Method for detecting a substance using chemiluminescence

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 (α) or an oxazole compound of general formula (I) is used as an enhancer, wherein R₁ represents hydrogen, C n H 2n+1 (where n is a positive integer of 1 to 4), XC n H 2n (where X represents F, Cl, Br or I, and n is a positive integer of 1 to 40), C n H 2n+1 CO₂ (where n is as defined above), phenyl, naphthyl, C n H 2n+1 C₆H₄ (where n is as defined above), YCoH₄ (where Y represents F, Cl, Br, I or phenyl), or XYC₆H₃ (where X and Y are each as defined above).

Description: Technical Field This invention relates to a method of high sensitivity 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 chemiluminescent 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. (1) 6-Hydroxybenzothiazole (Patent Publication No. JP-A-56-500252) (2) A certain kind of phenol having a substitution group (Patent Publication No. JP-A-59-171839) (3) A certain kind of aromatic amine (Patent Publication No. JP-A-61-54453) However, such enhancers have the following difficulties. The enhancer (1) is generally of less luminescence intensity and small signal-to-background ratio. The typical enhancer of the class (2) is p-iodophenol. Its luminescent signal is high, but the background 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 which do not show an enhancer effect, and it is difficult to choose an effective enhancer upon the foregoing theories. Also, for detection or quantification of a gene in a virulent microorganism, prostaglandin or any other physiologically active substance, luteinizing hormone (LH) and other anterior pituitary hormones, and cytokines such as interleukin in blood, it is required to determine a very small amount in body fluid. Thus, a high sensitivity detection system was called for, and for improvement of the detection sensitivity, an enhancer of higher efficiency has been desired. An object of the present invention is to provide a method of high sensitivity luminescent analysis which is characteristically carried out in the presence of a novel enhancer. Another object of the present invention is to provide a novel oxazole derivative which is effective as an enhancer and allows the luminescent analysis to be carried out with an enhancer of higher efficiency than the conventional enhancers used. Disclosure of the Invention According to the present invention a method for detecting or determining a substance by the chemiluminescence produced through reaction of (a) a peroxidase or a derivative thereof, (b) an oxidant and (c) luminol or isoluminol, or a derivative thereof, is characterized in that the luminescence inducing reaction is carried out in the presence of at least one compound chosen from the group comprised of 2-hydroxy-9-fluorenone, the compound expressed by the following formula and oxazole derivatives expressed by the formula wherein R₁ represents hydrogen, C n H 2n+1 , XC n H 2n , C n H 2n+1 CO₂, a phenyl group, a naphtyl group, C n H 2n+1 C₆H₄, YC₆H₄ or XYC₆H₃, wherein n is a positive integer from 1 to 4, X is F, Cl Br or I, and Y is F, Cl, Br, I or a phenyl group. Brief Description of Drawing Figs. 1 and 2 show the results of analysis of CA15-3 with an enhancer of the present invention, or 2-hydroxy-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 19, 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, [4-hydroxy-3-[3-(4-hydroxyphenyl)-1-oxo-2-propenyl]-2H-1-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 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. HHBP 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 described in the patent publication No. JP-A-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 [in the formula, R₁ representing hydrogen, C n H 2n+1 (here, n representing a positive integer of 1 to 4), XC n H 2n (here, X representing F, Cl, Br or I, and n being as defined above), C n H 2n+1 CO₂ (n being as defined above), C n H 2n+1 C₆H₄ (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-methylethyl, 1-chloropropyl, 2-chloropropyl, 3-chloropropyl, 1-chloro-1-methylethyl, 2-chloro-1-methylethyl, 1-bromopropyl, 2-bromopropyl, 3-bromopropyl, 1-bromo-1-methylethyl, 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-fluorobutyl, 1-fluoro-1-methylpropyl, 2-fluoro-1-methylpropyl, 3-fluoro-1-methylpropyl, 1-fluoromethylpropyl, 1-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-bromomethylmethyl-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-methylpropyl, 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, propoxycarbonyl, 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-ethylphenyl, 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-chlorophenyl, 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-dichlorophenyl. 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 [in the formula, R₂ representing XC n H 2n (here, X representing F, Cl, Br or I, and n representing a positive integer of 1 to 4), C n H 2n+1 CO₂ (n being as defined above), C n H 2n+1 C₆H₄ (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 [in the formula, R₁ representing hydrogen, C n H 2n+1 (here, n representing a positive integer of 1 to 4), XC n H 2n (here, X representing F, Cl, Br or I, and n being as defined above), C n H 2n+1 CO₂ (n being as defined above), phenyl group, naphthyl group, C n H 2n+1 C₆H₄ (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 C n H 2n+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, 1-methylpropyl, 2-methylpropyl and 1,1-dimethylethyl. 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 150°C, but for enhancing the yield of the objective product, a temperature of 50 to 120°C is preferable. The oxazole compound [I] may also be produced, when R₁ is XC n H 2n (X representing F, Cl, Br or I, and n representing a positive integer of 1 to 4), according to the formula (2), or when R₁ is YC₆H₄ (Y representing F, Cl, Br, I or a phenyl group) , according to the formula (3), set forth below. [in the formula, R₃ representing C n H 2n+1 (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 compound [I] may be used. Also, for R₃ used in the compound (IV), the same R₃ as that used in the compound [III] may be used. The reaction solvent used according to the present invention includes alcohols 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 150°C, but for increasing the yield of the objective product, a temperature of 60 to 90°C is preferable. [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 280°C, but for enhancing the yield of the compound [VI], a temperature of 150 to 250°C 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 50°C, but for enhancing the yield of the object, a temperature of 25 to 35°C 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 chloroacetonitrile 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-phthalazinedione and isoluminol (6-amino-2,3-dihydro-1,4-phthalazinedione) 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-ethylisoluminol (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-fluorenone purchased from the Aldrich Chemical Co. HHBP was prepared according to the method described in Patent Publication No. JP-A-50-46666. Mammary cancer related antigen (CA15-3) and anti-CA15-3 antibody (mouse monoclonal antibody) were provided by Centocor (Malvern, USA). The horse radish peroxidase labeled anti-CA15-3 antibody was prepared by the maleimide hinge 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, ⊘7.6 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 µg/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 EX90FTNMR, 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 spectrograph used. Example 1 Luminescent assay of peroxidase in use of luminol and 2-hydroxy-9-fluorenone 200 µl of luminol (100 mM DMSO solution 10 µl/10 ml 0.1M tris-hydrochloride buffer solution, pH 7.5), 200 µl of 2-hydroxy-9-fluorenone (100 mM DMSO solution 10 µl/10 ml 0.1M tris-hydrochloride buffer solution, pH 7.5), 10 µl 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 µl of hydrogen peroxide (1000 times dilution of a 9.1M 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 µl 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. Signal-to-background ratio (SN ratio) with 2,3-dihydro-1,4-phthalazinedione (DPD) DPD Luminescent intensity after 10 seconds (relative value) +HRP -HRP SN ratio Example 1 Luminol 449863 103 4368 Example 2 Isoluminol 9885 99 99.8 Example 3 N-(4-aminobutyl)-N-ethylisoluminol 17370 96 180.9 Reference 1 Luminol 149 133 1.12 Reference 2 Isoluminol 167 154 1.08 Reference 3 N-(4-aminobutyl)-N-ethylisoluminol 157 137 1.15 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 µl. Then, 300 µl 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 25°C 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 µl of luminol solution (100 mM DMSO solution 10 µl/10 ml 0.1M tris-hydrochloride buffer solution, pH 7.5), 200 µl of 2-hydroxy-9-fluorenone solution (100 mM DMSO solution 10 µl/ 10 ml 0.1M tris-hydrochloride buffer solution, pH 7.5) and 10 µl of hydrogen peroxide solution (1000 times dilution of a 9.1M 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 10 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-iodophenol, p-hydroxycinnamic acid, p-phenylphenol, 6-hydroxybenzothiazole 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. Example 5 Luminescent assay of peroxidase in use of luminol and HHBP 200 µl of luminol solution (100 mM DMSO solution 10 µl/10 ml 0.1M tris-hydrochloride buffer solution, pH 8.5), 200 µl of HHBP solution (100 mM DMSO solution 10 µl/10 ml 0.1M tris-hydrochloride buffer solution, pH 8.5), 10 µl 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 µl of hydrogen peroxide solution (1000 times dilution of a 9.1M 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 µl 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. 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. Signal-to-background ratios (SN ratios) with 2,3-dihydro-1,4-phthalazinedione (DPD) DPD Luminescent intensity after 1 minute (relative value) +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 100°C 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. 2°C IR (KBr, cm⁻¹): 3150, 1630, 1528, 1489, 1276, 1236, 1195, 1104, 1085, 816 NMR (δ, DMSO-d₆): 6.81 (dd, 1H), 7.03 (d, 1H), 7.53 (d, 1H), 8.46 (s, 1H), 9.75 (s, 1H) 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 µl of a luminol solution (100 mM DMSO solution 10 µl/10 ml, 0.1M tris-hydrochloride buffer solution, pH 8.5), 200 µl of a 6-hydroxybenzoxazole solution (100 mM DMSO solution 10 µl/10 ml, 0.1M tris-hydrochloride buffer solution, pH 8.5), 10 µl 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 µl of a hydrogen peroxide solution (1000 times dilution of a 9.1M 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 µl 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-ethylisoluminol (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 15 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. 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 µl. To each well, 300 µl 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 25°C 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 µl of luminol solution (100 mM DMSO solution 10 µl/10 ml 0.1M tris-hydrochloride buffer solution, pH 7.5), 200 µl of HHBP solution (100 mM DMSO solution 10 µl/ml 0.1M tris-hydrochloride buffer solution, pH 7.5) and 10 µl of hydrogen peroxide solution (1000 times dilution of a 9.1M 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 10 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. Analysis of CA15-3: Mean luminescent intensities after 10 seconds (relative values) Enhancer CA15-3 (U/ml) 0 25 50 100 200 300 Example 17 HHBP 602 8031 17045 30454 65824 91745 Example 18 2-Methyl-6-hydroxybenzoxazole 401 3370 6929 17223 27926 46613 Reference 15 None 154 148 165 165 152 182 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 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-hydroxybenzoxazole, and the yield of the reaction was 30.9%. m.p.: 148.5 to 153.7°C 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, 1H), 7.55 (d, 1H), 9.89 (s, 1H) MS (EI): 183 (M⁺), 148 High resolution MS (EI): C₈H₆O₂NCl 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 105°C 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 85°C was collected. The yield of dichloroethoxyethyl 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 90°C 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 89°C. 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 two-necked flask provided with a cooling condenser under argon atmosphere, and the mixture was stirred with heat at 100°C 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 200.2°C IR (Fig. 6, KBr, cm⁻¹): 3278, 1736, 1630, 1539, 1489, 1261, 1241, 1145, 116, 849 NMR (Fig. 7, DMSO-d₆): 1.35 (t, 3H), 4.41 (q, 2H), 6.97 (dd, 1H), 7.14 (d, 1H), 7.72 (d, 1H), 10.25 (bs, 1H) MS (EI): 207 (M⁺), 135 High resolution MS (EI): C₁₀H₉O₄N Calculated 207.0591 Observed 207.0561 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 145°C 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 1N 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 248.5°C IR (Fig. 8, KBr cm⁻¹): 3190, 1630, 1549, 1475, 1325, 1234, 1143, 1062, 835 NMR (Fig. 9, DMSO-d₆): 6.90 (dd, 1H), 7.12 (d, 1H), 7.66 (m, 2H), 7.71 (d, 1H), 8.15 (m, 2H), 9.94 (s, 1H) MS (EI): 289 (M⁺), 291 (M⁺+2), 210, 182 High resolution MS (EI): C₁₃H₈O₂NBr 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 215°C 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 230°C 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 137.5°C IR (Fig. 10, KBr, cm⁻¹): 3064, 1613, 1489, 1224, 1151, 729 NMR (Fig. 11, DMSO-d₆): 2.73 (s, 3H), 6.87 (dd, 1H), 7.11 (d, 1H), 7.42 (m, 3H), 7.64 (d, 1H), 8.06 (m, 1H), 9.86 (s, 1H) MS (EI): 225 (M⁺), 196, 168, 156, 116, 91, 79, 51, 39 High resolution MS (EI): C₁₄H₁₁O₂N Calculated 225.0790 Observed 225.0764 Example 23 Luminescent assay of peroxidase in use of luminol and 2-ethoxycarbonyl-6-hydroxybenzoxazole 200 µl of a luminol solution (100 mM DMSO solution 10 µl/10 ml 0.1M tris-hydrochloride buffer solution, pH 8.5), 200 µl of the 2-ethoxycarbonyl-6-hydroxybenzoxazole obtained in Example 3 (100 mM DMSO solution 10 µl/10 ml, 0.1M tris-hydrochloride buffer solution, pH 8.5) 10 µl 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 µl of an aqueous solution of hydrogen peroxide (1000 times dilution of a 9.1M aqueous solution) were introduced into a plastic cuvette, and the mixture was stirred for 3 seconds with a vortex mixer used, then the luminescent intensity after 1 minute was measured. Next, 10 µl 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-hydroxybenzoxazole 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, 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-methylphenyl)-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. 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 235°C 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 247.5°C IR (Fig. 12, KBr, cm⁻¹): 3146, 1630, 1599, 1551, 1477, 1328, 1232, 1145, 1062, 835 NMR (Fig. 13, DMSO-d₆): 6.90 (dd, 1H), 7.12 (d, 1H), 7.61 (m, 3H), 8.06 (m, 2H), 9.94 (s, 1H) MS (EI): 245 (M⁺), 247 (M⁺+2), 138, 122, 80, 52 High resolution MS (EI): C₁₃H₈O₂NCl 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 200°C 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 273.5°C IR (Fig. 14, KBr, cm⁻¹): 3138, 1632, 1618, 1485, 1236, 1143, 832 NMR (Fig. 15, DMSO-d₆): 6.87 (dd, 1H), 7.11 (d, 1H), 7.62 (m, 3H), 8.13 (d, 2H), 9.90 (s, 1H) MS (EI): 245 (M⁺), 247 (M⁺+2), 138, 122 High resolution MS (EI): C₁₃H₈O₂NCl 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 205°C 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 226.2°C IR (Fig. 16, KBr, cm⁻¹): 3050, 1620, 1485, 1450, 1303, 1141, 1116, 816, 752 NMR (Fig. 17, DMSO-d₆): 6.92 (dd, 1H), 7.17 (d, 1H), 7.64 (m, 3H), 8.08 (m, 4H), 8.74 (s, 1H), 9.92 (s, 1H) MS (EI): 261 (M⁺), 130 High resolution MS (EI): C₁₇H₁₁O₂N 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 247°C 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 212.8°C IR (Fig. 18, KBr, cm⁻¹): 3150, 1638, 1611, 1564, 1489, 1464, 1325, 1232, 1094, 822, 808 NMR (Fig. 19, DMSO-d₆): 6.92 (dd, 1H), 7.11 (d, 1H), 7.66 (m, 2H), 7.85 (dd, 1H), 8.12 (d, 1H), 9.97 (s, 1H) MS (EI): 279 (M⁺), 281 (M⁺+2), 283 (M⁺+4), 172, 139, 108, 80, 52 High resolution MS (EL): C₁₃H₇O₂NCl Calculated 278.9898 Observed 278.9876 Example 39 Luminescent assay of peroxidase in use of luminol and 2-(3-chlorophenyl)-6-hydroxybenzoxazole 200 µl of a luminol solution (100 mM DMSO solution 10 µl/10 ml, 0.1M tris-hydrochloride buffer solution, pH 8.5), 200 µl of the 2-(3-chlorophenyl)-6-hydroxybenzoxazole solution obtained in Example 35 (100 mM DMSO solution 10 µl/10 ml, 0.1M tris-hydrochloride buffer solution pH 8.5), 10 µl 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 µl of a solution of hydrogen peroxide (1000 times dilution of a 9.1M 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 µl 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 µl of a luminol solution (100 mM DMSO solution 10 µl/10 ml, 0.1 M tris-hydrochloride buffer solution pH 8.5), 200 µl of the 2-(4-chlorophenyl)-6-hydroxybenzoxazole solution obtained in Example 36 (100 mM DMSO solution 10 µl/10 ml, 0.1M tris-hydrochloride buffer solution pH 8.5), 10 µl 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 µl of a solution of hydrogen peroxide (1000 times dilution of a 9.1M 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 µl 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 µl of a luminol solution (100 mM DMSO solution 10 µl/10 ml, 0.1M tris-hydrochloride buffer solution pH 8.5), 200 µl of the 2-(2-naphthyl)-6-hydroxybenzoxazole solution obtained in Example 37 (100 mM DMSO solution 10 µl/10 ml, 0.1M tris-hydrochloride buffer solution pH 8.5), 10 µl 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 µl of a solution of hydrogen peroxide (1000 times dilution of a 9.1M 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 µl 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 µl of a luminol solution (100 mM DMSO solution 10 µl/10 ml, 0.1M tris-hydrochloride buffer solution pH 8.5), 200 µl of the 2-(2,4-dichlorophenyl)-6-hydroxybenzoxazole solution obtained in Example 38 (100 mM DMSO solution 10 µl/10 ml, 0.1M tris-hydrochloride buffer solution pH 8.5), 10 µl 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 µl of a solution of hydrogen peroxide (1000 times dilution of a 9.1M 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 µl 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. 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 µl. Then, 300 µl 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 25°C 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 µl of a luminol Na salt solution (18 times dilution of a solution of 12.6 mM, luminol Na salt/0.1M tris-hydrochloride buffer solution pH 8.5 with the same buffer solution), 100 µl of a 2-ethoxycarbonyl-6-hydroxybenzoxazole solution (100 mM DMSO solution 10 µl/10 ml, 0.1M tris-hydrochloride buffer solution pH 8.5) and 100 µl of a solution of hydrogen peroxide (18 times dilution of 16.2 µM hydrogen peroxide/0.01M 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 37°C 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 50 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. Industrial Applicability As described in the foregoing, the method of luminescence analysis of the present invention enables high sensitivity and prompt determination of substances.

Claims: A method for detecting or determining a substance by the chemiluminescence produced through reaction of (a) peroxidase or a derivative thereof, (b) an oxidant and (c) luminol or isoluminol or a derivative thereof, characterized in that the luminescence inducing reaction is carried out in the presence of at least one compound chosen from the group comprised of 2-hydroxy-9-fluorenone, the compound expressed by the following formula and oxazole derivatives expressed by the formula wherein R₁ represents hydrogen, C n H 2n+1 , XC n H 2n , C n H 2n+1 CO₂, a phenyl group, a naphtyl group, C n H 2n+1 C₆H₄, YC₆H₄ or XYC₆H₃, wherein n is a positive integer from 1 to 4, X is F, Cl, Br or I, and Y is F, Cl, Br, I or a phenyl group. The method according to Claim 1 wherein R₁ is hydrogen, a methyl group, a chloromethyl group, an ethoxycarbonyl group, a phenyl group, a bromophenyl group, a methylphenyl group, a chlorophenyl group, a naphtyl group or a dichlorophenyl group. An oxazole derivative having the formula wherein R₂ represents XC n H 2n , C n H 2n+1 CO₂, a naphtyl group, C n H 2n+1 C₆H₄, YC₆H₄ or XYC₆H₃, wherein n is a positive integer from 1 to 4, X is F, Cl, Br or I, and Y is F, Cl, Br, I or a phenyl group. An oxazole derivative as set forth in claim 3 wherein R₂ is a chloromethyl group, an ethoxycarbonyl group, a bromophenyl group, a chlorophenyl group, a naphtyl group, a dichlorophenyl group or a methylphenyl group.

Inventors: HOSAKA SHUNTARO, MAKINO TETSUYA, OYAMA YOSHIHIRO, HOSAKA, SHUNTARO, MAKINO, TETSUYA, OYAMA, YOSHIHIRO
Applicants: TORAY INDUSTRIES, TORAY INDUSTRIES, INC.
Application year: 1990
Main classification: C12Q1
IPC codes: C07D311, C07D263, G01N21, C09K11, C12Q1