JPH04339260A - Enzyme immunoassay - Google Patents

Abstract

PURPOSE:To rapidly perform highly sensitive enzyme immunoassay by using luciferase occurring from a sea firefly as labelling enzyme. CONSTITUTION:Sea firefly luciferase to be used is protein having an amino acid sequence and, for example, it is collected from the natural world or prepared from an artifically bred sea firefly or a host cell other than the sea firefly by a gene recombination technique or cell culture technique and, if necessary, said liciferase is purified from an anzyme solution having luciferase activity. This luciferase is modified by a functional group or molecule of every kind to form a labelled antibody and, when this antibody is used in enzyme immunoassay, luminescence based on a very small amount of a component at the beginning of reaction when antigen-antibody reaction is not sufficiently advanced can be detected.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme immunoassay. More specifically, the present invention relates to a rapid enzyme immunoassay using Cypridina luciferase, a bioluminescent enzyme that can be detected with high sensitivity.

0002

[Prior Art] Chemiluminescence and bioluminescence are generally used as methods for analyzing trace amounts of biological components, and are highly sensitive.
It is widely used in clinical chemical analysis by combining with enzyme systems that produce D, NADH, ATP, hydrogen peroxide, etc. Recently, the development of measuring instruments has progressed, and it has become possible to analyze many biological components at the picomole, femtomole, or attomole level. In addition to emission analysis in these solution systems, its high sensitivity allows it to be used for image analysis,
The applications of immobilized enzymes, enzyme immunoassays, DNA probe methods, biological tests, biomass measurements, luminescence analysis from living organisms, etc. are increasing (ed. Imai, "Bioluminescence and Chemiluminescence", Hirokawa Shoten, 1990) (Kasai) , Watanabe; Proteins/Nucleic Acids/Enzymes 32, 1234 (1987)).

Among luminescence analyses, bioluminescence is defined as chemiluminescence catalyzed by an enzyme system, but its quantum yield is overwhelmingly higher than that of ordinary chemiluminescence; It has excellent sensitivity, and its detection sensitivity is comparable to analytical methods that use radioisotopes. Furthermore, it is a very useful method from the viewpoints of safety, ease of operation, no special equipment is required as long as a photomultiplier tube is required as a measuring device, and no hazardous waste is involved. .

[0004] On the other hand, since immunoassay allows for specific selection from a mixture of complex composition by immune reaction, there is no need for pretreatment to separate the target substance from the sample, which is required for normal quantitative methods, and it is also highly efficient. It has the feature of being able to quantify with high sensitivity and precision. However, it has the disadvantage that the antigen-antibody reaction, which cannot be avoided in principle, takes time, and it cannot be said to be a rapid measurement method. Furthermore, among immunoassay methods, enzyme immunoassay, which is characterized by enzyme labeling, is less expensive and does not involve hazardous waste compared to radioimmunoassay using radioisotopes, and has better sensitivity and accuracy in measurement. Although it is gradually becoming more widely used because it is comparable, it still takes time to accumulate the products in order to ultimately quantify the products based on the enzymatic reaction, which has hindered speeding up.

[0005] When a luminescent enzyme is used as a label in this enzyme immunoassay method, it is 1) highly sensitive and can detect trace components, 2) has a wide range of quantitative performance, and has
Since no light source is required, there is no stray light, and ■Compared to the color method,
It is thought that the enzyme reaction is rapid and can be analyzed in a short period of time on the order of seconds. An enzyme immunoassay using firefly luciferase as a labeled enzyme had already been reported (Wan et al.
Nlund, J. et al. : Biochem
．． Biophys. Res. Commun. ,9
6, 440 (1980), Wannlund, J.
．． , DeLuca, M. : Anal. Bioc
hem. , 122, 385 (1982)), but the label reduces its activity by 60-90%, and the labeled product can only be stored for a short period of time, so it has not been put to practical use. To compensate for this drawback, many systems have been developed that enable highly sensitive detection by conjugating products generated from other labeling enzymes with firefly or bacterial luciferase reactions (Tanaka, K., Ishikawa).
, E. :Anal. Lett. ,17 (B18)
, 2025 (1984), Yabuuchi, M.
, Maeda, M. , Tsuji, A. : Analytical Chemistry, 34, 6 (1985), Fricke,
H. , Strasburger, J. , Woo
d, W. G. : J. Clin. Chem.
Clin. Biochem. , 20, 91 (19
82), Geiger, R. and Miska
, W. : J. Clin. Chem. Cli
n. Biochem. , 25, 23, 30 (198
7)) However, as a matter of course, these systems are more complex and include other enzymatic reactions, making rapid measurement difficult.

[0006] Myoglobin, which is one of the measurement targets, is a protein that plays a role in storing and transporting oxygen mainly in muscle tissue present in skeletal and cardiac muscles. It has been pointed out that there is a correlation between the degree of infarction, the extent of infarction, and prognosis. Also, the blood peak was 4 after infarction.
~12 hours later, and can be detected earlier than creatine kinase, which will be described later, which reaches its peak around 24 hours, so early diagnosis is desired. Currently, radioimmunoassay methods and semi-quantitative latex agglutination methods have been established, but from the standpoint of early diagnosis, it is desired to develop a rapid enzyme immunoassay method that can detect quantitatively using non-radioactivity. was.

[0007] Creatine kinase MB (hereinafter CK
-MB) is one of the three isozymes of creatine kinase, and M (muscle)
It is a dimer consisting of two types of subunits named B (brain) and B (brain). Creatine kinase MM (hereinafter referred to as CK-MM) is present in high concentrations mainly in skeletal muscles, creatine kinase BB (hereinafter referred to as CK-BB) in the brain, and CK-MB in myocardium. Clinical significance includes acute myocardial infarction, myocarditis, open heart surgery, muscular dystrophy, polymyositis,
CK-MB in the blood increases in dermatomyositis, viral myositis, malignant hyperthermia, Reye syndrome, etc., and is used as an effective means for early diagnosis of acute myocardial infarction (Takagi et al.: Clinical Examination, 32, 1309 (1988)).

Methods for measuring CK-MB in blood have already been developed, including a method for analyzing isozymes by electrophoresis and a method for measuring activity under immune inhibition using anti-M subunit antibodies. The former lacks rapidity, and the latter may show abnormally high values due to the presence of CK-BB, mitochondrial-derived creatine kinase, immunoglobulin-bound creatine kinase, etc. From the viewpoint of early diagnosis of acute myocardial infarction, the above-mentioned enzymes Among the specific detection methods using immunoassay, there has been a desire to develop a rapid method.

[0009]

[Problems to be Solved by the Invention] An object of the present invention is to perform an enzyme immunoassay using Cypridina luciferase as a labeling enzyme for an enzyme immunoassay, and furthermore, to perform an enzyme immunoassay in which the antigen-antibody reaction has sufficiently progressed. The object of the present invention is to provide a method for rapidly performing an enzyme immunoassay by detecting luminescence based on trace components in the initial stage of a reaction.

0010

[Means for Solving the Problems] The above objects can be achieved by the following present invention. That is, the present invention provides: a) a sample containing an antigen to be measured, an immobilized antibody in which an antibody capable of specifically reacting with the antigen to be measured is immobilized on a solid carrier, and a labeled antibody capable of specifically reacting to the antigen to be measured; b) a step of separating the immobilized antibody-antigen to be measured-labeled antibody complex produced in step a) from unreacted labeled antibody;
and c) an enzyme characterized in that Cypridina luciferase is used as a labeling enzyme in an immunoassay method comprising a step of detecting a complex immobilized on a solid support using a label contained in the complex. It is an immunoassay.

[0011] The Cypridina luciferase used in the present invention is a protein having the amino acid sequence disclosed in WO90/01542; Substitutions, deletions, insertions, etc. may be made.

[0012] Any method may be used to produce the Cypridina luciferase used in the present invention, such as a method of producing it using Cypridina luciferase collected from the natural world or an artificially cultivated Cypridina luciferase, or a method of producing Cypridina luciferase using genetic recombination technology or cell culture technology. Examples include a method in which the protein is produced by a host cell, or a method in which the protein is produced by protein synthesis technology.

[0013] Next, if necessary, luciferase is purified from the enzyme solution containing luciferase activity obtained by the above method. Purification of the luciferase was carried out by Thomp
The method disclosed by Thompson et al.
, E. M. et al: Proc. Nat
l. Acad. Sci. USA, 86, 656
7 (1989)), but more preferably the final product is further subjected to DEAE-HPLC and SD
The purity was confirmed by S-polyacrylamide electrophoresis.

[0014] To prepare the labeled antibody used in the present invention, Cypridina luciferase is preferably modified with various functional groups or molecules. In general, many methods have been reported for modifying purified proteins with appropriate functional groups or molecules, such as amino groups, carboxyl groups,
It can be carried out using hydroxyl groups, SH groups, sugar chains, etc. (Eiji Ishikawa "Enzyme immunoassay" 3rd edition, edited by Ishikawa et al., Igaku Shoin, 75 (1987), Kaichiro Ishibashi "Enzyme immunoassay" 3rd edition, Edited by Ishikawa et al., Igaku Shoin, 127 (19
87) ). Although the methods differ depending on the molecule you want to modify, what you need to be careful about when applying these methods is to choose a selective modification reaction that allows binding while retaining the activity of the modified substance and the activity of luciferase, and that the reaction is The process should be completed under mild conditions to avoid inadvertent deactivation of luciferase.

[0015] A preferred method for binding an antibody and Cypridina luciferase is to use a bivalent reagent having the same or different reactive groups at both ends with a spacer in between to form a protein-protein complex. can be formed. There are many reactive groups for divalent reagents, but representative examples include N-hydroxysuccinimide, imidoester, and nitroaryl halide, which react with the amino group on luciferase, and maleimide, which reacts with thiol groups. Pyridine disulfide, thiophthalimide, active halogen, amino group by ultraviolet irradiation.
Phenyl azides and diazo alkanes, which react non-selectively with any thiol group, can be used (Kaneyu Kino,
"Enzyme immunoassay" edited by Kitagawa et al., Kyoritsu Shuppan, 335 (1)
987) ). Among them, N-succinimidyl-
Maleimide is introduced into the amino group on luciferase using a bivalent reagent such as 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and reduced Ig
A complex can be formed by reacting with the thiol group of G or Fab.

[0016] The antibodies used in the present invention are appropriately selected depending on the object to be measured, and affinity-purified polyclonal antibodies, monoclonal antibodies, etc. are used.

[0017] The carrier for immobilizing the antibody is appropriately selected depending on the device for detecting bioluminescence, and commercially available plastic test tubes, microtiter plates, glass beads, plastic beads, membranes, magnetic beads, etc. are used. .

On the other hand, the enzyme immunoassay measurement system has many variations due to differences in the measurement target, the type of antigen-antibody reaction, the enzyme label, the labeling method, the enzyme activity measurement method, and the bound/free type separation method. Divided into methods (Yuichi Endo, Miyai)
Kiyoshi “Enzyme immunoassay” edited by Kitagawa et al., Kyoritsu Shuppan, 13 (
(1987)), but the enzymes used are limited to peroxidase, β-galactosidase, glucose oxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, etc. (Akio Tsuji, Masako Maeda, Enzyme immunoassay) ” Edited by Kitagawa et al., Kyoritsu Shuppan, 51 (
1987)). The complex using luciferase as a labeled enzyme used in the present invention can be used as an enzyme-labeled antibody in an enzyme immunoassay using the same method applied to the labeled enzyme described above. For example, the enzyme immunoassay method of the present invention can be used in both the so-called one-step method and the two-step method, but the one-step method is preferably used because rapid measurement can be performed.

Furthermore, in the present invention, it is possible to detect luminescence based on the amount of complexes formed immediately after the start of the reaction, when the antigen-antibody reaction has not yet reached a saturated state. Measurements can be made quickly without the need for The measurement immediately after the start of the reaction means the measurement within 30 minutes after adding the sample to be measured, more preferably within 30 seconds.

[0020]

[Examples] The present invention will be explained in more detail with reference to Examples below.

Example 1 IL-6 by luciferase/anti-IL-6 antibody complex
Rapid detection of 1. Obtaining Cypridina luciferase Natural luciferase was obtained by washing sea fireflies collected in Tateyama Bay, Chiba Prefecture with physiological saline. When 10 g of Cypridina was washed, crudely purified luciferase corresponding to an activity of 3.0×10 13 cps was obtained.

The luciferase activity in solution was determined by diluting the appropriate amount of enzyme into 300 μl of assay buffer.
μl of 33 μM Cypridina luciferin was mixed in a polystyrene test tube (1.2×3 cm), and the number of photons was immediately measured for 10 seconds using a luminometer (Biocounter M2010, Lumac, West Germany). The emission intensity is the average number of photons per second (cp
s).

2. Synthesis of Cypridina luciferin Cypridina luciferin was synthesized by S. Inoue, S.
Sugiura, H. Kakoi, T. Goto
:Tetrahedron Lett. , 1609
(1969).

3. Purification of Cypridina luciferase The crudely purified luciferase obtained by the above method is
Tsuji, F. I. : Methods Enz
ymol. 57, 364 (1987)) and further according to the method disclosed by Thompson et al. (Thompson, EM.
et al: Proc. Natl. Acad.
Sci. USA, 86, 6567 (1989)
) to proceed with refining, and finally DEAE manufactured by Tosoh Co., Ltd.
HP using 5PW (7.5mm x 7.5cm) column
LC, 25mM phosphate buffer, pH 5.8,
It was eluted with 0.5M NaCl and confirmed to be a single protein by SDS-polyacrylamide gel electrophoresis. It had a specific activity of 1.2 x 1013 cps/mg protein.

4. Application of luciferase/antibody complex to rapid EIA system As an example of application of the title complex to an EIA system, an example of detection of interleukin-6 (hereinafter referred to as IL-6) will be given. Antigen IL-6 and anti-IL-6 monoclonal antibody used in this example;
IG61 is Ida, N. et al; Bio
chem. Biophys. Res. Commu
n. , 165, 728 (1989). Further, according to a conventional method, a goat was immunized with this antigen to prepare an antiserum, and a polyclonal antibody was purified by IL-6 affinity column chromatography and used.

4-a. Preparation of maleimidated luciferase 100mM sodium phosphate, pH 7.0, 100
To 1 ml of 0.58 mg/ml purified luciferase solution dissolved in mM NaCl was added 10 mg/ml Pierce dissolved in N,N-dimethylformamide.
N-succinimidyl 4-(N-malei
midomethyl)-cyclohexane-1
4 μl of -carboxylate solution was added and slowly stirred at room temperature for 30 minutes. After the reaction, unreacted N-succinimidyl 4-(N-ma
leimidomethyl)-cyclohexan
e-1-carboxylate was excluded. Ishi
Kawa's method (Ishikawa E., et a
l;J. Immunoassay, 4, 209
(1983) ) was determined with reference to Luciferase 1.
The amount of maleimide groups introduced per molecule was 1.3. At this time, the specific activity of luciferase is 1.0×1013c
ps/mg protein.

4-b. Reduction of anti-IL-6 monoclonal antibody 100mM sodium phosphate, pH 6.0, 100
To 750 μl of 1.3 mg/ml anti-IL-6 monoclonal antibody:IG61 solution dissolved in mM NaCl, 5 mM sodium ethylenediaminetetraacetate, 75 μl of 0.1 M mercaptoethylamine dissolved in the same buffer was added, and the mixture was incubated at 37°C. It was kept warm for 60 minutes. After the reaction, apply to Pharmacia PD-10 column using the same buffer,
Unreacted mercaptoethylamine was removed.

4-c. Preparation of luciferase/antibody complex Maleimidized luciferase prepared by the above method and reduced anti-IL-6 monoclonal antibody were each used in an amount of 0.34 mg using Centricon-10 manufactured by Amicon.
It was concentrated to 0.62 mg/ml. 1.
3 ml of the maleimidized luciferase solution and 1.0 m
1 of the reduced anti-IL-6 monoclonal antibody solution were mixed and slowly stirred at 4° C. overnight. After the reaction, 500 μl of the solution was added using Amicon Centricon-30.
After concentrating as below, use a Tosoh G3000SW column (
Gel filtration HPLC using 0.78 mm diameter x 30 cm)
Two large molecular weight fractions (fraction numbers 43 and 46) having luciferase activity were separated. As confirmed by SDS-polyacrylamide gel electrophoresis, the molecular weights of these two fractions were approximately several 100,000 and approximately 200,000, indicating that a complex between the antibody and luciferase was clearly formed. .

4-d. Rapid detection of IL-6 using luciferase/antibody complexes Affinity-purified anti-IL
-6 polyclonal antibody in 10mM sodium phosphate, pH 7.2, 100mM NaCl for 2μ
g/ml, 240 μl was added to a polystyrene tube for measuring luciferase activity, and kept at 4° C. overnight. Immediately before use, add 3ml of 10mM Sodium Phosphate, pH 7.2, 100mM NaCl, 0.2
It was used after washing once with 5% (W/V) bovine serum albumin. Add 100 μl of 10 mM sodium phosphate, pH 7.2, 100 mM NaCl, 0 to this tube.
．． 0-1 dissolved in 25% (w/v) bovine serum albumin
Add 60 pg of IL-6 and stir for 1 second at room temperature (v
ortexing). Furthermore, the above luciferase/cell equivalent to 1 x 10 cps dissolved in 10 μl of the same buffer
The antibody complex (fraction number 43 or 46) complex was added and vortexed for 1 second at room temperature. 3ml 10mM sodium phosphate, pH 7.2
, 100mM NaCl, 0.05% (W/V) Tw
Results of measuring luciferase activity by adding 300 μl of 10 mM sodium phosphate, pH 7.2, 100 mM NaCl after washing once with EEN-20 and 4 times with 3 ml of 10 mM sodium phosphate, pH 7.2, 100 mM NaCl. As shown in Table 1, the amount of luminescence was obtained depending on the amount of antigen. The time required from the addition of IL-6 to the end of the measurement of luciferase activity was 1 minute and 30 seconds per tube.

[0030]

[Table 1]

Example 2 Rapid detection of myoglobin using luciferase/antibody complex The antigen myoglobin used in this example was purchased from Cambridge Medical, USA, the goat antiserum was purchased from Kappel, USA, and the anti-myoglobin monoclonal antibody was purchased from ICN, Israel. I used it. In addition, this antigen was immobilized with Affigel 10 manufactured by Bio-Rad (USA) according to a conventional method, and a polyclonal antibody was purified from the above-mentioned antiserum by affinity column chromatography and used. In addition, maleimidated luciferase was produced, anti-myoglobin monoclonal antibody was reduced, and luciferase/antibody complex was produced by the same method as in the above-mentioned IL-6 example.

[0032] Affinity-purified anti-myoglobin polyclonal antibody was added to 10mM sodium phosphate, p
H7.2, 2μg/m using 100mM NaCl
240 μl was added to a polystyrene tube for measuring luciferase activity and kept at 4° C. overnight. Immediately before use, add 3 ml of 10 mM sodium phosphate, p.
H7.2, 100mM NaCl, 0.25% (W/
V) It was used after washing once with bovine serum albumin. Add 100 μl of 10 mM sodium phosphate, pH 7.2, 100 mM NaCl, 0.25% (
W/V) 0 or 6 ng dissolved in bovine serum albumin
of myoglobin and a luciferase/antibody complex having a luciferase activity of 1×10 8 cps were added thereto, and the mixture was stirred at 180 rpm for 20 seconds to 60 minutes at room temperature. 3m
l of 10mM sodium phosphate, pH 7.2, 10
0mM NaCl, 0.25% (V/V) Tween
After washing twice with -20, 3 ml of 10 mM sodium phosphate, pH 7.2, and 5 times with 100 mM NaCl, 300 μl of 100 mM sodium phosphate,
As a result of measuring the luciferase activity by adding 100 mM NaCl at pH 7.2, the amount of luminescence corresponding to the antigen-antibody reaction time was obtained as shown in Table 2. Regardless of the antigen-antibody reaction time from 20 seconds to 30 minutes, a high luminescence amount was obtained when myoglobin was included.

[0033]

[Table 2]

Example 3 Antigen CK-MB and its isozyme CK-MB used in this example
MM or CK-BB, anti-CK-MB monoclonal antibody, and affinity-purified goat anti-CK-MM polyclonal antibody were purchased from Scripps, USA. In addition, in the same manner as in Examples 1 and 2, maleimidized luciferase was prepared and luciferase/goat anti-C
A K-MM polyclonal antibody complex was prepared.

Luciferase activity bound to the tube was determined using 300 μl of 10 mM sodium phosphate, pH 7.2.
After adding 2 μl of 33 μM Cypridina luciferin in 100 mM NaCl, the amount of luminescence for 10 seconds was integrated and measured using a luminometer (manufactured by Aloka, BLR-201).

[0036] Anti-CK-MB monoclonal antibody was added at 10 m
M Sodium phosphate, pH 7.2, 100mM
Adjust to 5 μg/ml using NaCl and add 250 μl
Polystyrene tube for luciferase activity measurement (
The mixture was added to a Star Tube (registered trademark) manufactured by NunC and kept at 4°C overnight. Immediately before use, add 5ml of 10m
M sodium phosphate, pH 7.2, 100mM Na
It was washed once with Cl and 1% (w/v) bovine serum albumin before use. Add 100μl of 10mM sodium phosphate, pH 7.2, 100mM NaCl to this tube.
0~ dissolved in 0.25% (W/V) bovine serum albumin
62.5 ng of CK-MB, CK-MM or CK-
BB and the above luciferase/antibody complex equivalent to 1×10 6 counts were added and stirred at room temperature for 30 seconds to 30 minutes. 5ml 10mM Sodium Phosphate, pH 7.2, 100mM NaCl, 0.05%
(W/V) Tween-20 5x5ml 10mM
Sodium phosphate, pH 7.2, 100mM N
After washing twice with aCl, 300 μl of 10 mM
Sodium phosphate, pH 7.2, 100mM NaC
As a result of measuring the luciferase activity by adding 1.
As shown in the table, the amount of luminescence was obtained depending on the amount of antigen. Third
From the table, even if the antigen-antibody reaction was performed for 30 minutes, CK-M
M and CK-BB were not detected, indicating that the detection system was specific to CK-MB.

[0037]

[Table 3]

[0038]

[Effects of the Invention] According to the present invention, it has become possible to utilize luciferase derived from Cypridina, which is a bioluminescent enzyme, in enzyme immunoassay, and it has become possible to perform enzyme immunoassay more quickly.

Claims (1)

[Claims] Claim 1: a) A sample containing an antigen to be measured, an immobilized antibody in which an antibody capable of specifically reacting with the antigen to be measured is immobilized on a solid carrier, and a label capable of specifically reacting with the antigen to be measured. a step of reacting with the antibody; b) a step of separating the immobilized antibody-antigen to be measured-labeled antibody complex produced in step a) from unreacted labeled antibody;
and c) an enzyme characterized in that Cypridina luciferase is used as a labeling enzyme in an immunoassay method comprising a step of detecting a complex immobilized on a solid support using a label contained in the complex. Immunoassay.