeatedly.

Thus, the use of such techniques is limited to qualified laboratories, while their more general use, for example at the sampling site or even in private homes, has so far been impossible. Although these traditional methods do not present any particular difficulties for the person skilled in the art, there are still problems in processing and interpreting the test results 5 which are very difficult, if not impossible, for the untrained person to solve.

For example, a technique known as "ELISA" (enzyme-linked immunosorbent assay) is known in the art. Immunochem., 8, 871-874, 1971). In addition to long reaction and incubation times and repeated washing procedures, this technique requires careful operation and handling of the reaction system.

15 On the other hand, the limitation of the "EMIT" technique is that it cannot identify molecules with a high molecular weight.

Finally, the method known in the art as "FIA" technology requires the use of specific determining instruments.

20

The above diagnostic techniques are described in detail, for example, in "Enzyme Immunoassay", E. Ishikawa, T. Kawai, K. Miyai, Igaku-Shoin-Tokyo-New York (1981).

In the light of the foregoing, it is apparent that a diagnostic technique based on an enzymatic immune response would be very useful, in which easy and rapid use would be combined with the sensitivity and specificity characteristic of said immunoenzymatic methods.

The present invention avoids the problems of prior art techniques by allowing the method and apparatus of the present invention to quantify clinical parameters of even arbitrary nature without having to resort to any particular procedure.

The method according to the invention is mainly characterized by what is set forth in the characterizing part of claim 1 and the device according to the invention is mainly characterized by what is set forth in the characterizing part of claim 10.

According to the invention, the biological fluid from which it is desired to determine clinical parameters is passed up or down through a suitably shaped carrier comprising: (a) an early or intermediate zone into which substances that may interfere with the experiment may be adsorbed; (B) a zone to which one or more antigens or enzyme-labeled antibodies that have been reversibly adsorbed against one or more epitopes specific for the clinical parameters to be determined (NR Jerne, Angew. Chem. Int. Ed. Engl. , 24; 1985, 810-816); (C) an additional zone adjacent to the preceding zone immobilized using antibodies or antigens constituting the clinical parameters to be determined using known techniques in an amount equal to or greater than the amount of enzyme-labeled antibodies or antigens. Antibodies raised against one or more epitopes (epitopes) recognized by the antibodies in zone (b) may also be used in this zone (layer assay). In this case, chromogenic substrates for enzyme-labeled antibodies of zone (b) should be bound to zone (c).

Alternatively, these chromogenic substrates can be reversibly adsorbed to the lower zone. Alternatively, an antibody specific for the antigen-antibody complex may be immobilized in this zone (c), in which case zone (b) is in turn divided into two parts, for example by adsorbing an enzyme-labeled antigen to the first part and a second part. contains a corresponding antibody or vice versa.

(d) A zone to which a chromogenic substrate for an enzyme conjugated to antigen or antibody present in zone (b) is adsorbed, or one or more carriers that allow the flow of biological fluids (layer assay).

* 85194

The biological fluid flowing up or down through the support by gravity, capillary force, chromatography, or diffusion is first contacted with zone (b), whereby any antigen or antibody present reacts with complementary enzyme-labeled antibodies or antigen. with.

The complex thus formed, or combined system, ascends or descends chromatographically along the support, passing through zone (c), in which zone (c) the excess ent-10 labeled antibody or antigen, if present, binds to the complementary, immobilized antigen or antibody. In the case of the layer experiment, this specific complex binds to form a layered whole.

After the sample fluid has passed through an intermediate zone in which potentially interfering substances are removed, for example by adsorption, chemical binding, digestion, etc., the enzyme-labeled complex between the antigen and the antibody reaches the chromogenic substrate, resulting in a color (20 positive test) can be used for both quantitative and 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 assay), then in one case the enzyme-labeled antigen or antibody binds completely to zone 25 (c) as a result of an immunochemical reaction, whereby it cannot reach the chromogenic substrate; and in the second case (layer assay), the enzyme-labeled antibody does not adhere to zone (c), and the flowing biological fluid rinses the dye formed as the antibody-conjugated enzyme progresses out of the zone (negative assay).

That is, the system is based on competition between an antigen or antibody present in a biological fluid and an immobilized antigen or antibody, which in the case of a negative response acts as a blocker of either the enzyme-labeled antibody or antigen, preventing it from migrating to the chromogenic substrate. zone; or, in another case, as a non-reactive material incapable of binding enzyme-labeled antibodies.

The carrier device of the invention may be in the form of a strip, tape, film, tube, template, bottle, blotting disc, etc. The various components may be adsorbed or immobilized either directly on the device-forming material or on a suitable solid carrier inside the device, or gel , in powder, granules, microbeads, beads, etc.

The materials of manufacture of the device and the solid carriers or excipients in the different 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, starches, polyols, blood cells, inorganic substances such as BaSO 4, TiO 2, kaolin, diatomaceous earth, etc. This instrument is preferably an opaque material except for the assay-20 zone, which is transparent.

The bond by which the antigen or antibody immobilizes said materials can be obtained by physical or chemical methods (ester or amide bond, etc.), as disclosed in U.S. Patent 4,003,988 and the following publications: B.K. Van Weemen and A.H.W. Schuurs, Febs Letters - Vol. 15, no. 3 - June, 1971, pages 232-5; P. Leinikki and Suvi Passila, J. Clin. Path., 1976, 29, pages 1116-20; B. R. Brodeur, F.E. Ashton et B.B. Day, The Journal of Medical Micorbiology - Vol. 15, no. 1, 1981, pages 1-9; A. Voller 1., 30 D.E. Bidwell 2, A. Bartlett 2, D.G. Fleck 3, M. Perkins 3 and B. Olade-hin 3, J. Clin. Path., 1976, 29, pages 150-3: Howard H. Weetall, Immobilized Enzymes, Antigens, Antibodies, and Peptides - Vol.

Binding of non-immobilized components by adsorption, on the other hand, is performed according to known techniques and is well known in the field of affinity chromatography.

6 85194

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

Enzyme labeling of antigens or antibodies can be accomplished by known techniques, for example, those disclosed in the aforementioned Enzyme Immunoassay, using any enzyme that produces a color reaction with a substrate, and include, for example, peroxidase, alkaline phosphatase, β- galactosidase and the like. Such enzymes can be conjugated to, for example, glutaraldehyde, dimaleimide, and their esters following the techniques described on pages 54-113 of the aforementioned "Enzyme Immunoassay".

Both antibodies and antigens can be attached to the carrier without limiting their amounts, which can vary in any case from 1 to 10 mg / cm 2, depending on the assay in question, provided only that the immobilized component and the enzyme-labeled component should be present. at least stoichiometrically equal amounts. As mentioned above, the quantitative determination is possible by determining the intensity of the color developed either arbitrarily or using a suitable device for measuring the reflection.

Finally, the separation zone at the beginning or in the middle stages may consist exclusively of a material capable of acting as a Kro-30 matrix carrier, on the surface of which substances can be adsorbed in advance which effectively remove compounds which interfere with the recognition reaction. Thus, the invention may use: ion exchange resins to chelate heavy metals; immobilized enzymes to degrade urea or other metabolites; antiprotein 35 antibodies to remove albumin; antibodies specific for molecules such as the antigen to be determined, etc.

7 85194

By means of the method and apparatus described above, the invention provides a means of determining many different clinical parameters in a simple, rapid and accurate manner. These parameters include, for example, peptide hormones such as HCG, LH, steroid -5 hormones such as progesterone, estrone glucuronide, pregnanediol glucuronide, as well as other clinically very interesting parameters such as streptolysin, immunoglobulins, viruses such as herpes viruses, HTLV 3 virus, etc. In addition, multiple enzyme-labeled antigens and / or antibodies can be attached to the same carrier together with 10 corresponding immobilized antibodies and / or antigens, allowing numerous clinical parameters to be determined simultaneously when a corresponding amount is present in different zones. chromogenic substrates that preferably produce different colors when reacting with different enzyme-labeled complexes.

15

In the accompanying drawings, which schematically illustrate some preferred embodiments of the invention as limiting examples in any way, the symbol denotes an antigen; the symbol 0 ”” 0 represents immobilized antigen; the symbol (y * '"' ^ 20 represents the antigen conjugated to the enzyme, while the symbols (') -O') - similarly represent the corresponding antibody, the immobilized antibody and the antibody conjugated to the enzyme. Finally, the symbol l ·· denotes a chromogenic substrate, (^ J · —means immobilized antibody, and the arrow indicates the direction of flow of the fluid introduced into the system.

In said drawings: Figure 1 schematically shows a system for determining antigen according to an embodiment of the invention, and a carrier (strip, tube or other device) comprising a first zone containing an enzyme-labeled antibody specific for the antigen to be determined, adjacent to the first zone. 35, which contains an immobilized antigen, followed by a separation zone and finally a zone to which a chromogenic substrate is attached. When 8 85194 tubes are used as a carrier, its ends can be closed with porous septa.

Figure 2 shows the case where the antibody is determined. In this embodiment, unlike the previous case, the biological five liquid is caused to flow downward direction of the arrow, so that it must successively into contact first with the enzyme labeled antigen, then the immobilized antibody and the last of the chromogenic substrate with which the substrate can be added into the output of the system give a solution of the instead of being adsorbed on a solid support.

Figure 3 shows, in the case of Figure 1, the different stages of movement under the conditions when the test is positive (+) or negative (-) (i.e. when the antigen in question is present or absent).

15

Figure 4 shows another embodiment similar to Figure 1, differing in that the immobilized antigen is pre-agglutinated with an enzyme-labeled antibody; thus, any antigen present in the biological fluid competes with the immobilized antigen to release the labeled antibody, the latter being able to migrate toward the chromogenic substrate.

Figure 5 shows a further modification of a suitable method for the determination of antigens. In this case, the first zone comprising the carrier contains an enzyme-labeled antigen, the corresponding antibody is adsorbed to the second zone comprising the carrier, and the third zone contains an immobilized antibody (e.g., Sepharose protein A or anti-IgC bound to PVC) which 30 is again followed by a chromogenic substrate; Again, only competition with the free antigen present in the biological fluid causes the enzyme-labeled antigen to reach the substrate, while the complex between the antibody and the antigen remains attached to the antibody.

Figures 6a and 6b show a cross-section and a plan view of a different type of device seen from above. The device comprises a support strip 2, 35 9 85194 with holes 1 through which the liquid can pass, and an impermeable transparent cover 3 which allows a visual inspection of the chromogen-containing layer 4. In this case, color can be assessed either by visual observation when the assay is kva-5 litative, or by a reflectometer when the assay is qualitative.

The embodiments shown are intended solely to illustrate the principles of the invention without limiting it in any way. Thus, for example, the size of the discrete zones may vary from case to case, and these sizes are generally from a few millimeters to a few centimeters. Also, certain details that make the carrier of the invention more useful are not described below. These details may be mentioned the applicant's kan-bearing members 15, means for receiving a biological fluid (aspiration), markings that indicate the applicant's side or the head to which the biological fluid is put, color gamut, which is disposed on one side of the zone containing the chromogenic substrate, etc.

The devices according to the invention can also be symmetrical devices in which an enzyme-labeled component is incorporated at each end, and in which these ends are followed by the successive zones described above, thus preventing the liquid from being improperly introduced into the device. In addition, for use in homes 25, the device can be made into simply packaged compact units that are sterile, stable, adapted to the conditions, etc., if necessary.

From the foregoing, it is clear that the method and device of the invention are very versatile and practical, and that they can be used in a wide variety of clinical assays without special equipment or necessary procedures. In addition, and above all, they provide analytical results in a short time, usually in minutes.

The invention is further explained with the aid of the following example, which is not intended to limit in any way the spirit or objects of the invention.

35 10 85 1 94

EXAMPLE

a. Preparation of HCG immobilized on PVC 5,100 grams of PVC with a particle size of 200 μ are treated overnight at 37 ° C in a solution of 50 units / ml in HCG prepared in phosphate buffer, pH 7.2. . The coated polymer is washed five times with the same buffer and dried at 37 ° C for 10 hours.

10 b. Preparation of peroxidase-labeled anti-HCG

Anti-HCG from rabbit (10 ml) is precipitated three times with 18% anhydrous sodium sulfate, and the product is recovered from 10 ml of distilled water after each precipitation. The product is then dialyzed overnight at +4 ° C against 10 mmol phosphate buffer, pH 7.2.

The dialyzed solution is treated with 100 milligrams of peroxidase RZ 20 3.00 and 25 milliliters of 25% glutaraldehyde for 48 hours at + 4 ° C. The solution is eluted through a Sephadex G150 gel equilibrated with sodium chloride, 50 mmol. Elute. . the complex between anti-HCG and peroxidase can be stored at -20 ° C.

25 c. Preparation of Labeled Antibody Adsorbed on Carrier 50 grams of glass is treated with 5 ml of anti-HCG-peroxidase complex diluted 1: 500 in phosphate-30 buffer, 10 mmol, pH 7.2, and the product is dried overnight ____ At 37 ° C.

d. Preparation of chromopene adsorbed on a carrier: 35 50 grams of glass are treated with 50 milligrams of tetramethylbenzidine dissolved in 5 ml of DMSO water.

i Π 85194 e. Filling the column

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

Both ends of the glass column are closed with a cotton ball or suitable porous plugs.

10 f. Performing the analysis

The column described above is contacted with the urine to be analyzed, which urine rises in the column as a result of capillary forces. If HCG is present in the urine, it binds to the anti-HCG peroxidase contained in the first part of the column; as it continues to rise in the column, it no longer produces an agglutination reaction with the immobilized HCG contained in the second part of the column, but continues to travel upwards to the last part of the column, which contains a chromogenic substrate forming a blue dye upon reaction with the ent-20 enzyme. In the case of the layer assay, the complex is specifically bound to zone (c) by an immunochemical reaction, and a blue color is induced as a result of the reaction between the enzyme and the chromogenic substrate, and this blue color is determined in zone (c). On the other hand, if HCG is not present in the urine, the anti-HCG peroxidase of the first part of the column 25 moves freely upwards and reaches the zone containing immobilized HCG in the column, which immobilized HCG binds the anti-HCG peroxidase as a result of the immunochemical reaction. cannot reach a chromogenic substrate. In this case, no color develops. In the case of the stratification test, the anti-HCG peroxidase does not bind to zone (c), but is leached from it, so that no color develops either.

The process described above can also be applied to downflow-based systems.

35

Claims (18)

A method for the determination of clinical parameters such as, for example, peptide hormones, steroid hormones, streptolysin, immunoglobulins and viruses in biological upstream or downstream fluids by an enzymatic immune process, characterized in that the method is sequentially contacted , with antibodies or antigens specific for the analyte; (b) antibodies, antigens, antibodies or antigen-antibody complexes immobilized on a different or the same solid support, the amount immobilized being at least stoichiometrically equal to the enzyme-labeled antibodies or antigens corresponding to the analyte; and (c) one or more chromogenic substrates capable of producing an expressive reaction with enzymes conjugated to antibodies or antigens. A method according to claim 1, characterized in that before the biological liquid is contacted with the chromogenic substrate, it is passed through a chromatographic material to which substances capable of removing compounds which interfere with said analysis may have been adsorbed. A method according to claim 1 or 2, characterized in that when used to determine an antigen, the biological fluid is sequentially contacted with: (a) a complementary enzyme-labeled antibody reversibly adsorbed on a solid support; ; (B) with the same antigen immobilized on a solid support in an amount at least stoichiometrically equal to the amount of 13 85194 antibody labeled with the enzyme; (c) with a chromogenic substrate. A method according to claim 1 or 2, characterized in that when used to detect an antibody, the biological fluid is sequentially contacted with: (a) a complementary enzyme-labeled antigen reversibly adsorbed on a solid support; (b) with the same antibody immobilized on a solid support in an amount at least stoichiometrically equal to the amount of antigen-labeled antigen; and 15 (c) with a chromogenic substrate. A method according to claim 1 or 2, characterized in that when used to determine an antigen, the biological fluid is sequentially contacted with: (a) reversibly adsorbed to the same enzyme-labeled antigen; (B) with a complementary antibody reversibly adsorbed on a solid support; (c) a complex specific for the complex between the antigen and the antibody immobilized on a solid support; and 30 (d) with a chromogenic substrate. A method for determining a plurality of clinical parameters simultaneously according to claim 1, characterized in that the corresponding enzyme-labeled antibodies or antigens and the corresponding immobilized antibodies or antigens are attached to the same solid support, and that for each separate enzyme used. 94 to which specific chromogenic substrates are attached to separate supports. Process according to any one of the preceding claims, characterized in that said carriers, which may be of the same or different material in the case of different components, are made of glass or silica derivatives, paper or cellulose derivatives, polymers, polysaccharides or polyols, kaolin, titanium dioxide TiO 2, barium sulphate BaSO 4, diatomaceous earth, metals or stabilized red blood cells. Method according to one of Claims 1, 2, 3, 5 to 7, characterized in that the method is used for the determination of peptide hormones (HCG, LH), steroid hormones, proteins, streptolysin and viruses. Method according to one of Claims 1, 2, 6 or 7, characterized in that it is used for the determination of immunoglobulins and other antibodies of diagnostic interest. 20 10. A device for determining clinical parameters such as, for example, peptide hormones, steroid hormones, streptolysin, immunoglobulins and viruses, which may be carried and based on upward or downwardly flowing biological fluids, in the form of a tube, model, strip, blotting disc, bottle or film , characterized in that said device consists of: (a) an initial or intermediate separation zone into which substances capable of removing potentially present compounds likely to interfere with the analysis have been adsorbed; (b) an antibody or antigen specific for the clinical parameters to be determined from a zone to which one or more enzyme-labeled enzymes have been pre-adsorbed; optionally, when determining the antigen, the antibody attached to the carrier has been agglutinated in advance with the enzyme-labeled antigen, i) the antibodies, antigens, antibodies, or antibody-antigen complexes are immobilized using known techniques in amounts at least equal to the amounts of enzyme-labeled antibodies or antigens; and (d) the zone to which the chromogenic substrates are adsorbed for enzymes conjugated to the anti-10 gene or antibody. A device according to claim 10, comprising one or more materials selected from glass, metals, synthetic or natural polymeric materials and paper, to which suitable carrier materials in the form of gels, powders, granules, microspheres or films can be attached. , which are the same or different material in each separate zone, and which support materials are selected from starch, diatomaceous earth, kaolin, barium sulfate BaSO 4 and titanium dioxide TiO 2. 20 Device according to Claim 10 or 11, characterized in that the zone (a) in the initial or intermediate stages comprises ion exchange resins, enzymes such as urease or protease, oxidizing or reducing agents, antibodies or antigens as interfering compounds. Device according to any one of claims 10 to 12, characterized in that when used for antigen determination it comprises: (a) a first zone to which a reversibly complementary enzyme-labeled antibody has been adsorbed; (b) a zone adjacent to the first zone to which the same antigen has been immobilized in an amount at least equal to the amount of antibody labeled with the enzyme; i6 85 1 94 (c) the next zone in which chromatographic separation and, optionally, removal of interfering compounds takes place; (d) a final end zone containing chromogen-5 for its substrate enzyme. Device according to any one of claims 10 to 12, characterized in that when used for the determination of antibodies it comprises: (a) a first zone to which a reversibly complementary enzyme-labeled antigen has been adsorbed; (b) a zone adjacent to the first zone to which the same antibody is immobilized in an amount at least equal to the amount of antigen-labeled antigen; (c) a separation zone; and 20 (d) a chromogenic expression zone. Device according to claims 10-12, characterized in that in determining the antigen, the device comprises a first zone containing the enzyme-labeled antigen, a second zone reversibly adsorbed on the antibody, a zone containing the immobilized antibody, and finally, a separating and chromogenic expression zone. Device according to one of Claims 10 to 15, characterized in that, when several clinical parameters are determined simultaneously, each of the separate zones (a) and (b), respectively, contains a corresponding number of enzyme-labeled antibodies or antigens and immobilized antigens or and that the device comprises a plurality of chromogenic expression zones. 35 Device according to one of Claims 10 to 16, characterized in that it can be used to determine the hormone HCG or LH or other 17 85 1 94 peptide hormones, progesterone, estrone glucuronide, pregnanediol glucuronide or other steroid hormones, streptolysin, immunoglobulin lines and / or viruses. Device according to one of Claims 10 to 17, characterized in that the device as a whole is made of an otherwise opaque material (s), with the exception of an assay zone made of transparent material (s). 85194 BC