JPS62235566A - Glucose-6-phosphoric acid dehydrogenated enzyme labelled composite and enzyme immunological measurement of erythropoietin - Google Patents

Abstract

PURPOSE:To enable highly sensitive measurement of erythropoietin (EPO) by coupling an immunochemically active substance having a thiol group in a molecule to a specified enzyme using a compound given by a specified formula to obtain a stable specified enzyme-labelled composite. CONSTITUTION:An immunocheimcally active substance having a thiol group in a molecule or bonded to a thiol group is coupled to a glucose-6-phosphoric dehydrogenated enzyme as label substance through a compound as given by the formula, wherein m represents an integer of 0-5, R, bivalent 6-member cyclic hydrogen carbon residual group and n, an integer of 0-1. Thus, an enzyme immunological measurement is done using a labelled composite in which erythropoietin or an antibody tended to react specifically with erythropoietin is coupled to a glucose-6-phosphoric acid dehydrogenated enzyme as label substance by a compound as given by the formula.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention relates to reagents used in immunochemical analysis, particularly enzyme immunoassay (hereinafter referred to as EIA), and erythropoietin (hereinafter referred to as EIA) using the reagents. This is related to EIA (sometimes abbreviated as EPO).

[Prior Art] EIA, along with radioimmunoassay (hereinafter abbreviated as RIA), is used as a clinical testing method that can quantify extremely small amounts of substances, such as in diagnosing diseases and understanding pathological conditions. However, since RIA uses radioactive isotopes, its widespread use is limited because it requires special equipment and there are problems with waste. On the other hand, since EIA uses enzymes for labeling, it does not require special equipment and has rapidly developed in recent years as a test method that can be easily performed anywhere.

EIA techniques are broadly classified into competitive methods and non-competitive methods; the former uses a labeled antigen labeled with an enzyme, and the latter uses a labeled antibody labeled with an enzyme. As enzymes used for labeling, peroxidase, β-galactosidase, alkaline phosphadase, etc. are known, but peroxidase-labeled antibodies, in which peroxidase and antibodies are bonded with a maleimide compound, have less nonspecific adsorption and can be used for EIA. It is said that extremely sensitive measurements can be made using a non-competitive method and the Sanderuch method (Japanese Unexamined Patent Publication No.
8-149700).

However, even in the sandwich method using peroxidase-labeled antibodies, if O-phenylenediamine, which is considered to be the most sensitive chromogen and is the substrate for peroxidase, is used, the measurement pack ground may sometimes be detected for unknown reasons. is known to increase, and measurement sensitivity decreases.

Additionally, peroxidase-labeled antibodies can usually be stored in a concentrated solution for several months to a year, but when used for Sanderchsch method measurements, the labeled antibodies can be stored several hundred times in i-buffer containing an appropriate stabilizing agent. It is used after being diluted several thousand times from
The labeled antibody in a diluted state retains its activity for only one week at room temperature and about one month at low temperature even in the state containing the stabilizer, and is extremely unstable, and there are many problems in practical use. I had a problem.

On the other hand, EPO is a hormone with a molecular weight of approximately 40,000 that is mainly produced in the kidneys and controls the differentiation of red blood cells, and the amount of EPO in body fluids may increase or decrease in various anemias and polycythemias. EP is known and found in body fluids.
Accurately measuring the amount of O is extremely important for diagnosing these blood diseases. The normal blood (or body fluid) concentration of EPO is thought to be 0.1 to O,jng/ml.
Highly sensitive measurement methods are required, such as bioassay methods (Clinical Testing, Vol. 22. No. 2.197t1) and mouse bone marrow cells! In vitro bioassay method measuring the uptake of 19F (J, Lab, C1
1n, Med,, Vol, 97°No, 2, 158~
169, 1981) has the drawbacks of low detection sensitivity, complicated operations, and high measurement costs. Therefore, it is desired to realize an EPO measurement method that is simple and can detect the amount of EPO with high accuracy.

[Problems to be Solved by the Invention] Under the circumstances described above, the present inventors have developed an antibody-bound insoluble support, EPO, in which an antibody that specifically reacts with EPO is bound to an insoluble support. Antibody-EP produced on an antibody-bound insoluble support by reacting a sample solution containing EPO with a labeled antibody that is labeled with an enzyme and an antibody that specifically reacts with EPO.
We have completed an enzyme immunoassay method for EPO by measuring O-labeled antibody conjugates, and have already applied for a patent (patent application filed in 1983).
O-39687).

However, even in this technique, since the titer of antibodies that specifically react with EPO is low, satisfactory sensitivity cannot always be obtained even with labeled antibodies labeled with peroxidase or β-galactosidase. The reason for the low titer of antibodies that specifically react with EPO is that EPO is poorly metabolized when non-human animals are immunized with EPO, and that the animal itself physiologically reacts to the administered EPO. It is thought that this can lead to symptoms of extreme anemia.

In view of the current situation, the inventors of the present invention have conducted extensive research into a measurement method that allows high-sensitivity measurement at all times, and reagents for the same.
Using glucose-6-phosphate dehydrogenase (hereinafter sometimes abbreviated as G6PDH) as a labeling enzyme, the following general formula (I) - (I) [where m is an integer of 0 to 5, R is Using a compound represented by a divalent 6-membered cyclic hydrocarbon residue, where n is an integer of 0 to 1, an immunochemically active substance having a thiol group (-SH) or By combining the enzyme with an immunochemical substance into which a thiol group has been introduced, an extremely stable G6PDH-labeled composite label can be used.
The present inventors have discovered that EPO can be measured with extremely high sensitivity by carrying out IA and measuring the enzyme activity using a bioluminescent reaction, and have completed the present invention.

[Means for Solving the Problems] That is, the present invention uses an immunochemically active substance that has a thiol group in its molecule or has a thiol group bonded thereto, and glucose-6-phosphate dehydrogenase as a labeling substance. , the following general formula (I) - (T ) [wherein m represents an integer of 0 to 5, R represents a divalent 6-membered cyclic hydrocarbon residue, and n represents an integer of O to 1] This is a glucose-6-phosphate dehydrogenase labeled complex in that it is bound via a compound represented by:

In addition, the method of the present invention provides an antibody-bound solid phase carrier in which an antibody that specifically reacts with erythropoietin, a test solution containing erythroboethylene, and an antibody that specifically reacts with enzyme-labeled erythropoietin are bound to a solid phase carrier. In the enzyme immunoassay method for erythropoietin in which a test solution containing erythropoietin and an enzyme-labeled antibody that specifically reacts with erythropoietin are reacted, the enzyme as a labeling substance is glucose-6-phosphate dehydrogenase, and the enzyme contains erythropoietin. ,
Or an antibody that specifically reacts with erythropoietin,
The following general formula (I) ... (I) [wherein m represents an integer of 0 to 5, R represents a divalent 6-membered cyclic hydrocarbon residue, and n represents an integer of 0 to 1, respectively. This is an enzyme-linked immunoassay method for erythropoietin, which is characterized in that it is carried out using a labeled complex bound with a compound represented by the following formula.

[Effect] Until now, there have been few reports on EIA using G6PDH as a labeling enzyme.
A technique has been reported that uses a bioluminescent reaction using a bacterial luciferase system to label 6PDH and measure 06PDH activity (Analytical Biochemistry,
Vol. 122.385, 1982). However, this technology utilizes the functional groups possessed by Habten to
6PDH is combined, and is different from the present invention in terms of wood quality. Therefore, it is no exaggeration to say that there have been no reports to date of the use of 06PDH as an enzyme for labeling antibodies in the Sand-Deutsch method.

G6PDH is an enzyme that exists widely in the animal and plant kingdoms.
In the present invention, animal organs, bacteria, and molds are also used.

G6PDH from any origin such as yeast may be used, but
In particular, it is preferable to use a material of bacterial origin, considering that it is easier to obtain and cheaper.

06PDH is an enzyme that catalyzes the reaction of formula (1) below, and its activity is based on the N produced by reduction of NAD (P).
The increase in AD (P)H is detected by measuring the absorbance at 340 nm.

p-glucose-6-phosphoric acid + NAD (P) = ioD-glucose-δ-lactone-6-phosphoric acid + NAD (P)H
・=-(1) However, in EIA, which quantifies extremely small amounts of substances,
The activity of G6PDH used as a labeling agent is preferably measured by a bioluminescence method.

The bioluminescence method is an analysis method that applies the luminescence phenomenon observed in living organisms, and due to its extremely sensitive analysis characteristics, it is expected to be applied to the recent field of clinical testing. For example, in the measurement of ATP using the firefly bioluminescence reaction, the amount of luminescence generated by the reaction of formula (2) below is measured in the presence of luciferin and luciferase u g 2- ions. Applying this reaction principle, in addition to ATP, AMP, AD
It is possible to measure P, pyruvate, creatine kinase, etc.

In an example in which the bioluminescent reaction of oxyluciferin + AMP + birophosphate decaluminescent bacteria is applied, in addition to measuring the activity of G6PD)I, bile acids, ATP, etc. are measured, and G6Pt
) H activity, NAD (P)l( produced by the reaction of formula (1)) can be measured by the amount of luminescence produced by the reactions of formulas (3) and (4) below.

FMN+NAD (P)H+H" FMNH2+NAD+ (P)... (3) Luciferase F M N Hx + 02 + R-CHO where F
MN is flavin sononucleotide.

FMN) (2 is reduced FMN, R-C) 10 is 7 carbons
~18 linear aldehydes are shown.

Immunochemically active substances that are used in the present invention and have a thiol group (-SH) in their molecules generally include proteins and peptides, but antibodies in particular have disulfide bonds (-S-S-) in their molecules. This is preferable because the thiol group can be easily exposed by reduction and the activity as an antibody does not decrease much. Antibodies are degraded by treatment with enzymes such as pepsin and papain, and then converted into lower molecular fragments by reducing agents such as mercaptoethylamine.

These reduced molecular weight fragments are F (a b'
)2, F a b', F a b, etc., and all of them retain their antigen-binding sites and have activity. On the other hand, for immunochemically active substances that do not have a thiol group in the molecule, the substance is reacted with S-acetylmercaptosuccinic anhydride, 2-iminothiolane, methyl-3-mercaptopropionimidate, etc. , can be used in the present invention by introducing a thiol group. These immunochemically active substances include peptide hormones such as insulin, glucagon and EPO, or tumor-associated antigens such as α-fetoprotein and CEA (embryonic antigen).

Next, these immunochemically active substances and G6PDH are combined with the general formula (RI-, (I) [where m is an integer of 0 to 5 and R is a divalent 6-membered cyclic hydrocarbon residue]. , n is an integer of O to 1] A method for bonding using compounds represented by the following will be described.

In the above general formula (1), the 6-membered cyclic hydrocarbon residue represented by R may be either saturated or unsaturated; an example of the former is cyclohexylene, and an example of the latter is phenylene. can be mentioned. These compounds, for example, The Journal of Biochemistry.

Vol, 79. 233 pages (1976), European Journal of Biochemistry.

Vol. 101, p. 395 (1979), etc., or according to these methods, but commercially available compounds can also be used.

First, a compound represented by general formula (1) and G6PDH are reacted in an appropriate buffer at 0 to 50°C for 10 minutes to 24 hours to obtain maleimidated G6PDH. As the buffer solution, a phosphate buffer solution with a pH of 7.0, a Tris-Mi buffer solution with a pH of 8.0, etc. are suitable.

Next, the maleimidized G6PDH and the immunochemically active substance are combined by reacting in an ionic solution at 0 to 40° C. for 1 to 24 hours.

Examples of the buffer include a phosphate buffer with a pH of 6.5.

The 06PDH-labeled complex thus obtained is used after being purified by gel chromatography or the like. Examples of carriers used in gel chromatography include Ultrogel AcA44 (manufactured by LKB) and Sephacryl S-
200 (manufactured by Pharmacia Fine Chemicals) and the like.

Next, a method for measuring EPO using the G6PDH6PDH label will be explained.

As mentioned above, EIA is broadly divided into competitive methods and non-competitive methods, but it can also be used in the method of G6PDH6PDH labeling according to the present invention. In other words, under competitive law, G6PD)
I-labeled EPO and EPO in the test solution are subjected to a competitive reaction with a specific antibody against EPO, and the G6PDH-labeled EPO that has bound to the specific antibody and the G6 that has not bound to the specific antibody are
Separate PDH-labeled EPO and collect either G6PDH
By measuring the enzyme activity, the amount of EPO in the test solution can be determined.

On the other hand, in the non-competitive method, for example, taking the Sand-Deutsch method as an example, a test solution containing EPO is added to an antibody-bound solid phase carrier in which an antibody that specifically reacts with EPO is bound to the solid phase carrier.
The amount of EPO in the test solution is determined by reacting a G6PDH-labeled antibody with G6PDH bound to an antibody that specifically reacts with EPO, and measuring the amount of G6PDH-labeled antibody bound to the antibody-bound solid phase carrier. You can ask for it.

Furthermore, in both competitive and non-competitive methods,
By measuring the activity of G6PDI using the bioluminescence method as described above, it is possible to measure EPO with high sensitivity.

[Example] The present invention will be explained below by exemplifying the non-competitive method, particularly the sandwich method, as a typical example. However, the present invention is not limited to these examples, and
It is also clear from the above-mentioned purpose that the case of labeling is also included in the technical scope of the present invention.

Example I A G6PDH-labeled anti-EPO antibody was prepared as follows.

Anti-EPO monoclonal antibody (JP-A-59-15539
(Refer to Publication No. 5), add 1 mg of pepsin to 20 mg,
After 24 hours of digestion at 7°C, Sephadex G-15
Gel filtration was performed at 0 to obtain the F(ab')2 fragment of anti-EPo monoclonal antibody.
b'), the fragment was reduced with 2-mercaptoethylamine to obtain Fab'. On the other hand, 5 mg of 06PDH (manufactured by Toyobo) was added to 1ff11 Tris buffer (pH 7.0).
) and N-hydroxysucciimidyl ester of N-(4-carboxycyclohexylmethyl)maleimide [in general formula (I), n=1. 2 mg of the compound R-cyclohexylene] was added, and the mixture was reacted at 37° C. for 3 hours, and the reaction solution was gel-filtered through Sephadex G-25 to obtain G8PD)I into which a maleimide group had been introduced. The maleimidated 06PDH and Fab' of the anti-EPO monoclonal antibody were reacted at 4°C for 200 hours, and the reaction solution was gel-filtered through Kultrogel AcA44.
Fractions with high activity were collected to obtain a G6PDH-labeled anti-EPO monoclonal antibody. The elution pattern of gel chromatography in this case is shown in FIG.
The absorbance (-·-) is also shown as 1 at 0 nm.

G6PDH activity was measured as follows.

60mM-N to 2.7 ml of 55mM-1 Lithium chloride solution (pH 7.8, containing 3.3mM magnesium chloride)
AD to cotinamide dinucleotide) 0.1 ml
and 0.1 ml of 0.1 mM glucose-6-phosphate was added, and the G6PDH solution diluted appropriately with the above buffer solution was diluted with 0.1 ml of 0.1 mM glucose-6-phosphate.
1 ml was added, the reaction was started at 30°C, and the absorbance at 340 nm of NADH (reduced nicotinamide dinucleotide) produced every minute was measured. Activity was considered as increased absorbance per minute.

Example 2 Measurement of EPO was carried out as follows.

Polyclonal anti-EPO antibody (manufactured by Kanin Dairy Products) 0.3 m
g was dissolved in 15 ml of 0.1 M NaHCOs,
100 polystyrene spheres (diameter 6.4 mm) were added and left overnight at 4°C to obtain anti-EPO antibody-bound polystyrene spheres. Polystyrene tube (12x75m+a)
Take one polystyrene sphere bound to the anti-EPO antibody, and
A test solution containing O was added at 300 µC, and the reaction was carried out at 37 C for 2 hours. It was diluted to SOO times with a buffer solution, 300 μm of the solution was added to the above reaction solution, and the mixture was reacted for 2 hours at 37°C and washed.
, 8, 3,3mM magnesium chloride) 2.7 m
l, 60mM NAD O,1a+1.0.1M
300 µ of a substrate solution consisting of 0.1a+1 -glucose-6-phosphoric acid was added and reacted at 37°C for 1 hour. The reaction solution Q, lm was added to luciferase O, QIU 7m11N.
A D H-FMN oxidoreductase 0.3υ/m
It was immediately added to 09 ml of a luminescent substrate consisting of 1% FMNl mM and 0.1% n-decylaldehyde, and the amount of luminescence was measured using a Lumiphotometer TD4000 (Labo Science Group). The results are shown in FIG.

[Effects of the Invention] As described above, according to the present invention, a G6PDH6PDH label complex that enables high-sensitivity measurement in EIA can be obtained, and the 06PDH label complex can be used to perform EP
O was able to be measured with high sensitivity.

[Brief explanation of drawings]

Figure 1 is a graph showing the elution pattern in gel chromatography of the reaction product of G6PDH obtained in Example 1 and the Fab' fragment of anti-EPO monoclonal antibody, and Figure 2 is the EPO standard curve in Example 2. .

Claims (4)

[Claims] (1) An immunochemically active substance having a thiol group in its molecule or bonded with a thiol group and glucose-6-phosphate dehydrogenase as a labeling substance are combined using the following general formula (I) ▲Math. Chemical formulas, tables, etc. are available▼...(I) [In the formula, m represents an integer of 0 to 5, R represents a divalent 6-membered cyclic hydrocarbon residue, and n represents an integer of 0 to 1. ] A glucose-6-phosphate dehydrogenase labeling complex, characterized in that it is bound via a compound represented by the following. (2) The complex according to claim 1, wherein the immunochemically active substance is an antibody that specifically reacts with erythropoietin. (3) The complex according to claim 1, wherein the immunochemically active substance is eryrospoietin. (4) Antibody binding in which an antibody that specifically reacts with erythropoietin is subjected to a competitive reaction with a test solution containing erythropoietin and enzyme-labeled erythropoietin, or an antibody that specifically reacts with erythropoietin is bound to a solid phase carrier. In an enzyme immunoassay method for erythropoietin in which a solid phase carrier is reacted with a test solution containing erythropoietin and an enzyme-labeled antibody that specifically reacts with erythropoietin, the enzyme as a labeling substance is glucose-6-phosphate dehydrogenase, Erythropoietin or an antibody that specifically reacts with erythropoietin is added to the enzyme using the following general formula (
I) ▲Mathematical formulas, chemical formulas, tables, etc.▼...(I) [In the formula, m is an integer from 0 to 5, R is a divalent 6-membered cyclic hydrocarbon residue, and n is 0 to 1. An enzyme-linked immunosorbent assay for erythropoietin, characterized in that it is carried out using a labeled complex bound with a compound represented by: