JPS62501989A - Measuring method of clinical parameters by enzyme immunoassay - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for Measuring Clinical Parameters by Enzyme Immunoassay Qualitative and/or quantitative determination of clinical parameters using affinity chromatography The present invention relates to a clinical testing method and device for quantitative measurement.

This invention is useful in biological fluids such as urine, plasma, blood, exudates, lfl fibrous extracts, etc. It is particularly advantageous for the analysis of biological substances.

Immune enzyme clinical testing techniques have been known for a long time. When they come into contact with a suitable substrate, When testing, conjugate ( It is based on the reaction between a conjugated antibody and its antigen. Then enzyme label By contacting the antigen-antibody complex with the measurement substrate, the antigen or antibody in question is cause a reaction that can be used for qualitative or quantitative measurement of

The enzyme immunotechniques currently most commonly used in clinical testing are complicated and difficult to use. This essentially results in a lengthy incubation time with a complicated process and repeated washing of the reaction system. Being hairy has many disadvantages to be faced.

And the practice of such techniques is limited to experienced experimenters.

(and more general use, such as mobile use or even at home) Conventionally, this was not possible. In fact, ordinary methods are of no use to experts. Even if it does not pose special difficulties such as Deciphering is difficult and difficult, if not impossible, for non-experts to overcome. It was.

For example, ELISA (Enzyme-linked Immonosorben tassay, lnu.

ochet, 8,871-874.1971). In addition to the intermediate equipment and reaction time and repeated cleaning operations, it requires skilled operation and small workmanship. It is essential.

On the other hand, the EMIT technology has the limitation that it is impossible to measure high molecular weight molecules. Yes.

Finally, the technology known to experts as FIA technology also requires special decoding equipment. It requires the use of a

For detailed explanations of the clinical testing techniques mentioned above, please refer to "Enzyme Immunoassay JE.

Ishikawa, T., Endai, K., Yoshii, Igakushoin-Tokyo-New York (1981) Quote.

From the above points, enzyme immunoassay testing technology is of great interest because it is easy and quick to operate. It seems to be a combination of response sensitivity and special characteristics unique to these immunoenzymatic methods. It seems to me.

The present invention allows clinical parameters of any nature to be measured without resorting to any special tricks. To provide a method and apparatus that can perform even quantitative measurements of data. This overcomes the problems associated with the prior art.

or passed downwards: a) starting zone or medium in which substances are adsorbed that can prevent interference with the analysis; Interband; b)? l! lI one or multiple epitopes specific to the clinical parameter to be determined one or more antigen or enzyme-labeled antibodies sensitized to (N, R, Jer ne, Angew, Chem, Int, Ed, Eng l,, 24 ;1985.81 (1-81tli) is around O Reversibly adsorbing band C) The antibody or antigen forming the clinical parameter to be measured is an enzyme-labeled antibody or An amount equal to or greater than the amount of antigen is immobilized (Im+nbollzed) using known techniques. ) is the band following the preceding band. And in this band, the antibody in band (b) for one or multiple epitopes different from the recognized epitope (recognition site) It is possible to use expressed antibodies (sandwich measurement). In this case The chromogenic material for the enzyme-labeled antibody in zone (b) is coupled to zone (c).

Alternatively, chromogenic substances can also be reversibly adsorbed in the lower band. Wear. Separately, this zone (C) has an anti-antibody specific for the antigen-antibody complex above it. You may have an immobile body. In this case, band (b) is instead two The first part contains, for example, the enzyme-label adsorbed thereon. The second part has the corresponding antibody.

and on the contrary d) Zone (b) or antigen- or is the band where the chromogenic substance for the antibody-enzyme complex is adsorbed (sand inch measurement) By gravity, capillary action, chromatography or permeation, the support is The biological fluid that can pass downwards first contacts zone (b) and The antigens and antibodies present will react with complementary antibodies or antigens labeled with enzymes. Ru.

The complex or combined system thus produced passes through zone (C) to the carrier. will rise or fall chromatographically.

and in zone (C) excess enzyme-labeled antibody or antigen is optionally immobilized. binds to complementary antibodies or antigens. In the case of this sand char measurement method, its specific complexes form a "sandwich" arrangement.

Following passage through an intermediate zone where interfering substances are removed (e.g. adsorption, chemical binding, decomposition, etc.) ) The enzyme-labeled antigen-antibody complex acquires chromogen and develops color. that and can be used for qualitative and quantitative measurements, e.g. by comparison with color scales. (bozitib test).

On the other hand, when the clinical parameter is not present in the biological solution (negative test) The enzyme-labeled antigen or antibody is completely immobilized in zone (C) by the immune reaction. If this occurs, the acquisition of chromogenic substances is prevented. In other cases (sandwich measurement), the enzyme-labeled antibody is not immobilized in zone (C). and the antibody complex The color produced during the passage of the enzyme is washed out of the zone by the flow of biological fluid. (negative test).

In other words, this system combines antigens or antibodies present in biological fluids with immobilized It is based on competition between antigen or antibody. And the immobilized antigen or antibody is In the first case, in the case of a negative response, enzyme-labeled antibodies or inhibitors for the antigen may be used. As a stopper, it acts to prevent its movement into the chromogenic substance-containing zone. It is. In this second case, the working block is a non-reactive material that does not bind the enzyme label. , specimine, flask, pad, etc.

Various constituents may be present on or in the materials forming the device. It is adsorbed directly or immobilized on a suitable solid support inside the device.

Alternatively, it is immobilized on a surface in the form of a gel, powder, granule, microsphere, sphere, etc.

The materials of the apparatus and the solid supports or carriers in each reaction zone may be the same. It's okay to be different. Examples of suitable materials and carriers are glass, silica derivatives, paper or Cellulose derivatives, metals, polymers such as polyvinyl chloride, polystyrene, polypropylene Polysaccharides or polysaccharides such as tagene, nylon, polyacrylamide, methacrylate, etc. starch, stabilized human or animal red blood cells, organic substances such as BaSO4, These include TiO2, kaolin, diatomaceous earth, etc. The device is opaque except for the transparent reaction zone. Preferably, it is a material.

The binding that immobilizes the antigen or antibody on the above material can be carried out by physical or chemical methods. This can be done using the techniques cited in U.S. Patent No. 4,003,988 and the references below. Ru. Namely, B, K, van Bemen and A, H, W.

Schuls, Febs Letters, Vol. +5, No. 3-June 1971, 23 Pages 2-5; p.

Reinicki and Subi Parasila, J., ClIn, Path, 197Ei, 2 9. pp, IIIG-20; B, R, Projua, F, B, Ashton et al. B, B, Jeana, The Journa lof Medical Mic orbology-15, No. I, 198111-9; A, Bora-1, , D, E, Bidwell 2, A, Bertread 2, D, G, Fleck 3, M, Par. Kins 3 and B, Oladehin 3, J, Cl1o, Path, 197[i, 29. pp. 150-3; Howard H, Utal, Immobilizing enzymes, antigens, antibodies and peptides. Chido volume 1.

Adsorptive binding of non-immobilized components, on the other hand, This is done by known techniques such as those well known in the art.

The immobilized antibodies and enzyme-labeled antibodies that can be used in the present invention are polyclonal, or It may be the entire monoclonal or its Φ fragments, such as Fab' and F( ab')2, which may be mutually adhered. And that They can be specific for one or many of the active sites of the antigen.

Enzyme labeling of antigens or antibodies can be performed using known techniques, such as the above-mentioned text "Enzyme immunoassay". Enzymes capable of developing chromogenic reactions on substrates by techniques published in Using peroxide, alkaline phosphatase, β-galactosidase, etc. It can be carried out. Such enzymes include, for example, glutaraldehyde, sialeimide and their esters, as published in the above-mentioned “Enzyme Immunoassay J, pages 54-113. It can be combined with other technologies.

Both the antibody and the antigen can be brought into contact with the support. And the amount is analyzed Depending on the model, it may vary between 1-10 mg/sq (there is no particular quantitative limit. stomach.

However, the immobilization component and the enzyme label component are present in at least stoichiometrically corresponding amounts. There must be. Quantitative measurements can be made by intermediary methods or with a suitable reflex reader. This is possible by preparing 11 levels of color development.

Finally, the first or intermediate separation zone removes substances that interfere with the identification reaction. A chromatographic uniform support that can pre-adsorb substances, which is useful for It is sufficient that it is made of a material that has the function of This way, heavy metals are removed ion exchange resins; immobilizing enzymes that degrade urea or other metabolic products; Anti-protein antibodies that remove albumin, antibodies that are similar to the antigen to be measured Child-specific antibodies can be used.

The method and apparatus disclosed above enable the present invention to easily, quickly and accurately perform a large number of Clinical parameters (e.g. HCG, peptide hormones like LH, progesterone Steroids such as telone, estrone, glucuronide, pregnanediol, Hormones, streptolysin, herpes virus, HTLV-3 virus, etc. other parameters of broad clinical interest such as immunoglobulin It provides a means by which it can be measured. Moreover, the same support has some Enzyme-labeled antigens and/or antibodies are attached together with the corresponding immobilized antibodies and/or antibodies. (in which case different bands, preferably different enzyme labels) a corresponding number of chromogenic substances capable of reacting with the complex to produce different colors; This allows several clinical parameters to be measured simultaneously. Ru.

The accompanying drawings illustrate a clinical testing method according to a preferred embodiment of the present invention. However, the present invention is not limited thereto.

In the figure, the symbol ◆ represents the antigen, the symbol ◇←-0 represents the immobilized antigen, and the symbol ◇--E indicates enzyme-conjugated antigen. Figure 1 shows the embodiment of the present invention. Therefore, this is a schematic diagram of a device that can measure antigens, and at the same time a support The first band is the one to be measured. Contains an enzyme-labeled antibody specific to the antigen of interest. Also, the second band is band 1. adjacent to the zone, containing the immobilized antigen, the separation zone and finally the chromogenic substance. Continuing with the carried band. When using a tube as a support, The ends are closed with porous septa.

FIG. 2 shows the case of measuring antibodies. In this embodiment, the previous case Unlike, biological fluids are allowed to percolate downwards, as indicated by the arrows. and then contact the enzyme-labeled antigen in the first region, then contact the immobilized antibody, And finally, it comes into contact with a chromogenic substance. The chromogenic substance is then adsorbed onto the solid support. Alternatively, it may be added to the solution exiting the end of the device.

Figure 3 shows the case of Figure 1 when the plus (+) and test are negative (-). It shows the various migration steps under the conditions (presence or absence of the antigen in question). present).

FIG. 4 shows another embodiment similar to FIG. 1, the difference being that the immobilization The point is that the antigen is pre-coagulated with an enzyme-labeled antibody. Thus, biological Any antigen present in the liquid will compete with the immobilized antigen, releasing the labeled antigen and labeling. This allows the antigen to move towards the chromogenic substance.

FIG. 5 shows another modification of the antigen assay method. In this case, the support is the enzyme The first layer contains the labeled antigen, the second layer has the corresponding antibody adsorbed, and then the immobile antibody (e.g., Sepharose protein A or anti-I conjugated to PCT) gC), followed by a chromogenic substance. Again, the chromogenic It is only through competition with free antibodies present in the biological fluid that it reaches the desired quality. be. The antibody-antigen complex remains trapped on the antigen-antibody. It is.

As can be seen from the cross section and the plan view from above, Figures 6a and 6b respectively It shows a different type of device.

This device allows the hole 1 through which the liquid flows out and the layer 4 containing the chromogen to be examined with the naked eye. a support strip 2 with a water-impermeable transparent coating 3 having a similar shape; There is. In this case, readings are taken by eye for qualitative measurements and by countermeasure for quantitative measurements. This can be done using a radiation reader.

The embodiments shown are only detailed illustrations of the invention and do not limit the invention thereto. It is not intended to be determined. Thus, for example, the size of each individual band is can vary depending on the case, and usually lies between the values of the number ■ and the number C1l. On top of that, Although some details are not shown, it is clear that the support of the present invention For general use, e.g. support handles, aspiration collection methods for biological fluids, Indication on the side where the liquid is added, coloring strip marked on one side of the zone containing the chromogenic substance. Kale et al.

The device according to the invention is a symmetrical device with an enzyme label component provided at both ends. The latter can be followed by a continuous zone or an interval in which the liquid It is designed so that it cannot be inserted incorrectly. Moreover, so that it can be used at home, Provides a conveniently packaged, compact unit that is sterile, stable, and has the right characteristics. I can provide it.

From the foregoing, it can be seen that the method and apparatus according to the present invention have a wide range of uses. It is also practical and can be used in a wide range of clinical applications without the need for special equipment or operations. Can be used for analysis. Furthermore, especially when obtaining analytical results in a short time, usually within a few minutes, I can do it.

The invention will be further explained below with reference to Examples. However, they are by no means the essence of the present invention. It does not limit God or its scope.

100 g of PVC with a particle size of 200 μm in phosphate buffer with 50 units of HCG/m 1 solution at pH 7.2 at 37° C. overnight. This coated polymer was applied 5 times. Wash with the same buffer and dry at 37°C for 10 hours.

b. Anti-HC, G from rabbits with peroxylase label ( 10 ml) was precipitated three times with 18% anhydrous sodium sulfate, and the product The precipitates were collected in each case from 1 liter of distilled water and the product was then incubated overnight at +4'C for 7.2 Dialyze against pH phosphate buffer, 10 ggMol.

This dialysate was mixed with 100+ig of Veroxinder RZ3,000 and 25mci of 2 Treat with 5% glutaradehyde for 48 hours at +4°C. Add this solution to sodium chloride. Elute with Sephadex G150 balanced with 50 wNols . The eluted anti-peroxidase complex can be stored at -20°C.

C. Label - Notification Glass, 50g with phosphate buffer (10mMols, pH 7,2) 1=500 Anti-HGC peroxidase diluted in 5I! Processed with.

The product was then dried at 37°C overnight.

d,, - Romone's report Add 50g of glass to 501g of tetramethylbenzidine in 5i1 of DMSO. - Dissolved in water and treated.

e, column backing Glass column (inner diameter 3.5mm 1 height 15c+s) to material: Adsorption anti-H CG peroxidase, immobilized HCG, and adsorbed chromogen prepared as above. Fill from bottom to top.

The glass column is closed at the ends with a wire or a suitable porous plug.

10 analysis examples conducted The column described above is brought into contact with the urine to be analyzed. Urine rises up the column by capillary action.

When HCG is present in the urine, the anti-HC,G protein contained in the first part of the column – Binds to oxidase. Continue the upward movement up to the ascending column section. So The chromogenic substrate then reacts with the enzyme to produce a blue color. sandwich In the method, the conjugate binds specifically to zone (C) by an immunochemical reaction. stop is generated by reaction with an enzyme and a chromogen substrate and is read in band (C). Expand the blue color. On the other hand, when no HcG is present in the urine, the The presence of anti-HCG molecules increases due to oxidase and contains immobilized HCG. and is bound thereto by an immunochemical reaction. and there is blocked from reaching chromogen substrates. In this way, no coloring occurs. sun In the case of the Deutsch method, anti-HCG peroxise is not bound in zone (C); washed away from the band. In this way, color development does not occur. The above method is a liquid It can also be applied to downstream systems.

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Claims (1)

[Claims] (1) (a) Enzyme-labeled antibody or antigen reversibly adsorbed onto a solid support (b) at least stoichiometrically equivalent amounts of the enzyme-labeled antibody or antigen on a solid support; Antibodies, antigens, anti-antibodies, or antibody-antigen complexes that are immobilized on the body (c) capable of causing a detection reaction with the enzyme complexed with the antibody or antigen; Enzyme consisting of contacting a biological fluid sequentially with one or multiple chromogenic substances Method for measuring clinical parameters in biological fluids using immunological methods (2) Biological fluids The process of removing compounds that interfere with the analysis of chromogenic substances before the body comes into contact with them. The biological liquid is passed over a chromatographic material that has adsorbed the resulting substance. A method according to claim 1 comprising: (3) Biological fluid (a) Complementary enzyme-labeled antibody reversibly adsorbed onto a solid support. (b) adsorb on a solid support at least stoichiometrically equivalent amounts of the enzyme-labeled antibody; The same antigen that has been (c) Chromogenic substance A patent claim for use in measuring an antigen, comprising sequentially contacting with (4) Method according to Scope 1 or 2 of (a) a complementary enzyme-labeled antibody reversibly adsorbed onto a solid support; (b) one or more complementary enzyme-labeled antibodies; immobilized antibody and solid support, reversibly adsorbed or immobilized. (c) one or more chromogenic substances with which biological fluids can flow; support The method according to claim 1 or 2, which is used for antigen measurement by contacting with (5) Biological fluid (a) Supplementary enzyme-labeled antigen reversibly adsorbed onto a solid support. (b) stoichiometric equivalents of the enzyme-labeled antigen are immobilized on a solid support; same antibody (c) Chromogenic substance The method according to claim 1 or 2 used for antibody measurement, which comprises sequentially contacting with (6) Enzyme-labeled antibody and immobilized antigen are coagulated on the same solid support. (7) The method of claim 3 comprising: (a) the same enzyme-labeled antigen reversibly adsorbed onto a solid support; (b) a solid support. (c) a complementary antibody that is reversibly adsorbed onto the support; (c) immobilized on the solid support; Antibodies specific for antigen-antibody complexes (d) Chromogenic substance Claim 1 or Method 2 (8) Corresponding enzyme-labeled antibody or antigen and corresponding immobilized antibody or antigen The enzymes were contacted with the same solid support and specific for each enzyme used. Several types of chromogenic substances are supported on their respective supports. Method (9) according to claim 1 for use in simultaneously measuring bed parameters For the various components, the solid support, which may be the same or different, is glass or Silica derivatives, paper, cellulose derivatives, polymers, polysaccharides or polyols, carbonaceous Phosphorus, Ti02, BaSO4, diatomaceous earth, metals, stabilized red blood cells are growing The method according to any one of claims 1 to 8 above. (10) Peptide hormones (HCG, LH), steroid hormones, protein The above-mentioned claims are used for measuring streptolysin, streptolysin, and viruses. Any one of the methods in boxes 1, 2, 3, 6 to 9 (11) To measure immunoglobulin and other antibodies that are problematic in clinical tests A method according to any one of claims 1, 2, 5, 8 or 9 used for (12) having a form capable of moving biological fluids upward or downward; Available in tube, specimen, strip, pad, flask, and film shapes. , (a) to be able to remove all compounds that may interfere with the analysis; initial or intermediate separation zone capable of adsorbing substances (b) one or more oxygen-labeled antibodies specific to the clinical parameter to be measured; (c) forming a clinical parameter; The antibody, antigen, anti-antibody or antibody-antigen complex to be measured is the enzyme-labeled antibody. or the previous zone has been immobilized by known techniques in an amount at least equivalent to the amount of antigen. followed by another band (d) Zone where chromogenic substances are adsorbed for use with antibodies or antigen-conjugating enzymes. (13) A device for measuring clinical parameters consisting of a biological fluid directed upwardly or downwardly. It is a movable format that can be used for tubes, specimen, strips, and patches. It has the shape of a glass, a flask, and a film. (a) to be able to remove all compounds that may interfere with the analysis; initial or intermediate zone for adsorption or separation of substances (b) one or more enzyme labels specific for the clinical parameter to be measured; A band that has previously adsorbed antibodies (sensitized to one or multiple epitopes) area (c) one or more antibodies (episodes recognized by the enzyme-conjugated antibody of band (b)); sensitized to one or more epitopes different from the epitope) has been immobilized. and also the zone in which the chromogenic substance is reversibly adsorbed or immobilized. (d) a zone with one or more supporting materials through which biological fluids can flow; (14) A device for measuring clinical parameters consisting of a Can also be different, starch, diatomaceous earth, kaolin, BaSO4, Ti02 Suitable supporting materials selected from: in the form of gel, powder, granules, microspheres, films one selected from glass, metal, synthetic or natural polymeric materials, and paper to be brought into contact. Device according to claim 12 or 13 consisting of one or more materials (15) first or the middle zone (a) is an ion exchange resin, such as urease or protease. Enzymes, oxidizing or reducing substances, antibodies or antigens are included as substances capable of removing interferences. The device according to claim 12 or 14, comprising: (16) (a) First zone that is complementary and reversibly adsorbs enzyme-labeled antibodies. (b) transfer the same immobilized antibody to the enzyme-labeled antibody at least stoichiometrically; band (c) which follows the first band and which is equivalent to the chromatographic separation. a subsequent band of effective and possibly interference-cancelling interference, and (d) used for measuring antigens consisting of the last band, which carries the chromogenic substance for the enzyme; The device according to any one of claims 12-15 (17) (a) First zone reversibly adsorbing complementary, enzyme-labeled antigen. (b) at least a stoichiometric amount of the same immobilized antibody to its enzyme-labeled antigen; Bands following the first band that have logically equivalent, immobilized (c) a separation band; and (d) Chromogenic substance band Any one of claims 12 to 15 used for measuring an antibody containing one device (18) The antibody attached to the support is pre-coagulated with its enzyme-labeled antigen. , a patent for measuring antigens consisting of an initial band, a separation band and a chromogenic substance band The device of claims 12-15 (19) first zone with enzyme-labeled antigen, antibody a second zone having reversibly adsorbed and having an immobilized anti-one antibody; and finally a claim for antigen determination consisting of separation and chromogenic substance bands. 12-15 devices (20) Each band (a) and (b) corresponds to a corresponding number of enzyme labels. contains fixed antibodies or antigens and immobilized antigens or antibodies, and contains several chromogenic material bands. Claim 1 for simultaneously measuring several clinical parameters Device according to any one of 2-19 (21) The device contains HCG, LH or other peptide hormones, progesterone, Esterone, glucuronide, predananediol, glucuronide or other substances For measuring terrorism hormones, streptolysin, immunoglobulins, and viruses. Apparatus according to any one of claims 12-20 suitable for (22) The device is entirely made of emulsion material, except for the reading zone, which is made of transparent material. Detailed description of the device invention according to any one of claims 12-21 consisting of Akira