CA2032154C - Method of high sensitivity luminescence analysis - Google Patents

Abstract

An analytical method by which a substance can be rapidly analyzed with a high sensitivity, wherein 2-hydroxy-9-fluorenone, a compound of formula (.alpha.) or an oxazole compound of general formula (I) is used as an enhancer, wherein R1 represents hydrogen, CnH2n+1 (where n is a positive integer of 1 to 4), XCnH2n (where X represents F, Cl, Br or I, and n is a positive integer of 1 to 40), CnH2n+1CO2 (where n is as defined above), phenyl, naphthyl, CnH2n+1C6H4 (where n is as defined above), YCoH4 (where Y re-presents F, Cl, Br, I or phenyl), or XYC6H3 (where X
and Y are each as defined above).

Description

Summary This invention relates to a method of high sensi-tivity luminescence analysis using, as an enhancer,

2-hydroxy-9-fluorenone, the compound expressed by the following formula OH O
~OH

or an oxazole compound expressed by the formula HOJ~o~Rl [ ]

[in the formula, Rl representing hydrogen, CnH2n+l (here, n representing a positive integer of 1 to 4), XCnH2n (here, X representing F, Cl, Br or I, and n representing a positive integer of 1 to 4), CnH2n+lC02 (n being as defined above), a phenyl group, a naphthyl group, CnH2n+1C6H4 (n being as defined above), YC6H4 (Y
representing F, Cl, Br, I or a phenyl group) or XYC6H3 (X
and Y being as defined above)].
By using the luminescent system of the present invention, high sensitivity and prompt determination of substances is enabled.

3~

-Description Method of High Sensitivity Luminescence Analysis Technical Field This invention relates to a method of high sensi-tivity analysis through chemiluminescence measurement in use of the enzymatic reaction, antigen-antibody reaction and nucleic acid hybridization in the areas of clinical laboratory test, food inspection~ environmental analysiS~
inspection of animals and plants, and manufacturing process control check.

Background Art The luminescent reaction using oxidation of luminol, isoluminol or a derivative thereof [abbreviated as chemi-luminescent DPD (2,3-dihydro-1,4-phthalazinedione) in the following] by a peroxidase is used for immunoassay, analysis of elastase, analysis of glucose and analysis of oxidants.
It is known that for improving the luminescent intensity of said luminescent reaction, it is effective to add an enhancer such as shown below to the reaction system.
(l) 6-Hydroxybenzothiazole Al JG pci n e 5e yPatent Publication No. SHO 56-5000252) (2) A certain kind of phenol having a substitution ~,,~
a ~es~
group (~atent Publication No. SHO 59-171839) (3) A certain kind of aromatic amine Jo. ~c Gl n esc ~atent Publication No. SHO 61-54453) However, such enhancers have the following difficulties.
The enhancer (1) is generally of less luminescence sity and small signal-to-background ratio-The typical enhancer of the class (2) is p-iodo-phenol. Its luminescent signal is high, but the back-ground is also high, therefore the signal-to-background ratio is relatively low.
In the case of the typical enhancer of the class (3) or N,N,N',N'-tetramethylbenzidine, rise to the luminescent peak is slow, and much time is required for measurement.
For reaction mechanism for the enhancer effect, there are reports proposing requirement for efficient formation of luminol semiquinone radical [Thorpe, G.H.C. and Kricka, L.J., "Bioluminescence and Chemiluminescence: New Perspectives," Scholmerich, J., Andreesn, R., Kapp, A., Ernst, M. and Woods, W.G. (Eds), John Wiley, Chichester, pp. 199-208 (1987)] and requirement for efficient formation of phenoxy radical [Hodgson, M. and Jones, P., Journal of Bioluminescence and Chemiluminescence, Vol. 3, pp. 21-25 (1989)].
However, the phenol derivatives include a number of compounds whlch do not show an enhancer effect, and it is dlfflcult to choose an effectlve enhancer upon the foregolng theorles.
Also, for detectlon or quantlflcatlon of a gene ln a vlrulent mlcroorganlsm, prostaglandln or any other physlologl-cally active substance, luteinizlng hormone (LH) and other anterlor pltultary hormones, and cytoklnes such as lnterleukln ln blood, lt ls requlred to determlne a very small amount ln body fluld. Thus, a hlgh sensitlvity detection system was called for, and for improvement of the detectlon sensltlvity, an enhancer of hlgher efflclency has been deslred.
An ob~ect of the present invention ls to provlde a method of hlgh sensltivlty luminescent analysls whlch is characterlstlcally carrled out ln the presence of a novel enhancer.
Another ob~ect of the present invention is to provlde a novel oxazole derlvative which is effectlve as an enhancer and allows the lumlnescent analysis to be carried out with an enhancer of hlgher efflclency than the conventlonal enhancers used.
Dlsclosure of the Invention In d~tectlng or determlning a substance by the use of chemllumlnescence produced through reaction of (a) a peroxi-dase or a derlvatlve thereof, (b) an oxldant and (c) . ~
u~u 66623-193 luminol or isoluminol, or a derivative thereof, the present invention relates to a method of a luminescent analysis characterized in that the luminescence inducing reaction is carried out in the presence of at least one compound selected from the group consisting of (i) 2-hydroxy-9-fluorenone of the formula:

~ ~ H

(ii) a compound of the following formula:

HO ~ ~

O HO
and (iii) ixazole derivatives of the formula:

~ ~ Rl [I]
HO

[wherein Rl represents hydrogen, CnH2n+l (in which n represents an integer of 1 to 4), XCnH2n (in which X represents F, Cl, Br or I; n is as defined above), CnH2n+lC02 (in which n is as defined above), phenyl~ naphthyl~ CnH2n+1C6H4 (in which n is as defined above), YC6H4 (in which Y represents F, Cl, Br, I or phenyl), or XYC6H3 (in which X and Y are as defined above)].

J"~

Another aspect of the present invention provides novel oxazole derivatives of the formula:

HO ~ ~ R2 [II]

[wherein R2 represents XCnH2n- (in which X represents F, Cl, Br or I, and n represents an integer of 1 to 4), CnH2n+lC02- (in which n is as defined above), naphthyl, CnH2n+lC6H4- (in which n is as defined above), YC6H4- (Y represents F, Cl, Br, I or phenyl) or XYC6H3- (in which X and Y are as defined above)].
Brief Description of Drawing Figs. 1 and 2 show the results of analysis of CAl5-3 with an enhancer of the present invention, or 2-hydroxy-~ !} - 4a -9-fluorenone, used against those with the conventional enhancers used, Fig. 1 showing the SN ratios of luminescent intensity after 5 seconds and Fig. 2 showing those after 10 seconds.
Fig. 3 shows the results of analysis of CA15-3 with and without addition of the enhancer of the invention to the luminescent system.
Fig. 4 is an IR spectrum of 2-chloromethyl-6-hydroxybenzoxazole obtained in Example l9, and Fig. 5 is an NMR spectrum of the same.
Fig. 6 is an IR spectrum of 2-ethoxycarbonyl-6-hydroxybenzoxazole obtained in Example 20, and Fig. 7 is an NMR spectrum of the same.
Fig. 8 is an IR spectrum of 2-(3-bromophenyl)-6-hydroxybenzoxazole obtained in Example 21, and Fig. 9 is an NMR spectrum of the same.
Fig. 10 is an IR spectrum of 2-(2-methylphenyl)-hydroxybenzoxazole obtained in Example 22, and Fig. 11 is an NMR spectrum of the same.
Fig. 12 is an IR spectrum of 2-(3-chlorophenyl)-6-hydroxybenzoxazole obtained in Example 35, and Fig. 13 is an NMR spectrum of the same.
Fig. 14 is an IR spectrum of 2-(4-chlorophenyl)-6-hydroxybenzoxazole obtained in Example 36, and Fig. 15 is an NMR spectrum of the same.

Fig. 16 is an IR spectrum of 2-(2-naphthyl)-6-hydroxybenzoxazole obtained in Example 37, and Fig. 17 is an NMR spectrum of the same.
Fig. 18 is an IR spectrum of 2-(2,4-dichlorophenyl)-6-hydroxybenzoxazole obtained in Example 38, and Fig. 19 is an NMR spectrum of the same.
Fig. 20 shows the values of SN ratio obtained in Example 51 and Reference 22.
Best Mode for Carring Out the Invention The chemiluminescence enhancing effect of the enhancer of the present invention or 2-hydroxy-9-fluorenone/

OH O
OH

[4-hydroxy-3-[3-(4-hydroxyphenyl)-1-oxo-2-propenyl]-2H-l-benzopyrane-2-one (abbreviated as HHBP in the following]
or an oxazole derivative expressed by formula [I] can be confirmed as the luminescent signal-to-background ratio obtainable in the peroxidase/oxidant/chemiluminescent DPD
system is greatly improved when the enhancer of the present invention is added to the system. The term "background" used here refers to the luminescent intensity 2032 1 5~

in the absence of the peroxidase or derivative thereof.
The peroxidase used according to the present invention is not particularly limited but is preferably a plant peroxidase such as horse radish peroxidase. For the peroxidase derivative, there may be listed, for example, a peroxidase-antibody conjugate, peroxidase-antigen conjugate, peroxidase-streptoavidin conjugate and biotin bonded peroxidase. The antibody used here is not particularly limited, but there may be preferably used such partial structures as Fab' and F(ab)2 in addition to intact IgG and IgM. The antigen is also not limited, and low molecular haptens such as fluorescein and steroid hormone and high molecular substances such as polypeptides, proteins and polysaccharides are as well usable.
For the nucleic acid hybridization, linear and cyclic DNA and RNA of 10 or more bases are usable.
As an oxidant, hydrogen peroxide is preferably used, but perborates and hypochlorites are also usable.
The chemiluminescent DPD used according to the present invention includes luminol, isoluminol and N-(4-aminobutyl)-N-ethylisoluminol, and antigens, antibodies and nucleic acids bonded thereto. Of these, luminol is particularly preferable.
HHsP used as an enhancer according to the present invention can be produced through condensation of 3-acetyl-4-hydroxycumarin and para-hydroxybenbenzoaldehyde in the presence of an amine, according to the method J~a~ese described in the~patent publication No. Sho 50-46666.
As described in the foregoing, the present invention relates to a method of high sensitivity luminescent analysis which is characteristically carried out in the presence of an oxazole derivative expressed by the formula HO~o~Bl [I]

[in the formula, R1 representing hydrogen, CnH2n+1 (here, n representing a positive integer of 1 to 4), XCnH2n (here, X representing F, Cl, Br or I, and n being as defined above), CnH2n+1CO2 (n being as defined above), CnH2n+1C6H4 (n being as defined above), phenyl group, naphthyl group, YC6H4 (Y representing F, Cl, Br, or a phenyl group) or XYC6H3 (X and Y being as defined above)].
Specifically, for R1 in the compound [I] of the present invention, there may be listed hydrogen and, as alkyl group of the carbon atom number of 1 to 4, such groups as methyl, ethyl, propyl (n- and iso-), butyl (n-, iso-, sec- and tert-) and phenyl.
Also, for the halogenated alkyl group, there may be listed such groups as fluoromethyl, 1-chloromethyl, bromomethyl, iodomethyl, 1-fluoroethyl, 2-fluoroethyl, 1-chloroethyl, 2-chloroethyl, 1-bromoethyl, 2-bromoethyl, 1-iodoethyl, 2-iodoethyl, 1-fluoropropyl, 2-fluoropropyl, 3-fluoropropyl, 1-fluoro-1-methylethyl, 2-fluoro-1-methyl-ethyl, 1-chloropropyl, 2-chloropropyl, 3-chloropropyl, 1-chloro-1-methylethyl, 2-chloro-1-methylethyl, 1-bromo-propyl, 2-bromopropyl, 3-bromopropyl, 1-bromo-1-methyl-ethyl, 2-bromo-1-methylethyl, 1-iodopropyl, 2-iodopropyl, 3-iodopropyl, 1-iodo-1-methylethyl, 2-iodo-1-methylethyl, 1-fluoropropyl, 2-fluorobutyl, 3-fluorobutyl, 4-fluoro-butyl, l-fluoro-l-methylpropyl, 2-fluoro-1-methylpropyl, 3-fluoro-1-methylpropyl, l-fluoromethylpropyl, l-fluoro-2-methylpropyl, 2-fluoro-2-methylpropyl, 3-fluoro-2-methylpropyl, 1-fluoromethyl-1,1-dimethylmethyl, 1-chlorobutyl, 2-chlorobutyl, 3-chlorobutyl, 4-chlorobutyl, 1-chloro-1-methylpropyl, 2-chloro-1-methylpropyl, 3-chloro-1-methylpropyl, 1-chloromethylpropyl, 1-chloro-2-methylpropyl, 2-chloro-2-methylpropyl, 3-chloro-2-methylpropyl, 1-chloromethyl-1,1-dimethylmethyl, 1-bromobutyl, 2-bromobutyl, 3-bromobutyl, 4-bromobutyl, 1-bromo-1-methylpropyl, 2-bromo-1-methylpropyl, 3-bromo-1-methylpropyl, 1-bromomethylpropyl, 1-bromo-2-methylpropyl, 2-bromo-2-methylpropyl, 3-bromo-2-methylpropyl, 1-bromo-methylmethyl-1,1-dimethylmethyl, 1-iodobutyl, 2-iodobutyl, 3-iodobutyl, 4-iodobutyl, 1-iodo-1-methylpropyl, 2-iodo-1-methylpropyl, 3-iodo-1-methylpropy, 1-iodo-2-methyl-propyl, 2-iodo-2-methylpropyl, 3-iodo-2-methylpropyl and 1-iodomethyl-1,1-dimethylmethyl.
For the alkoxycarbonyl group, there may be listed such groups as methoxycarbonyl, ethoxycarbonyl, propoxy-carbonyl, 1-methylethoxycarbonyl, butoxycarbonyl, 1-methylpropoxycarbonyl, 2-methylpropoxycarbonyl and 1,1-dimethylethoxycarbonyl.
For the alkyl substituted phenyl group, there may be listed such groups as 2-methylphenyl, 3-methylphenyl,

4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethyl-phenyl, 2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-(1-methylethyl), 3-(1-methylethyl), 4-(1-methylethyl), 2-butylphenyl, 3-butylphenyl, 4-butylphenyl, 2-(1-methylpropyl)phenyl, 3-(1-methylpropyl)phenyl, 4-(1-methylpropyl)phenyl, 2-(2-methylpropyl)phenyl, 3-(2-methylpropyl)phenyl, 4-(2-methylpropyl)phenyl, 2-(1,1-dimethylethyl)phenyl, 3-(1,1-dimethylethyl)phenyl and 4-(1,1-dimethylethyl)phenyl.
For the halogen substituted phenyl group, there may be listed such groups as 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chloro-phenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-iodophenyl, 3-iodophenyl and 4-iodophenyl.
For the dissubstituted phenyl group, there may be -listed such groups as 2,4-dichlorophenyl and 3,5-dichloro-phenyl.
Of these, preferable substitution groups are hydrogen, methyl group, phenyl group, chloromethyl group, ethoxycarbonyl group, 2-methylphenyl group, 3-bromophenyl group, 4-chlorophenyl group and 2,4-dichlorophenyl group.
Of the compounds expressed by the formula [I], any oxazole derivative expressed by the formula HO~O~ 2 [in the formula, R2 representing XCnH2n (here, X
representing F, Cl, Br or I, and n representing a positive integer of 1 to 4), CnH2n+lC02 (n being as defined above), CnH2n+1C6H4 (n being as defined above), naphthyl group, YC6H4 (Y representing F, Cl, Br, or I or a phenyl group) XYC6H3 (X and Y being as defined above), is a novel substance.
The compound [I] of the present invention can be produced by synthesis according to the reaction formula -NH+Z-. ~ OH

RlC(OR~), OH ~ ~ R, (1) Base HO O
[~ [I]

[in the formula, R1 representing hydrogen, CnH2n+l (here, n representing a positive integer of 1 to 4), XCnH2n (here, X representing F, Cl, Br or I, and n being as defined above), CnH2n+1CO2 (n being as defined above), phenyl group, naphthyl group, CnH2n+1C6H4 (n being as defined above), YC6H4 (Y representing F, Cl, Br, I or a phenyl group) or XYC6H3 (X and Y being as defined above);
R3 representing CnH2n+1 (n being as defined above); and Z
representing F, Cl, Br or I].
Here, R1 used in the compound [III] is specifically the same with R1 used in the compound [I].
Also, for the alkyl group R3, there may be listed such groups as methyl, ethyl, propyl, 1-methylethyl, butyl, l-methylpropyl, 2-methylpropyl and 1,1-dimethyl-ethyl.
The base used according to the present invention includes carbonates such as sodium hydrogencarbonate, potassium hydrogencarbonate, sodium carbonate and potassium carbonate, amines such as trimethylamine, triethylamine and t-butylamine, and aromatic heterocyclic compounds such as pyridine and quinoline, but sodium hydrogencarbonate is particularly preferable.
The reaction does not require a solvent and proceeds at a temperature of 20 to 150C, but for enhancing the yleld of the objective product, a temperature of 50 to 120C is preferable.
The oxazole compound [I] may also be produced, when Rl is XCnH2n (X representing F, Cl, Br or I, and n representing a positive integer of 1 to 4), according to the formula (2), or when Rl is YC6H4 (Y representing F, Cl, Br, I or a phenyl group), according to the formula (3), set forth below.

NH+Z-~ OH

XCnH2nC - OR3 OH HO ~ ~ CnH2nX (2) NH+CI-t~] [I ]

[in the formula, R3 representing CnH2n+l (n representing a positive integer of 1 to 4), and X and Z representing F, Cl, Br or I respectively].
For the halogenated alkyl group used in the compound -[IV], the same halogenated alkyl group as that used in the compoundCI~ may be used. Also, for R3 used in the compound ~IV~, the same R3 as that used in the compound tIIIl may be used.
The reaction solvent used according to the present invention includes alcoho~ls such as methanol, ethanol, propanol and butanol, ethers such as ethyl ether and THF, and aprotic polar solvents such as DMF and DMSO. Of these, ethanol is particularly preferable.
The reaction proceeds at a temperature of 20 to 150C, but for increasing the yield of the objective product, a temperature of 60 to 90C is preferable.

NH+Z-~ OH

Y OC OH \\ O ~ ~ C6H4Y

[ V ] .~C6~ [

- Base ,~ ~ C6H~Y ( 3 ) Solvent HO O

- ~I"]

[in the formula, Y representing F, Cl, Br, I or a phenyl group, and Z representing F, Cl, Br or I].
For the substituted phenyl group used in the compound [V], the halogenated phenyl group or phenylphenyl group used in the compound [I] may be used.
The first stage of the reaction does not require a solvent and proceeds at a temperature of 100 to 280C, but for enhancing the yield of the compound [VI], a temperature of 150 to 250C is preferable.
The base used for hydrolytic reaction of the compound [VI] includes sodium hydroxide~ potassium hydroxide and sodium carbonate, but sodium hydroxide is particularly preferable. For the solvent, water, alcohols such as methanol and ethanol and ethers such as THF are usable, but a mixture of water/ethanol is particularly preferable.
The hydrolytic reaction proceeds at a temperature of 20 to 50C, but for enhancing the yield of the object, a temperature of 25 to 35C is preferable.
The enhancer of the present invention is usable for determination of a substance in use of a luminescent system comprising a peroxidase, chemiluminescent DPD and oxidant but is preferably usable for determination through enzymatic immunoassay and DNA hybridization.
The present invention will now be described in detail with reference to examples.

The reagents and apparatus used in the examples are as described below.
Reagents 4-Aminoresorcinol hydrochloride and chloroaceto-nitrile were purchased from the Aldrich Co.; ethanol, DMSO, diethyl oxalate, 3-bromobenzoyl chloride, 2-methylbenzoyl chloride, 4-chlorobenzoyl chloride, 2,4-dichlorobenzoyl chloride, luminol (5-amino-2,3-dihydro-1,4-phthalazine-dione and isoluminol (6-amino-2,3-dihydro-1,4-phthalazine-dione) purchased from the Tokyo Kasei Co.; phosphorus pentachloride and sodium hydrogencarbonate purchased from the Kanto Kagaku Co.; hydrogen chloride cylinder purchased from the Tsurumi Soda Co.; N-(4-aminobutyl)-N-ethyl-isoluminol (ABEI) and tris(hydroxymethyl)aminomethane purchased from the Sigma Chemical Co.; horse radish peroxidase (HRP) purchased from the Boehringer Mannheim GmbH; powder PBS purchased from the Nissui Seiyaku Co.;
bovine serum albumin (BSA) purchased from the Seikagaku Kogyo Co.; and 2-hydroxy-9-fluorenne purchased from the Aldrich Chemical Co.
HHBP was prepared according to the method described in Patent Publication No. SHO 50-46666.
M~mm~ry cancer related antigen (CA15-3) and anti-CA15-3 antibody (mouse monoclonal antibody) were provided by Centocor (Malvern, USA). The horse radish peroxidaselabeled anti-CA15-3 antibody was prepared by the maleimide hingé method [Yoshitake, S. et al., J. Biochem., 92, 1413-1424 (1982)] and isolated and purified by Pharmacia's FPLC using a hydroxy-apatite column (Mitsui-Toatsu, HCA-column, 07.6 mm x L100 mm).
Anti-CA15-3 antibody coated polystyrene beads were prepared by immersing 6 mm diameter polystyrene beads (Meiwa Fusso Shokai, surface roughness #80) in a 10 ~g/ml PBS solution of anti-CA15-3 antibody overnight.
Analytical Apparatus The chemiluminescent reaction was carried out in a disposable 3 ml plastic tube of 12 mm~ x 47 mm. The generated light was detected by a luminometer (Biolumat LB9500T), product of the Berthold Co.
The infrared spectrum (abbreviated as IR in the following) was measured by an FT/IR-5000, product of the Nippon Bunko Kogyo Co.
The nuclear magnetic resonance spectrum (abbreviated as NMR in the following) was measured by an EX9OFTNMR, product of the Nippon Denshi Co.
The mass spectrum (abbreviated as MS in the following) was measured by the direct introduction method using a JMS D300 spectrograph. The high resolution MS was determined with a JMS DX-303 spectro-graph used.

7fQde moir k Example 1 Luminescent assay of peroxidase in use of luminol and 2-hydroxy-9-fluorenone 200 ~1 of luminol (100 mM DMSO solution 10 ~1/10 ml O.lM tris-hydrochloride buffer solution, pH 7.5), 200 ~1 of 2-hydroxy-9-fluorenone (100 mM DMSO solution 10 ~1/
10 ml O.lM tris-hydrochloride buffer solution, pH 7.5), 10 ~1 of HRP [100,000 times dilution of 1111 unit/mg with a PBS buffer solution containing 1 g/l of BSA (pH 7.4)], and 10 ~1 of hydrogen peroxide (1000 times dilution of a 9.lM aqueous solution) were introduced into a plastic cuvette, and the mixture was stirred for 3 seconds with a vortex mixer used, and the luminescent intensity after 10 seconds was measured. Next, 10 ~1 of a PBS buffer solution containing no HRP (pH 7.~4) and the foregoing amounts of luminol, 2-hydroxy-9-fluorenone and hydrogen peroxide were mixed and stirred, and the luminescent intensity after 10 seconds was measured. The ratio of the former to the latter is shown as a signal-to-background ratio (SN ratio) in Table 1.
Example 2 The procedure of Example 1 was repeated with isoluminol used in place of luminol. The result is shown in Table 1.

Example 3 The procedure of Example 1 was repeated with N-(4-amino-butyl)-N-ethylisoluminol used in place of luminol.
The result is shown in Table 1.
References 1 to 3 The procedures of Examples 1, 2 and 3 were followed except that 2-hydroxy-9-fluorenone was not used. The results are also shown in Table 1.

Table 1 Signal-to-background ratio (SN ratio) with 2,3-dihydro-1,4-phthalazinedione (DPD) Luminescent intensity after 10 seconds (relative value) DPD
+HRP -HRP SN ratio Example 1 Luminol 449863 103 4368 Example 2 Isoluminol 9885 99 99.8 Example 3 N-(4-aminobutyl)-17370 96 180.9 N-ethylisoluminol Reference 1 Luminol 149 133 1.12 Reference 2 Isoluminol 167 154 1.08 Reference 3 N-(4-aminobutyl)- 157 137 1.15 N-ethylisoluminol 2032 1 54 ^ - ~

Example 4 Luminescent assay of CA15-3 antigen with 2-hydroxy-9-fluorenone and various enhancers used Diluting a CA15-3 antigen solution (615 U/ml) with a phosphate buffer solution (PBS) containing bovine serum albumin into solutions of the concentrations of 300 U/ml, 200 U/ml, 100 U/ml, 50 U/ml, 25 U/ml and 0 U/ml (PBS
containing bovine serum albumin), these were taken as standard CA15-3 solutions.
Two samples of each of the standard CA15-3 solutions of the foregoing concentrations were poured into the wells of a tray (25 wells), each in 20 ~ l. Then, 300 ~ i of a peroxidase labeled anti-CA15-3 antibody (mouse) was added to the respective wells. To each well, an antibody coated bead having the adhering liquid soaked up with filter paper was added with a pincette used.
Applying a tray cover seal, the tray was lightly tapped for admixture of the components in the respective wells, and each mixture was allowed to react at 25C for 2 hours. After completion of the reaction, the beads were washed 3 times, each time with 5 ml of physiological saline, with a bead washer used. After washing, each bead in the tray was transferred to a test tube then to a plastic cuvette for measurement with a luminometer.
200 ~l of luminol solution(100 mM DMSO solution 10 1~ 1/10 ml O.lM tris-hydrochloride buffer solution, pH 7.5), 200 ~1 of 2-hydroxy-9-fluorenone solution (100 mM
DMSO solution 10 ~1/ 10 ml O.lM tris-hydrochloride buffer solution, pH 7.5) and 10 ~1 of hydrogen peroxide solution (1000 times dilution of a 9.lM aqueous solution) were added to each plastic cuvette, and the luminescent intensities after 5 and 10 seconds were measured. The results are shown in Tables 2-1 and 2-2.
The ratios of the luminescent intensities after 5 and seconds of the standard CA15-3 antigen solutions (300 U/ml, 200 U/ml, 100 U/ml, 50 U/ml and 25 U/ml) to those after 5 and 10 seconds of 0 U/ml (SN ratios) were obtained, as shown in Figs. 1 and 2.
References 4 to 8 The procedure of Example 4 was followed with p-iodo-phenol, p-hydroxyc; nn~m; c acid, p-phenylphenol, 6-hydroxy-benzothiazole and N,N,N',N'-tetramethylbenzidine used in place of 2-hydroxy-9-fluorenone. The luminescent intensities are shown in Tables 2-1 and 2-2, and the SN
ratios shown in Figs. 1 and 2.

able 2-1 Luminescent intensities after 5 seconds with various enhancers added (relative values) CA15-3 (U/ml) Enhancers Reference 4 p-Iodophenol 168610314 2846367438 142810238017 Reference 5 p-Hydroxycinnamic acid 17454661 773610314 17851 28246 Reference 6 p-Phenylphenol 5907023 1052521541 31672 49180 Reference 7 6-Hydroxybenzothiazole 9123471 68439719 15868 22810 Reference 8 N,N,N',N'-tetramethylbenzidine203 99 174 298 1785 3968 Example 4 2-Hydroxy-9-fluorenone 47213554 2449241803 64033116066 Table 2-2 Luminescent intensities after 10 seconds with various enhancers added (relative values) CA15-3 (U/ml) Enhancers Reference 4 p-Iodophenol 17961266438123 85089153034 242580 Reference 5 p-Hydroxycinnamic acid 16304670 7663 10363 15525 27940 Reference 6 p-Phenylphenol 6757003 10487 21288 35650 52421 Reference 7 6-Hydroxybenzothiazole 8113438 6819 9696 18092 22644 ir~

Reference 8 N,N,N',N'-tetramethylbenzidine 423 84 354 290 3127 6900 Example 4 2-Hydroxy-9-fluorenone 6431365224248 40907 63885 116905 ` -Example 5 ~uminescent assay of peroxidase in use of luminol and HHBP
200 ~ 1 of luminol solution (100 mM DMSO solution 10 ~1/10 ml O.lM tris-hydrochloride buffer solution, pH
8.5), 200 ~ 1 of HHBP solution (100 mM DMSO solution 10 ~ 1/10 ml O.lM tris-hydrochloride buffer solution, pH 8.5), 10 ~1 of horse radish peroxidase (HRP) solution (100,000 times dilution of 1111 unit/mg with a PBS buffer solution (pH 7.0) containing 1 g/l of BSA) and 10 ~1 of hydrogen peroxide solution (1000 times dilution of a 9.lM
aqueous solution) were introduced into a plastic tube and stirred for 3 seconds with a vortex mixer, then the luminescent intensity after 1 minute was measured.
Next, 10 ~1 of the buffer solution (pH 7.0) not containing HRP and the foregoing amounts of luminol, HHBP
and hydrogen peroxide solution were mixed and stirred, and the luminescent intensity after 1 minute was measured.
The ratio of the former to the latter is shown as signal-to-background ratio (SN ratio) in Table 3.
Examples 6 and 7 As Examples 6 and 7, the luminescent intensities were measured similarly to Example 5 except that isoluminol (Example 6) and ABEI (Example 7) were used in place of luminol in Example 5, as shown in Table 3.

2032 1 ~4 References 9 to 11 Luminescent intensities were measured similarly to Examples 5 to 7 except that HHBP was not used, as shown in Table 3.
From Examples 5 to 7 and References 9 to 11, it has become apparent that the method of the invention is a distinguished method of luminescence analysis.

Table 3 Signal-to-background ratios (SN ratios) with 2,3-dihydro-1,4-phthalazinedione (DPD) Luminescent intensity after 1 minute (relative value) DPD
+HRP -HRP SN ratio Example 5 Luminol 453039 88 5148 Example 6 Isoluminol 7524 24 313.5 Example 7 ABEI 21073 31 679.8 Reference 9 Luminol 550 507 1.08 Reference 10 Isoluminol 160 137 1.17 Reference 11 ABEI 188 180 1.04 Example 8 Synthesis of 6-hydroxybenzoxazole 2.0 g of 4-aminoresorcinol hydrochloride (12.4 mmol), 8.5 ml of methyl orthoformate (51.4 mmol) and 1.07 g of sodium hydrogencarbonate (12.8 mmol) were introduced into a two-necked flask provided with a cooling condenser, and the mixture was stirred in argon atmosphere at 100C for a whole day and night. After completion of the reaction, the reaction solution was cooled. Then, hexane was added, and the reaction product was filtered. The crystal was washed with water to remove the inorganic salt then filtered again.
The obtained crystal was dissolved in acetone, and with activated carbon added, the solution was refluxed with heat for 1.5 hours. Removing the activated carbon, and evaporating acetone , the obtained crystal was recrystallized with acetone-water. There was obtained 0.28 g of a white powder. The reaction yield was 16.7%.
m.p.: 183.5 to 185.2C
IR (KBr, cm 1):
3150, 1630, 1528, 1489, 1276, 1236, 1195, 1104, 1085, 816 NMR (~, DMSO-d6):
6.81 (dd, lH), 7.03 (d, lH), 7.53 (d, lH), 8.46 (s, lH), 9.75 (s, lH) MS (EI): 135 (M ), 52, 31 High resolution MS (EI): C7H5O2N
Calclulated: 135.0344 Observed : 135.0332 Luminescent assay of peroxidase in use of luminol and 6-hydroxybenzoxazole 200 ~l of a luminol solution (100 mM DMSO solution 10 ~l/10 ml, 0.1M tris-hydrochloride buffer solution, pH
8.5), 200 ~l of a 6-hydroxybenzoxazole solution (100 mM
DMSO solution 10 ~l/10 ml, 0.lM tris-hydrochloride buffer solution, pH 8.5), 10 ~l of a horse radish peroxidase (HRP) solution (10,000 times dilution of 1111 unit/mg with a PBS buffer solution (pH 7.0) containing 1 g/l of BSA) and 10 ~l of a hydrogen peroxide solution (1000 times dilution of a 9.lM aqueous solution) were introduced into a plastic cuvette and stirred for 3 seconds with a vortex mixer used, then the luminescent intensity after 1 minute was measured.
Next, 10 ~l of the buffer solution not containing HRP
and the foregoing amounts of luminol, 6-hydroxybenzoxazole and hydrogen peroxide solution were mixed and stirred, and the luminescent intensity after 1 minute was measured.
The ratio of the former to the latter is shown as signal-to-background ratio (SN ratio) in Table 4.

Examples 9 and 10 As Examples 9 and 10, the luminescent intensities were measured similarly to Example 8 except that isoluminol (Example 9) and N-(4-aminobutyl)-N-ethyl-isoluminol (abbreviated as ABEI in the following) (Example 10) were used in place of luminol in the luminescent assay in Example 8, as shown in Table 4.
Example 11 The luminescent intensity was measured similarly to Example 8 except that 2-methyl-6-hydroxybenzoxazole was used in place of 6-hydroxybenzoxazole in Example 8, as shown in Table 4.
Examples 12 and 13 As Examples 12 and 13, the luminescent intensities were measured similarly to Example 11 except that isoluminol (Example 12) and ABEI (Example 13) were used in place of luminol in Example 11, as shown in Table 4.
Example 14 The luminescent intensity was measured similarly to Example 8 with 2-phenyl-6-hydroxybenzoxazole used in place of 6-hydroxybenzoxazole in Example 8, as shown in Table 4.
Examples lS and 16 The luminescent intensities were measured similarly to Example 14 except that isoluminol (Example 15) and ABEI
(Example 16) were used in place of luminol in Example 14, as shown in Table 4.
References 12 to 14 As References 12 to 14, the luminescent intensities were measured similarly to Examples 8 to 10 except that 6-hydroxybenzoxazole was not used, as shown in Table 4.

Table 4 Signal-to-background ratios through combination of enhancers and 2,3-dihydro-1,4-phthalazinedione (DPD) Luminescent intensity after 1 minute (relative value) Enhancer DPD
+HRP -HRPSN ratio Example 8 6-Hydroxybenzoxazole Luminol664522 81 8204.0 Example 9 6-Hydroxybenzoxazole Isoluminol10277 25 411.1 Example 10 6-Hydroxybenzoxazole ABEI 26481 26 1018.5 Example 11 2-Methyl-6-hydroxybenzoxazole Luminol 975682 78 12508.7 ~ Example 12 2-Methyl-6-hydroxybenzoxazole Isoluminol 15028 28 536.7 w r~
Example 13 2-Methyl-6-hydroxybenzoxazole ABEI 46109 26 1773.4 o Example 14 2-Phenyl-6-hydroxybenzoxazole Luminol 2764970* 82 33719.1* r~
Example 15 2-Phenyl-6-hydroxybenzoxazole Isoluminol 54033 50 1080.7 ~n Example 16 2-Phenyl-6-hydroxybenzoxazole ABEI 92167 50 1843.3 Reference 12 None Luminol 622 357 1.7 Reference 13 NoneIsoluminol 459 193 2.4 Reference 14 None ABEI 1371 226 6.1 * Value measured with HRP diluted 10 times and the luminescent intensity after 1 minute multiplied 10 times.

Example 17 Luminescent assay of CA15-3 antigen in use of HHBP
A CA15-3 antigen solution (615 U/ml) was diluted with a phosphate buffer solution (PBS) containing bovine serum albumine into solutions of the concentrations of 300 U/ml, 200 U/ml, 100 U/ml, 50 U/ml, 25 U/ml and 0 U/ml (PBS
containing bovine serum albumine), and these were taken as standard CA15-3 solutions.
Two samples of each of the standard solutions of the foregoing concentrations were introduced into the wells of a tray (25 wells), each in 20 ~1. To each well, 300~1 of a peroxidase labeled marker anti-CA15-3 antibody (mouse) was added. Then, an antibody coated bead having the adhering liquid soaked up with filter paper was added to each well.
Applying a tray cover seal and lightly tapping for mixture, the reaction was made at 25C for 2 hours. After completion of the reaction, washing was made 3 times with

5 ml of physiological saline with a bead washer used.
After washing, each bead in the~tray was transferred to a test tube then to a plastic tube for measurement with a luminometer.
Adding 200 ~1 of luminol solution (100 mM DMSO
solution 10 ~ 1/10 ml O.lM tris-hydrochloride buffer solution, pH 7.5), 200 rl of HHBP solution (100 mM DMSO

solution 10~ l/ml O.lM tris-hydrochloride buffer solution, pH 7.5) and 10 ~l of hydrogen peroxide solution (1000 times dilution of a 9.lM aqueous solution) to the respective plastic tubes, the luminescent intensities after 10 seconds were measured. Mean luminescent intensities of two samples are shown in Table 5.
The ratios of the mean luminescent intensities after seconds of the standard CA15-3 antigen solutions (300 U/ml, 200 U/ml, 100 U/ml, 50 U/ml and 25 U/ml) to the mean luminescent intensity after 10 seconds of 0 U/ml (SN
ratios) were obtained, as shown in Fig. 3.
Example 18 Luminescent assay of CA15-3 antigen in use of 2-methyl-6-hydroxybenzoxazole Following the procedure of Example 17 except for the use of 2-methyl-6-hydroxybenzoxazole in place of HHBP in Example 17 the luminescent intensities were measured. The mean luminescent intensities are shown in Table 5, and the SN ratios shown in Fig. 3.
Reference 15 Following the procedure of Example 17 except that HHBP and 2-methyl-6-hydroxybenzoxazole were not used, the measurement was made similarly. The mean luminescent intensities are shown in Table 5, and the SN ratios shown in Fig. 3.

Table 5 Analysis of CA15-3: Mean luminescent intensities after 10 seconds (relative values) CA15-3 (U/ml) Enhancer Example 17 HHBP 602803117045 3045465824 91745 2-Methyl-6-Example 18 hydroxy- 40133706929 1722327926 46613 benzoxazole Reference 15 None154 148 165 165 152 182 Example 19 Synthesis of 2-chloromethyl-6-hydroxybenzoxazole 26.1 g (345.7 mmol) of chloroacetonitrile, 20 ml of absolute ethanol, and 70 ml of ether were introduced into a three-necked 300 ml flask provided with stirring vanes, and while stirring gently, hydrogen chloride gas was blown into the flask at room temperature for about 40 minutes.
A white precipitate was produced which was then filtered and washed with ether. There was obtained 37.5 g (237.3 mmol) of ethyl chloromethylimidate hydrochloride. The yield of the reaction was 68.7%.

1.78 g (11.3 mmol) of said ethyl chloromethylimidate hydrochloride, 1.22 g (7.55 mmol) of 4-aminoresorcinol hydrochloride, and 20 ml of ethanol were introduced into a _ 2032 1 54 two-necked flask provided with a Dimroth condenser, and the mixture was refluxed with heat in argon atmosphere for a whole day and night. After completion of the reaction and cooling, the crystal was filtered and washed with hexane, then the object was vacuum dried. There was obtained 0.40 g (2.33 mmol) of 2-chloromethyl-6-hydroxy-benzoxazole, and the yield of the reaction was 30.9%.
m.p.: 148.5 to 153.7C
IR (Fig. 4, KBr, cm 1):
3076, 1615, 1572, 1491, 1334, 1238, 1141, 837 NMR (Fig. 5, DMSO-d6):
4.97 (s, 2H), 6,84 (dd, 2H), 7.08 (d, lH), 7.55 (d, lH), 9.89 (s, lH) MS (EI): 183 (M ), 148 High resolution MS (EI): C8H6O2NC1 Calculated: 183.0015 Observed : 183.0051 Example 20 Synthesis of 2-ethoxycarbonyl-6-hydroxybenzoxazole 61.3 g (420 mmol) of diethyl oxalate and 90.2 g (433 mmol) of phosphorous pentachloride were introduced into a three-necked 500 ml flask provided with stirring vanes and a cooling condenser and were stirred with heat at 105C for 17 hours.
The remaining liquid was vacuum distilled under 11 mmHg, and 53.2 g (265 mmol) of the fraction distilling at 77 to 85C was collected. The yield of dichloroethoxy-ethyl acetate was 63.0%.
85 ml of dehydrated ether, 35.5 ml of absolute ethanol and 53.2 g (265 mmol) of dichloroethoxyethyl acetate were introduced into a three-necked 500 ml flask provided with stirring vanes, a cooling condenser and a dropping funnel in argon atmosphere, and while stirring, 49.5 ml of dehydrated pyridine was added dropwise from the dropping funnel over 1 hour.
After stirring at room temperature for 2 hours, pyridine hydrochloride was removed by filtering and washed with 50 ml of dehydrated ether. Distilling off ether from the filtrate, the remaining solution was stirred at 90C
for 1.5 hours. The reaction solution was cooled, had ether added and washed with 3N-sulfuric acid and then an aqueous solution of sodium hydrogencarbonate. Drying the ether layer with magnesium sulfate and distilling off ether, the remaining solution was vacuum distilled at 7 mmHg, and there was collected 40.1 g (182 mmol) of the fraction distilling at 83 to 89C. The yield of produced triethoxyethyl acetate was 68.7%.
Taking 4 ml of said triethoxyethyl acetate, 1.00 g (6.19 mmol) of 4-aminoresorcinol hydrochloride and 5.05 mg of sodium hydrogencarbonate, they were introduced into a 2~32~ 54 two-necked flask provided with a cooling condenser under argon atmosphere, and the mixture was stirred with heat at 100C for a whole day and night.
The reaction solution was cooled to produce a precipitate which was then filtered. Dissolving the precipitate in acetone, and adding activated carbon, the solution was refluxed with heat for 1.5 hours. Filtering out the activated carbon, the filtrate was run through a silica gel column for concentration. The produced crystal was vacuum dried, and there was obtained 1.07 g (5.17 mmol) of 2-ethoxycarbonyl-6-hydroxybenzoxazole. The yield of the producut was 83.5%.
m.p.: 199.5 to 200.2C
IR (Fig. 6, KBr, cm ):
3278, 1736, 1630, 1539, 1489, 1261, 1241, 1145, 116, 8~9 NMR (Fig. 7, DMSO-d6):
1.35 (t, 3H), 4.41 (q, 2H), 6.97 (dd, lH), 7.14 (d, lH), 7.72 (d, lH), 10.25 (bs, lH) MS (EI): 207 (M ), 135 High resolution MS (EI): C1oHgO4N
Calculated: 207.0591 Observed : 207.0561 20321 5~

Example 21 Synthesis of 2-(3-bromophenyl)-6-hydroxybenzoxazole 14.1 ml (107 mmol) of 3-bromobenzoic chloride and 1.7 g (10.5 mmol) of 4-aminoresorcinol hydrochloride were introduced into a three-necked flask provided with a thermometer and a cooling condenser, and the mixture was heated at 90 to 145C for 30 minutes. To the residue, an aqueous solution of sodium hydroxide and methanol were added into a homogeneous solution which was then stirred at room temperature for 2 hours. The reaction solution was extracted twice with ethyl acetate and washed with lN
hydrochloric acid then with a saturated sodium chloride solution. Distilling off the excessive ethyl acetate, the obtained precipitate was recrystallized with THF/water.
There was obtained 0.68 g (2.35 mmol) of 2-(3-bromophenyl)-6-hydroxybenzoxazole, and the yield was 16.6%.
m.p.: 248.0 to 248.5C
IR (Fig. 8, KBr cm ):
3190, 1630, 1549, 1475, 1325, 1234, 1143, 1062, NMR (Fig. 9, DMSO-d6):

6.90 (dd, lH), 7.12 (d, lH), 7.66 (m, 2H),

7.71 (d, lH), 8.15 (m, 2H), 9.94 (s, lH) MS (EI): 289 (M ), 291 (M +2), 210, 182 High resolution MS (EI): C13H82NBr Calculated: 288.9738 Observed : 288.9711 Example 22 Synthesis of 2-(2-methylphenyl)-6-hydroxybenzoxazole 14 ml (107.3 mmol) of 2-methylbenzoyl chloride and 1.7 g (10.5 mmol) of 4-aminoresorcinol hydrochloride were introduced into a three-necked flask provided with a thermometer and a cooling condenser, and the mixture was heated at 144 to 215C for 1 hour. Removing the excessive acid chloride by distillation, the system was vacuumed to 1 to 1.5 mmHg, and the fraction distilling at 88 to 230C
was collected. It was 6.2 g.
With ethanol added, the foregoing fraction was recrystalized, and there was obtained 2.32 g (6.76 mmol) of the crystal. To 500 mg (1.46 mmol) of the crystal, 772 mg (13.6 mmol) of KOH, 30 ml of THF, 5 ml of water and 10 ml of methanol were added, and the mixture was stirred at room temperature for a whole day and night. The reaction solution was extracted with ethyl acetate, the extract was washed with saturated sodium chloride solution, and ethyl acetate was distilled off. Upon drying the residue under reduced pressure, there was obtained 320 g of (1.42 mmol) of 2-(2-methylphenyl) -6-hydroxybenzoxazole. The yield was 62.7%.
m.p.: 133.5 to 137.5C

2~32t 54 IR (Fig. 10, KBr, cm 1):
3064, 1613, 1489, 1224, 1151, 729 NMR (Fig. 11, DMSO-d6):
2.73 (s, 3H), 6.87 (dd, lH), 7.11 (d, lH), 7.42 (m, 3H), 7.64 (d, lH), 8.06 (m, lH), 9.86 (s, lH) MS (EI): 225 (M ), 196, 168, 156, 116, 91, 79, 51, High resolution MS (EI): C14HllO2N
Calculated: 225.0790 Observed : 225.0764 Example 23 Luminescent assay of peroxidase in use of luminol and 2-ethoxycarbonyl-6-hydroxybenzoxazole 200 ~l of a luminol solution (100 mM DMSO solution 10 ~l/10 ml 0.lM tris-hydrochloride buffer solution, pH 8.5), 200 ~1 of the 2-ethoxycarbonyl-6-hydroxy-benzoxazole obtained in Example 3 (100 mM DMSO solution 10 ~l/10 ml, 0.1M tris-hydrochloride buffer solution, pH 8.5) 10 ~l of a horse radish peroxidase (HRP) solution [10,000 times dilution of 1111 unit/mg with a PBS buffer solution (pH 7.0) containing 1 g/l of BSA], and 10 ~l of an aqueous solution of hydrogen peroxide (1000 times dilution of a 9.lM aqueous solution) were introduced into a plastic cuvette, and the mixture was stirred for 3 2~32t 54 seconds with a vortex mixer used, then the luminescent intensity after 1 minute was measured.
Next, 10 ~l of a PBS buffer solution (pH 7.0) containing no HRP and the foregoing amounts of luminol and 2-ethoxycarbonyl-6-hydroxybenzoxazole were admixed and stirred, and the luminescent intensity after 1 minute was-measured. The ratio of the former to the latter is shown, as signal-to-background ratio (NS ratio), in Table 6.
Examples 24 and 25 As Examples 24 and 25, the procedure of Example 23 was followed except that isoluminol (Example 24) and N-(4-aminobutyl)-N-ethylisoluminol (abbreviated as ABEI in the following) (Example 25) were used in place of luminol in Example 23, and the luminescent intensities were measured, as shown in Table 6.
Example 26 The procedure of Example 23 was followed except that 2-(3-bromophenyl)-6-hydroxybenzoxazole obtained in Example 21 was used in place of 2-ethoxycarbonyl-6-hydroxy-benzoxazole in Example 23, and the luminescent intensity was measured, as shown in Table 6.
Examples 27 and 28 As Examples 27 and 28, the procedure of Example 26 was followed except that isoluminol (Example 27) and ABEI
(Example 28) were used in place of luminol in Example 26, _ ~032 1 54 and the luminescent intensities were measured, as shown in Table 6.
Example 29 Similarly to Example 23 except that 2-chloromethyl-6-hydroxybenzoxazole obtained in Example 19 was used in place of 2-ethoxycarbonyl-6-hydroxybenzoxazole in Example 23, the luminescent intensity was measured, as shown in Table 6.
Examples 30 and 31 As Examples 30. and 31, the luminescent intensities were measured similarly to Example 29 except that isoluminol (Example 30) and ABEI (Example 31) were used in place of luminol in Example 29, as shown in Table 6.
Example 32 Similarly to Example 23 except that 2-(2-methyl-phenyl)-6-hydroxybenzoxazole obtained in Example 22 was used in place of 2-ethoxycarbonyl-6-hydroxybenzoxazole in Example 20, and luminescent intensity was measured, as shown in Table 6.
Examples 33 and 34 As Examples 33 and 34, the luminescent intensities were measured similarly to Example 32 except that isoluminol (Example 33) and ABEI (Example 34) were used in place of luminol in Example 32, as shown in Table 6.

References 16 to 18 As References 16 to 18, luminescent intensities were measured similarly to Examples 23 to 25 except that 2-ethoxycarbonyl-6-hydroxybenzoxazole was not used, as shown in Table 6.

Table 6 Signal-to-background ratios (SN ratios) through combination of 2,3-dihydro-1,4-phthalazinedione (DPD) with enhancers Luminescent intensity after 1 minute (relative value) Enhancer DPD
~HRP -HRPSN ratio Example 23 2-Ethoxycarbonyl-6- Luminol798373 86 9283.4 hydroxybenzoxazole Example 24 2-Ethoxycarbonyl-6- Isoluminol40492 28 1446.1 hydroxybenzoxazole Exam le 25 2-Ethoxycarbonyl-6- ABEI 114290 32 3571.6 P hydroxybenzoxazole Example 26 2-(3-Bromophenyl)-6- Luminol47370* 246 192.6 Example 27 2-(3-Bromophenyl)-6- Isoluminol602566 104 5793.9 Example 28 2-(3-Bromophenyl)-6- ABEI 780934 131 5961.3 Exam le 29 2-Chloromethyl-6- Luminol360133 77 4677.1 P hydroxybenzoxazole * 10 times value of the luminescent intensity obtained by 10 times dilution of HRP.

Table 6 (Continues) Luminescent intensity after 1 minute (relative value) Enhancer DPD
+HRP -HRPSN ratio Example 30 2-Chloromethyl-6- Isoluminol5842 30 194.7 hydroxybenzoxazole 2-Chloromethyl-6-Exam~le 31 . ABEI 15880 32 496.3 hydroxybenzoxazole Example 32 2-(2-Methylphenyl)-6- Luminol1938090* 78 24847.3 hydroxybenzoxazole Example 33 2-(2-Methylphenyl)-6- Isoluminol33186 49 677.3 r~
hydroxybenzoxazole Example 34 2-(2-Methylphenyl)-6- ABEI 56238 48 1171.6 r~
hydroxybenzoxazole ~n Reference 16 None Luminol 867 461 1.9 Reference 17 NoneIsoluminol 694 122 5.7 Reference 18 None ABEI 344 167 1.2 * 10 times value of the luminescent intensity obtained by 10 times dilution of HRP.

Example 35 Synthesis of 2-(3-chlorophenyl)-6-hydroxybenzoxazole 4.5 ml (35.2 mmol) of 3-chlorobenzoyl chloride and 1.0 g (6.19 mmol) of 4-aminoresorcinol hydrochloride were introduced into a three-necked flask provided with a thermometer and a cooling condenser, and the mixture was heated at 170 to 235C for 1 hour, then the excessive acid chloride was removed by distillation. To the residue, 1.5 g (38.5 mmol) of NaOH, 20 ml of THF, 20 ml of water and 10 ml of ethanol were added, and the mixture was stirred at room temperature for about 2 hours. The reaction solution was extracted with ethyl acetate, the extract was washed with saturated sodium chloride water solution, and ethyl acetate was distilled off.
Recrystallizing the residue with water/ methanol/THF and drying the crystal, there was obtained 250 mg (1.02 mmol) of 2-(3-chlorophenyl)-6-hydroxybenzoxazole. The yield was 16.5%.
m.p.: 246.0 to 247.5C
IR (Fig. 12, KBr, cm 1):
- 3146, 1630, 1599, 1551, 1477, 1328, 1232, 1145, 1062, 835 NMR (Fig. 13, DMSO-d6):
6.90 (dd, lH), 7.12 (d, lH), 7.61 (m, 3H),

8.06 (m, 2H), 9.94 (s, lH) MS (EI): 245 (M ), 247 tM +2), 138, 122, 80, 52 High resolution MS (EI): C13H8O
Calculated: 245.0193 Observed : 245.0218 Example 36 Synthesis of 2-(4-chlorophenyl)-6-hydroxybenzoxazole 25 g (142.8 mmol) of 4-chlorobenzoyl chloride and 3.0 g (18.6 mmol) of 4-aminoresorcinol hydrochloride were introduced into a three-necked flask provided with a thermometer and a cooling condenser, and the mixture was heated at 170 to 200C for 1 hour, then the excessive acid chloride was removed by distillation. To the residue, 8.4 g (210 mmol) of NaOH, 60 ml of THF, 60 ml of water and 30 ml of methanol were added, and the mixture was stirred at room temperature for about 2 hours. The reaction solution was extracted with ethyl acetate, the extract was washed with saturated sodium chloride water solution, and ethyl acetate was distilled off. Recrystallizing the residue with water/ methanol/THF and drying the crystal, there was obtained 1.24 g (5.05 mmol) of 2-(4-chlorophenyl)-6-hydroxybenzoxazole. The yield was 27.2%.
m.p.: 272.8 to 273.5C
IR (Fig. 14, KBr, cm 1):
3138, 1632, 1618, 1485, 1236, 1143, 832 NMR (Fig. 15, DMSO-d6):
6.87 (dd, lH), 7.11 (d, lH), 7.62 (m, 3H), 203~ 1 54 8.13 (d, 2H), 9.90 (s, lH) MS (EI): 245 (M ), 247 (M +2), 138, 122 High resolution MS (EI): C13H8O
Calculated: 245.0178 Observed : 245.0211 Example 37 Synthesis of 2-(2-naphthyl)-6-hydroxybenzoxazole 20 g (104.9 mmol) of 2-naphthyl chloride and 2.5 g (15.5 mmol) of 4-aminoresorcinol hydrochloride were introduced into a three-necked flask provided with a thermometer and a cooling condenser, and the mixture was heated at 135 to 205C for 1 hour, then excessive acid chloride was removed by distillation. To the residue, 8.3 g (207.5 mmol) of NaOH, 60 ml of THF, 60 ml of water and 30 ml of methanol were added, and the mixture was stirred at room temperature for about 2 hours. The reaction solution was extracted with ethyl acetate, the extract was washed with saturated sodium chloride water solution, and ethyl acetate was distilled off.
Recrystalizing the residue with water/ methanol/THF and drying the crystal, there was obtained 1.92 mg (7.36 mmol) of 2-(2-naphthyl)-6-hydroxybenzoxazole. The yield was 47.5%.
m.p.: 226.0 to 226.2C
IR (Fig. 16, KBr, cm 1):
3050, 1620, 1485, 1450, 1303, 1141, 1116, 816, NMR (Fig. 17, DMSO-d6):
6.92 (dd, lH), 7.17 (d, lH), 7.64 (m, 3H), 8.08 (m, 4H), 8.74 (s, lH), 9.92 (s, lH) MS (EI): 261 (M ), 130 High resolution MS (EI): C17HllO2N
Calculated: 261.0825 Observed : 261.0807 Example 38 Synthesis of 2-(2,4-dichlorophenyl)-6-hydroxybenzoxazole

9.9 ml (70.5 mmol) of 2,4-dichlorobenzoyl chloride and 2.0 g (12.4 mmol) of 4-aminoresorcinol hydrochloride were introduced into a three-necked flask provided with a thermometer and a cooling condenser, and the mixture was heated at 172 to 247C for 1 hour, then excessive acid chloride was removed by distillation. To the residue, 6.47 g (161.8 mmol) of NaOH, 60 ml of THF, 60 ml of water and 30 ml of methanol were added, and the mixture was stirred at room temperature for about 2 hours. The reaction solution was extracted with ethyl acetate, the extract was washed with saturated sodium chloride water solution, and ethyl acetate was distilled off.
Recrystallizing the residue with water/ methanol/THF and drying the crystal, there was obtained 915 mg (3.27 mmol) of 2-(2,4-dichlorophenyl)-6-hydroxybenzoxazole. The yield was 26.3%.

m.p.: 212.5 to 212.8C
IR (Fig. 18, KBr, cm 1):
3150, 1638, 1611, 1564, 1489, 1464, 1325, 1232, 1094, 822, 808 ~MR (Fig. 19, DMSO-d6):
6.92 (dd, lH), 7.11 (d, lH), 7.66 (m, 2H), 7.85 (dd, lH), 8.12 (d, lH), 9.97 (s, lH) MS (EI): 279 (M ), 281 (M +2), 283 (M +4), 172, 139, 108, 80, 52 High resolution MS (EI): C13H7O2NCl Calculated: 278.9898 Observed : 278.9876 Example 39 Luminescent assay of peroxidase in use of luminol and 2-(3-chlorophenyl)-6-hydroxybenzoxazole 200 ~l of a luminol solution (100 mM DMSO solution

10 ~1/10 ml, 0.1M tris-hydrochloride buffer solution, pH
8.5), 200 ~1 of the 2-(3-chlorophenyl)-6-hydroxy-benzoxazole solution obtained in Example 35 (100 mM DMSO
solution 10 ~1/10 ml, 0.lM tris-hydrochloride buffer solution pH 8.5), 10 ~1 of a horse radish peroxidase (HRP) solution [10,000 times dilution of 1111 unit/mg with a PBS
buffer solution (pH 7.0) containing 1 g/l of BSA] and 10 ~1 of a solution of hydrogen peroxide (1000 times dilution of a 9.lM aqueous solution) were introduced into - 203~ 54 a plastic cuvette, and after stirring for 3 seconds with a vortex mixer used, the luminescent intensity after minute was measured.
Next, 10 ~l of a PBS buffer solution (pH 7.0) not containing HRP and the foregoing amounts of luminol and 2-(3-chlorophenyl)-6-hydroxybenzoxazole were admixed and stirred, and the luminescent intensity after 1 minute was measured. The ratio of the former to the latter is shown, as signal-to-background ratio (SN ratio), in Table 7.
Examples 40 and 41 As Examples 40 and 41, luminescent intensities were measured similarly to Example 39 except that in place of luminol in Example 39, isoluminol (Example 40) and N-(4-aminobutyl)-N-ethylisoluminol (Example 41) were used, as shown in Table 7.
Example 42 Luminescent assay of peroxidase in use of luminol and 2-(4-chlorophenyl)-6-hydroxybenzoxazole 200 ~l of a luminol solution (100 mM DMSO solution 10 ~1/10 ml, 0.1 M tris-hydrochloride buffer solution pH 8.5), 200 ~1 of the 2-(4-chlorophenyl)-6-hydroxy-benzoxazole solution obtained in Example 36 (100 mM DMSO
solution 10 ~l/10 ml, O.lM tris-hydrochloride buffer solution pH 8.5), 10 ~l of a horse radish peroxidase tHRP) solution [10,000 times dilution of 1111 unit/g with a PBS

buffer solution (pH 7.0) containing 1 g/l of BSA] and 10 ~1 of a solution of hydrogen peroxide (1000 times dilution of a 9.lM aqueous solution) were introduced into a plastic cuvette, and after stirring for 3 seconds with a vortex mixer used, the luminescent intensity after minute was measured.
Next, 10 ~1 of a PBS buffer solution (pH 7.0) not containing HRP and the foregoing amount of luminol and 2-(4-chlorophenyl)-6-hydroxybenzoxazole were mixed and stirred, and the luminescent intensity after 1 minute was measured. The ratio of the former to the latter is shown, as signal-to-background ratio (SN ratio), in Table 7.
Examples 43 and 44 As Examples 43 and 44, luminescent intensities were measured similarly to Example 42 except that in place of luminol in Example 42, isoluminol (Example 43) and N-(4-aminobutyl)-N-ethylisoluminol (ABEI) (Example 44) were used, as shown in Table 7.
Example 45 Luminescent assay of peroxidase in use of luminol and 2-(2-naphthyl)-6-hydroxybenzoxazole 200 ~1 of a luminol solution (100 mM DMSO solution 10 ~1/10 ml, O.lM tris-hydrochloride buffer solution pH
8.5), 200 ~1 of the 2-(2-naphthyl)-6-hydroxybenzoxazole solution obtained in Example 37 (100 mM DMSO solution 10 ~1/10 ml, O.lM tris-hydrochloride buffer solution pH 8.5), 10 ~1 of a horse radish peroxidase (HRP) solution [10,000 times dilution of 1111 unit/mg with a PBS buffer solution (pH 7.0) containing 1 g/l of BSA] and 10 ~1 of a solution of hydrogen peroxide (1000 times dilution of a 9.lM aqueous solution) were introduced into a plastic cuvette, and after stirring for 3 seconds with a vortex mixer used, the luminescent intensity after 1 minute was measured.
Next, 10 ~1 of a PBS buffer solution (pH 7.0) not containing HRP and the foregoing amounts of luminol and 2-(2-naphthyl)-6-hydroxybenzoxazole were admixed and stirred, and the luminescent intensity after 1 minute was measured. The ratio of the former to the latter is shown, as signal-to-background ratio (SN ratio), in Table 7.
Examples 46 and 47 As Examples 46 and 47, luminescent intensities were measured similarly to Example 45 except that in place of luminol in Example 45, isoluminol (Example 46) and N-(4-aminobutyl)-N-ethylisoluminol (Example 47) were used, as shown in Table 7.
Example 48 Luminescent assay of peroxidase in use of luminol and 2-(2,4-dichlorophenyl)-6-hydroxybenzoxazole 200 ~1 of a luminol solution (100 mM DMSO solution 10 ~1/10 ml, O.lM tris-hydrochloride buffer solution pH 8.5), 200 ~1 of the 2-(2,4-dichlorophenyl)-6-hydroxy-benzoxazole solution obtained in Example 38 (100 mM DMSO
solution 10 ~1/10 ml, O.lM tris-hydrochloride buffer solution pH 8.5), 10 ~1 of a horse radish peroxidase (HRP) solution [10,000 times dilution of 1111 unit/g with a PBS
buffer solution (pH 7.0) containing 1 g/l of BSA] and 10 ~1 of a solution of hydrogen peroxide (1000 tlmes dilution of a 9.lM aqueous solution) were introduced into a plastic cuvette, and after stirring for 3 seconds with a vortex mixer used, the luminescent intensity after minute was measured.
Next, 10 ~1 of a PBS buffer solution (pH 7.0) not containing HRP and the foregoing amounts of luminol and 2-(2,4-dichlorophenyl)-6-hydroxybenzoxazole were mixed and stirred, and the luminescent intensity after 1 minute was measured. The ratio of the former to the latter is shown, as signal-to-background ratio (SN ratio), in Table 7.
Examples 49 and 50 As Examples 49 and 50, luminescent intensities were measured similarly to Example 48 except that in place of luminol in Example 48, isoluminol (Example 49) and N-(4-aminobutyl)-N-ethylisoluminol (ABEI) (Example 50) were used, as shown in Table 7.

References 19 to 21 As References 19 to 21, luminescent intensities were measured quite similarly to Examples 39 to 41 except that 2-(3-chlorophenyl)-6-hydroxybenzoxazole was not used, as shown in Table 7.

Table 7 Signal-to-background ratios (SN ratios) through combination of 2,3-dihydro-1,4-phthalizinedione (DPD) with enhancers Luminescent intensity after 1 minute (relative value) Enhancer DPD
+HRP -HRP SN ratio Exam le 39 2-(3-Chlorophenyl)-6- Luminol 10253700* 246 41681.7 P hydroxybenzoxazole Example 40 hydroxybenzoxazole Isoluminol802093 85 9436.4 Example 41 2-(3-Chlorophenyl)-6- ABEI 1570750* 100 15707.5 hydroxybenzoxazole Example 42 hydroxybenzoxazole Luminol 20805800~ 374 55630.5 Example 43 hydroxybenzoxazole Isoluminol582270 94 6194.4 Example 44 hydroxybenzoxazole ABEI 796149 96 8293.2 Example 45 2-(2-Naphthyl)-6- Luminol 6168950 350 17625.6 * lO times value of the luminescent intensity obtained through 10 times dilution of HRP.
** 20 times value of the luminescent intensity obtained through 20 times dilution of HRP.

Table 7 (Continues) Luminescent intensity after l minute (relative value) Enhancer DPD
+HRP -HRPSN ratio Example 46 2-(2-Naphthyl)-6- Isoluminol446699 106 4214.l hydroxybenzoxazole Example 47 2-(2-Naphthyl)-6- ABEI 591743 115 5145.6 hydroxybenzoxazole Example 48 2-(2,4-Dichloro)-6- Luminol2923520~ 129 22662.9 Example 49 2-(2,4-Dichloro)-S- Isoluminol706751 62 11399.2 r~

Example 50 2-(2,4-Dichloro)-S- ABEI 935112 68 13751.6 r~

Reference l9 None Luminol 583 377 l.5 Reference 20 NoneIsoluminol 463 160 2.9 Reference 21 None ABEI 273 174 l.6 ** 20 times value of the luminescent intensity obtained through 20 times dilution of HRP.

Example 51 Luminescent assay of CA15-3 antigen in use of 2-ethoxycarbonyl-6-hydroxybenzoxazole Diluting a CA15-3 antigen solution (615 U/ml) with a phosphate buffer solution (PBS) containing 0.25% bovine serum albumin into solutions of the concentrations of 300 U/ml, 200 U/ml, 100 U/ml, 50 U/ml, 25 U/ml and 0 U/ml (PBS containing 0.25 bovine serum albumin), these were taken as standard CA15-3 solutions.
The standard CA15-3 solutions of the foregoing concentrations were introduced to the wells of a tray (25 wells), each in 200 ~ 1. Then, 300 ~1 of a peroxidase labeled anti-CA15-3 antibody (mouse) was added to the respective wells. To each well, an antibody coated bead, having the adhering liquid soaked up with filter paper, was added with a pincette used.
Applying a tray cover seal, the tray was lightly tapped for admixture of the components in the respective wells, and each mixture was allowed to react at 25C for 2 hours. After completion of the reaction, the beads were washed 3 times, each time with 5 ml of physiological saline, with a bead washer used. After washing, each bead in the tray was transferred to a test tube then to a plastic cuvette for measurement with a luminometer.
100 ~1 of a luminol Na salt solution (18 times dilution of a solution of 12.6 mM, luminol Na salt/O.lM
tris-hydrochloride buffer solution pH 8.5 with the same buffer solution), 100 ~1 of a 2-ethoxycarbonyl-6-hydroxy-benzoxazole solution (100 mM DMSO solution 10 rl/10 ml, O.lM tris-hydrochloride buffer solution pH 8.5) and 1001~1 of a solution of hydrogen peroxide (18 times dilution of 16.2 l~M hydrogen peroxide/O.OlM disodium hydrogen phosphate-citric acid buffer solution pH 5.2 with the same buffer solution) were added to a plastic vial, and after heating at 37C for 10 minutes, the luminescence was measured for 60 seconds. 1/6 Values of the cumulative values of luminescent intensities of from 50 seconds to 60 seconds are shown in Table 8.
Then, the ratios of the 1/6 values of the cumulative values of luminescent intensities of from 50 seconds to 60 seconds of the standard CA15-3 solutions (300 U/ml, 200 U/ml, 100 U/ml, 50 U/ml and 25 U/ml) to the 1/6 value of the cumulative value of luminescent intensity of from seconds to 60 seconds of 0 U/ml (SN ratios) were obtained, as shown in Table 8 and Fig. 20.
Reference 22 According to the procedure of Example 35 except that 2-ethoxycarbonyl-6-hydroxybenzoxazole was not used, the luminescent intensities and SN ratios were obtained, as shown in Table 8 and Fig. 20.

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Industrial Applicability As described in the foregoing, the method of lumine-scence analysis of the present invention enables high sensitivity and prompt determination of substances.

Claims (21)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of detecting or determining a substance by the use of chemiluminescence produced by reacting (a) a peroxidase or a derivative thereof, (b) an oxidant and (c) luminol or isoluminol or a derivative thereof, wherein the chemiluminescence is produced in the presence of a chemi-luminescence enhancing effective amount of at least one compound selected from the group consisting of (i) 2-hydroxy-9-fluorenone, (ii) a compound of the following formula:

and (iii) oxazole derivatives of the formula:

(I) [wherein R1 represents hydrogen, CnH2n+1-, XCnH2n-, CnH2n+1CO2-, phenyl, naphthyl, CnH2n+1C6H4-, YC6H4- or XYC6H3-;

X represents F, Cl, Br or I;
Y represents F, Cl, Br, I or phenyl; and n represents an integer of 1 to 4].

2. A method as set forth in claim 1, wherein an oxazole of formula [I] is employed in which R1 is hydrogen, methyl, chloromethyl, ethoxycarbonyl, phenyl, bromophenyl, methylphenyl, chlorophenyl, naphthyl or dichlorophenyl.

3. An oxazole derivative of the formula:

[II]

[wherein R2 represents XCnH2n- (in which X represents F, Cl, Br or I, and n represents an integer of 1 to 4), CnH2n+1CO2- (in which n is as defined above), naphthyl, CnH2n+1C6H4- (in which n is as defined above), YC6H4- (Y represents F, Cl, Br, I or phenyl) or XYC6H3- (in which X and Y are as defined above)].

4. An oxazole derivative as set forth in claim 3, wherein R2 is chloromethyl, ethoxycarbonyl, bromophenyl, chlorophenyl, naphthyl, dichlorophenyl or methylphenyl.

5. A method as set forth in claim 1, wherein an oxazole compound of the formula [I] is employed in the production of chemiluminescence.

6. A method as set forth in claim 1, wherein 2-hydroxy-9-fluorenone is employed in the production of chemiluminescence.

7. A method as set forth in claim 1, wherein a compound of the formula:

is used in the production of chemiluminescence.

8. The compound 2-chloromethyl-6-hydroxybenzoxazole.

9. The compound 2-ethoxycarbonyl-6-hydroxybenzoxazole.

10. The compound 2-(3-bromophenyl)-6-hydroxybenzoxazole.

11. The compound 2-(2-methylphenyl)-6-hydroxybenzoxazole.

12. The compound 2-(3-chlorophenyl)-6-hydroxybenzoxazole.

13. The compound 2-(4-chlorophenyl)-6-hydroxybenzoxazole.

14. The compound 2-(2-naphthyl)-6-hydroxybenzoxazole.

15. The compound 2-(2,4-dichlorophenyl)-6-hydroxybenzoxazole.

16. A method as set forth in claim 1, 2, 5, 6 or 7, wherein:
the component (a) is a plant peroxidase or a derivative thereof selected from the group consisting of a peroxidase-antibody conjugate, a peroxidase-antigen conjugate, a peroxidase-streptoavidin conjugate and a biotin-bonded peroxidase;
the component (b) is a hydrogen peroxide, a perborate or a hypochlorite; and the component (c) is (c-i) a member selected from the group luminol, isoluminol and N-(4-aminobutyl)-N-ethylisoluminol or (c-ii) a member selected from the group consisting of an antigen, an antibody and a nucleic acid, each bonded to the member (c-i).

17. A method as set forth in claim 16, wherein the component (a) is the peroxidase-antigen conjugate in which the antigen is a member selected from the group consisting of a low molecular weight hapten and a high molecular weight substance selected from the group consisting of a polypeptide, a protein, a polysaccharide and a linear or cyclic DNA or RNA having at least 10 bases; and the component (c) is the member (c-i).

18. A method as set forth in claim 17, wherein the antigen is a cancer-related antigen.

19. A method as set forth in claim 16, wherein the component (a) is the peroxidase-antibody conjugate; and the component (c) is the member (c-i).

20. A method as set forth in claim 19, wherein the antibody in the peroxidase-antibody conjugate is a cancer-related antibody.

21. A process for producing an oxazole derivative of the formula [II] as defined in claim 3, which comprises (1) reacting an orthoformate of the formula:
R2C(OR3)3 [III]
(wherein R2 is as defined in claim 3, and R3 is CnH2n+1 in which n is as defined in claim 3) with an acid addition salt of 4-aminoresorcinol of the formula:

[VII]

(wherein Z- is an anion derived from an acid) in the presence of a base at a temperature of 20 to 150°C;
(2) to produce an oxazole derivative of the formula [II] in which R2 is XCnH2n-, reacting an imidate hydrochloride of the formula:

[IV]

(wherein X and n are as defined in claim 3, and R3 is as defined above with respect to the formula [III]) with an acid addition salt of 4-aminoresorcinol of the formula [VII] defined above in a reaction-inert solvent at a temperature of 20 to 150°C; or (3) to produce an oxazole derivative of the formula [II] in which R2 is YC6H4-, reacting a halobenzoyl chloride of the formula:

YC6H4COCl [V]

(wherein Y is as defined in claim 3) with an acid addition salt of 4-aminoresorcinol of the formula [VII] defined above at a temperature of 100 to 280°C, thereby producing an intermediate of the formula:

[VI]

and then subjecting the intermediate [VI] to a hydrolysis in the presence of a base in a solvent at a temperature of 20 to 50°C.