JPS60105965A - Method of determining heterogeneous system digoxin immunity - 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 The present invention provides an improved method for the immunoassay of ζ digoxin, and in particular a non-competitive assay using a labeled or divalent anti-digoxin antibody as an indicator and an immobilized ouabain column as a separation plate. Concerning heterogeneous immunoassay methods.

There is a large and widespread market for laboratory diagnostic tests that can rapidly and accurately measure the modification of digoxin present in biological fluids. Digoxin is often present in nanomolar or sub-molar concentrations.

In recent years, a number of immunoassay techniques have been developed for the measurement of clinically important ligands. Typically, competitive binding immunoassays consist of a conjugate of a labeled substance bound to a binding moiety that participates in a binding reaction, producing two labeled conjugates, one bound and one free. The relative amounts of labeled conjugate, bound and free, are a function of the concentration of the ligand to be detected in the test sample.

If the bound and free labeled conjugates are not inherently distinguishable by the means used to measure the labeled substance, the bound and free labeled conjugates must not be physically separated. Must not be.

This type of test is called a heterogeneous system.

The two most widely used heterogeneous immunoassays are radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (
ICL Engineering E3A). In RIA, a sample containing an unknown amount of antigen is mixed with known amounts of radiolabeled antigen and antibody. The system is allowed to react to a point near equilibrium and then antibody-bound antigen is separated from unbound antigen. The sample antigen competes with the labeled antigen for a limited number of antibody binding sites, so the more antigen in the sample, the less labeled antigen will be in the bound fraction (or the unbound fraction). (increasingly). This method is generally time consuming (1-3 hours) and labor intensive.

More recently, R1 has been automated by immobilization of antibodies on porous supports. After a sample suspected of containing antigen is mixed with a known amount of labeled antigen, the sample is passed through a column containing a limited number of immobilized antibody binding sites.

Free or bound label can be quantified.

Although rapid, this assay requires a strict fl! of the antibody to make it reproducible. ! Requires an amount of I.

RIAs have two major disadvantages. The first used labeling substances are radioisotopes which have many problems regarding their handling, storage and disposal. Second, RI
A is performed in a competitive manner (i.e., the analyte and labeled analyte compete for a limited number of binding sites on the antibody), and therefore the antibody affinity constant represents the sensitivity of the assay. is limited to a range of 10-8M1 to 10-''Ml.

EL SA is similar in principle to RIA, except that the labeling agent is an enzyme rather than a radioisotope. It is also limited in that sensitivity is still a strict function of the antibody affinity constant.

Other labeling substances have been described in addition to isotopes and enzymes. These include fluorophores, coenzymes, bioluminescent substances, and enzyme inhibitors.

Various methods of implementing the separation step in heterogeneous immunoassays are known. These include filtration, centrifugation, chromatography, and others.

The use of affinity columns to carry out separation steps is described in French Patent Application No. 7915992. It describes the use of a gel that has an affinity for the labeling substance and also has molecular sieving properties or has a Gant coupled thereto. has an affinity for the ligand of interest rather than the labeling substance;
The use of gels with molecular sieving properties is also disclosed. The assays described can be performed in a competitive or non-competitive manner.

U.S. Pat. No. 4,298,687 discloses a heterogeneous immunoassay method in which the substance to be measured is reacted with a label-subbound corresponding component and the amount of unreacted bound corresponding component is is measured by absorption into a solid soil that provides specific binding for its primary binding counterpart. This −order bond corresponding component is present in a limited amount.

U.S. Pat. No. 3,654,090 discloses a non-competitive heterogeneous immunoassay for human chorionic gonadotropin (HOG), which requires an excess of enzyme-labeled conjugate to carry out the separation step. using titrated antibodies and an immobilized HOG column. This assay uses 1 molar HCG-conjugated antibody and HO
It is limited in its sensitivity by the fact that it cannot distinguish between antibodies without G-coupling. Both species are retained by the affinity column.

US Pat. No. 4,134,792 discloses a heterogeneous immunoassay method in which a label-specific binding counterpart for a ligand of interest is present in excess. The label-specific binding counterpart is a divalent antibody, which has the same disadvantages as described above.

47, p. 269 (1981) discloses a two-site immunoradiometric assay (RMA) for coagulation factor ■, in which monovalent Fab antibody fragments are used. There is. These results show that 10 times higher sensitivity is obtained when using monovalent antibodies rather than bivalent antibodies.

(U.S. Pat. No. 4,200,436 discloses an immunoassay method using labeled-valent antibodies, but in the case of using immobilized antigen (the same antigen as that to be measured) The fraction and the free fraction are fractionated.
Since it is technically a bound fraction, this assay is usually performed in a competitive format.

Sensitivity is therefore limited by the affinity constant of the antibody when the assay is performed in the preferred manner.

In some cases, the analyte in the immunoassay can be replaced by an analyte congener. In general, performance is expected to be equal with no difference between the use of analyte and analyte congeners. Surprisingly, it has been discovered that in the digoxin assay of the present invention, replacing digoxin with ouabain as the immobilized antigen significantly improves the sensitivity and accuracy of the assay. Although the exact mechanism leading to this improvement is unknown, it is thought to be due to the nature of the antigen-antibody reaction.

There is a need in the art for a heterogeneous immunoassay of digoxin whose sensitivity and accuracy are not limited by the affinity constants of the antibody.

The non-competitive heterogeneous immunoassay method of the present invention involves the following steps: (a) forming a reaction mixture by contacting a molar excess of labeled or divalent anti-digoxin antibody with a test sample suspected of containing digoxin; (b) the reaction mixture is present in an amount sufficient to bind all of the free antibody, such that one side of the antibody forms a complex with digoxin and the other side remains free; The free antibody is separated from the reaction mixture by contacting the solid phase with ouabain immobilized on a solid support, and the amount of complex eluted from the solid phase is determined by measuring the (0) label. includes doing. The amount of digoxin in the sample can then be determined by comparing the standard curve.

In other embodiments, competitive heterogeneous immunoassays are provided that involve the following steps: (=) molar excess of a sample suspected of containing digoxin immobilized in a solid medium. (b) contacting the reaction mixture with a labeled or divalent anti-digoxin antibody in a molar excess relative to digoxin, but less than a molar relative to ouabain; Let me.

(c) carrying out a reaction whereby one side of the antibody forms a first complex with digoxin and a second fraction with immobilized ouabain; (b)) from the second fraction; Separate the first fraction and <)
It includes measuring the amount of label present in either the first fraction or the second fraction.

It is generally desirable to immunologically purify antibodies before using them in immunoassays. Methods for the isolation of IgG from animal serum, ascites or tissue culture media and its immunological purification by affinity chromatography are also known in the art. Briefly, the IgG fraction is prepared by ammonium sulfate precipitation. (2) The gG fraction is then prepared by ion exchange, gel filtration or protein containing chromatography. Affinity purification is performed by elution from an antigen column.

Antibodies may be either polyclonal or monoclonal. -valent antibodies can be prepared by methods known in the art. For example, Fab fragments are obtained by papain digestion of engineered gG, and 'Fab' fragments are obtained by pezocine digestion of engineered g().
') Obtained by disulfide reduction of the 27 fragment.

Any of a number of methods can be used for coupling a label to an antibody. The labeling substance can be an enzyme, a radioisotope, a chromophore, a fluorophore, or any other substance capable of producing a detectable signal alone or in combination with other reagents. Generally, at least one label should be coupled to each antibody, preferably covalently and in a manner that preserves the immunoreactivity of the antibody and the activity of the label. The free sulfhydryl group present in the F'ab'7 fragment provides a specific reactive group for covalent attachment of the label.

Heterotrifunctional cross-linking reagents with maleimide or thiopyridyl groups are useful for this purpose. Generally, to ensure retention of immunological activity, it is desirable that the final step in the synthesis of labeled antibodies be an immunological purification step.

Ouabain or its conjugate is e.g. rBio
hemietry [Vol. 9, p. 331 (1970)], by methods known in the art, on a suitable carrier. Generally, the carrier is selected for its flow properties and examples can include beaded agarose, beaded dextodin, polyacrylamide or glass. Ouabain can be covalently attached to this carrier directly or via a spacer arm which can be a protein, a polyamino acid or a synthetic linking moiety. Typically, the affinity column material is discarded after one use, but it can be recycled if desired. It is generally undesirable for the carrier to have molecular sieving properties. This is because if the bradycardia antibodies dissociate from the sample analyte, the molecular sieve will tend to reduce the likelihood that they will encounter each other again.

In a non-competitive format, the assay of the invention is as follows:
7 can be implemented. A known volume of patient sample, usually 5 μa to 500 An of serum containing an unknown amount of d-xin, is combined with a solution containing a labeled monovalent or divalent antibody in an amount clearly in excess of digoxin. Mix with.

Normally labeled antibodies are about 10 to 1
00 molar excess. Digoxin and antibodies are incubated at a constant temperature between 4° C. and 45° C., usually 3° C., for a specified length of time, usually at least 5 minutes, and less than 60 minutes.
Preincubate at 7°C. A known volume of this solution containing digoxin-bound and unbound antibodies (usually 5 to 500 μF) is passed through a column consisting of ouabain immobilized on a porous support, preferably having dimensions of 2×10 μm. let Enough ouabain-coupling carrier is used to bind all free labeled antibody.

The column was run at a flow rate of 0.2 to 5.0 m11 min, typically 1 to
5-All appropriate roses 71! Elute at −. The fraction eluted from the column contains labeled antibodies complexed with digoxin from the patient's serum. Label activity in this fraction is then measured. Alternatively, this fraction can be discarded and chaotropic
c) Retained antibodies can be eluted from the column by agents or extreme pH values. In the first case the amount of label is directly proportional to the digoxin rotation in the sample.

In the second case it is inversely proportional.

The assay of the invention can be performed manually or it can be adapted to a variety of automatic or semi-automatic devices, such as the ACA[F] Independent Clinical Analyzer (DuPont). In this case, the patient sample and excess labeled or divalent antibody are preincubated outside the device. This mixture of known 61 is injected automatically into an analytical test pack (described in Re 29.725, which is hereby incorporated by reference) at the filling station of this equipment and then into the final pack. Inject enough cap buffer to bring the volume to 5-. The sample mixture is passed through a column of ouabain immobilized on a porous support placed in the pack header and eluted directly into the pack. This eluted fraction contains labeled antibodies complexed with the analyte in the patient's serum. This puncture automatically adds the necessary reagents for the signal generation reaction in either Breaker/Mixer I or Breaker/Mixer II at 67 DEG C., and reads out the signal photometrically.

Although the assays of the invention can also be performed in a competitive format,
This means that the antibody is exposed simultaneously, rather than sequentially, to the sample digoxin.

In the competitive format, the assay is performed as follows.

An aliquot of a sample suspected of containing digoxin, typically 10-100 μλ, is added to a sample tube containing a molar excess of ouabain immobilized on a solid matrix (generally an affinity column resin such as cross-linked agarose or dextran). 100-1000μλ filling capacity). A monovalent or divalent labeled anti-digoxin antibody solution is then added in a 9 molar excess over the maximum amount of digoxin expected in the sample, but not more than 9 molar over ouabain. The amount of this antibody solution is generally 5-50 μλ. The reaction mixture thus produced is heated at 26°C to 45°C, preferably at 6°C.
Incubate at a temperature of 7° C. for 15 to 60 minutes, preferably 15 minutes, with gentle stirring. The antibody-digoxin complex is then separated from the antibody/immobilized ouabain complex. If affinity resins are used, centrifugation at 2000 Xf for about 6 minutes is preferred. The supernatant fluid is then aspirated. Either the amount of label adsorbed on the solid set or the amount of label in the supernatant fluid can be measured to determine the amount of digoxin originally present in the test sample. If the label is an enzyme, the measurement can be performed by reacting the enzyme with its substrate to produce a detectable product.

Non-enzymatic labels such as fluorophores, chromophores, and radioisotopes can be measured by techniques well known in the art.

The advantage of using ouabain rather than digoxin on a solid state system is illustrated by the following example. In these examples, both digoxin (analyte) and ouabain (analyte congener) columns were prepared under various experimental conditions, and their performance in immunoassays using digoxin affinity columns was determined by the background, Evaluated for sensitivity and accuracy.

Although the optimal conditions for the synthesis of each resin are different,
The best ouabain resin consistently outperformed the best digoxin resin.

Although only ouabain is specifically exemplified herein, equivalents are contemplated such as digitoxin, deslanonde, digoxigenin, and strophanthin.

Example 1 Synthesis of A-valent antibody-enzyme conjugate ouabain-
Digoxin-specific antibodies were directly immunopurified from the gold serum used for the ISA immunoadsorbent.

Ouabain was bound to the degarose matrix via a protein (ISA1 human serum albumin)-ther arm. The first step involves the synthesis of the ouabain-albumin conjugate. Ouabain (1) (56 mmol dissolved in 20 mmol of water) was oxidized with sodium metaperiodate (1.02 mmol) for 1 hour at room temperature in the dark.

Quantitative oxidation is ethyl acetate: methanol: H2O (75:
25:1) on silica gel G plates. Excess periodate was removed by passing the aqueous mixture over a 3-column of DOWEX AG-I X8 ion exchange resin. Quantitative recovery of ouabain was demonstrated by tracking radiolabeled (tritiated) ouabain.

The solution of ouabain oxide was buffered to pH 9.5 by adding 0.4-5% NazOOs and 20 tnl of H8A i solution (28 nq/rn
1. ) combined. After 45 minutes, this conjugate was
Reduction was achieved by adding freshly dissolved 0.61 storium borohydride to the water of the barge. After 3 hours, 8-1 molar formic acid was added to lower the pH to 6.5. After 1 hour at pH 6.5, 1 mol, NH4OH
The pH was raised to 7.5.

The entire reaction mixture was thoroughly dialyzed against distilled water and finally
．． 015 molar sodium phosphate buffer (pH 7,8)
, 0.15 molar Na0j2. The conoconjugate was concentrated on an Amicon PM-30 membrane at 14 °C.
2η/−. Protein concentration was determined by the Rho9-method known in the art.

Ouabain-H8A conjugate 'ji-rBio-
It was immobilized on Affigel 10 (manufactured by Bio-Rad Laboratories) using the method described in RadmanualJ. 25-Affigeno L/@) 10 to 75
- Washed with ice-cold water. This gel was dialyzed with Ouabain-H8.
Added to A conjugate and allowed to mix overnight on a rocker at 4°C. Excess active ester groups were quenched by adding 0.1 units of 1 molar ethanol (pHaO) for 1 hour at room temperature.

Finally, rinse this gel thoroughly with distilled water and then in turn
-0.5 mol Naoffi, 400m600.1-
ek glycine (pH 2,5), 300rnl of 25M NH45cN.

Washed with 1000 mg of phos7ate buffered brine. Ouabain hydrophilic resin was packed into a column (0.7X 15 crn) to a bed volume of 6- and equilibrated with phosphate buffered saline.

Antiserum (10 ml of Kabell anti-digoxin serum, a 4,5η/me monospecific antibody) was applied at a flow rate of 1 me min or less. The column was washed with phosphate buffered saline until its 280 nm absorbance reached baseline (<0.01). Then 60me of 3M NH45ON (pH 7
The antibody was eluted from this column at 5) and immediately dialyzed four times against 22-phosphate buffered saline at 4°C.

This 27-
The affinity-purified anti-digosin antibodies were concentrated in 27 vessels. The final protein concentration was 10η/-. sample to 0
．． 1M sodium acetate (pH 4,5) 1000-4
Dialysis was performed at ℃ for 4 hours. After dialysis, 20 μλ of a 1 omy/lnt pepsin solution dissolved in the same sodium acetate buffer was added, and the temperature was raised to 37° C. and maintained for 20 hours.

After this digestion time, the sample was clarified by brief centrifugation and then [Sephadex equilibrated in 1015 molar sodium phosphate (pH 7,4), 0.15 molar Na01 (phosphate buffered saline)]. G-150
It was subjected to O mudography on a column (1,5×90 crn). Identified by gel electrophoresis (Fab'
) The column fractions containing the 27 fragments were pooled 1
9.2m)% then pressure filtration (PM-3Q Amicon membrane)
It was concentrated to 2.71 rLt. After concentration, this (Fab 2
Noragnont 55. Adding a 1M dithiothreitol solution of d and reducing the corresponding Fab')2
It was made into a component. Reduction was carried out for 90 minutes at 25°C.

Then at 4℃ 0.15 Mok NaC! I1. ．． 0.0
15 mo/l/sodium phosphate, pH 5,6 (2X 1
000-).

The Fab' 7 fragment thus prepared was then reacted with a 20-fold molar excess of m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS). 85 pi of a 79 mmol MBS solution in tetrahydrone run was added to the solution of the 2-0 Fab'7 fragment and reacted for 1 hour at 25° C. under argon. This mixture was mixed with Sephadex G in phosphate buffered saline.
-25 (cross-linked bead dextran, with exclusion limit of 5000 daltons) column (1,5X4Don)
Desalted above. The derivatized Pa b' fragments eluted into the void volume were pooled and incubated with 12 my/- β-galacto7 in phosphate-buffered saline at 4°C.
combined with Dase2rnt. After 16 hours, the solution was concentrated to 2-2 in an Amicon PM-30 pressurized perturbation cell and then injected into Sepharose 4B-OL (bead-shaped cross-linked macrocells with Dalton exclusion limits of 1-5X10'' in 1,5X90 Locazim). The Fab'-β-galactosidase conjugate was eluted with free i-galactosidase. All peaks of enzyme activity were pooled and then immunized on an ouabain affinity column. The immunological purification method was as follows: Sepharose 4B-
The pooled column fractions from the OL column were eluted through an Ouabain affinity column (1, OX 7. Dcm) and then eluted with 100-phosphate buffered saline.

The Fab'-β-galact7dase conjugate was then eluted with 23 mmol ouabain 50-in phosphate-buffered saline. This eluate represented the final reagent, which was dialyzed six times against 4 L of phosphate buffered saline at 4°C.

B. Synthesis of ouabain and digoxin resins Oouabain and digoxin were prepared separately in various ratios as an organic spacer component triethylenetetamine (TJllTA).
was coupled to Sephadex()-10 (a beaded dextran available from 7Alma Fine Chemicals, Inc.) via .

Dissolve ouabain (20-325 nv) in distilled water,
It was then oxidized for 2 hours at 25° C. by adding a 5-fold molar excess of sodium metaperiodate. At the end of this time, the reactions were stopped by passing each solution through a 5-0 column of Dowexney X8 (0J2-ion exchange resin) to remove excess periodate. The eluate was collected and strained to 0.1 by addition of a stock solution.
The final concentration was molar sodium phosphate (pH 7.0).

Tl! Add 1 TA (50-125 TA) to each solution (Table 1
(see A) and the pH of the final solution was adjusted to pH 7, [+]. The solution was incubated for 1 hour at 25°C, and at the end of this time 60~ of sodium cyanoborohydride was added. The resulting solution was stirred at 25°C for 48 hours.

The same method described above for ouabain was performed for digoxin except that digoxin was dissolved in 30% ethanol. Experimental details are shown in Table 1B.

Each conjugate (90 units of ouabain-TFliTA and 90 units of digoxin-TETA) was then coupled to Sephadex G-10 by the following method. Sephadex G-10 at 210F was swollen in distilled water at 1C. The resin was then washed six times with one portion of water. Finally, resin 11! It was oxidized by making it suspensible with sodium chloride (IC1/λ).

After 1 hour at 25 °C, the resin was allowed to settle, the supernatant fluid was removed, and the resin was washed on a sintered glass funnel using 0.25 molar sodium phosphate (pH 7, D) of approximately 3β. . Divide the obtained fat into 25ml portions,
and a portion thereof was mixed into each lot of the synthetic conjugate. After 1 hour of mixing, sodium cyanoborohydride (30 ni) was added to each portion of the conjugate-loaded resin. The resulting suspension is allowed to mix at 25° C. for 644 hours, during which time the resin is allowed to settle, the supernatant fluid is removed,
and resin (each section separately) in 300 - 300 b of water
0.5 mol Naaj2 of m and 0,15 of 500 m1
Washed with molar sodium phosphate (pH 7.1). Each resin section was separated into an ACA Independent Clinical Analyzer analytical column (0,5
X8crn1 column (1.8 rnl) was packed.

The column was then loaded with 7 μm of O-nitrophenyl-β-D.
-ac containing galactopyranoside (0NI)G)
a [F] was placed at 6 dimples into the water conduit of the Independent Clinical Analyzer Analysis Test Pack.

C. Comparison of ouabain-TFtTA-Sephadex and digoxin-TFliTA-Sephadex (as above)
Each resin lot synthesized in was evaluated on an aca Independent Clinical Analyzer according to the following protocol. 10 pmol of β-galactosidase-labeled digoxin Fab′ synthesized in (A) above was added to 0 or 5nr at 100μ℃.
/- to human pool serum samples containing digoxin. After incubation for 10 minutes at 25°C, the antibody-sample mixture was automatically injected into the ACA ■ Independent Clinical Analyzer Analysis Test Pack and eluted through the column in the pack water tube. The sample was followed by a stream of 2-0.15M sodium phosphate (pH 7,8). Column flow rate is 34 μ2
/second. This pack was then filled with an additional 2.9-mL of water at needle position 2 (force 2 m bypass). 0NP
G was discharged from the breaker/mixer ■ after about 3% minutes. Enzyme activity was measured at 405 nm at 29 and 46 seconds after substrate addition.

Table ■ compares the performance of all 18 resin lots in terms of pack ground (Table 11A) and sensitivity (Table JIB). Pack ground was defined as the change in absorbance at 405 μm when the sample contained Qnt/ml digoxin. Under these conditions all labeled antibody should remain bound to the column. Sensitivity is as a separation, ie QnP/rn1. Defined as the change in absorbance at 405 μm between digoxin and 5nr/ml:) goxin.

Packground generally uses digoxin-TET columns.
Although it was lower when the column was made of A-Sephadex, the sensitivity was always better when the column was made of Ouabain-TFiTA-Sephadex. The best ouabain resin had a sensitivity of 0.158 absorption rates/min, while the best digoxin resins exhibited only 0.110 absorption rates/min. Those resins marked with an asterisk in Table 11B were selected for further study.

D. The resins marked with an asterisk in the Accuracy Table ■, namely one consisting of Ouabain-TIIITA-Sephadex and the other consisting of Digokin-TETA-Sephadex, were further evaluated for testing accuracy against digoxin. did. Packs were operated exactly as above using samples containing 0.1 or 14 nt/- digoxin. At least 13 packs were run, two at each system concentration. Then, the mean 11α), the standard deviation M (S,D,) and the coefficient of variation e%C0-V,) were determined. These are shown in Table ■.

The medically determined concentration for this drug is 1nr/ml
:) The accuracy of measurements in cockroaches was significantly improved for the ouabain column compared to the digoxin column.
ζ. When using a digoxin column, a person with a serum concentration of 11/- digoxin is easily misidentified. and may lead to over-dosing (or under-dosing) of the drug.

Digoxin is ineffective at concentrations below Int/-;
And since it is toxic at concentrations significantly above that, the consequences of such measurement errors can be significant. For this reason, the use of an ouabain column instead of a digoxin column forms the basis of the invention.

Table 1 person 0U-A 325 125 0 [IT-B 195 75 0U-013050 0U-D 125 125 0U-1ii 75 75 0U-F 50 50 0U-G 50 125 0U-H3075 0U-Eng 20 50 Table 1B DG- A 525 125 DG-B 195 75 DG-013050 DG-D I25 125 DG-E 75 75 DG-F 50 50 DG-[) 50 125 DG-H3075 DG-Engineering 20 50 Table 11a AO,2340,042 B O ,2540,108 CD,261 0.318 D O,1110,044 n O,1210,206 y O,1240,311 e O,1640,137 HO,1660,224 Eng 0.159 0.268 Table ■B A O,0300,008 B 0.019 a O,0150,013 D O,158* 0.012 E O,1480,011 F' 0.148 0.047 () 0.106 0.033 HD, 108 0. 087 E 0.112 0.110* Table ■ Comparison of resin 0U-D and DG-machining: Accuracy i Cnr/d) 0.83 0.96S, D,
(1', zQ) 0.14 0.530, v, (%) (
n-) 17 55-/X (nr/rn+ri 3.4
3.4 E1. D, (nr/mg) o, 09 0.37a
, v, (x) (nf%/) 2.5 11 rows 2 A, , Synthesis of bivalent (F(ab2) antibody enzyme conjugates) 1rnl of anti-digoxin F(a) manufactured by Affinity N
t/ ) 27 fragments (prepared as described in Example 1) [2,85 mr/- of protein - 0,015 molar sodium phosphate, 0.15 molar Na0A, 1 mmol FiDT
A (pH 1Q) 60 mmol succinimide #4-(N-maleimido-methyl)cyclohexane-1-carboxylate (5M0O) dissolved in amide m
mixed with liquid. After 60 minutes, the same sodium phosphate-'
The reaction was stopped by desalting the solution on a Sephadex G-25 plate (1,5 x 3[]6n) equilibrated with NaO[beta]-EDTA solution. The eluted protein in the void volume was collected and concentrated to 1- using an Amicon stirred cell concentrator (PM-301 version). 1-0.05 mol Tris HOA, 0.15 mol N
aOR, β-galactosidase 24η dissolved in 1 mmol Mg0f12 (pH 7,5) was added to this F(absu2-8M0O adduct and reacted for 20 h at 4°C. 0.1 The reaction was quenched by the addition of 10 μM of 2-mercaptoethanol.
Sepha'Rose 4B column (t5
This F(a
The bri2-ρ-ga2ctosidase conjugate was separated from unreacted β-gadictosidase.

B. Synthesis of ouabain resin and digoxin resin Ouabain and digoxin were each synthesized with bovine serum albumin (BSA).
) to Sephadex G-10 in optimal ratios.

1) Ouabain-BEIA is 500 - hot distilled water (70
℃) to dissolve 52 ouabain octahydrate and K! 2 were prepared. After cooling the solution to 25°C, Z6
Add sodium periodate and then leave in the dark for 25 minutes.
Continuous mixing was continued for 2 hours at °C. This mixture was then heated to 250
The oxidation was stopped by passing through a bed of 1y1g of Dowex (1-X8) anion exchange resin. The eluate was collected and added to 1 molar sodium phosphate buffer (pH 7).
Bovine serum albumin solution (1og15
Combined with 00-n. After incubation for 1 hour at 25℃, 0.6
4r of sodium 71 noborobide 2ide was added with stirring and the mixture was incubated at 25°C for 72 hours.

The uncoupled ouabain is ouabain-B.
The SA conjugate solution was removed from this mixture by dialysis for 24 hours against running distilled water and then against 20 volumes of 0.015 molar phosphoric acid sodium phosphate Balanoa-H7,0) at 4°C. The final ionic strength of this conjugate solution was increased to 0.0 by adding NaCR14,6f tl- to the MfJ coupled to the Sephadex resin.
The amount was adjusted to 25 mol.

2) Ouabain-BSA to Sephadex G-10 II yJ' Sephadex G-10 (420F) (Pharmacia)
(manufactured by Fine Kebakals) for more than 1 hour, 2000m
1 in distilled water. The resin particulates were removed by decanting and resuspending in 5x2oooy water.

The leaves were then oxidized by resuspending them in water containing 200 mg of dissolved sodium metaperiodate. After 10 minutes, the resin was washed with 5 x 2000 g of water, then 4000 g of 0.25 molar sodium phosphate buffer.
(pH 7.0). Decanted resin to 101
00O uano2ine-BSA solution (from above)
resuspended at 25 °C for 1 hour, then 0.6
Mixed with 6f Sodium Acupoint I:f/% Idonoid. After 72 hours, it was thoroughly washed with 4000 tnl of 00.1% sodium dodecyl sulfate in water, 12 A of distilled water, then 4000 tnl of 0.15 molar sodium phosphate, S tuffer (pH 7.1). The final resin was loaded as a slurry into a small column (0.5 m x 7 t:m) used for automated analysis in a DuPont ACA Independent Clinical Analyzer.

6) Digoxin-BSA is 1.2 to 75 grams of ethanol
Dissolve 5 t of digoxin and then dissolve this solution in 1.
Water containing 83F sodium metaperiodate 150-
It was prepared by combining.

After stirring for 2 hours at 25°C, oxidation was stopped by passing the mixture through a bed of Dowex (1-X8) anion exchange resin (1o OJ). The eluate was dissolved in 0.1 molar sodium phosphate (pH 5). A solution of dissolved bovine serum albumin (2,5F) was combined.

Sodium cyanoborohydride (0,241) was then added and the mixture was mixed for 48 hours at 25°C. Free, unconjugated digoxin was
It was removed by dialysis against running distilled water for 1 day and then against 20 volumes of 0.015 molar sodium phosphate puffer (pH 7.0) at 4°C.

4):) Coupling of Goxin-BSA to Sephadex G-10 Sephadex G-10 (5B) for over 1 hour at 250
- swelled in distilled water. The resin particulates were removed by decanting and resuspending. The resin was then oxidized by resuspending it in 250° of water containing 52° of dissolved sodium metaperiodate. After 10 minutes, add 5x250 side fat.
- of water, then 0.1 molar sodium phosphate buffer (
Washed with pH 8,5) 1000-. The decanted resin (~250 ml of sedimentation volume) was then mixed with 125 ml of sodium cyanoborohydride.
Slurried in 1 molar sodium phosphate buffer (pH 5). 3X50 resin after 24 hours of constant mixing
0- of water, 500- of 0.15 molar sodium phosphate (p
H7,8) and resuspended in 125-00.15 molar sodium phosphate (pHa5) K at 4°C. Acetic anhydride (1.25m) was added to the slurry and reacted at 4°C with mixing for 60 minutes. Resin 1 to 0.5 mol Na
0It, 4i, +7) Washed thoroughly with distilled water and 2β 0.15M sodium phosphate (pH 7,1). The final resin was packed in slurry form into a small column (0,5 cm x 8 cm) designed to be placed in the water tube of the acaI3 Independent Clinical Analyzer Test Kit.

Digoxin-BSA-Sephadex resin and ouabain-BSA- using C1 divalent antibody enzyme conjugate
Comparison of Sephadex Resin Both resin lots were compared under the same conditions. F(ab'12-β-galactonase (20
Various amounts of digoxin (0,0,5,1,5,5,5 and 5,0
200μ normal human serum standard solution containing nr/ml)
Added to °C. After an incubation period of 10 minutes at 25°C,
The whole antibody-sample mixture was automatically injected into the acaI31 Independent Clinical Analyzer test pack and eluted through the column in the pack water tube. 2rn following the sample
1 of 0.15 molar sodium phosphate pH 7.8k was flowed.

Column flow rate was 64 microseconds. Then, at needle position 2 (force 2 m bypass), the pack had a total of 2.6 m/! filled with water.

0NPG was discharged from the breaker/mixer ■ after approximately 3.5 minutes. Enzyme activity was measured at 405 nm and after substrate addition 2
Measured at 9 and 46 seconds.

Figure 1 and Table ■ compare the performance of two optimized digoxin-BsA-Sephadex and Ouabain-BSA-Sephadex resins in terms of pack ground and slope sensitivity (Figure 1) and precision (Table ■). do.

Both resin lots allow linear dose sensitivity for the F(ab')2-β-galactosidase conjugate, but the ouabain resin has a lower packing ground (32° lower) and greater slope sensitivity. (47% larger). Lower pack ground and higher slope sensitivity are preferred for better assay performance and precision. Furthermore, the assay accuracy was significantly better for ouabain resin than for digoxin resin (Table ■). Individual pack assays were carried out exactly as before with two digoxin resins %D, 5nr/go and 1.5nf/-.

At least 12 packs were created for each drug concentration. Then, the mean value (X), % standard deviation (El, D,) and coefficient of variation (support) (0, V,) were measured and determined.

Table ■ Comparison of ouabain resin and digoxin resin with respect to accuracy X (nf/mt) [1,470,43seD, (nr
7Qri D, [140,07C,V, (%) (nl/1
tti) 8. 'I 14.2X (nr/d) 1.
52 1.54 S, D, (n7/+ri α05 0.200, v,
f%) (nr/m/) 3.0 15.1

[Brief explanation of the drawing]

The attached drawing is a graph showing the slope sensitivity when using the Jigokin 7 column and the ouabain column.

Claims (1)

Claims: I and one fraction remains free; (1)) this reaction mixture is contacted with a solid phase having immobilized ouabain present thereon in an amount sufficient to bind all of the free antibody, thereby Non-competitive immunization for determining digoxin in a test sample consisting of binding an antibody onto this solid phase and (C) determining the amount of complex eluted from the solid phase by measuring the labeling activity. Quantitative method. 2) The immunoassay method according to claim 1, wherein the label is an enzyme, a radioisotope, a chromophore or a fluorophore. 3) - If the antibody is a Fab fragment or a Fab' fragment
2. The immunoassay method according to claim 1, which is a immunoassay method. 4) The immunoassay method according to claim 1, wherein the bivalent antibody is an F(ab')27 fragment. 5) The immunoassay method according to claim 1, wherein the solid phase is beaded agarose, beaded dextran, polyacrylamide or glass. 6) The immunoassay method according to claim 5, wherein the solid phase is filled into a container. The titer antibody is a Fab'7 fragment, and the label is I
- The immunoassay method according to claim 1, which is galact7dase. 8) The immunoassay method according to claim 1, wherein the solid state is beaded dextofur, the divalent antibody is an F(ab')2 fragment, and the label is β-galact7dase. 9) The aforementioned patent claim, wherein the measuring step of (0) is carried out by contacting the eluted β-galactosidase-labeled Fab anti-digoxin antibody/digoxin complex with O-nitrophenyl-β-D-galactobyranoside. The immunoassay method according to item 7. 10) The aforementioned patent claim, wherein the measuring step of (0) is carried out by contacting the eluted β-galactosidase labeled mF(ab')z anti-digoxin antibody/digoxin complex fo-nitrophenyl-β-D-galactopinoside. The immunoassay method according to item 8. 11% a) forming a reaction mixture by contacting a sample suspected of containing digoxin with a molar excess of ouabain immobilized in solid soil; (C) A reaction is allowed to occur so that a fraction of the antibody forms a first complex with digoxin. and (d) separating the first fraction from the second fraction, and (e) causing the second fraction to form a complex with the immobilized ouabain.
A competitive immunoassay method for determining digoxin in a test sample comprising a series of steps consisting of determining the amount of label present in either one of the first or second fractions. 12) The immunoassay method according to claim 11, wherein the label is an enzyme, a radioisotope, a chromophore or a fluorophore. 16) The -valent antibody is a Fab fragment or fc is a Fab'
12. The immunoassay method according to claim 11, which is a 7-ragment. 14) The immunoassay method according to claim 11, wherein the bivalent antibody is F(ab')2 noragment. 15) The immunoassay method according to claim 11, wherein the solid material is beaded agarose, beaded dextofurne, polyacrylamide or glass. 16) The immunoassay method according to claim 11, wherein the solid material is beaded dextran, the -valent antibody is a Fab' fragment, and the label is β-galact7dase. 17) The solid version is beaded dextran, the -valent antibody is Il' (ab2 fragment) and the label is β
- The immunoassay method according to claim 11, which is galactosidase. 18) The immunoassay method according to claim 16, wherein the measuring step (e) is carried out by bringing the fraction to be measured into contact with o-2)lophenyls-β-D-ga2ctopyranoside. 19) The immunoassay according to claim 17, wherein the measuring step (e) is carried out by contacting the fraction to be measured with O-nitrophenyl-β-D-ganocytoside. Law. 20) A test kit for measuring the amount of digoxin in a liquid sample, comprising (a) a labeled amount of digoxin antibody, (b) a solid having ouabain bound thereto, and (C) instructions for using said kit.