JPH07227298A - Method for immunoassay - Google Patents

Abstract

PURPOSE:To measure a target measurement substance in high sensitivity by an enzyme cycling by reacting a liposome having immobilized an antigen against an antibody of a target to be measured to a membrane and containing an enzyme with a complement, a coenzyme such as a thio NAD, an NAD, etc., a dehydrogenase and a specimen. CONSTITUTION:A liposome which has immobilized an antigen against an antibody of a target measurement substance to the surface of a membrane, contains a detectable enzyme in the interior and is subjected to a membrane dissolving action with a complement is reacted with the complement, at least one of a thio NADP a thio NAD, a NADP, a NAD, etc., as a coenzyme, a dehydrogenase and a target measurement substance-containing specimen to be tested. Activity of the enzyme in the liposome made measurable by the dissolution reaction of the liposome following the activation of the complement is measured by supplying a substance formed by action of the enzyme to an enzyme cycling system of the formula (A1 is thio NANDP, thio NAD, NADP or NAD; A2 is a reduction type product of A1; B1 is a reduction type coenzyme different from A1; B2 is oxidation type product of B1 to measure the target measurement substance in the specimen to be tested in high sensitivity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly sensitive immunoassay method and reagent utilizing the membrane lysis reaction of liposomes with complement and the enzyme cycling method.

[0002]

2. Description of the Related Art Conventional immunoassay methods include, for example, a radioimmunoassay method (RIA method) and an enzyme immunoassay method (EIA method). The RIA method uses a radioactive isotope, although it is quantitative. Limited space,
Problems such as special facilities and equipment are required, EIA
The method has a problem that the normal operation is complicated. In addition, there is a problem in that any of the above measuring methods requires a long time for analysis.

On the other hand, the immunoassay method (complement immunoassay method) which utilizes the lytic action of liposomes for complement is a homogeneous assay system and has the advantage that it can be easily and quickly quantified. Therefore, the immunoassay has important and potential utility in clinical diagnosis. One of the usefulness is that the antigen-antibody reaction and the measurement of the marker substance released from the dissolved liposome can be carried out simultaneously in the same measurement solution, and such a homogeneous immunoassay has a liquid phase and a solid phase. This is in contrast to conventional immunoassay methods such as the RIA method and the EIA method, which include a step of separating the phases (B / F separation).

As an immunoassay utilizing such a homogeneous liposome lysis reaction, there is a method by Cole et al. (US Pat. No. 4,342,826), which utilizes a liposome having an antigen immobilized on the surface and an enzyme encapsulated therein. An immunoassay is disclosed. The liposome is immunospecifically destroyed in the presence of an antibody and an active complement, and further the released enzyme marker is detected by utilizing a homogeneous phase reaction. The phenomenon mediated by this complement has been of interest in many literatures (Lipo
somes, MJ.Ostro, MARCEL DEKKER, INC; p209, 1983
reference). Since such a liposome lysis reaction does not involve B / F separation, no special equipment is required. By selecting the marker substance to be encapsulated in the liposome, an automatic analyzer used in clinical laboratories can be selected. Can be applied. However, the reagent reaction time in such an automatic analyzer is usually about 5 to 20 minutes, and a method of inducing a larger signal response in a short time even when utilizing a liposome lysis reaction has been desired. In addition, it is known that the amount of antibody used is reduced in order to construct a highly sensitive immunoassay, but when the amount of antibody used is reduced, the signal also decreases accordingly, making measurement impossible. There were many cases. Therefore, a system that increases a slight signal has been required in order to highly sensitively measure a measurement target that is present in serum and plasma at low concentrations.

Recently, in the field of biochemistry, low molecular weight compounds such as glucose 6-phosphate, myo-inositol, hydroxybutyric acid, malic acid, oxaloacetic acid, and carnitine are treated with thio-NAD.
(P) s, NAD (Ps), and a method for highly sensitive measurement using an enzyme cycling system that reacts with dehydrogenase have been disclosed, and the possibility of detecting the enzyme activity that produces small molecules is also suggested. (JP-A-4-335898, JP-A-4-126099, JP-A-4-15)
8799, JP-A-4-179498, and JP-A-3-251196).

This method is an excellent method in which conventional enzyme cycling, which requires a multi-step process of enzyme cycling reaction, reaction termination, and detection of an indicator substance, is continuously carried out in one solution. However, in these patents, although the application to the EIA method involving B / F separation is suggested, the application to the immunoassay method making use of the characteristic of this method that the enzyme cycling can be performed in one solution is shown. It has not been.

[0007]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention An object of the present invention is a substance (hereinafter abbreviated as an enzyme product) produced by the action of an enzyme contained in the liposome, which can be measured by the dissolution reaction of the liposome. , As it is, Thio NAD (P) type,
Introducing NAD (P) s, an enzyme cycling system that uses dehydrogenase, to provide an immunoassay method intended for highly sensitive automated analysis by obtaining a larger signal response in a shorter time compared to conventional methods. is there.

[0008]

In order to solve the above problems, the present invention has the following constitution. `` (1) A liposome immobilizing an antigen against an antibody that is a substance to be measured on the surface of a membrane and encapsulating a detectable enzyme inside the membrane, which undergoes a membrane-dissolving action by complement activity, and (2) complement, ) As a coenzyme, thionicotine amidene adenine nucleotide phosphates (hereinafter thio NADP
) And thionicotinamid adenine nucleotides (hereinafter thio)
At least one selected from the group consisting of NADs) and at least one selected from the group consisting of nicotine amido adenine nucleotide nucleophosphates (hereinafter referred to as NADPs) and nicotine amino adenine nucleotide nucleosides (hereinafter referred to as NADs); 4) dehydrogenase, and (5) reacting a test sample containing the substance to be measured, the activity of the enzyme encapsulated in the liposome, which can be measured by the dissolution reaction of the liposome accompanying the activation of complement, An immunoassay method characterized in that a substance produced by the action of the enzyme is measured by subjecting it to an enzyme cycling system of the following formula 1 and the substance to be measured in a test sample is quantified based on the obtained activity value. .

[Formula 5] (In the formula, A1 represents thioNADPs, thioNADs, NADPs or NADs, and A2 represents a reduced product of A1. B1 represents A
When 1 is thio-NADPs or thio-NADs, it indicates reduced NADPs or reduced-NADs, and when A1 is NADPs or NADs, it indicates reduced-thioNADPs or reduced-thioNADs, and B2
Indicates an oxidized product of B1. ) "(1) An antigen or a part of the substance to be measured is immobilized on the surface of the membrane, and a liposome that has a detectable enzyme inside and is subjected to membrane lysis by complement activity, and (2) measurement As an antibody against the object, (3) complement, and (4) coenzyme,
A test sample containing at least one selected from the group consisting of thio-NADPs and thio-NADs and at least one selected from the group consisting of NADPs and NADs, (5) dehydrogenase, and (6) a substance to be measured. And reacting the enzyme with the activity of the enzyme encapsulated in the liposome, which can be measured by the dissolution reaction of the liposome associated with the activation of complement, and subjecting the substance to the enzyme cycling system of the following formula 1 An immunoassay method characterized by quantifying a substance to be measured in a test sample based on the obtained activity value.

[Formula 6] (In the formula, A1 represents thioNADPs, thioNADs, NADPs or NADs, and A2 represents a reduced product of A1. B1 represents A
When 1 is thio-NADPs or thio-NADs, it indicates reduced NADPs or reduced-NADs, and when A1 is NADPs or NADs, it indicates reduced-thioNADPs or reduced-thioNADs, and B2
Indicates an oxidized product of B1. ) ”(1) A liposome immobilizing an antibody against an antigen, which is a substance to be measured, on the surface of a membrane and encapsulating a detectable enzyme inside, which is subjected to a membrane-dissolving action by complement activity, and (2) complement. (3) As a coenzyme, at least one selected from the group consisting of thioNADPs and thioNADs and at least one selected from the group consisting of NADPs and NADs, (4) dehydrogenase, (5) measurement target A substance that produces the activity of an enzyme encapsulated in a liposome, which can be measured by the dissolution reaction of the liposome accompanying activation of complement, by reacting with a test sample containing the substance by the action of the enzyme is represented by the following formula 1 The immunoassay method characterized by quantifying the substance to be measured in the test sample based on the obtained activity value.

[Formula 7] (In the formula, A1 represents thioNADPs, thioNADs, NADPs or NADs, and A2 represents a reduced product of A1. B1 represents A
When 1 is thio-NADPs or thio-NADs, it indicates reduced NADPs or reduced-NADs, and when A1 is NADPs or NADs, it indicates reduced-thioNADPs or reduced-thioNADs, and B2
Indicates an oxidized product of B1. ) ”(1) An antibody against a substance to be measured or a part of the antibody is immobilized on the surface of the membrane and has a detectable enzyme inside, and is subjected to a membrane-dissolving action by complement activity; An antibody against the object, (3) complement, and (4) as a coenzyme, at least one selected from the group consisting of thio-NADPs and thio-NADs, and at least one selected from the group consisting of NADPs and NADs, (5) dehydrogenase, (6) react with a test sample containing a substance to be measured, the activity of the enzyme encapsulated in the liposome, which can be measured by the dissolution reaction of the liposome with the activation of complement, An immunoassay method characterized in that a substance produced by the action of the enzyme is measured by subjecting it to an enzyme cycling system of the following formula 1 and the substance to be measured in a test sample is quantified based on the obtained activity value. .

[Formula 8] (In the formula, A1 represents thioNADPs, thioNADs, NADPs or NADs, and A2 represents a reduced product of A1. B1 represents A
When 1 is thio-NADPs or thio-NADs, it indicates reduced NADPs or reduced-NADs, and when A1 is NADPs or NADs, it indicates reduced-thioNADPs or reduced-thioNADs, and B2
Indicates an oxidized product of B1. ) "

That is, the inventors of the present invention have conducted diligent research to solve the above-mentioned problems, and as a result, in a homogeneous immunoassay method using liposomes, the marker to be encapsulated in the liposomes can be an enzyme and can be measured by the lysis reaction of the liposomes. The obtained enzyme activity is regarded as the amount of the enzyme product produced by the action of the enzyme, and the enzyme product is analyzed as thioNAD.
(P), NAD (P), by using an enzyme cycling system that uses a dehydrogenase, the measurement system remains homogeneous,
They found that a large signal can be obtained in a short time, and completed the present invention.

The present invention uses an enzyme as a marker in the complement immunoassay known per se, and the resulting enzyme product is used as an enzyme using thio-NAD (P) s, NAD (P) s and dehydrogenase. It is an invention characterized by increasing the signal in a cycling system, and other operations and reagents to be used are the same as known complement immunoassay methods, enzyme cycling method operations and reagents, etc. It's enough. The liposome used in the present invention is not particularly limited as long as it is composed of a membrane that is dissolved by complement activity, and the composition, preparation method, antigen, antibody and the like can be arbitrarily selected.

That is, as the method for preparing the liposome used in the present invention, for example, the conventionally known vortex swing method, ultrasonic method, surfactant removal method, reverse phase evaporation method, etc.
(REV method), ethanol injection method, ether injection method, pre-
Pre-Vesicle method, French Press Extrusion method, Ca ²⁺ fusion method,
Annealing method, freeze-thaw fusion method, W / O / W emulsion method, etc., and recently SMGruner et al. (Bioche
Mistry, Vol. 24, 2833, 1985) Stabl
e Plurilamellar Vesicle method (SPLV method), a method of using lipopolysaccharide as a part of membrane constituents (Japanese Patent Laid-Open No. 63-107742)
No. gazette) and the like. In addition, as its main membrane constituents, natural lecithin (for example, egg yolk lecithin, soybean lecithin, etc.) and dipalmitoylphosphatidylcholine (DPPC), dimyristoyl which are used as membrane constituents in ordinary liposome preparation Phosphatidylcholine (DMPC), Distearoylphosphatidylcholine (DSPC), Dioleoyl phosphatidylcholine (DOP)
C), dipalmitoylphosphatidylethanolamine
(DPPE), Dimyristoylphosphatidylethanolamine (DMPE), Egg yolk phosphatidylglycerol, Dipalmitoylphosphatidylglycerol (DPPG), Dimyristoylphosphatidic acid (DMPA), Dipalmitoylphosphatidic acid (DPPA) , One or more phospholipids such as palmitoyl oleoylphosphatidylcholine (POPC), or a mixed system of these and cholesterols,
Alternatively, all of these may be combined with lipopolysaccharide and the like.

A method for sensitizing (immobilizing) an antigen or an antibody on the surface of a liposome membrane is also known as a crosslinking method (Bioche).
mistry, Volume 20, 4229-4238, 1981, J. Biol. Bioche
m., Vol. 257, pp. 286-288, 1982 etc.), lipid activation method and the like, and other methods may of course be used. In addition, the antigen to be immobilized includes not only the antigen itself but also a part of the antigen containing a specific antigen recognition site, and the antibody to be immobilized is not only the antibody itself (F
It goes without saying that ab) ₂ , Fab ', Fab, etc. may be used.

The enzyme contained in the liposome may be such that the enzyme product can reversibly react with dehydrogenase in the presence of coenzymes such as thio-NAD (P) s and NAD (P) s. Examples thereof include kinases such as hexokinase, pyruvate kinase and glycerol kinase, and hydratases such as fumarate hydratase and aconitate hydratase. In addition, as the dehydrogenase for performing the cycling reaction, liposomes such as glucose 6-phosphate dehydrogenase, malate dehydrogenase, lactate dehydrogenase, glycerol 3-phosphate dehydrogenase, and isocitrate dehydrogenase. What is necessary is just to use the product of the enzyme encapsulated in the above as a substrate, and use thio NAD (P) s and NAD (P) s as coenzymes.

Table 1 shows typical combinations of the enzyme, the enzyme product, and the dehydrogenase to be encapsulated in the liposome.

[0015]

[Table 1]

Among the above-mentioned combinations, as a combination in which a stable titer can be easily obtained, hexokinase is used as the enzyme to be encapsulated in the liposome, and glucose 6-phosphate dehydrogenase is used as the enzyme for enzyme cycling. Desirably mentioned. In this case, the solution of hexokinase used when preparing the liposome is usually 5
00-4000 U / ml, preferably 750-250
An enzyme solution having a concentration of 0 U / ml may be used. The amount of glucose 6-phosphate dehydrogenase used for enzyme cycling may be 5 U / ml to 300 U / ml, preferably 50 U / ml to 200 U / ml. ThioNAD and NADH, thioNADP and NADPH, thioNADP and NADH, thioNAD and N are coenzymes required for enzyme cycling.
A combination of ADPH is conceivable, but it may be appropriately selected in consideration of the substrate specificity of the dehydrogenase to be used, reaction conditions and the like. Regarding the concentrations to be used, it is sufficient to use concentrations known per se, but in the case of the combination of glucose 6-phosphate dehydrogenase, thio NAD and NADH, thio NAD
0.05 mM to 0.5 mM, NADH 0.1 mM to 1 m
A concentration of M is desirable.

The pH of the reaction solution is such that an antigen-antibody reaction, a complement reaction and an enzyme cycling reaction can occur.
For example, in the case of a combination of glucose 6-phosphate dehydrogenase, thio NAD and NADH, p is selected.
H6-8, preferably 6.5-7.

When the amount of the liposome used in the present invention is shown by the concentration in the final reaction solution, the amount of phospholipid contained in the liposome is usually 0.1 to 50 nmol / ml.
It is preferably 1 to 10 nmol / ml.

As the complement used in the present invention,
For example, any complement commonly used in this field, such as those obtained from blood of animals such as humans, guinea pigs, horses, goats, sheep, and rabbits, which have been appropriately purified according to a conventional method, can be mentioned without exception.

The present invention is not limited to the method used, but can be sufficiently applied to a measurement system using an automatic analyzer, and the measurement can be performed easily and quickly. There are no particular restrictions on the combination of reagents, etc. when performing measurements using an automatic analyzer, depending on the model, or reagents considered to be the best considering other factors. A combination may be appropriately selected and used.

The present invention will be described in more detail below with reference to experimental examples and examples, but the present invention is not limited to these examples.

[0022]

【Example】

Example 1. Enhancement of Response by Enzyme Cycling Liposomes encapsulating hexokinase were used as microcapsules, and phenobarbital, an antiepileptic drug, was measured by combining this with an enzyme cycling reaction by glucose 6-phosphate dehydrogenase. A phospholipid derivative of phenobarbital [Clin. Chem., 38 , 808 (199
2)] as a membrane component was prepared as follows. Dimyristoylphosphatidylcholine 72 μmol,
Dimyristoylphosphatidylglycerol 8 μmol,
5 ml of 80 μmol cholesterol and 0.8 μmol phosphatidylethanolamine derivative of phenobarbital
Was dissolved in chloroform and dried under reduced pressure. To this, a solution of hexokinase in Tris-HCl (0.01mol / l, pH 7.8)
7.5 ml of 1100 U / ml was added and mixed, and the lipid hydration solution thus obtained was sized through a 0.2 μm membrane. Next, the enzyme not encapsulated in the liposome was removed by repeating ultracentrifugation (100000xg). The prepared liposome is 0.1m
ol / l Tris-HCl (containing 0.03mol / l NaCl, pH 7.8)
It was suspended in and stored in a refrigerator. (Reaction solution) 50 mM MES buffer (pH 6.5) 10 mM magnesium chloride 0.375 mM thio NAD 0.375 mM NADH 100 U / ml glucose 6-phosphate dehydrogenase (Leuconosto)
c Mesenteroides origin) 0.5 mM glucose 1.5 mM ATP rabbit anti-phenobarbital antibody 640-fold diluted phenobarbital liposomes 1000-fold diluted 5% guinea pig serum (manipulated) anti-phenobarbital antibody, thio NAD, N
200 μl of a buffer solution containing ADH, glucose 6-phosphate dehydrogenase, glucose and ATP was added. After heating at 37 ℃ for 5 minutes, a buffer solution containing phenobarbital-sensitized liposomes containing hexokinase and guinea pig complement 200
μl was added, and the mixture was reacted at 37 ° C. for 5 minutes, and the change in absorbance at 405 nm derived from the hexokinase activity in the liposome, which became measurable by complement activity, was measured. Further, as a comparison with this method, there is a conventional method which does not carry out an enzyme cycling reaction, that is, a method of detecting an increased absorption of thioNADH at 405 nm by combining glucose 6-phosphate dehydrogenase and thioNAD. The results are summarized in FIG. As a result, it can be seen that the response is increased by using the enzyme cycling system.

Example 2. Quantification of digoxin Digoxin, which is one of the cardiac glycosides, was measured by using a liposome encapsulating hexokinase as a microcapsule and combining it with an enzyme cycling reaction by glucose 6-phosphate dehydrogenase. A liposome containing hexokinase and having a phospholipid derivative of digoxin [see Japanese Patent Publication No. 62-501800] as a membrane component was prepared as follows. Dimyristoylphosphatidylcholine 72 μmol, dimyristoylphosphatidylglycerol 8 μmol, cholesterol 80 μmol and phosphatidylethanolamine derivative of digoxin
0.8 μmol was dissolved in 5 ml of chloroform and dried under reduced pressure. To this, hexokinase tris-HCl (0.01 mol
/ l, pH 7.8) 7.5 ml of 1100 U / ml solution was added and mixed. Then, the obtained lipid hydration solution was sized by passing through a 0.2 μm membrane. Next, the enzyme not encapsulated in the liposome was removed by repeating ultracentrifugation (100000xg). The prepared liposomes were 0.1 mol / l Tris-HCl (0.03 mol / l
It was suspended in NaCl containing pH 7.8) and stored under refrigeration. (Reaction solution) 50 mM MES buffer (pH 6.5) 10 mM magnesium chloride 150 mM sodium chloride 0.375 mM thioNAD 0.375 mM NADH 100U / ml glucose 6-phosphate dehydrogenase (Leuconosto)
c Mesenteroides origin) 0.5mM glucose 1.5mM ATP goat anti-digoxin antibody 80000 times diluted liposomes 1000 times diluted 10% goat serum (manipulation) 10 μl of standard solution of digoxin, anti-digoxin antibody, thio-NAD, NADH, glucose 6-phosphate dehydrogenase Buffer containing glucose, glucose and ATP (pH 6.5) 20
0 μl was added, and the mixture was reacted at 37 ° C. for 5 minutes. Next, hexokinase-encapsulated liposomes (lipid concentration
(5 nmol / ml) and 200 μl of a buffer solution containing goat complement were added and reacted at 37 ° C. for 5 minutes. Hexokinase activity in liposomes solubilized by anti-digoxin antibody and complement was detected at the absorption maximum wavelength of thioNADH at 405 nm. The result is shown in FIG. As is clear from this result, it can be seen that digoxin can be measured with high sensitivity by using the present invention. In addition, the conventional reaction system that does not utilize the enzyme cycling system has a small response and it was impossible to prepare a calibration curve for digoxin.

[0024]

INDUSTRIAL APPLICABILITY The present invention uses a marker to be encapsulated in a liposome as an enzyme in a complement immunoassay using serum, plasma or the like as a sample, and the enzymatic activity that can be measured by the lysis reaction of the liposome The amount of the enzyme product produced by the action is regarded as
By carrying out a signal-enhancing reaction by subjecting it to an enzyme cycling system that uses enzymes, NAD (P) s, and dehydrogenase to obtain a large signal in a short time while maintaining a homogeneous assay system. It provides a measuring method. As a result, it becomes possible to measure low-concentration substances that could not be measured in the past, and a homogeneous measurement system can be used for items that are conventionally measured by fluorescence immunoassay or EIA for B / F separation. It is an invention whose effect is recognized in that it can be measured by an automatic analyzer.

[0025]

[Brief description of drawings]

FIG. 1 shows the change in absorbance (Abs. × 10000) of the reaction solution at 405 nm obtained in Example 1 on the horizontal axis for each reaction time (minutes).
It is plotted for each.

FIG. 2 shows each digoxin concentration (ng / m 2) obtained in Example 2.
It is a calibration curve obtained by plotting the absorbance (Abs. × 10000) at 405 nm against l).

[Explanation of symbols]

In FIG. 1,-++-represents the result obtained by the method according to the present invention,
Further, − □ − indicates the results obtained by the conventional method in which the enzyme cycling reaction is not performed.

Claims (8)

[Claims] 1. (1) A liposome having an antigen against an antibody, which is a substance to be measured, immobilized on the surface of a membrane and having a detectable enzyme incorporated therein, which is subjected to a membrane-dissolving action by complement activity, and (2) complement. And (3) as the coenzyme, at least one selected from the group consisting of thionicotine amidene adenine nucleotide phosphates (hereinafter referred to as thioNADPs) and thionicotine amidane adenine nucleotides (hereinafter referred to as thioNADs) and nicotineamido Includes at least one selected from the group consisting of adenine nucleotide phosphates (hereinafter referred to as NADPs) and nicotine amidane adenine nucleotites (hereinafter referred to as NADs), (4) dehydrogenase, and (5) a substance to be measured It is measured by reacting with a test sample and measuring the dissolution reaction of liposomes accompanying complement activation. The activity of the enzyme encapsulated in the enabled liposomes was measured by subjecting the substance produced by the action of the enzyme to the enzyme cycling system of the following formula 1, and based on the obtained activity value, An immunoassay method characterized by quantifying a substance to be measured. [Formula 1] (In the formula, A1 represents thioNADPs, thioNADs, NADPs or NADs, and A2 represents a reduced product of A1. B1 represents A
When 1 is thio-NADPs or thio-NADs, it indicates reduced NADPs or reduced-NADs, and when A1 is NADPs or NADs, it indicates reduced-thioNADPs or reduced-thioNADs, and B2
Indicates an oxidized product of B1. ) 2. (1) A liposome which has an antigen, which is a substance to be measured, immobilized on the surface of a membrane, and which has a detectable enzyme inside and which is subjected to a membrane-dissolving action by complement activity, and (2) for the substance to be measured. Antibodies, (3) complement and (4) coenzyme as thioNA
At least one selected from the group consisting of DPs and thio-NADs, at least one selected from the group consisting of NADPs and NADs, and (5) a dehydrogenase, (6) a test sample containing a substance to be measured. By subjecting a substance produced by the action of the enzyme to the activity of the enzyme encapsulated in the liposome, which is made measurable by the reaction of the reaction and the dissolution reaction of the liposome accompanying the activation of complement, to the enzyme cycling system of the following formula 1. An immunoassay method, which comprises measuring and quantifying a substance to be measured in a test sample based on the obtained activity value. [Formula 2] (In the formula, A1 represents thioNADPs, thioNADs, NADPs or NADs, and A2 represents a reduced product of A1. B1 represents A
When 1 is thio-NADPs or thio-NADs, it indicates reduced NADPs or reduced-NADs, and when A1 is NADPs or NADs, it indicates reduced-thioNADPs or reduced-thioNADs, and B2
Indicates an oxidized product of B1. ) 3. (1) A liposome immobilizing an antibody against an antigen, which is a substance to be measured, on the surface of a membrane and encapsulating a detectable enzyme therein, and having a membrane-dissolving action by complement activity, and (2) complement. And (3) as coenzymes, thioNADPs and thioNA
At least one selected from the group consisting of Ds, and at least one selected from the group consisting of NADPs and NADs,
(4) dehydrogenase, (5) reacting with a test sample containing the substance to be measured, the activity of the enzyme encapsulated in the liposome, which can be measured by the dissolution reaction of the liposome accompanying the activation of complement, An immunoassay method characterized in that a substance produced by the action of the enzyme is measured by subjecting it to an enzyme cycling system of the following formula 1 and the substance to be measured in a test sample is quantified based on the obtained activity value. . [Formula 3] (In the formula, A1 represents thioNADPs, thioNADs, NADPs or NADs, and A2 represents a reduced product of A1. B1 represents A
When 1 is thio-NADPs or thio-NADs, it indicates reduced NADPs or reduced-NADs, and when A1 is NADPs or NADs, it indicates reduced-thioNADPs or reduced-thioNADs, and B2
Indicates an oxidized product of B1. ) 4. (1) A liposome immobilizing an antibody against a substance to be measured on the surface of a membrane and encapsulating a detectable enzyme inside, which is subjected to a membrane-dissolving action by complement activity, and (2) an antibody against the substance to be measured. And (3) complement and (4) coenzyme
At least one selected from the group consisting of NADPs and thio-NADs, at least one selected from the group consisting of NADPs and NADs, and (5) dehydrogenase, (6) a test sample containing a substance to be measured. By subjecting a substance produced by the action of the enzyme to the activity of the enzyme encapsulated in the liposome, which is made measurable by the reaction of the reaction and the dissolution reaction of the liposome accompanying the activation of complement, to the enzyme cycling system of the following formula 1. An immunoassay method, which comprises measuring and quantifying a substance to be measured in a test sample based on the obtained activity value. [Formula 4] (In the formula, A1 represents thioNADPs, thioNADs, NADPs or NADs, and A2 represents a reduced product of A1. B1 represents A
When 1 is thio-NADPs or thio-NADs, it indicates reduced NADPs or reduced-NADs, and when A1 is NADPs or NADs, it indicates reduced-thioNADPs or reduced-thioNADs, and B2
Indicates an oxidized product of B1. ) 5. The method according to any one of claims 1 to 4, wherein the thioNADP is thionicotine amido adenine nucleotide phosphate (thio NADP) or thionicotine amido hypoxanthine nucleotide phosphate. 6. The thio NADs are thionicotine amidene adenine nucleotides (thio
NAD) or thionicotine amide hypoxanthine nucleotidide, according to any one of claims 1 to 4. 7. The NADPs are nicotine amidene adenine nucleotide phosphates.
(NADP), acetylphosphoric adenine nucleotide phosphate (acetyl NADP),
The measuring method according to any one of claims 1 to 4, wherein the measuring method is at least one selected from the group consisting of acetylphosphonium hypoxanthine nucleotide phosphate and nicotine amidiohypoxanthine nucleotide phosphate (deamino NADP). 8. The NADs are nicotine amidene adenine nucleotides (NAD),
Acetyl pyridine adenine nucleotide (acetyl NAD), acetyl phenylene phoxanthine nucleotide and nicotine amido phoxanthine nucleotide (deamino N)
5. The measuring method according to claim 1, wherein the measuring method is at least one selected from the group consisting of AD).