WO1986004683A1

WO1986004683A1 - Determination of clinical parameters by enzyme immunoprocess

Info

Publication number: WO1986004683A1
Application number: PCT/EP1986/000055
Authority: WO
Inventors: Ennio Iaccheri; Paola Piro; Claudio Manzati
Original assignee: Boehringer Biochemia Robin S.P.A.
Priority date: 1985-02-08
Filing date: 1986-02-02
Publication date: 1986-08-14
Also published as: ES557802A0; ES557803A0; ES557662A0; ES8802256A1; FI85194C; RO100056A2; FI873398A0; OA08640A; EP0243370A1; JPS62501989A; RO100056B1; ES551760A0; IT1200382B; ES8802258A1; IT8519443A0; AU5511886A; MW5287A1; HUT56632A; ES8802260A1; DD245727A5

Abstract

A method and a device for the determination of clinical parameters in biological liquids by enzyme immunoprocess, wherein the biological liquid is contacted sequentially with: a) enzyme labelled antibodies or antigens that are absorbed on a solid support in a reversible manner; b) antibodies, antigens, anti-antibodies or antigen-antibody complexes that are immobilized on a solid support in a quantity stoichiometrically equivalent to the enzyme-labelled antibodies or antigens; c) one or more chromogen substrates capable of producing evidencing reaction with the antibody or antigen-conjugated enzymes.

Description

Determination of clinical parameters by enzyme im unoprocess

This invention relates to a diagnostic method and to a device for the qualitative and/or quantitative deter¬ mination of clinical parameters by enzyme immunoprocess using the affinity chromatography. The invention is particularly useful for the ana¬ lysis of biological substances in biological liquids such as urines, plasma, blood, exudates, tissue extracts, etc.

The immunoenzyme diagnostic techniques have been known for a long time. They are based on the reaction between an antibody and its antigen one of which is con¬ jugated to an enzyme capable of giving a detecting reac¬ tion (of chromogen or other nature) when contacted with a suitable substrate. Thereafter, the enzyme-labelled anti¬ gen antibody complex, will give rise, by contact with the detector substrate, to a reaction that can be utilized for the qualitative or quantitative determination of the anti¬ gen or the antibody of interest.

The enzyme immunotechniques most currently used in diagnostic are all suffering from a number of deficiencies that are substantially imputable ^'.to the complicated and delicate procedures involved, and the long incubation times with intervening repeated washing of the reactive system.

Thus, the practice of such techniques is limited to qualified laboratories, whereas a more generalized use thereof, such for example as the ambulatory and even the domiciliary use, have hitherto not been possible. In fact, even though the conventional methods do not imply any particular difficulties for one skilled in the art, the handling and reading 'of the test results involve problems that are hard, if not impossible, to be overcome by un- skilled people.

For instance, a technique designated under the abbreviation name "ELISA" (Enzyme-linked immunosorbent assay, Immunochem. , 8, 871-874, 1971) is known which, in addition to long incubation and reaction times and reite- rate washing operations, also requires a skillful opera¬ tion and manipulation.

On the other hand, the "EMIT" technique has limita¬ tion in that it does not permite 'the determination of high molecular weight molecules. Finally, a technique known in the art as the "FlA" technique again requires use to be made of special reading instruments.

For a detailed description of the above-recited diagnostic techniques reference may be made to "Enzyme Jmmunoassay", E. Ishikawa, T. Kawai, K. Miyai, Igaku- Shoin-Tokyo-New York (1981).

It appears, from the above, that an enzyme immune- based diagnostic technique of a very great interest would be one that combines easy and fast operation with the response sensitiveness and the specifity which are pecu¬ liar to these immunoenzymatic methods.

This invention overcomes the problems encountered in the prior art techniques by providing a method and a device which enable to carry out, even on a quantitative basis, the determination of clinical parameters of what- ever nature, without recourse having to be made to any particular manipulations.

According to the invention, a biological liquid containing the clinical parameter(s) to be determined is made to pass in a upward or downward direction along a conveniently shaped support which comprises: (a) an initial or intermediate zone on which substances may be adsorbed which are able to prevent interferences with the assay; (b) a zone onto which one or more antigens or enzyme-la¬ belled antibodies raised against one or more epitopes (N.R. Jerne, Angew. Chem. Int. Ed. Engl., 24; 1985, 810- 816), specific for the clinical parameter to be determin¬ ed, have previously adsorbed in a reversible manner; (c) a further zone, adjacent the preceeding zone, on which the antibodies or antigens forming the clinical parameters to be determined, are by known techniques immobilized in a quantity equivalent to, or greater than, that of .the enzy¬ me-labelled antibodies or antigens. In this zone it is also possible to use antibodies raised against one or more epitopes different from the epitope(s) recognized by the antibodies of zone (b) (sandwich-assay). In this case the chromogenic substrate(s), for the enzyme-labelled antibo¬ dies' of zone (b), should be bound to zone (c). Alternatively the chromogenic substrate(s) could be adsor¬ bed in reversible manner on a zone laying below. As an alternative, this zone (c) may have an anti-antibody immo¬ bilized thereon which is specific for an antigen-antibody complex, in which case the zone (b) is in turn divided , into two portions, with a first portion having, for exam- pie, the enzyme-labelled antigen adsorbed thereon and a second portion carrying the corresponding antibody, or conversely.

(d) A zone on which there is adsorbed a chromogenic sub- strate for the antigen- or antibody-conjugated enzyme present in zone (b) or one or more support capable of allowing the flow of biological liquids (sandwich-assay).

The biological liquid flowing up or down through the support by gravity, capillarity, chromatography or diffusion, will be caused to contact the zone (b) the first: the antigene or antibody that may be present will react with the complementary, enzyme-labelled antibodies or antigens.

The thus formed complex, or combined system, will rise (or fall) chromatographically along the support to pass through the zone (c) where the excess enzyme-labelled antibody or antigene, if any, will be bound to the comple¬ mentary, immobilized antigene or antibody. In the case of the sandwich-assay the specific complex will be bound forming a "sandwich" arrangement.

Subsequent to its passing through the intermediate zone, where any interfering substances may be removed (as, for example, by adsorption, chemical bond, degradation, etc.), the enzyme-labelled antigen-aritibody complex at- tains the chromogenic substrate to give rise to color (positive test) which can be , used for both quantitative and qualitative determination, for example, in comparison with a colorimetric scale. On the other hand, when no clinical parameter is present in the biological liquid (negative test), then the enzyme-labelled antigen or anti- body, in one case will be entirely fixed on the zone (c) by immunochemical reaction, thereby preventing it from attaining the chromogen substrate; in the other case (sandwich-assay) the enzyme-labelled antibody will not be fixed on the zone (c), and the colour formed during the passage of antibody-conjugated enzyme will be washed from the zone (negative test) by the flow of biological liquid. In other words, the system is based on a competi¬ tion between the antigen or antibody present in the biolo- gical liquid and the immobilized antigen or antibody whi¬ ch, in case of a negative response, functions in the first case as a barrier for the enzyme-labelled antibody or antigen to prevent migration thereof to the chromogen substrate-containing zone; in the second case as an un- reactive material which will not be able to bound the enzyme-labelled antibodies.

The support device according to the invention may take the form of a strip, band, film, tube, specimen, flask, pad, etc. The various constituents may be either adsorbed or immobilized directly ^"onto the material forming the device or an appropriate solid carrier located inside the device, or fixed on the surface thereof in the form of a gel, powder, granules, microspheres, spheres, etc.

The materials of the device, and the solid supports or carriers, at the various reaction zones, may be the same or different. Examples of suitable materials and carriers are glass, silica derivatives, paper or cellulose derivative*, metals, polymers such as polyvinyl chloride, polystyrene, polybutadiene, nylon, polyacryla ides, metha- crylates etc., polysaccharides or polyols, starch, stabϊ-^~ lized human or animal red blood cells, organic substanc such as BaSO , TiO , kaolin, diatomaceous earth, etc. Th device is preferably of an opaque material except the re ding-zone which is transparent. The immobilizing bond of the antigen or antibo on said materials may be achieved through physical chemical process (ester or amide bonding, etc.) accordi to the teachings in U.S. Patent No. 4,003,988 and t following references: B.K. Van Wee en and A.H.W. Schuur Febs Letters - Vol. 15, No. 3 - June, 1971, pp. 232-5; Leinikki and Suvi Passila, J. Clin. Path., 1976, 29, p 1116-20; B.R. Brodeur, F.E. Ashton et B.B. Diena, T Journal of Medical Microbiology - Vol. 15, No. 1, 198 pp. 1-9; A. Voller 1., D.E. Bidwell 2, A. Bartlett 2, D. Fleck 3, M. Perkins 3 and B. Oladehin 3, J. Clin. Path 1976, 29, pp. 150-3; Howard H.^" eetall, Immobilized Enz mes, Antigens, Antibodies, and Peptides - Vol. 1.

The adsorption bond of the not immobilized const tuents may, on the other hand, occur by known technique matter as it is well known from the affinity chromatogr phy art.

Both the immobilized and the enzyme-labelled¹ ant bodies useful to the invention can be polyclonal or mon clonal in toto or in fragments thereof, as for exampl Fab' and F(ab')2„, possibly in admixture with one anothe and they may be specific for one or more active sites the antigen.

Enzyme-labelling of the antigens or antibodies c be effected by known techniques such as those disclosed the above-mentioned text "Enzyme Immunoassay", by usi any enzyme capable of developing a chromogen reaction wi respect to , a substrate, such for example as peroxidas alkaline phosphatase, β-galactosidase or the like. Suc enzymes may, for example, be conjugated by means of gluta- raldehyde, dimaleimide and the esters thereof according t the techniques disclosed in the word "Enzyme Immunoassay" , pp. 54 to 113, mentioned above.

Both the antibodies and the antigens may be atta ched to a support without restriction as to the quantit which may, in any case, be varied from 1 to 10 mg/sq c according to the concerned type of analysis, with only th provision that the immobilized component and the enzyme labelled component should be present in at least stoichio metrically equivalent amounts. As already said, a quanti tative determination is possible by determining the leve of the developed color either in an arbitrary way or b means or a suit'able reflection reader.

Finally, the initial or intermediate separatio zone may merely consist of a material capable of function ing as a chromatographic support onto which such substan¬ ces are enabled to be previously adsorbed as are effective to remove compounds interfering with the identification reaction. Thus, use may be made of: - ion-exchange resins to sequester heavy metals; immobilized enzymes to degrade urea or other metabolites; antiprotein antibodies to remo¬ ve albumin; antibodies that are specific for molecules having similarity to the antigen to be determined, etc.. By the above-disclosed method and device, the in¬ vention provides a means enabling to determine in a sim¬ ple, fast and accurate manner a great deal of different clinical parameters: for example, peptide hormones such as HCG, LH, steroid hormones such as progesterone, estrone - _ -* -»

- 8 - glucuronide, pregnanediol glucuronide, or other 'parameters of broad clinical interest such as streptolysin, immune globulins, viruses such as herpes viruses, HTLV-3 virus etc. Moreover, attached to the same support may be several enzyme-labelled antigens and/or antibodies along with the corresponding immobilized antibodies and/or antigens the¬ reby permitting a number of clinical parameters to be determined in a simultaneous manner due to the presence, at different zones, of a corresponding number of chromogen substrates that are preferably such as to produce diffe¬ rent colors by reaction with different enzyme-labelled complexes.

In the accompanying drawings which show in a dia¬ grammatic manner, by way of non restrictive examples, some preferred embodiments of the ^• invention, the sym¬ bol ^^ designates the antigen; the symbol <^> Q indicates the immobilized antigen; the symbol

indicates the enzyme-conjugated antigen while the sym¬ bols /—— - / _) , H, in a similar manner designate the antibody, the immobilized antibody and the enzyme-con¬ jugated antibody, respectively. Finally the sym- , bol (—•indicates the chromogen substrate, N—Q the immobilized anti-antibody, and the arrow designates the direction of the incoming liquid. In said drawings:

- Figure 1 diagrammatically shows an arrangement enabling, according to one embodiment of the invention, to determine an antigen and including a support (strip, tube or other means) with a first zone containing the enzyme-labelled antibody that is specific for the antigen to be determin¬ ed, a second zone adjacent the first one which contains the immobilized antigen and which is followed by a sepa ration zone and, finally, a zone carrying the chromoge substrate thereon. When a. tube is used as the support, th tube ends may be closed by porous septa. - Figure 2 shows the case when an antibody is to be deter mined. In this embodiment, unlike the previous case the biological liquid is caused to percolate in a downwar direction as indicated by an arrow, to successively conta ct the enzyme-labelled antigene in a first place, then th immobilized antibody and, in a last place, the chromoge substrate which instead of being adsorbed onto the soli support, may be added to the solution going out of th bottom end of the device.

- Figure 3 shows, for the case in Figure 1, the variou migration steps .under condition^'s of positivity (+) and-o negativity (-) of the test (presence or absence of th antigen concerned) .

- Figure 4 shows another embodiment similar to that i Fig. 1 from which it differs in that the immobilized anti gen is preagglutinated with the enzyme-labelled antibody thus, any antigen present in the biological liquid wil compete with the immobilized antigen to release the la belled antibody thereby enabling this latter to migrate t the chromogen substrate. - Figure 5 shows a further variation of the method o determining antigens. In this case, the support comprise a first zone containing an enzyme-labelled antigen, second zone having a corresponding antibody adsorbed ont it, and then a zone containing an immobilized anti-antibo dy (for example, sepharose protein-A or anti-IgC bound t PVC) again followed by the chromogen substrate; in thi case too, it is only the competition with the free antige present in the biological liquid that causes the enzyme labelled antigen to attain the substrate, whereas th antibody-antigen complex is kept trapped on the anti-anti body.

- Figures 6a and 6b show a different type of device, a seen in cross-sectional view and in plan view from th .above, respectively. The device comprises a support stri 2 provided with holes 1 for a liquid to pass therethroug and with an impervious transparent coating 3 which permit the chroroogen-containing layer 4 to be inspected visually In this case, reading may be effected either visually, fo qualitative determination, or by the aid of a reflectio reader, in the case of a quantitative determination.

. The embodiments shown are to be intended as bein merely illustrative of the principle of the inventio without any limitation thereto. Thus, for example, th sizes of the individual zones may vary in each particula case and are commonly in the range of the few mm to few c values. Moreover, certain details have not been show which could make for a more practical use of the inventiv support, such for example as support handles, means fo taking off (by suction) of biological liquid, marking indicative of the side to which the biological liquid i to be admitted, colorimetric scales arranged to one sid of the zones containing the chromogen subs ate, etc..

The devices according to the invention may also b symmetrical devices having the enzyme-labelled componen arranged at both ends thereof, these latter being followe by the above-described sequential zones, in order to pre¬ vent the liquid from being uncorrectly admitted. Moreover, for domiciliary use, conveniently packaged compact units may be provided which possess the sterile, stable, condi- tioned characteristics as required, etc..

From the above, it is clear that the method and device according to the invention are versatile and prac¬ tical in use and can be used for a large range of clinical analysis without the need for particular equip- ments or operations being involved. They are moreover, and above all, capable of giving analysis results in a short period of time, usually of the order of the minutes.

The invention will now be further explained by the aid of the following Example which is intended to be no restrictive to any extent as to the spirit and scope of the invention.

EXAMPLE a. Preparation of HCG immobilized on PVC

PVC, 100 g, 200 p particle size, is treated in a 50 units/ml solution of HCG in phosphate buffer at pH 7.2, 37°C, overnight. The coated polymer is washed 5 times with the same buffer and dried at 37°C for 10 hours. b. Preparation of peroxidase-labelled anti-HCG

Anti-HCG (10 ml) from rabbit, is precipitated 3 times with 18% anhydrous sodium sulfate and the product recovered from 10 ml distilled water following to each precipitation. Then, the product is dialyzed one night at +4°C against phosphate buffer at 7.2 pH, 10 mMols.

The dialyzed solution is treated with 100 mg of peroxidase RZ 3.00 and 25 mcl of 25% glutaraldehyde for 48 hours at +4°C. The solution is eluted on Sephadex G150 balanced with sodium chloride, 50 mMols. The eluted anti- HCG - peroxidase complex may be stored at -20°C. c. Preparation of the labelled, support-adsorbed antibody Glass, 50 g, is treated with anti-HGC peroxidase, 5 ml, 1:500 diluted in phosphate buffer, 10 mMols, pH 7.2, and the product dried overnight at 37°C. d. Preparation of the support-adsorbed chromogen

50 Grams of glass are treated with 50 mg of tetra- methylbenzidine dissolved in 5 ml of DMSO-water. e. Column packing

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

The glass column is closed at both ends with cotton wool or suitable porous stoppers. f. Carrying out of the analysis

The above-described column is contacted with urine to be analysed which will rise up the column by capillari¬ ty. When HCG is present in urine, it will be bound to the anti-HCG peroxidase contained in the first column portion; in proceeding on its rising path,it will no longer produce any agglutination reaction with the immobilized HCG conta- ined in the second column portion so that it continues its way up to the last column portion containing the chromogen substrate which will develop a blue color by reaction with the enzyme; in the case of sandwich-assay, the complex will be specifically bound to zone (c) by immunochemical reac- tion, and the blue colour will be developed by the reaction with the enzyme and the chromogen substrate, developing a blue colour which will be read in the zone (c). On the other hand, when no HCG is present in urine, the anti- HCG peroxidase in the first column portion, in going up in a free manner, will attain the column zone containing the immobilized HCG to be bound thereto by immunochemi- cal reaction thereby being prevented from reaching the chromogen substrate. Thus, no color is developed; in the case of sandwich-assay, the anti-HCG peroxidase will not be bound in zone (c) and washed from the zone, in this way there will not be developed of colour.

The above-described process may apply as well to falling liquid systems.

Claims

1. A method of determining clinical parameters in biological liquids fay enzyme immune process wherein a biological liquid is sequentially contacted with:

(a) enzyme-labelled antibodies or antigens that are reversibly adsorbed onto a solid support;

(b) antibodies, antigens, anti-antibodies or anti- gen-antibody complexes which are immobilized on a solid support in a quantity at least stoichiometrically equiva¬ lent to the enzyme-labelled antibodies or antigens; and

(c) one or more chromogen substrates capable of producing a detecting reaction with the enzymes conjugated to the antibodies or the antigens.

2. The method according to claim 1, wherein the biolo¬ gical liquid before contacting the chromogen substrate is made to pass through a chromatographic material onto which substances may have been adsorbed which are capable of removing compounds interfering with the analysis concern¬ ed.

3. The method according to claim 1 or 2, when used for the determination of antigen, wherein the biological liquid is sequentially contacted with: (a) the complementary, enzyme-labelled antibody that is adsorbed on a solid support in a reversible man¬ ner;

(b) the same antigen that is immobilized on a solid support in a quantity at least stoichiometrically equivalent to the enzyme labelled antibody? - ' (c) a chromogen substrate. :^~

4. The method according to claim 1 or 2, when used for the determination of an antigen, wherein the biological li_^ quid is contacted with a) the complementary enzyme-labelled antibody that is adsorbed on a solid support in a reversible manner; b) one or more immobilized antibodies and chromogen sub¬ strate adsorbed in reversible manner or immobilized on a solid support; c) one or more support capable of allowing the flow of biological liquids.

5. The method according to claim 1 or 2, when used for the determination of an antibody, wherein the biological liquid is sequentially contacted with: (a) the complementary, enzyme-labelled antigen that is adsorbed on a solid support in a reversible man¬ ner;

(b) the same antibody which is immobilized on a solid support in an amount at least stoichiometrically equivalent to the enzyme-labelled antigen; and

(c) a chromogen substrate.

6. The method according to claim 3, wherein the enzy¬ me-labelled antibody and the immobilized antigen are ag¬ glutinated on the same solid support.

7. The method according to claim 1 or 2, when used for the determination of an antigen, wherein the biological liquid is sequentially contacted with:

(a) the same, enzyme-labelled antigen that is adsorbed on a solid support in a reversible manner; (b) the complementary antibody that is adsorbed in - IS -

a reversible manner on a solid support;

(c) an anti-antibody specific for the antigen-an¬ tibody complex, which is immobilized on a solid support; and (d) a chromogen substrate.

8. The method according to claim 1, as used -for deter¬ mining several clinical parameters in a simultaneous man¬ ner, wherein attached to the same solid supports are the corresponding enzyme-labelled antibodies or antigens and the corresponding immobilized antibodies or antigens, and wherein the chromogen substrates specific for each indivi¬ dual enzyme used are carried on individual supports.

9. The method according to any preceding claim, where¬ in said supports, which may be the same or different for the various components, are comprised of glass or silica derivatives, paper or cellulose derivatives, polymers, polysaccharides or polyols, kaolin, TiO , BaSO , diatoma- ceous earth, metals, stabilized red blood cells.

10. The method according to any one of the claims 1, 2, 3, 6 to 9, wherein the method is used for the determina¬ tion of peptide hormones (HCG, LH), steroid hormones, proteins, streptolysin , viruses.

11. ,The method according to any of the claims 1, 2, 5, 8 or 9, wherein it is used for the determination of immune _ι globulins and other antibodies of diagnostic interest.

12. A device for the determination of clinical parame¬ ters, of the type capable of being travelled along by rising or falling biological liquids, and taking the form of tube, specimen, strip, pad, flask, film, wherein said device is comprised of: (a) an initial or intermediate zone of separation onto which such substances may have been adsorbed as are able to remove any compound likely to interfere with the concerned analysis; (b) a zone onto which there have been previously adsorbed one or more enzyme-labelled antibodies or anti¬ gens which are specific for the clinical parameter to be determined;

(c) a further zone adjoining the former zone, on which the antibodies, antigens, anti-antibodies or antibo¬ dy-antigen complexes forming the clinical parameters to be determined, have been by known techniques immobilized in a quantity at least equivalent to that of the enzyme-label¬ led antibodies or antigens; and (d) a zone onto which chromogen substrates have been adsorbed for use by the antigen or antibody-conjugat¬ ed enzymes.

13. A device for the determination of clinical parame¬ ters, of the type capable of being travelled along by ris- ing or falling biological liquids, and taking the form of tube, specimen, strip, pad, flask, film, wherein said devi_ ce is comprised of:

(a) an initial or intermediate zone of separation onto which such substances may have been adsorbed as are able to remove any compound likely to interfer with the concerri ed analysis; b) a zone onto which there have been previously adsor¬ bed one or more enzyme-labelled antibodies (raised against one or more epitopes) which are specific for the clinical parameter to be determined; c) a zone on which one or more antibodies (raised aga¬ inst one or more epitopes different from the epitopes re¬ cognized by the enzyme-conjugated antibodies of zone b) are immobilized and, chromogenic substrate(s) are adsorb- ed in reversible manner or immobilized; d) a zone with one or more support materiale capable of allowing the flow of biological liquids.

14. The device according to claim 12 or 13 comprising one o more materials selected from among glass, metals, synthe- tic or natural polymer materials, paper, to which there may be attached, in the form of gel, powders, granules, microspheres, films, suitable support materials that are the same or different for each individual zone and selec¬ ted from among starch, diatomaceous earth, kaolin, BaSO , Ti0₂.

15. The device according to claim 12 or 14, wherein the initial or intermediate zone (a) contains ion-exchange resins, enzymes such as urease or protease, oxidizing or reducing substances, antibodies or antigens, as the sub- stances capable of removing interferences.

16. The device according to any of the claims 12 to 15 when used for the determination of an antigen, which com¬ prises:

(a) a first zone having the complementary, enzyme- labelled antibody reversibly adsorbed thereon;

(b) a zone adjacent to the first zone having the same antigen immobilized thereon in a quantity at least equivalent to the enzyme-labelled antibody;

(c) a subsequent zone effective to cause chromato- graphic separation and, optionally, interference removal; •_

- 19 - ^~~ - and

(d) a last end-zone carrying the chromogen substra¬ te for the enzyme.

17. The device according to any of the claims 12 to 15, wherein for the determination of antibodies the device includes:

(a) a first zone having the complementary, enzyme- labelled antigene reversibly adsorbed thereon;

(b) a zone adjacent to the first one, having the same antibody immobilized thereon in an amount at least equivalent to the enzyme-labelled antigen;

(c) a separation zone, and

(d) a chromogen detecting zone.

18. The device according to claims 12 to 15 wherein for the determination of an antigen the device comprises a first zone on which the support-attached antibody is pre- agglutinated with the enzyme-labelled antigen, a separa¬ tion zone, and a chromogen detecting zone.

19. The device according to claims 12 to 15 wherein for the determination of an antigene the device comprises a first zone provided with the enzyme-labelled antigen, second zone having the antibody reversibly adsorbed there¬ on, a zone provided with the immobilized anti-antibod and, finally, the separation and chromogen detecting zone.

20.' The device according to any of the claims 12 to 19 wherein for determining several clinical parameter simul¬ taneously, each of the individual zones (a) and (b) re spectively contains a corresponding number of the enzyme labelled antibodies or antigens and the immobilized anti gens or antibodies, and wherein several chromogen detect ing zones are provided.

21. The device according to any of the claims 12 to 20, wherein the device is suitable for the determination of HCG, LH or other peptide hormones, progesterone, estrone glucuronide, pregnanediol glucuronide or other steroid hormones, streptolysin, immune globulins, viruses.

22. The device according to any of the claims 12 to 21, wherein all the device is of opaque material(s) except- for • the zone of reading which will be of trasparent material(s)