JPH07509059A - Immunoassay using dye-conjugated enzyme conjugates - 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] Background of the invention of immunoassay using dye-conjugated enzyme conjugate 1. Field of invention The present invention generally relates to compositions and methods of their preparation and biological assay systems. Regarding the use of More particularly, the present invention relates to specific binding substances, enzymes and The present invention relates to target-specific labeling complexes comprising conjugated dye molecules.

The use of chromogens and fluorochromes as “label agents” in biological assay methods is Are known. Typical assay methods involve determining the analyte or fraction in a biological sample. The assay here requires the direct or indirect attachment of a dye label to an analyte analog. The presence or absence of dye at a particular stage of the process is visually determined and added to the sample. is related to the amount of analyte initially present. A wide range of specific assay methods There is a law.

Of particular interest to the present invention is that the assay is naturally colored as a label. or by utilizing stained particles, where the particles are conjugated with antibodies or other specific is bound to a binding substance. Suggested particles include colored latex beads, colored Including colored liposomes, red blood cells, metal sols and the like. like that The colored particles in the complex act as visible markers, where the separation of the particles , capture or agglutination is intervened through the binding of antibodies or other specific binding substances. identification analysis in which the amount of label separated during the assay step is initially present in the sample. related to the amount of things.

Various methods have been proposed for preparing such antibody-particle compositions. So Methods such as these typically use latex beads, liposomes or similar produce colored particles by coloring the relies on binding of the particles to the antibody, typically by passive adsorption or covalent bonding. do.

Although generally useful, methods for preparing such antibody-particle compositions are Relatively complex, typically involving particle preparation, particle coloring, binding of particles to antibodies, and immobilization. A multi-step procedure involving blocking of particles for use in assays It takes. Furthermore, loss of antibody binding capacity is often due to particle binding. sometimes, Those particles are not compatible with the antibody selected for a particular application.

Improved labeling compositions that allow use of any antibody for any application It would be desirable to provide methods for their preparation.

The composition should be of low profile and relatively easy to prepare, and should have uniform characteristics. Should have. In particular, the composition should retain antibody activity to a significant degree. death However, the methods and compositions of the present invention may differ in each or any of their aspects. They need not be superior to the prior art, but rather they are Preferably, it is a general advantage that can be provided over certain prior art methods and products. Delicious.

2. Description of background technology U.S. Pat. No. 4,863,875 discloses that the dye is A composition comprising at least 10 dye molecules or monomers covalently bonded to the body. Describe things. U.S. Pat. No. 4,452,886 discloses that proteins, e.g. Covalent attachment of light absorbing or emitting polymers to antibodies and antigens is described. American patent no. No. 4.373.932 applies to aqueous dispersions of hydrophobic dyes or colorants or such dyes. Alternatively, the labeling of the ligand by means of a pigment coated polymer layer is described.

U.S. Pat. A solid phase assay device is described that relies on the specific binding of conjugates, wherein said label is Particulates such as sacks, red blood cells, red blood cell ghosts, liposomes or polymeric materials Disclosed as microcapsules. U.S. Patent No. 4.943.522 is , describe a solid phase lateral flow assay using red blood cells as labels. american patent No. 4.861.711 discloses an enzyme-antibody conjugate and an enzyme-antibody conjugate held separately on a suction pad. A solid phase lateral flow assay using a support is described. Also, U, K, Patent No. 2. 204.398; EP Patent No. 306,722; and EP Patent No. 276,15 See also No. 2. It is colored by the reaction between the enzyme that binds to the target component and the support. Enzyme assays and immunohistochemical staining techniques that produce pigments are known.

Summary of the invention The present invention can be used in biological assays, such as the lateral flow assay described hereinafter. Provided are improved target-specific labeling complexes for labeling. Its target - specific A complex is a specific binding substance component, such as an enzyme component that is bound to an antibody or antigen, and an enzyme and/or in an amount sufficient to impart a visually discernible color to the complex. It comprises a dye molecule conjugated to a specific binding substance. The complex is composed of an enzyme due to either covalent or non-covalent binding of the dye to the molecules or both.

The conjugated dye molecules do not significantly block the active site on the specific binding substance; Therefore, it can be used to bind analytes or other target molecules in biological assays. survive so that it can be used. Typically, the enzyme will have either native enzymatic activity or the labeling of the present invention. Before use as a conjugate, it will be inactivated to be removed. convenient , the conjugated dye molecules are lyophilized and processed as described below. It remains intact even when introduced into an aqueous anti-LL medium.

The number of complexed dye molecules or components can range from 10 per complex to 10 per complex. 10' particles per day, and the number of particles in the biological assay in which it is used. The main requirement is that there is sufficient dye to visualize the offspring. Usually, pieces Each complex comprises 10" to 10' dye molecules or components, and each The individual molecular weights of the components are between 100 and 1000 Daltons, provided that the molecular weight is 1 The aggregate size of the composite is typically 10”. ~1011 Daltons, more usually 10@~1010 Daltons.

According to the method of the invention, the target-specific labeling complex comprises an enzyme and a specific binding complex. formed in an aqueous medium by reacting a substance component with an enzyme substrate; Here the enzyme converts the substrate into a dye. so formed in an aqueous medium The labeled dye complexes into the conjugate, and the enzymatic reaction produces this color of the labeling complex. sufficient for easy observation when used in assays such as those described below. It will stop at a certain point. Conveniently, said reaction is carried out by depletion of substrate and/or can be stopped by changing the reaction conditions. The method also inactivates the enzyme After chromatinization and/or imparting the desired color intensity, the composite can be lyophilized. It consists of.

In another aspect of the invention, the test article comprises a support matrix, typically a Preferably, a non-absorbent material that can direct liquid flow in a single direction in the matrix. The mark is impregnated by freeze-drying in a strong or highly absorbent solid matrix. target-specific labeling complex. In this method, the liquid sample is applied to the solid matrix, and the impregnated labeling complex is “downstream” flow through the matrix so that it can be transported by the liquid flow to the location. good capital In some cases, the labeling complex of the present invention can be transported by a fluid stream as described below. It has been found that in some cases, it remains intact.

Typically, the assay involves the intervening of a labeling complex by a specific binding agent. It can be performed using a capture-based test product. Typically, the test product area is labeled Incorporating an immobilized capture reagent that can directly or indirectly bind the binding complex. cormorant. The assay result will then depend on the appearance of the label within this capture area. .

Labeling complexes, on the other hand, are labeled with antibodies or other binding substances specific for the analyte. can be initially bound to a capture reagent within a "capture zone".

Then, by applying the sample to the support matrix, the result of the capture competition binding is determined. As a result, the labeling complex will be moved.

Therefore, the color initially present in the capture area is proportional to the amount of analyte present in the sample. and it will disappear.

In yet another aspect of the invention, the presence of an analyte in a liquid sample The assay device for detecting the area, capture area and adsorption area. a sample is applied to the receiving area, and into the device where it combines with the labeling complex in the labeling zone or before Remove the labeling complex from the capture area. In the first case, the labeling complex binds to or competes with the analyte in the sample, and the label Depending on the amount of analyte initially present in the pull is bound within the capture zone. second place In this case, a labeling zone is not required and the analytes in the sample are labeled in the adsorption zone. Move the ring complex. In both cases, the adsorption zone is the area where the liquid sample is inside the device. serves to receive the liquid sample as it flows through the previous zone.

Drawing description FIG. 1 illustrates an assay device incorporating the principles of the invention, a specific example of which follows the details in the experimental section.

Description of specific aspects The invention relates to compositions, methods of preparing such compositions, test products and biological assays. B. Provides a test device, particularly for use in performing immunoassays. The composition Objects are visually distinguishable, typically colored, and then detailed Colors that are easily visible when aggregated or accumulated in the capture area as described It comprises a target-specific labeling complex capable of providing a signal. Further composition the substance comprises a specific binding substance that mediates such capture within the capture zone; The compositions are applicable to a wide variety of specific assay types and procedures. The composition is While valuable for use in lateral flow assay instruments, they are equally widespread. It may also be used for other assay types of this type.

The composition of the invention comprises a conjugate comprising an enzyme component and a specific binding substance component, wherein The enzyme component is added to the conjugate as it is produced in an aqueous medium. selected to confer conversion of the appropriate substrate to the dye substance that will be complexed. Ru. A suitable dye should have high avidity for the conjugate and Pigments that match are preferred. Dyes that are not covalently bound are particularly sensitive to protein components of complexes. complex through hydrophobic, ionic and other non-covalent mechanisms resulting in strong binding to Should interact with coalescence. specific enzymes and groups to produce specific pigments The choice of quality system will depend on the specific application, desired color and the like. Dew.

Representative enzyme-substrate systems are shown in the table below.

Table A A specific binding substance specifically has spatial and/or polar characteristics that allow it to bind to other compounds. It is probably a compound that has certain characteristics. Specific binding agents useful in the invention include certain compositions, For example, analytes, anti-analyte substances and binding as part of a biological assay method. Chairman: <Specific substances selected or prepared to bind specifically to other target substances that may be implemented. will be done. Natural specific binding pairs are antigens and antibodies, lectins and carbohydrates. , hormones and hormone receptors, enzymes and enzyme substrates, biotin and ahidin , vitamins and vitamin binding proteins, complementary polynucleotide sequences, drugs and and receptors, enzymes and inhibitors, apoproteins and cofactors, growth factors and and receptors, and the like. biotin and avidin derivatives, and For example, biotin analogues/avidin, biotin/streptavidin and biotins. Mimics/streptavidin can also be used. Natural specific binding substances exist If not, it can be prepared. For antigenic and paten target substances, antibodies can be prepared by well known techniques.

For polynucleotides, complementary DNA or RNA fragments are also suitable. They can be prepared by well known synthetic techniques.

The preferred specific binding substances are antibodies, antigens and haptens, and the remaining discussion is mainly will be directed towards those substances. However, the preliminary method is as above can be easily adapted to provide compositions with other specific binding agents.

Furthermore, the test products and test devices of the present invention can detect specific binding substances other than antibodies and antigens. can be easily modified to use target-specific complexes with The assay method can also be improved.

Enzymes include oxidase, peroxidase, phosphatase, diaphorase, and galaxase. tosidase, lytic enzyme or oxidoreductase, and representative enzymes are shown in the table above. Enzymes are usually covalently linked to specific binding substances, but through an indirect bond, e.g. pyotin-avidin bond, or through a specific Other homologous members of binding pairs may also be used. The specific binding substance is a polypeptide or In the case of proteins, e.g. antibodies, the enzymes contain various components, e.g. hydride, hydroxyphenyl, amino, carboxyl, indole, or other functional groups through groups, using conventional conjugation chemistry as described in the scientific and patent literature Can be combined. Binding occurs when the active site on the specific binding substance is unblocked and The cleavage provided by such means can be utilized for binding to the desired target substance. be. In the case of antibodies, binding preferably involves the region determining complementarity being linked to the desired substance. will be made available for binding to. for binding to enzymes Specific techniques for derivatizing antibodies to ice and Theory of Enzyme Immunoassay ”.

Laboratory Techniques in Biochemistr y and Molecular Biology.

Vol, 15. Burdon and van Knipponberg, e ds, l985. Elsevier.

Ams terdam, which is incorporated herein by reference. Ru.

Immediately after the specific binding agent-enzyme conjugates are formed, they are combined with the appropriate enzyme substrate ( (see Table A) and convert the substrate to a colored dye product in an aqueous medium. The reaction will be carried out under conditions sufficient to do so. The reaction conditions further include fermentation. The dye molecules produced by the elementary conversion form a complex with the conjugate, and sufficient dye or selected to be incorporated into the composite to impart a visually distinguishable color. It will be done.

The reaction conditions necessary to effect conjugation depend on the enzyme selected for the particular application. and depends on the nature of the substrate. Individual enzymes are designed to optimize the turnover and production of pigment products. and have unique reaction conditions for their production. Ideal candidate enzyme, chromogenic substrate and reaction conditions (e.g. pi, ionic strength, temperature, cofactors and the like) , “Practice and Theory of Enzyme Immun enzyme-antibody delivery system described in Chapter 17, supra. It is used for scientific staining.

Enzyme conversion and conjugation reactions occur until sufficient dye is produced and conjugated into a conjugate. and the resulting complexes are allowed to aggregate within the capture zone, as described hereinafter. When assembled or combined, perform until it has a sufficiently intense color that it can be visually detected. will be done. Reactions and conjugations can be stopped by a variety of approaches. flight Advantageously, the amount of substrate to be reacted can be reduced if the substrate has sufficient complex color density. , may be limited to being consumed. On the other hand, enzymatic conversion of the substrate to the pigment product results in color Change reaction conditions or use enzyme inhibitors at selected points to stop enzyme production. can be stopped by adding .

As a second option, to stop the reaction and/or remove the soluble dye, in the reaction medium, for example by using gel permeation chromatography. It is possible to simply remove or separate the particles from the matrix.

Usually, but not necessarily, enzyme activity is inhibited after conjugation is complete. exposure to bitters, denaturing conditions (the conditions chosen do not denature the specific binding substance and should not be inactivated), depletion of enzyme cofactors, or any similar means. will be inhibited by either. The ability of the target-specific labeling complex of the present invention is to , does not derive from the enzyme's ability to turn over the substrate, but rather from the complexed It originates from the color imparted by dye molecules.

Unreacted substrate is preferably quenched, typically by chromatography. It will be removed from the labeling complex by separation techniques, such as gel permeation.

When the enzyme is inactivated, virtually no substrate remains in the subsequent assay step. It is essential to separate to spin additional pigment production. However, because of this, Usually the enzyme is inactivated to further avoid the possibility of unintended pigment formation. There will be.

In a preferred technique, the target-specific labeling complex of the invention is placed on a solid phase matrix. and more preferably lyophilized within a matrix. solid phase matato Rix may be highly absorbent or non-absorbable (continuing application 07/639.967 ), and generally through which fluid flow can be directed. and, as a result, the liquid can interact with the impregnated labeling complex. .

Such matrices generally include porous membranes, absorbent pads, and the like. from various materials of the type used in the immunoassay industry for the preparation of Become. Particularly preferred are polyester, acrylonitrile copolymers, It is a woven fabric composed of fiberglass, cellulose, cellulose, and blends thereof. . In certain embodiments, generally described in continuing application N1107/639.967 Such fabrics are treated with blocking agents to make them non-absorbent. It may be desired to process objects.

Another impregnation in the solid phase matrix is the Lahelig complex of the present invention. The particles can be dried into powder form and lyophilized.

Such lyophilized labeling complexes are then generally known in the art. It can be used as a means of understanding such as those described.

Referring to FIG. 1, a typical test apparatus constructed in accordance with the principles of the present invention is illustrated. It will be posted. Test device IO is described in continuing application Nα07/639, 967 A non-absorbing sample and reagent flow area may be used as described above. however , strongly absorbing sample and reagent flow areas, or non-absorbing and strongly absorbing areas. Other test devices using the combination include the use with target-specific labeling complexes of the present invention. It would be appropriate to configure it for use.

The test device IO includes a sample receiving area 12, a label area 14, a capture area 16, and an adsorption area 1. Includes 8. In the sample receiving area, the label area 14 and the capture area 16 contain liquid sump. and other liquid reagents, and laterally, i.e. receiving area 12. Is the material capable of transporting such samples and liquid reagents from the pond towards the adsorption zone 18? It will consist of The adsorption zone 18 can receive the same liquid sample and reagent and It will be made of materials that can be accommodated. In this case, it is first applied to sample area 12. The liquid sample or other liquid reagent is transferred laterally from the sample receiving area I2 to the label area. 14, into and through the capture area 16, and finally the suction Flow into the adsorption zone 18 with upward or downward action (wick or 5 ink) so that the entire sample or reagent volume correctly completes the assay. 12.14 and 16 above.

In the preferred case of a non-absorbable assay device IO, the sample receiving area 12 and the label area 1 4 are both made of non-absorbent material that allows liquid flow, where the liquid is dissolved. all of the dispersed or dispersed components at substantially equal rates and relatively attenuated. (specific binding to the capture reagent as discussed below) (in the absence of Suitable non-absorbable materials include essentially non-absorbent materials, such as porous polyethylene or essentially absorbent materials such as paper, nitrocellulose, polyethylene, etc. Includes esters, acrylonitrile, copolymers, rayon and blends thereof. They include blocking agents, such as surfactants and materials that contribute to their absorbent properties. Application of proteins that inhibit the molecular forces exerting will be converted to

The sample receiving area 12 is typically 20μI! Small sample volumes in the ~200μl range and act to receive the flow and initiate a lateral flow into the label area 14. act. The label area 14 is typically impregnated or fixed with the inventive material. Target-specific labeling complexes will be included. Typically, the labeling complex is It will be lyophilized as described above for the preparation of the test product. sample or other The flow of reagent moves (dissolves) the labeling complex and moves it toward the capture zone 16. and transport the complex therein.

A sump mediating the binding of the labeling complex to the labeling complex in the capture zone 16. It may be appropriate for there to be an interaction between the analyte within the sample. such mutual Effects can take a variety of forms, typically 'sandwich' labeling ( Here, the labeling complex contains an anti-analyte that binds to the analyte and a capture zone The immobilized anti-analyte can then capture the labeling complex through the analyte. ) or “competitive” labeling (where the labeling complex is the analyte or analyte analog and the binding of the label to the anti-analyte within the capture zone 16 is similar to that of the original sample. (inversely proportional to the amount of analyte in the sample). Basic sandwich and competitive appetizer Various modifications to the Sei type are known in the art and are exemplified herein. may be adapted for use in test equipment identical or similar to .

The capture zone 16 can also be prepared from strongly absorbent or non-absorbent materials, and Preferably wood. Particularly suitable materials for the capture area are It is nitrocellulose which is blocked to spin a highly absorbent interaction.

When used, the binding of the target-specific labeling complex of the invention within the capture zone 16 is , will provide a visible signal or indication indicative of the assay results. Capture area 16 The binding of a sufficient amount of labeling complex within a defined position in the capture area 1 6 will allow the user to visually read the results.

The following examples are illustrative and do not limit the invention.

experiment The following examples include antibody-enzyme conjugates, suitable enzyme substrate systems, and enzyme inhibitors and/or or from any use of stabilizers, human coriogonal ropine (h) in body fluids CG) shows the preparation of pre-dyed complexes for the detection of CG).

Monoclonal anti-hCG ascites fluid was added to sodium dextran at 50% salt saturation. Membrane lipidation by sulfate and calcium chloride followed by ammonium sulfate treatment fractionated at 0-4 °C and loaded onto a 025 column in 10 mM Tris buffer ( pH 8.0). Antibodies were further incubated at 0-0.3 M in the same buffer. Q-Sepharose@FF resin using sodium chloride salt gradient Purified above. Monitor fractionation at 280 μm and collect protein peaks. 0.1M sodium phosphate (pH 7.0) on a 025 column. The solution was exchanged. The obtained anti-hCG antibody was concentrated to 5 μ/ml by ultrafiltration. did.

The maleimide group is generally used in Enzyme Immunoassays, Ish ikawa etal, eds, Tgakushoin, Tokyo - maleimide benzoyl as described in New York, 1981. Add Ru-N-hydroxysuccinimide to a final concentration of 100 μg/ml. , the reaction mixture was incubated at 25°C for 45 minutes and then transferred to a G25 column. by buffer exchange with 0.1 M sodium phosphate (pH 6.0). Therefore, to prepare an antibody-enzyme conjugate in an anti-hCG antibody, the following enzyme was used: Glucose oxyguse (GO), horseradish peroxidase ( HRP), diaphorase I (DIA), and bovine intestinal alkaline phosphatase (ALP).

To introduce the SH group, the enzyme was mixed with 0.5 mM 2-mercaptoethanol. 5 mg/ml in O, IM sodium phosphate (pH 8,0) (GO .

DIA or ALP) or 10 mg/mt’ (HRP), 1.28 mg Incubate for 45 min at 25 °C with 2-iminothiolane at a final concentration of /mj’. and then 0.1 M sodium phosphate (pH 7.3) on a 025 column. The buffer was exchanged by

Maleimide-containing monoclonal anti-hCG antibody (section 1.1 above) and S The H-derivatizing enzyme was reacted for 2 hours at 25°C, followed by 5ephacrylO It involved binding the enzyme-antibody conjugate onto 3300HR resin.

0, 196 (w/v) NaNi, 0.03% (w/v) MgCj7!　 ・6HzO10,00396(w/v)ZnSO4・7)+20 and 0.005 % (w/v) 0.05M sodium phosphate buffer (pH 0.45 μm of filtered conjugate elution buffer consisting of used. The fractionation was monitored at 280 μm and the antibody-enzyme conjugate fraction was I did a rule. Subsequently, antibody-enzyme conjugate (excluding anti-hCG/ALP conjugate) , 025 kg in the following buffer supplemented with 50 Mg gentamicin/ml. Buffer-exchanged on a ram: (i) t(RP conjugate: 0.05M Tris/HC 1 buffer (pH 7,5); (ii) GO conjugate: 0. IM HEPES buffer (pi(8,O); and (iii) DIA conjugate: O,IM sodium phosphate (pH 8,0).゛Lateral flow non-absorbable assay test strip, continuing application Nα 07/639, 967. Test equipment (Figure 1) has three active zones and a wicking or wicking system to receive the sample flow from the active zones. contains a fourth adsorption zone which has a sinking effect. The active area is sampled as shown below. The trap receiving area includes an intermediate area 14 and a trapping area 16.

A “wet” assay, not illustrated, involves dye-labeling prepared in test tubes. A sample is added to the complex. The resulting mixture is then subjected to a test strain. Applied to the lip. The test strip was constructed as follows: 1.2゜　Preparation of sample reception and intermediate area. ntara@0-100 DuPont 0rlon@5punlac fabric? Prepared from The fabric was saturated with methylated bovine serum albumin (BSA). This made it non-absorbable. Conversion to non-absorbable materials is BSA! 0■/m This was achieved by treatment with 38 μl/al for 5 minutes at room temperature. next, 5 ontaraO pads were frozen at -70°C for at least 1 hour. Next, 5o ntarao was lyophilized overnight on Virtis Freezemobi Ie. . Cut the sample receiving pad and intermediate area pad into a rectangle of IOX 4mm, and place it here. The five punlace fabrics are parallel to the long sides of the individual pads.

1, 2. 2 Preparation of capture zone membrane To prepare a non-absorbing capture zone, goat anti-hCG serum was added to Nato at 50% salt saturation. Lithium dextrans sulfate and calcium chloride followed by ammonium sulfate The membrane is lipidated by treatment, fractionated at 0-4°C, and phosphorylated on a G25 column. Desalination was performed using a buffer solution (pH 7.2; PBS). Further, the anti-hCG antibody hCG-3 using O,IM glycine buffer (pl+2.3) as elution medium. Purified on epharoseO affinity resin.

The fractionation was monitored at 280 μm and the monospecific anti-hCG protein peak was collected. buffer-exchanged with PBS on a G25 column. Finally, got The antibody was concentrated by ultrafiltration to more than 2 ml/ml.

Nitrocellulose with a pore size of 8 μm was fixed to an X-Y chart recorder. Place the hCG capture band using a drafting pen operating in manual mode. 2/me goat anti-hCG antibody was used to form parallel lines 2 cm apart.

Those lines were reactive with hCG contained in the sample. 10 minutes at room temperature After air drying, the nitrocellulose membrane was soaked in blocking buffer ( Placed in a tray containing 10/ml of BSA in the Tris buffer described above. the above The membrane is removed, plotted, air dried, and subsequently assembled or assembled into the device. and stored in a desiccator at room temperature.

1, 2. 3. Assembly of the device Fix a 20X4mm strip of captured acid film in the center on the adhesive transparent strip. Ta. The transparent strip is a 70X4m overhead project transparent film. It was made adhesive with double-sided adhesive tape. Next, inside The interspace was fixed next to the capture area pad with one order of overlap. Next, The sample receiving pad was placed next to the middle pad with a 1 mm overlap.

Next, the device is equipped with a sorbent vat, which has a one-order overlap. It was a 20 x 4 mm rectangular piece of cellulose paper that was fixed to the end of the capture acid membrane. .

Uninhibited predye containing anti-hCG monoclonal antibody-GO conjugate The labeled conjugate was treated with phenazine methosulfate and phenazine methosulfate as chemical intermediates. Using colorless hydrophilic tetrazolium salts as electron receptors (“GO substrates”) , formed by PAD-mediated electron transfer. This reaction occurs in body fluid samples. yields a pigmented enzyme-antibody conjugate suitable for the analyte in the analyte.

A 1.0 ml incubation reaction was added to a 0.0 ml incubation solution containing 50°C g gentamicin. Sequential addition of the following components to 500 μl of 1M HEPES buffer (pH 8,0) (a) 50% (V/V) dimethic acid in deionized water Dissolution of 8 μm of MTT tetrazolium salt per 1 me of diformamide (DMF) 100 μl (b) of the same 1 (0.5 M D-(+)-gluco in EPES buffer) -50μi? (c) 50μl of 0.5μ PMS solution per lrd of deionized water ; and finally, (d) the same 1 (in EPES buffer) to initiate complex formation. , 300 μl of anti-hCG antibody-Go conjugate at a final concentration of 1.7-50° C.g/bark. 0 At time intervals specified in Table 1, multiple aliquots of 15 μl were drawn and h CG standard (final hc of either O or 500 m1U of hCG /- 0.5% (w/v) bovine at 0 or 800 ml U/rR1 to obtain c concentration. Samples prepared in phosphate buffer containing serum albumin (BSA) Mix with 25 μl and immediately as described in section 1.2. The test strip was applied to a 4M wide lateral flow assay strip prepared as follows. 5 minutes later, Results based on the appearance of visually distinguishable positive signals in the capture area are shown in Table 1. It has been reported.

30-+ 1: + = clearly distinguishable appearance of colors in the capture area: +/- = in the capture area and - the absence of a clear appearance of colors in the two capture areas.

Uninhibited precolor containing anti-hCG monoclonal antibody-ALP conjugate The purified label complex is converted into a colored phenolic product (“ALP product”). Enzyme-catalyzed hydrolysis of colorless organophosphates (“ALP substrates”) to More formed. Incubate a 550 μl reaction with the following components: 0.1% ( w/v) of NaNt in 0.1 M AMPD (2-amino-2-methyl-1, Continuously add to 250 μl of 3-propanediol) buffer (pHio, 5). (a) The same buffer combined with 1-naphthol BGP (Fast Bordeaux G diazonium) salt) solution 200 μm and (b) conjugate elution buffer 45 μm! and finally, the complex (C) 2.75 μg or 5 μg/ml of the mixture to form a Anti-hCG-ALP conjugate in conjugate elution buffer at a final concentration of 55 μM , after 5 min incubation, 5μ! or multiple aliquots of 30 μl to obtain a final concentration of hCG of 0 or 550 ml U/ml, respectively. , 25μl or lOμ! of hCG standard! Mix with aliquot and immediately Above section! Prepared for “wet” assays as described in , 2. It was applied to a fabricated 4 inch width lateral flow assay strip. After 15 minutes, go to the capture area. The results are based on the appearance of visually distinguishable positive signals in Table 2. It is.

Table 2 hCG concentration in ALP-Ab standard 11:+=clear color in capture area Distinct appearance: and - = absence of distinct appearance of color in the capture area.

The uninhibited pre-pigmented label complex containing the antibody-ALP conjugate was It was also prepared by the indigo formation/tetrazolium salt technique. 600 μl of in The incubation reaction was carried out in O, 1M AMP containing 0.1% (w/v) NaN5. By sequential addition of the following components to 250 μl of D buffer (11H10,5) Performed at °C: (a) 0.1 M A containing 0.1% (w/v) NaNt MP (2-amino-2-methyl-1-propatur) buffer (puto, 15 ) 2.16 mp per IP (3-indoxyl phosphate disodium (b) 50% (V/v) DMF in deionized water 1 10 μl of 5 μl MTT solution per me and finally, to initiate complex formation. (C) at a final concentration of 0.25 μg/me to 4.58 μg/me. Anti-hCG antibody-ALP conjugate 100 μl infusion buffer for 10 min. After incubation, 15μ! or withdraw multiple aliquots of 30 μl; To obtain a final concentration of hCG of 0 or 500+n[U/me, respectively, Mix with a 25 μl or 10 μl aliquot of the sample and immediately add the above Prepared for “wet” assays as described in Section 1.2 was applied to a 4 m wide lateral flow assay strip. After 15 minutes, go to the capture area. The results are based on the appearance of visually distinguishable positive signals in Table 3. .

Table 3 hCG concentration in ALP-Ab standard 11:+=clear color in capture area and - the absence of a distinct appearance of color in one capture area.

Contains anti-hCG% clonal antibody-Diaphorase (DIA) conjugate Comparison of uninhibited pre-pigmented label complexes in the visible region NA of an appropriate organic hydrogen acceptor substrate to a product that exhibits an absorption spectrum that can It was formed by enzyme-catalyzed reduction involving the DH cofactor.

1677, 5 μl incubation with 50 Mg gentamicin/ml Add the next composition to 445 μl of O, 1M sodium phosphate buffer (pH 8,0) containing (a) 15% (v) in deionized water. /v) 67.5 μl of 4.1 ■ MTT solution in DMF; (b) 10 mg of BSA (bovine serum albumin per liter of the same buffer) 67.5 μl; (c ) 10mM NADI+64.5μl in the same buffer and finally the complex form (d) 50 Mg of anti-hCG monoclonal in the same buffer to start the synthesis. antibody-〇IA conjugate 30 μm, after 3 min incubation, 15 μf ( 7) Pull out multiple (7) 7 recoats, 0.150 or 500 m1 LI no hCG Mix with 25 μl of a sample of hCG standard to obtain a final hcc a degree of /me. and immediately perform a “wet” assay as described in Section 1.2 above. applied to a 401111 width lateral flow assay strip prepared for . Results based on the appearance of a visually distinguishable positive signal in the capture area after 3 minutes are shown in Table 4 below.

150m1U/ml+ 500m1U/m10 1, + = clearly distinguishable appearance of color in the capture area: and -2 in the capture area Absence of clear appearance of color.

Uninhibited precolor containing anti-hCG monoclonal antibody-HRP conjugate Periolation of the purified label complex to a colored product, which is a type of colorless electron donor. Formed by oxide-mediated enzymatic conversion.

Add 2016 μl of incubation reaction to 3 μl of 4 μl per ml of methanol. - by successive addition of the following components to a solution of 360μ, 1 of chloro-1-naphthol Performed at 25°C: (a) 0.05M 50Mg gentamicin/ml Tris/HCI buffer (pH 7,5) 54 μf; (b) 0.027.5 ) 1548 μl; and finally, to initiate complex formation, (C) 50 Mg 0.05 M Tris/HCI buffer (pH 7,5) containing gentamicin/- 0.27 μ of anti-hCG antibody/) IRP conjugate solution 54 μ! 015 minutes later , the following reagents (quenched to 4°C) were added: (a) 50 Mg gentamicin; 126 μl of 0.05 M Tris/HCI buffer (pH 7.5) containing : (b) 0.0296 (V/V) ■202.0.1mM EDTA and 50 Gentamicin/ml of Mg in 0.05 M Tris/II (J’ buffer ( pi-17,5) + 26μf; and (c) 0.05M Tris/HtJ' buffer (pH 8,0), 100μ solution of BSA/+n/252μ10 multiple 15μ Remove l aliquots and add final concentrations of 0.150 and 500 ml hCG/ml. mix with a 25 μl sample of hCG standard to obtain prepared for a “wet” assay as described in Section 1.2. Applied to a 4M wide lateral flow assay strip. After 3 minutes, vision in the capture area The results, based on the appearance of a positively distinguishable positive signal, are shown in Table 5 below.

0m1U/m+1- 151-15O/ml+ 500m 10/ml + 1: + = clearly distinguishable appearance of color in the capture area; and - = in the capture area Absence of clear appearance of color.

The uninhibited pre-pigmented complex containing the antibody-HRP conjugate is also It was prepared by reaction with an electronic carbazole derivative.

+1RP substrate was added to 3-amino-9-ethyl dissolved in dimethylformamide. 50 μl of 0.40% (V/V) solution of Rubazole and 50 mM sodium acetate Mix 940 μl of buffer (pH 5, 0), followed by 0, 3 in the same buffer. 96 (v/w) hydrogen peroxide by adding 10 μl.

Anti-hCG antibodies prepared as described in this section above, /HR A 5 μl aliquot of P conjugate (0.34 mg/ml”) was added to 1 (RP substrate solution Added to 228 μp. After 4 hours incubation at 25°C, lOμf and immediately as described in Section 12 above. 4 mm wide lateral flow assay strip assembled for a “wet” assay. Applied to lips. hCG samples prepared in pooled male urine 20 Apply 0.100 or 400 ml onto the sample receiving strip pad. A final standard concentration of UhCG/y++1 was obtained. Finally, not spiked (unspiked), apparently negative male urine boule lOμm sample received The study was concluded. After 8 minutes, there is no visible brightness in the capture area. The results based on the appearance of sexual signals are shown in Table 6 below.

'100m1U/-+ 400m [U/ml+ 1, + = clearly distinguishable appearance of color in the capture area; and -2 in the capture area Absence of clear appearance of color.

The inhibited pre-pigmented label complex for the detection of hCG in body fluids Once the pre-pigmented anti-hCG complex is formed, the complex is To show that it is not necessary to have enzymatic activity for the detection of hCG in It was configured as follows. The inhibited pre-pigmented complex is the enzyme in the initial antibody-enzyme conjugate. The primary activity is combined with the appropriate enzyme substrate to form a pre-pigmented label complex. After incubation of the antibody-enzyme conjugate, the complex is inhibited. .

The inhibited prepigmented complex containing the anti-hCG antibody/HRP conjugate was as follows: It was prepared as follows. Add 2016 μl of the incubation reaction to methanol Ing. Sequential addition of the following components to 360 μl of a solution of 3 μl of 4-chloro-I-naphthol: (a) 0.0000 mg containing 50 Mg gentamicin/-; 54 μl of 05M Tris/HCI buffer (p117.5); (b) 0.0 2% (v/v) H2O 1,0,1mM EDTA (ethylenediaminetetraacetic acid ) and 0.05M Tris/1cjl' containing 50Mg gentamicin/rd. 1548 μl of buffer (pH 7.5); and finally, to initiate complex formation. (C) 0.05 M Tris/HC1 containing 50 Mg gentamicin/- Solution 5 of 0.27 μ of anti-hCG antibody/HRP conjugate in buffer (pH 7.5) After a 15 min incubation at 4 μm, the following reagents (quenched to 4°C) were added: Added: (a) 0.05M Tris/me containing 50Mg gentamicin/me; IC! ! 125 μm solution of 2M NaNz in buffer (pH 7,5); (b ) 0.02% (v/v) H2O2, O, ImM EDTA and 5011 g of 0.05 M Tris/HCI buffer (pH 7, 5) Medium, 1091 w/v) c7) PVP (7) solution 126μi'; and 0.0 5M (7) Tris/HC! ! 100 mg BSA/m in buffer (pH 8,0) j' solution 252μ1. Next, remove the 15μf (7) multiple (7) 71J: T-to. aliquot, to obtain a final concentration of 0.150 and 500 ml hCG/ml. Mix with 25 μl sample of CG standard; and immediately ” applied to a 4-inch width lateral flow assay strip prepared for the assay. . After 3 and 60 minutes, the appearance of a visually distinguishable positive signal in the capture area The results are shown in Table 7 below.

100m1U/　m1+　+ 500m 10/me++ 1 + = clearly distinguishable appearance of color in the capture area; and -2 in the capture area Absence of clear appearance of color.

The device for the “one-step” assay, the latter intermediate region, was prepared as follows. “Wet” two-step assay by displacement with pre-pigmented label pads was prepared as described above.

Pre-pigmented label complex containing antibody-1 (RP conjugate) was dissolved in 1 m methanol Add the following ingredients to 360 μl of a solution of 3μ/l of 4-chloro-1-naphthol. Prepared in 2016 μl incubation reactions at 25°C by continuous addition. : (a) 0.05M Tris/HCI containing 50Mg gentamicin/rd! 54 μl of buffer (pH 7,5); (b) 0.02% (v/v) H2O2 , 0.05 containing ImM EDTA and 50 Mg gentamicin/rnl. Tris of M/HCf! 1548 μl of buffer (pH 7.5); and finally, To initiate coalescence formation, (c) 0 containing 50 Mg gentamicin/ml , 0.27μ anti-hCG anti-hCG in 05M Tris/HC1 buffer (pH 7.5). body/F (54 μf of solution of RP conjugate, after 15 min incubation, Reagents (quenched to 4°C) were added: (a) 50 Mg gentamicin/ ml of 0.05M Tris/HCl buffer (pH 7,5) 125μ! !　 (b) H601,0, ImM EDTA and 0.0296 (v/v) and 0.05M Tris/1Icj7 containing 50Mg of gentamicin/ml. buffer solution (pH 7.5) 126 μm; and (c) 0.05M Tris/HCI buffer. 252μ10 solution of 100mg mBSA/ml in buffer solution (pH 8, O) The mixture was poured at 38 ul/al onto 5 ontara 5 punlace fabric.

The matrix was kept at room temperature for 20 minutes and then left in the lyophilization flask for at least 1 hour. It was suddenly frozen at -70°C with a flash. Virti the resulting composition Lyophilize overnight on Freezemobile and intermediate pre-pigmentation Cut the labeling pad into a rectangle of IOX 4m, and place 5 punlace here. The fabric is parallel to the long side of the batt. The pads are then subjected to a “wet” two-step assembly. In a manner similar to that described above for istrips, a “single stage” device has an intermediate region. and compiled it.

Assay by adding 0.150 or 500 ml hCG/ml to the strip. I did it more. After 3 and 60 minutes, visually distinguishable positive signals in the capture area. The results based on the occurrence of nulls are shown in Table 8 below.

100m1U/+++1+ + 500m1U/ml' + + 1:+ = clearly distinguishable appearance of color in the capture area; and -1 in the capture area Absence of clear appearance of color.

Although the foregoing invention has been described in detail in an exemplary and perverse manner for clarity of understanding, It is obvious that changes and modifications may be practiced within the scope of the invention.

IG 1 Continuation of front page (72) Inventor: Pronovost, Alan Dee.

Salmon River, San Diego, California 92129, United States - Load

Claims (1)

[Claims] 1. Enzymes that are bound to specific binding substances; and enzymes that can be used for binding to target molecules. imparts a visually distinguishable color to the complex while preserving the active site of the specific binding substance. a targeting feature comprising a dye molecule conjugated to the conjugate in an amount sufficient to confer a heterologous labeling complex in which the dye molecule is present in the aqueous phase of the substrate; It is characterized by being formed by a reaction with an enzyme that is awaited by the specific binding substance. complex. 2. The labeling complex according to claim 1, wherein the specific binding substance is an antibody. . 3. 3. The labeling complex of claim 2, wherein said enzyme is covalently linked to said antibody. Combined. 4. The oxygen is oxidase, peroxidase, phosphatase, diaphora oxidoreductase, galactosidase and oxidoreductase. The labeling enzyme according to item 1. 5. The labeling complex of claim 1, wherein said enzyme is inactivated. 6. The label of claim 1, wherein the complex contains 10 to 10 dye molecules. complex. 7. The labeling complex of claim 1, wherein said complex is lyophilized. 8. Support matrix: and comprising a target-specific labeling complex impregnated within said support matrix. a test product, wherein said complex comprises: (i) a conjugate of an enzyme and a specific binding substance; and (ii) an active portion of a specific binding substance available for binding to the target molecule. in an amount sufficient to impart a visually distinguishable color to the complex while remaining A test product characterized in that it comprises a dye molecule complexed to said conjugate. 9. The target-specific labeling complex is lyophilized within a support matrix. A test product according to claim 8. 10. The support matrix is a solid matrix that induces non-absorbent flow of the liquid sample. 9. The test product according to claim 8, which is Rix. 11. The support matrix is a solid matrix that induces a strong absorbent flow of the liquid sample. 9. The test product of claim 8 which is Trix. 12. The support matrix is polyester, acrylonitrile copolymer, A fabric made of rayon, glass fiber, cellulose and blends thereof. The test product described in Item 8 of the Scope of Request. I3. the fabric is treated with a blocking agent to make it non-absorbent A test product according to claim 12. 14. 9. A test product according to claim 8, wherein said specific binding substance is an antibody. 15. 15. The test product of claim 14, wherein said enzyme is covalently linked to said antibody. . 16. The enzyme may include oxidase, peroxidase, phosphatase, diaphosate, etc. The group consisting of enzymes, galactosidases, lytic enzymes, and oxidoreductases 9. A test product according to claim 8, selected from: 17. 9. The test product of claim 8, wherein said enzyme is inactivated. 18. The test according to claim 8, wherein the complex contains 10 to 10 7 dye molecules. product. 19. 20. The test preparation according to claim 8, wherein said complex is lyophilized. Target special 1. A method for preparing a heterolabeling conjugate, comprising: an enzyme and a specific binding agent; wherein the enzyme converts the substrate into a colored dye product. and the specific binding agent is capable of binding to a target molecule; and The conjugate and the substrate are combined into a complex of the conjugate and the colored dye product. A process comprising reacting in an aqueous medium under conditions that result in 21. The reaction conditions are such that they promote turnover of the substrate by the enzyme. 21. The method according to claim 20, wherein the method is selected as follows. 22. The reaction between the conjugate and the substrate resulted in the complex obtaining the desired color intensity. 21. The method of claim 20, which is subsequently stopped. 23. 21. The method of claim 20, wherein the reaction is stopped by substrate depletion. 24. Claim No. 1, wherein the reaction is stopped by changing the reaction conditions. The method according to item 20. 25. Claims wherein said reaction is stopped by inactivating said enzyme. The method according to paragraph 20. 26. 21. The method of claim 20, wherein the specific binding substance is an antibody. 27. 27. The method of claim 26, wherein said enzyme is covalently linked to said antibody. 28. The enzyme may include oxidase, peroxidase, phosphatase, diaphosate, etc. from the group consisting of galactosidase, galactosidase lytic enzyme and oxidoreductase 21. The method of claim 20, wherein the method is selected. 29. An apparatus for detecting the presence of an analyte in a liquid sample, comprising: (a) a sample comprising a solid support matrix that induces lateral flow of a liquid sample; (b) specifically binds to or competes with the analyte; at least one assay label and a solid that induces lateral flow of the liquid sample. a labeling zone comprising a body support matrix, in contact therewith, (c) a lateral flow; said assay in a solid support matrix and at least a portion thereof inducing a capture zone comprising at least one capture reagent capable of binding label or analyte; , adjacent thereto, (d) an adsorption zone; Conjugates of enzymes and specific binding substances: and the properties available for binding to target molecules. Adds a visually distinguishable color to the complex while preserving the active site of the heteroconjugate. an assay comprising a dye molecule conjugated to said conjugate in an amount sufficient to A device characterized in that it comprises an illustrator. 30. 30. The device of claim 29, wherein the specific binding substance is an antibody. 31. 31. The device of claim 30, wherein the enzyme is covalently linked to the antibody. 32. The enzyme may include oxidase, peroxidase, phosphatase, diaphosate, etc. from the group consisting of galactosidase, galactosidase lytic enzyme and oxidoreductase 30. The apparatus of claim 29, wherein the apparatus is selected. 33. 30. The device of claim 29, wherein said enzyme is inactivated. 34. 30. The device of claim 29, wherein said complex comprises 10 to 10 dye molecules. Place. 35. 30. The apparatus of claim 29, wherein the complex is lyophilized.