JPS587561A - Enzyme immunity analyzing method - 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 relates to an enzyme immunoassay for determining or quantifying the presence of a substance in a medium suspected of containing the substance.

Various enzyme immunoassay techniques are known;
These include methods in which an enzyme is coupled to an antibody or antigen whose presence is to be detected. Enzyme immunoassay method, especially homogeneous enzyme immunoassay method U, patent issued d4tr No. 2
, 043.245 A. This particular release states that there is an enzyme and an irreversible inhibitor for #A, and that the irreversible enzyme inhibitor is conjugated to an analogue of the ligand to be detected to form a conjugate. It relates to homogeneous enzyme immunoassays that can react with the enzyme to form a covalent bond that irreversibly inhibits the enzyme's activity by changing the structure of the enzyme. There are also binding proteins and ligand conjugates capable of binding the ligand to be detected, but the conjugates are inactivated when bound to the binding protein. The amount of ligand to be detected influences the binding between the conjugate and the binding protein, and thus the effect the conjugate has on the activity of the enzyme. The enzyme activity is then measured and compared to the activity in the absence of the ligand to quantify the amount of ligand present.

IFKBS Volume 16, July 1980, pages 285 to 288, also describes such a system, but also suggests some alternatives to the homogeneous immune system on the same principle. are doing. However, these are not illustrated.

It has been suggested that such a system may include the use of an enzyme activator such as an antibody to wild-type colon-β-galactosidase.

The present invention is a novel homogeneous enzyme immunoassay for the quantification of a substance believed to be present, which is based on the activation of an inactive enzyme and in which the degree of activation of the enzyme depends on the amount of the substance present. It is about law.

Accordingly, the present invention provides a method for determining or quantifying the presence of a substance in a medium suspected of containing the substance, the method comprising: a) '! mixing together a conjugate of a substance bound to an antibody capable of activating a qualitatively inactive enzyme, a binding protein or antibody specific for the substance, and an excess of the substantially inactive enzyme and a vehicle; The order of addition of its components is such that any substance in the medium
The enzyme is added so that it does not mix with the conjugate before it has had a chance to react with the agent-specific binding matrix or antibody.

b) Add a substrate for the enzyme and measure the enzyme activity It consists of things.

In one preferred embodiment, the invention comprises: a) adding a medium to a mixture of a binding protein or antibody specific for the substance with an excess of a substantially inactive enzyme; C) adding a conjugate of the substance bound to an antibody capable of activating the enzyme; and C) adding a substrate for the enzyme and quantifying the activity of the enzyme in a medium suspected of containing the substance. provides a method for determining or quantifying the presence of.

In a further preferred embodiment, the present invention comprises adding a binding protein or antibody specific for (C1) conjugated to an antibody capable of activating a substantially inactive enzyme; In addition, it provides a method for determining or quantifying the presence of a substance in a medium suspected of containing the substance, which comprises (1) adding a substrate for the enzyme and quantifying its enzymatic activity; ing.

Desirably, the antibody capable of activating a substantially inactive enzyme is a monoclonal antibody.

A binding protein or antibody specific for the substance can bind to the substance when the substance is unconjugated or when conjugated to an activated antibody. When an antibody or binding protein specific for the substance is attached to the conjugate, this reduces the level of enzyme activation observed. Suitably, the antibodies have been purified by standard techniques, eg, by fractionation, including precipitation with ammonium sulfate, followed by column chromatography on ion-exchange cellulose. It is appropriate that this purified antibody be substantially free of other proteins, that is, the content of other proteins should be less than 10%.

Yet another antibody capable of activating a substantially inactive enzyme is added to the enzyme prior to adding it to any of the other ingredients.
It is appropriate to mix it with cables.

A substantially inactive enzyme is one whose activity when activated is less than 10, suitably less than 5, preferably less than 1, that of β-galactosidase.
means an enzyme such as an inactive mutant of lactosidase. The more β-lactosidase mutant is preferably prepared by culturing a mutant strain of E. coli.

Many types of substantially inactive β-lactosidase produced by culturing mutant strains of E. coli have been found to be unstable. It has also been discovered that many types of substantially inactive β-galactosidase produce a high background noise, i.e., a color reaction when mixed with a substrate without the presence of an activating antibody. Therefore, the substantially inactive β-galactosidase suitable for inclusion in the method A is stable (i.e., retains the ability to be activated for months and catalyzes linear reactions for many minutes). ) without producing significant background noise. A particular type of β-lactosidase is the WM 298 strain of E. coli, namely A11ele:
lac aba (527,-r 7 W, 1lesser of the Kusblanc Institute 1Berlin 3
3 (DAHIJM).

It is produced from a single strain obtained from P. hnestragse 66-73.

The activation rate clonal antibodies used in this technique are suitably intact immunoglobulins, although fragments of immunoglobulins may also be used. The antibody or binding protein specific for the substance to be tested is preferably a high titer polyclonal antibody or a monoclonal antibody specific for this substance.

The present invention relates to a method particularly suitable for the determination of substances in biological fluids such as serum, plasma, spinal fluid, and urine. Substances suitable for detection and quantification by the method of the invention include hormones, proteins, vitamins, poisons, drugs or drug substitutes.
Including i-things. The method of the invention is particularly suitable for the detection of medical substances such as steroids, carbon, proteins and protein fragments, folic acid, serotonin, prostaglandi, adrenaline, noradrenaline, opium, Theophyllic acid, cilanthine, pulpyrate, aminoglycoside antibiotic, acyclovir, and antiviral agent. Desirably, the substance is an antiepileptic, an antibiotic, an antiviral, a hormone or a protein. This method uses diphenyl hydantoin,
The determination of cortisol, acyclovir, fibrinogen, and fidelinol has been found to be useful in tcIK. The method of the invention is suitable for high molecular weight substances such as proteins and protein fragments, i.e.
The fact that it is suitable for the detection of related substances from 0 to i, o o o, and o o o is considered to be particularly valuable.

The substance to be formulated will usually be present in a concentration of 1 nanomolar or more. In the case of cortisol, a concentration of 40 nanomolar or more, in the case of acyclovir, a concentration of 250 nanomolar or more, in the case of diphenylhintoin, a concentration of 10 micromolar or more, and in the case of fibrinogen and fibrinogen breakdown products, a concentration of 60 nanomolar or more. The appropriate concentration is

Conjugates of the substance and antibodies capable of activating a substantially inactive enzyme are prepared by traditional methods well known to those skilled in the art. The substance will be attached to the antibody by conventional bridging groups. For both cortisol, asic, and diphenyl hydantoin, a suitable method is to introduce calginate groups into these ligands and then easily conjugate them to the appropriate groups of the monoclonal antibody. The purpose was to convert it into the N-hydroxy succimide ester. For the Fidelino degradation product DJC, a special Fc isolated method is to introduce the imaged thiol group into the D fragment, introduce it into a maleivalent monoclonal antibody, and then introduce the thiol residue into The aim was to remove the protecting group from the base and mix the two proteins to facilitate coupling. Other suitable conjugation methods include enzyme immunoassay C by Kabakoff;
R, C1Proms.

Compiled in H, T. Maggio eds. 1980.71-104.

The assays of the present invention will normally be carried out in an aqueous liquid medium at a temperature of -4 to 5 to 10, preferably about -7.4. The assay is conveniently stopped by adding a chelating agent for magnesium, such as IcDTA, up to about -10 and a lipid solubilizer, such as sodium cholate or sodium deoxycholate. . Preferably, an alkanol or polyol is present in the aqueous liquid medium. Methanol, ethanediol, propane-1,2-diol, mannitol, flubitol and D-arabitol are particularly suitable for inclusion in this aqueous medium. , seal〈should be D-
It has been discovered that arabitol increases the sensitivity of the assay and at the same time decreases the length of time required to perform the assay by a mechanism that is different from, and additional to, that of methanol. The temperature of the liquid medium during carrying out the process of the invention is usually between 10°C and 45°C, advantageously 37°C.

In order to generate an appropriate immunological reaction, the conjugate and the antibody or binding protein specific for the substance and, if present, react with the substance in the medium, and the enzyme It will be clear to the person skilled in the art that a certain amount of time is required (incubation period) in order for the microorganism to reach its proper activation state. The exact steps at which incubation must be carried out will depend on the particular circumstances of the analysis. For example, depending on the order of addition of reagents, incubation will always occur before addition of substrate; a person skilled in the art will be able to judge when special incubation steps are appropriate. .

The substance, conjugate enzyme and antibody, are usually soluble in the medium in which the method is carried out. Although the substrate may be soluble or insoluble in the medium, it is more convenient if it is soluble in the medium. In some situations it may be desirable to provide an insoluble synthetic substrate or to use an insoluble natural substrate.

For example, standard sheep immunization is used in the assays of the present invention to neutralize the effects of antibodies that may be incidentally present in the medium and interact with antibodies that may activate a substantially inactive enzyme. It may be advantageous to include an excess of standard immunoglobulin, such as globulin. It is known that mutations in β-lactosidase contain a thiol group essential for its activity. When such bases are used in the assays of the present invention, dithiolthreitol or similar reagents are usually included to prevent oxidation of the thiol groups present. It is convenient and advantageous to add a suitable preservative, such as sodium azide, during the analysis of the present invention.

In the method of the present invention, a specific bond exists between the substance to be quantified, if it exists, and a conjugate consisting of the substance and an antibody capable of activating a substantially inactive enzyme. Competition occurs for binding to the antibody or binding protein. Since the level of enzyme activation depends precisely on the degree of association of the conjugate with a specific antibody or binding protein, the enzyme activity of a sample depends on the amount of substance in the sample.

The method for analyzing enzyme activity varies as necessary.

Spectrophotometric or fluorescent measurements are conveniently used to measure enzyme activity levels.

Conjugates in which the substance to be quantified is bound to an antibody capable of activating a substantially inactive enzyme are novel and form an important part of the present invention.

The present invention also provides a test kit consisting of reagent 1: an antibody specific for the substance to be analyzed mixed with a substantially inactive enzyme, 2: a conjugate reagent, 6: a substrate reagent for the enzyme, 4: a stop agent. provide.

Suitably, reagent 1 comprises an antibody capable of activating a substantially inactive enzyme. The substantially inactive enzyme is β-
Suitably, it is a variant of lactosidase, in which case reagent 1 contains dithiothreitol or an equivalent reagent for the prevention of oxidation of the thiol groups normally contained in β-galactosidase. Conveniently, Test 41 also includes standard immunoglobulin, and preferably standard sheep immunoglobulin. Suitably, reagents 1 and 2 contain one of the polyols, preferably C<D-alator.

Reagent 2 suitably comprises standard immunoglobulin, preferably standard sheep immunoglobulin.

Preferably, reagent 6 includes one of the alkanols, such as methanol. The terminating agent is a mixture of a high buffer solution, a chelating agent for magnesium, and a lipid solubilizer. - The buffer solution contains sodium hydroxide in glycine, the chelating agent is KDTA and the lipid solubilizing agent is cholic acid. The preparation of the components of the test method and the operation of the test method are illustrated below: Enzyme Purification Melchers and Messer
Biochem, 17 (1970), 267) A strain of Escherichia coli that produced an activated mutant β-lactosidase, as developed by K., was supplemented with sodium succinate and thiamine. Grown in It section medium (
FOW18r, J, Bacterial, 112
(1972), 856), and furthermore b″ow1er (upper 8
(self reference) A3245 strain such as No.1ie41KLf was grown to provide enzymatically active wild-type β-lactosidase.

Bacteria were harvested by centrifugation and stored frozen until needed. After thawing and radiofrequency digestion, β-galactosidase and its various antibody-activatable mutants were isolated by Oraven et al.
B1C11, Ohem, 240 (1965), 24
68) and Tenu et al. (l1iur0.T, Bioche
It was purified by classical chromatographic methods as described by M2Q, (1981), 663). Affinity chromatography can also be used, but has the disadvantages of high cost of column solvent synthesis and low capacity. In a typical purification, 60 liters of bacterial growth medium is harvested, the bacteria are then radiofrequency digested, and the suspension is centrifuged. Approximately 1 liter of supernatant containing all the β-galactosidase from the degeneracy is obtained and the enzyme is partially purified by collecting the precipitated material by saturation between 15 and 45 volumes with ammonium tA acid. Concentrate. DI enzyme after dialysis or rP.
Apply to a column of BAE-cellulose and wash out with a gradient. Fractions from the DEAR column binding the enzyme are pooled and applied directly to a hydroxyapatite column and eluted with a phosphoric acid gradient. The enzyme-containing fractions are again pooled and then applied directly to a DEAR-Sepharose 0L4B column. Elution in a certain gradient is 500-1000
# of homogeneous β- produced lactosidase. The hydroxide apatite column can be used with Sephacryl 56 without loss of efficiency.
Can be replaced by 000 columns.

The 'dIg'enzyme can be prepared without loss of substantial or potential catalytic activity by suspending it in a buffer solution of 50 ml of saturated ammonium sulfate containing mercazotoethanol or other sulfhydryl preservative. You can try to save even months.

Antibody-producing mice were incubated with purified wild-type β-galactosidase, and the mice were incubated with Kohler and Miletein (Bur
, J.

The pancreas was lysed along with a bone marrow cell line according to the method derived from Eng. Lnunox; (5 (1975), 511). The thawed cells were cultured in vitro in multiwell plates, and the supernatant solution was probed for anti-β-lactosidase antibodies that activated the otherwise inactive mutant enzyme. Cells from wells that produced the desired antibody were replicate cloned by limited dilution. Monoclonal antibody cell lines were thus formed and grown as needed either in vitro using tissue culture or in vivo by inoculating mice intraperitoneally with ascites fluid. Antibodies were purified from culture media or ascites fluid by chromatography on ion-exchange cellulose.

Four cell lines producing antibodies were formed, BG-18,
They were numbered k3G-19, BG-79, and J3G-81. The rabbit geloprine produced by these strains is an inactive purified mutation! ! When mixed with 11 enzymes, β-
conferred galactosidase activity.

Antibodies from the BG-79 strain showed special properties.

When mixed with antibodies from any of the other six strains and added to the inactive mutant enzyme, there was a synergistic increase in enzymatic activity that would be expected from the saturation for any of the antibodies used alone. It has been discovered that it is possible to produce greater activity than Its activity is increased by the use of hamethanol, ethanediol, propane-1°2-diol or other compounds containing a hydroxyl function, and additionally by another mechanism sugar alcohols, especially sorbitol and mannitol. But it is especially increased by D-arabitol.

The observed synergy and the effect of added methanol are the first
It is shown in the table.

According to Table 41 BG-79 7.6 Shihada 16 4.
6±0.4BG-810,86±0.04 0.
2±0.0048G-18゛ 12.5±0.0! +
32±0.258G-191,0±o,oi
o, ss ±0.003BG-790BG
-8128± 1.2 46 ± 1.8BG
-790BG-1910,9±0.2 16.
9±0.1BG-79+BG-1842,5±0.5
95.5±6.5BG-180BG-1918
- Enzyme activity in the presence of excess antibody increases in μmole (7)
It is expressed as O-nitrophenol/n mole enzyme/min and is quantified using O-nitrophenyl-β-D-galactone as substrate.

Monoclonal antibody numbers BG-18, BG-19, BG-
79, BG-81 is Wellcomea Diagno
tics.

Available from Dartford, UK.

Antibody Preparation Monoclonal antibodies stored as lyophilized powders were rehydrated and then fractionated by precipitation with 50 ml of saturated ammonium sulfate. After resuspension and gel filtration of the harvested precipitate, the clonal antibodies were then transferred to a DB A-cellulose column or a Procion-6B column replacing the Blo-Gel DB in a 1QmM phosphate buffer bath. Applied at p)l 7.0.

The bathed antibody is a substantially homogeneous resuspended lyophilized powder 1
The yield of antibodies was up to 9 out of 45 per 0-.

Purified clonal antibodies were displaced by treatment with N-hydroxysuccinimide ester or other suitable active derivative of the ligand in question. For Cortisol and Tortoise, the optimal concentration of 10 molecules of N (cortisol-21
-hemisuccinyl)-succinate with purified antibodies of each molecule and 10 volumes of dimethylformamide at 0°C for 16-20 hours.
[1. Reacted with pi-17,4 in a buffer solution containing saline.

Displaced antibodies were precipitated with 70% saturated ammonium sulfate, washed by resuspension in 70 g of saturated ammonium sulfate, and centrifuged at least twice to remove all cortisol-21-hemisuccinate except for protein-bound. Traces were removed by filtration. Dilute the product appropriately,
Freeze-drying methods could be substituted for storage frozen.

For diphenylhydantoinni,! 20 molecules of N-hydroxysuccinimide ester of 1-(N-carboxylmethyl)-diphenylhydantoin were incubated with each molecule of purified antibody at room temperature (20°C) for 16 hours at 0.01 phosphoric acid.
The reaction was carried out at pal 7.4 in a slow bath solution containing M and sodium chloride. The substituted antibody was converted from free 3-(N-carboxymethyl)-diphenylhydantoin to BiOgel.
200-40 rp filtration using a P1Q column.
Fibrinogen and fibrinogen degradation products were separated using a column equilibrated with 0.15 M saline in 14 samples of 6-Pesodocorium at 0 mesh. The D fragment was reacted with 8-acetylmercaptosuccinic anhydride to introduce a protected thiol group. Monoclonal antibodies were prepared using succinimidyl 4-(N-?L/nomitomethyl)cyclohexane-1 to introduce the maleimide residue.
- reacted with carboxylates. For coupling, the tethered thiol groups in fibrinone or Dfi pieces were released by treatment with hydroxylamine 25mM and -7 for 1 hour at room temperature. The thiol proteins or fragments were then mixed with maleimide-monoclonal antibodies. - After day and night couplings, excess maleimide residue is blocked by treatment with conjugate mercaptoethanol and then N-ethylmaleimide is treated to block excess thiol groups. The conjugate was then purified by chromatography on Sepharose CL-4B for the fiderinone antibody and Sephacryl 8-300 for the single antibody, but the initial eluted protein is useful.

Chemical name: 9-(2-hydroxy-ethoxymethyl)guanine Antiviral agent acyclovir
ir), N- of succinyl acyclovir
1. Ten molecules of droxytechnimide ester were added to each molecule of purified antibody and phosphoric acid was added for 16 hours at room temperature (20°C).
The reaction was carried out at -7.4 in a buffer solution containing 0.1 M and sodium chloride. Displaced antibodies are converted from free small molecules to BiO
By Delu filtration using a column of gel P i Q,
6wt pet volume 11 with 200-400 mesh
The 1d sample was separated on a column equilibrated with 0.15 M monosaline.

A sample of serum or urine 50a# believed to contain 0 to 1000 picomolea of cortisol was directed against cortisol in 1 pmol of cortisol-monoclonal B()-79 antibody conjugate, 20 μj. Purification from antiserum: g060 pmol, 2mM sodium 8-anilino-1-naphthylenesulfonate 50μIc? 7.6). 37
Approximately 1 pmol of activatable β-galactosidase, already mixed with 130 nmol dithiothreitol and 20 pmol of the second unsubstituted activating antibody BG-81, was incubated for 30 min at °C in 10 methanol. -7mM in 100mM phosphate buffer solution containing -7,40
200 μ of o-nitrophenyl-β-D-ka9/)cytosubstrate! added to. Continue the incubation for another 30 minutes and then reduce the optical density to 420 nm.
The reading was recorded and the reaction was stopped with 0.4 M sodium carbonate containing 10 dimethyl-sulfoxide or other organic solvent or 11 sodium deoxycholate. Standards were prepared over the appropriate range, and blanks (no anti-cortisol, no substituted antibodies) were also prepared for each sample. The results are shown in the second section below.
shown in the table.

Presence of cortisol Optical density 420 (p
mol ) 0 0.6B5
0.9510
1.0720 1
．． 1240 1.18
60 1.21100
1.251000
Analysis for 1.36fIJ2-diphenylhydantoin 0.5 to 2.5 n
A sample of 25 µl of serum believed to contain diphenylhydantoin of M. moba was prepared with 4 picomoles of diphenylhydantoin-singular BG-79 antibody conjugate in 25 µl and 25 µl of the antiserum directed against diphenylhydantoin! It was mixed with picomole of refinery gG90Q. The tube was placed incubating at 67°C and 500 nmoles of dithiothreitol and 2
Six moles of activatable β-galactosidase enzyme were added to 0 pmol of the second non-substituted activating antibody BG-81, which was already mixed. -7,4 in 100 mM phosphate buffer solution of 7,4
200μ of galactoside substrate was then added for a total incubation volume of 325μ. Incubation lasted for 15 minutes at 6 °C, then 1 part of 2M sodium carbonate containing 11 parts of sodium deoxycholate.
The reaction was stopped at - and the optical density was measured at 420 nm. 5 to 20 μg/diphenylhydantoin standard
A suitable range of lrt sera were prepared and blanks (no anti-phenytoin, no diphenylhydantoin-conjugated antibodies) for each sample were also prepared. The results are shown in Table 3 below.

oO,175 50,225 100,241 200,247 Control: No antibody 0.328 Control: No conjugate 0.074 A 10 μl sample of serum believed to contain 0.6 to 24 pmol of fragment D'g was mixed with Reagent 1 at room temperature. The mixture was mixed and reacted for 5 minutes. Next, 20 μl of reagent 2 was added and incubation was started at 67° C. for 10 minutes. Then 500 μl of substrate was added and incubation continued for 5 minutes at 67°C. The reaction was then stopped by the addition of 100 μl of stopper.

The optical density of the bath solution was read at 420 nm. Fragment standards were prepared over the appropriate range 5 to 200 μg/+d serum. The reagents used and the results obtained are shown in Tables 4 and 5, respectively.

Table 4 Analytical reagents for fibrinogen degradation product DK Dithiothreitol 250 mM in water 400 μl Reagent 2 Conjugate of cloned gG-D fragment = 800 μl, arabic,
Toll 6001 Rfi/l, 115 μl - standard sheep immunoglobulinnia, in 200 μl buffer solution.

Buffer solutions used for reagents 1 and 2 and substrate 50mM) Lith 25mM Sodium chloride 10mM Magnesium chloride 1mM II! DTA-7,5 substrate 2.5 0-nitrophenyl β-D- in a buffer solution containing moral methanol is lactose IP2.5119/s
g Stop reagent 2M glycine 0.2 Sodium cholate Table 5 Fragment Dμg/ld Optical density 420
nm0 0.4041
5 0.40925
0.41240
0.42665
0.464100
0.548200
0.566 Example 4 - Analysis for Fibrinogen A 20 μl sample of serum believed to contain 0 to 31 Jv/- of fibrinogen was purified from an antiserum directed against fibrinogen.
- mixed with 20 μl of the solution. 20 μl of conjugated fiderinoden-egG(79), optical density 28”hm
+ 0.22 K then enzyme mixture 20 µl c#l of ammonium sulfate suspension 6 µ/, 10 da/- dithiothreitol 100 µJ, 250 mM Tris mild fl solution 1- and flask growth rate clonal antibody BG-811m
g) was added. The mixed solution was incubated at 67°C for 5 min and then 7 m O-nitrophenyl-β-D-galactoside in Tris buffer containing 10 methanol was added.
200 μl' of M was added. Incubation Y2
Continue at 37°C for 0 min and then react with 0.4 M sodium carbonate containing 1 ml of sodium deo-sicolate.
Stopped at 00 μl. Standards were prepared over the appropriate range and blanks containing no substituted monoclonal antibodies for each sample were also prepared.

The results are shown in Table 6 below.

Table 6 Fibrinone standard curve 0 0.518 0.031 0.626 0.063 0.667 0.125 0.769 0.25 0.892 0.50 0.955 1.0 1.102 The trusted disintegration was diluted 4 times in dk buffer solution, then 10 μl of the sample was mixed with 20 μl of Trial 4A at room temperature and allowed to react for 5 minutes. Then 20 μm of reagent B
was added and incubation continued for 10 minutes at room temperature. 500 μl of substrate, previously warmed to 37° C., was added and incubation was started for 5 minutes in a 67° C. water bath.

The reaction was then stopped by the addition of 100 μg of stop reagent and the optical density of the solution was read at 420 nm.

Acyclovir standards were prepared over the appropriate range from 1 to 10 μM in serum. The reagents used and the results obtained are shown in Tables 7 and 8, respectively.

Analytical reagent for No. 7 Cyclovir Reagent A D(+)arabitol 1.2
g buffer solution 6000μ!
Reagent B O, 11 v/- conjugate of single clone gG-acyclovir at 20 μg/- in buffer bath with standard sheep immunoglobulin, buffer solution used for reagents A and B and substrate 50
25mM sodium chloride [3mM magnesium chloride 1mM EDTA; -6 stop reagent 2M lysine 2 Q Q m M EDTA 0.2 Sodium cholate Adjust aMi to 110.1 p) No. 8
Table Acyclovir standard curve 0 0.529 0.345 2.5 0.3910.595 5 0.569 0.575 7.5 0.6450.629 10 0.675 0.665 Agent Asamura Kangai 4 people

Claims (1)

[Scope of Claims] (1) A method for determining or quantifying the presence of a substance in a medium presumed to contain the substance, comprising: a) an antibody capable of activating a substantially inactive enzyme; A conjugate consisting of a substance bound to the substance, a 41-membered antibody or binding protein to the substance, and an excess of a substantially inactive enzyme are mixed together with the medium, and any substance in the medium is specific for the substance. b) adding the components in an order such that the enzyme does not mix with the conjugate before it has had a chance to react with the binding protein or antibody; and b) adding a substrate for the enzyme and measuring the activity of the enzyme. , a method consisting of. +21&) Adding medium to a mixture of a binding protein or antibody specific for the substance and a substantially inactive enzyme. -1)) '*adding a conjugate of that substance conjugated to an antibody capable of activating a qualitatively inactive enzyme, and C) dI
(31!L) A conjugate consisting of a substance bound to an antibody capable of activating a substantially inactive enzyme: and the substance together with a t!!iJA-like binding protein or antibody and a medium, b) adding an excess of a substantially inactive enzyme, and C) adding a substrate for the enzyme to increase enzyme activity. (4) Any one of the above paragraphs 1X(1) to ta), wherein the antibody capable of activating a substantially inactive enzyme is a focal clonal antibody. Two ways. (5) Substantially inert #! Jl that activates e and fulfills
! Mix another antibody with the enzyme before it is added to any of the other ingredients (from paragraph 11 above).
4) - Any one of the methods. (6) β- has an activity less than one of the activity of lactosidase when the substantially inactive # is activated.
Any one of the above-mentioned methods from (11% to (5)), in which β- is a mutant type 14 of lactosidase. (6) above as
Section method. (8) The method according to any one of items (1) to (7) above, wherein the lipid to be tested is an antiepileptic drug, an antibiotic, an antiviral agent, a hormone, or a protein. (9) Is the analysis constrained by 7.4, alkanol or? The method of the above IE [(any one of paragraphs 11 to (8)) carried out at between 10 °C and 45 °C in the presence of D-arapital. Any one of the methods from item 1) to item (9). (1υ The above method (1) carried out in the presence of methanol.
Any one method from item 01 to item 01. (to) The analysis changed the - to about -10 and the chelating agent and lipid solubilizer for magnesium to 09
Any one way to the term. U A conjugate consisting of the substance to be measured as defined above in which a substantially inactive enzyme is linked to an activatable antibody. Reagent 1: An antibody specific for the substance to be analyzed mixed with a substantially inactive enzyme Reagent 2: Conjugate reagent 3 consisting of a substance bound to an antibody capable of activating a substantially inactive enzyme : A test kit consisting of: a substrate reagent for a substantially inactive enzyme; 4: a stop agent.