JPH0767696A - Method for reducing back ground luminescence - Google Patents

Abstract

PURPOSE:To reduce only back ground luminescence and to enable measurement in high sensitivity by making a specific compound exist in measuring a peroxidase based on luminous reaction comprising a dihydrophthalazinedione derivative as a substrate. CONSTITUTION:Luminescence occurring by treatment of a 2,3-dihydro-1,4- phthalazinedione derivative (e.g. luminol) with hem or a peroxidase (preferably one derived from horseradish) in the presence of an oxidizing agent (e.g. hydrogen peroxide) is measured in the presence of one or more compounds (e.g. citric acid) which contain a functional group of formula I to formula IV (X is H, OH or COOH; (n) is 1-3) or its salt, does not contain aromatic hydrocarbon containing OH and has chelating action. The measurement is carried out preferably at pH 8-9.5 and a boric acid-based buffering solution is preferable as the buffer solution. In the case of measuring hem or a peroxidase in an extremely low concentration, a combined use of an enhancer such as 6- hydroxybenzothiazole is preferably.

Description

Detailed Description of the Invention

[0001]

The present invention relates to 2,3-dihydro-
The present invention relates to a method for reducing background luminescence when a heme or peroxidase, which is an oxidation catalyst, is measured based on a luminescent reaction due to the catalytic action of a 1,4-phthalazine compound and an oxidizing agent.

[0002]

2. Description of the Related Art Recently, enzyme immunoassay (EIA) has been used to quantify a very small amount of a substance in a living body, but a fluorescent substrate is mainly used for measuring a labeled enzyme. There is. However, in the measurement of fluorescent substances, high-sensitivity measurement is not always easy due to the influence of excitation light, etc., and an enzyme-linked immunosorbent assay (CLE) using a luminescent substrate that enables higher-sensitive measurement
IA) has been proposed.

Examples of enzymes used in CLEIA include alkaline phosphatase and peroxidase. In the case of a luminescent substrate used for alkaline phosphatase, it is destabilized by hydrolyzing the phosphate group of the luminescent substrate containing a dioxetane structure, It is designed to emit light when disassembled.

In the case of peroxidase, when luminol is reacted alone with an oxidizing agent such as hydrogen peroxide, short-time luminescence is observed up to several ftomoles as the absolute amount of the enzyme, but when the amount is less than that, background emission occurs. Measurement is difficult because it is hidden by light emission. To this problem
Rpe et al. reported that the addition of a compound (enhancer) such as a phenol derivative or hydroxybenzothiazole to the above system markedly enhanced the luminescence and maintained the luminescence for a long time (Methods in Enzymology 133, p
331-353, 1986). These series of enhancers have the effect of reducing the background luminescence generated when the oxidant and luminol are mixed, and at the same time enhancing the luminescence due to a catalyst such as peroxidase, which allows peroxidase to measure up to several tens attomoles. You can do it. CLEI using peroxidase for labeling
In A, various measurement items can be quantified with higher sensitivity than before by utilizing this enhancement reaction.

[0005]

In the case of the luminescent substrate used for measuring alkaline phosphatase as described above, non-enzymatic hydrolysis of phosphate ester of these luminescent substrates is likely to occur at the optimum pH of alkaline phosphatase. There is a problem that background light emission due to this is likely to occur. When Backbrand luminescence occurs, the minute luminescence derived from a trace amount of alkaline phosphatase cannot be distinguished from the background, so that the measurement sensitivity cannot be increased.

Also, the luminescent substrate and enhancer used for the peroxidase measurement have the same problems as in the case of the alkaline phosphatase when the trace amount of peroxidase is to be measured. This is because even if an enhancer is added, the background light emission is still at a high level, and light emission below that level cannot be captured.

As described above, the development of a method for suppressing the high background emission to a low level was indispensable for achieving high-sensitivity measurement.

[0008]

The present inventors have completed the present invention as a result of intensive studies on a method for reducing background luminescence in a system consisting of luminol, an oxidizing agent and an enhancer. That is, the present invention relates to 2,3-dihydro-
Luminescence generated by treating a 1,4-phthalazine derivative with heme or peroxidase in the presence of an oxidant is expressed by the following chemical formulas 5 to 8 (where X is H, OH, COOH, n = 1 to 1).
3) The measurement is carried out in the coexistence of one or more compounds having a chelating effect, which have a functional group represented by 3) or a salt thereof in the structure and do not contain an aromatic hydrocarbon having a hydroxyl group in the structure. It is a characteristic method for reducing background luminescence in the measurement of heme or peroxidase. The present invention will be described in detail below.

[0009]

[Chemical 5]

[0010]

[Chemical 6]

[0011]

[Chemical 7]

[0012]

[Chemical 8]

When a 2,3-dihydro-1,4-phthalazinone derivative typified by luminol and an oxidizing agent such as hydrogen peroxide are mixed in a weakly alkaline solution, it is possible to add an oxidizing catalyst such as heme or peroxidase. A certain level of luminescence is observed. This is the background emission in the present invention. It was confirmed that this background luminescence was caused by a small amount of metal ions present in the reaction system. Therefore, in the present invention, one or more compounds having a chelating effect, which have the above-mentioned four kinds of functional groups or salts thereof in the structure and contain no aromatic hydrocarbon having a hydroxyl group in the structure, coexist. This reduces background light emission.

Examples of compounds having such properties include trans-1,2-Diaminocyclohexane-N, N, N ', N'-tetra
acetic acid monohydrate and its salt, N, N-Bis (2hydro
xyethl) glycine and its salts, 1,3-Diamino-2-hydroxypro
pane-N, N, N ', N'-tetraacetic acid and its salt, Diethyl
enetriamine-N, N, N ', N``, N''-pentaacetic acid and its salts, Ethylenediamine-N, N'-diacetic acid and its salts, Ethylenediamin-N, N'-dipropionic acid, dihydroch
loride and its salt, N- (2-Hydroxyethyl) ethylenediamin
eN, N ', N'-triacetic acid and its salts, O, O'-Bis (2-ami
noethyl) ethyleneglycol-N, N, N ', N'-tetraacetic acid
And its salts, 1,6-Hexamethylethylenediamine-N, N, N ',
N'-tetraacetic acid and its salt, N- (2-Hydroxyethyl) i
minodiacetic acid and its salt, Iminodiacetic acid and its salt, 1,2-Diaminopropane-N, N, N ', N'-tetraacetic
acid and its salt, Nitrilotriacetic acid and its salt, Nitrilotriporopionic acid and its salt, Triethyl
enetetramine-N, N, N ', N'',N''', N '''-hexaacetic acid
And salts thereof, N- (2-acetoamido) iminodiacetic acid and salts thereof, O, O'-Bis (2-aminophenyl) ethyleneglycol-N, N,
N'.N'-teraacetic acid and its salt, citric acid and its salt, etc. can be illustrated.

The concentration of the compound added to the system is 0.
01 mM to 5 mM, preferably 0.05 mM to 1.0 mM. When used at a concentration higher than this, light emission due to catalytic activity of heme, peroxidase, etc. may be inhibited, so it is advisable to conduct preliminary experiments and determine the concentration conditions for addition prior to implementation.

Even if the compound has a functional group as described above, a chelating agent containing an aromatic hydrocarbon having a hydroxyl group in its structure, such as Ethylenediaminehydroxyphenylacetic acid and its salt, is derived from the catalytic activity of heme and peroxidase. Light emission may be hindered.

2,3-dihydro-used in the present invention
Examples of 1,4-phthalazione derivatives include luminol and its derivatives, isoluminol and its derivatives, and 7-Di.
methylaminonaphthalene-1,2-dicarboxylic acid hydra
Examples include zide. Examples of the oxidizing agent include hydrogen peroxide and sodium perborate.

It is not necessary to add an enhancer for the purpose of reducing only background luminescence, but in a situation where a lower background luminescence is desirable, that is, when measuring extremely low concentration of heme or peroxidase, it is effective to use an enhancer together. Target. The enhancer is, for example, p
Examples include position-substituted phenol derivatives (p-iodophenol, 4-phenylphenol, etc.), 2-hydroxycinnamic acid, firefly luciferin and its derivatives, 6-hydroxybenzothiazole, 4- (4-hydroxyphenyl) thiazole, and the like.

The present invention is applicable to peroxidases of various origins, but is particularly suitable for horseradish-derived peroxidases. Among horse radish peroxidase isomers, it is particularly suitable for the basic isomer, and the pH at this time is weakly alkaline, from pH 8.0.
The pH is good at 9.5, and the buffer solution is preferably amine-based such as trishydroxymethane or boric acid-based.

[0020]

INDUSTRIAL APPLICABILITY According to the present invention, the background luminescence in the system can be reduced, and the luminescence derived from the oxidation catalyst is not hindered, that is, the background luminescence is specifically reduced. -Ze can be measured. By using the present invention, the background luminescence can be suppressed to be lower than that of the conventional one, so that the signal luminescence due to heme or peroxidase, which is conventionally buried in the background luminescence, can be measured with high sensitivity. Further, the present invention can be applied to an enhanced luminescence enzyme immunoassay (Enhanced-CLEIA) using peroxidase or the like as a labeled substance of an antibody. In this case, it can be applied not only to the measurement of peroxidase and the like but also to highly sensitive measurement of various biological trace substances.

[0021]

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

Example 1 0.2 mM 4-phenylphenol, 1.0 mM hydrogen peroxide, 0.
A 0.1 M Tris-hydrochloric acid buffer solution (pH 8.5) containing 5 mM luminol Na salt and 0.1 mM of the compounds shown in Table 1 was prepared, and the background luminescence (count / minute) was measured. For comparison, 0.1 M Tris-HCl buffer solution (pH 8.5) containing only 4-phenylphenol, hydrogen peroxide, and luminol Na salt
A solution was prepared and background emission was measured in the same manner.
Luminescence measurement was performed using a commercially available measuring device (BLR-30 manufactured by Aloka Co., Ltd.
Using 1), the amount of luminescence was integrated for each sample of 200 μl from 30 seconds to 1 minute after reagent preparation. The results are shown in Table 1.
Shown in. In Table 1, CyDTA is Trans-1,2-Diamin.
ocyclohexane-N, N, N ', N'-tetraacetic acid, monohydrat
e, DHEG is N, N-Bis (2hydroxyethl) glycine, EDDA is
Ethylenediamine-N, N'-diacetic acid, EDTA is Ethyle
nediamine-N, N, N ', N'-tetraacetic acid, EGTA is O, O'
-Bis (2-aminoethyl) ethyleneglycol-N, N, N ', N'-tetraac
etic acid, IDA is Iminodiacetic acid, NTA is Ni
trilotriaceticacid, Arg. for arginine, Asp. for aspartic acid, Cys. for cystine, Glu.A for glutamic acid, His. for histidine, Orn. for ornithine.
Tyr. Is tyrosine, 2,3DC Pyri. Is 2,3-Dicarboxypyridin
e, NM2,3DC Pyri. is 1-2,3-dicarboxypyridine, 2,
3DCPyra represents 2,3-Dicarboxypyradine, respectively. Of these compounds, CyDTA, DHEG, EDDA, EDTA, EGTA,
IDA and NTA are those satisfying the properties of the compound of the present invention, that is, a compound having a chelating effect which does not contain an aromatic hydrocarbon having a hydroxyl group in the structure and containing the above-mentioned functional group or a salt thereof in the structure. is there.

From Table 1, CyDTA, DHEG, EDDA, EDTA,
It can be seen that the background emission is obviously reduced when EGTA, IDA or NTA is added, as compared with the case where no addition is added or the case where other is added.

[0024]

[Table 1]

Example 2 Compounds in which reduction of background luminescence was observed in Example 1, CyDTA, DHEG, EDDA, EDTA, EGTA, IDA or
NTA was tested to see if these compounds interfere with the catalysis of peroxidase.

A 0.1 M Tris-HCl buffer solution (pH 8.5) containing 0.2 mM 4-phenylphenol, 1.0 mM hydrogen peroxide, 0.5 mM luminol Na salt, and 0.1 mM of the compounds shown in Table 2 was prepared. Attomole (10 μl) horseradish peroxidase solution was added. Luminescence was measured for 30 minutes to 1 minute after the addition in the same manner as in Example 1. The results are shown in Table 2. From Table 2, CyDTA, DHEG, EDDA, EDTA, EGTA, IDA or NT
It can be seen that A has the effect of reducing background luminescence and does not interfere with the oxidizing action of peroxidase.

[0027]

[Table 2]

Example 3 The same operation as in Example 1 was carried out except that the compounds shown in Table 3 were used at a concentration of 0.1 mM or 1 mM, and the background reducing effect was investigated. The results are shown in Table 3. In Table 3
ADA is N- (2-acetoamido) iminodiacetic acid, DS is De
xtran Sulfatesodium, MOPS is 3- (N-Morpholino) Propa
ne-sulfonic Acid, 2,2Bipy is 2,2Bipyridy, ο-
Phn indicates ο-Phenanthrolin, respectively. In Table 3, ADA or citric acid satisfying the properties as the compound of the present invention, that is, containing the above-mentioned functional group or a salt thereof in the structure and not containing an aromatic hydrocarbon having a hydroxyl group in the structure -A compound having a gallant action.

It can be seen from Table 3 that the background emission is obviously reduced when ADA, citric acid or DS is added, as compared with the case where no addition is added or the case where DS is added.

[0030]

[Table 3]

Example 4 The ADA, DS and citric acid used in Example 3 were tested to see if these compounds actually interfered with the peroxidase catalysis.

A 0.1 M Tris-HCl buffer (pH 8.5) solution containing 0.2 mM 4-phenylphenol, 1.0 mM hydrogen peroxide, 0.5 mM luminol Na salt and 0.1 mM of the compounds shown in Table 4 was prepared and Attomole (10 μl) of horseradish peroxidase solution was added, and the light emission was measured for 30 seconds to 1 minute after the addition as in Example 1. The results are shown in Table 4.

From Table 4, it can be seen that ADA and citric acid, which are the compounds of the present invention, have the effect of reducing background luminescence and do not interfere with the oxidizing action of peroxidase. It can be seen that although it is effective, it also interferes with the oxidative action of peroxidase.

[0034]

[Table 4]

Example 5 A monoclonal antibody against thyroid stimulating hormone (TSH) was digested with pepsin to form F (ab) '2, which was then purified by gel filtration and hydrophobic chromatography and reduced by dithiothreitol to form Fab. . The Fab fragment was purified by gel filtration, and SMCC (Succin
imidyl4-(N-maleimidomethyl) cyclohexane-1-carboxyl
ate) modified horseradish peroxidase 37
After reacting for 1 hour at ℃, the enzyme-labeled antibody (conjugate) fraction was collected by gel filtration.

The thus-obtained conjugate was stored at 4 ° C. after measuring the absorption at UV280 nm. Anti-TSH F in the reaction vessel
(Ab) Magnetic beads with immobilized antibody (φ = 1.4 mm)
Twelve TSE zero serum or known concentration (4.81μ)
(IU / ml) Serum 100 μl was added, reacted at 37 ° C for 5 minutes with stirring, and then B / F separation was performed. A solution containing 0.1 M NaCl, 50 mM Tris buffer, and 0.5% Tween 20 (pH 8.5). After washing with 100 μl, 100 μl of the previously prepared conjugate (diluted 200 times with the diluting solution) was added, and further reacted at 37 ° C for 10 minutes. Then, after B / F separation and washing five times, the luminescence detector (BLR-301 manufactured by Aloka Co.) was set, and 0.2 mM 4-
Phenylphenol, 1.0 mM hydrogen peroxide, 0.5 mM luminol Na
200 μl of a luminescence reagent containing salt, 0.1 mM CyDTA and 0.1 M Tris-HCl buffer (pH 8.5) was added, and the luminescence amount from 30 seconds to 1 minute after addition was integrated in the same manner as in Example 1. The measurement is
The same test solution was repeated 5 times. Table 5 shows the measurement results (average value, standard deviation, variation value, and lower limit concentration of detection by the 2SD method).

From Table 5, the S / N ratio of the CyDTA-added system (the amount of luminescence in the positive serum / the amount of luminescence in the zero serum) is 851, whereas the S / N ratio in the system without CyDTA added is 147. , S / N
It can be seen that the improvement is about 6 times. In addition, the lower limit of detection when CyDTA was added was 0.004, whereas
The value was 0.017 when this was not added, indicating that the lower limit of detection concentration was also improved by about 6 times.

[0038]

[Table 5]

Claims (1)

[Claims] 1. A luminescence produced by treating a 2,3-dihydro-1,4-phthalazione derivative with heme or peroxidase in the presence of an oxidant is represented by the following chemical formulas 1 to 4 (where X is H, OH, COOH, n = 1 to 3) and one or more compounds having a chelating effect, which have in their structure a functional group or a salt thereof and which do not contain an aromatic hydrocarbon having a hydroxyl group in the structure. A method for reducing background luminescence in the measurement of heme or peroxidase, which comprises measuring in the presence of [Chemical 1] [Chemical 2] [Chemical 3] [Chemical 4]