JP2735205B2 - Fluorescence analysis method - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a fluorescence analysis method, and more particularly to a fluorescence analysis method suitable for performing a fluorescence analysis by an automatic analyzer employing a direct fluorescence photometry method. [Prior Art] Conventionally, an automatic analyzer for performing an immunoassay or the like has mainly been a system for measuring the absorbance of a measurement solution. However, in recent years, there has been an increasing demand for analyzing trace components contained in a measurement solution, and attention has been paid to fluorescence photometry, which is said to be capable of high-sensitivity analysis. As a means for adopting this fluorescence photometry method in an automatic analyzer, a flow cell method in which a reaction solution is sucked into a flow cell fixed to the apparatus and photometry is performed is the simplest. However, according to this flow cell method, the photometry is performed by repeatedly supplying and discharging a plurality of types of samples to and from the flow cell, so that the carry-over rate between the samples is high. Need to be washed. Further, a reaction liquid suction mechanism to the flow cell is required, which is expected to increase the cost. In addition, it is difficult to control the temperature of the outer periphery of the flow cell with high accuracy required for clinical analysis. As a means for solving these problems, there is a direct fluorescence photometry system. In this method, the samples are filled into different husks and photometry is performed. Other known direct fluorescence photometry methods include a method of measuring fluorescence polarization and a so-called top / top photometry method in which fluorescence is extracted from the same liquid surface as the liquid surface on which the sample enters the excitation light. [Problems to be Solved by the Invention] However, in the method of photometry using the above-mentioned hubet, when a large number of samples are continuously analyzed, the hubet is often washed and used repeatedly. For this reason, as the number of times of use increases, the cover is damaged. Also, it is difficult to supply all the huskets completely intact even in the husk supply stage. And, the scratches generated on these kits greatly affect the fluorescence intensity. Further, in the case of performing continuous measurement using a large number of packets, it is required that the light source be arranged to irradiate the cell at the time of measurement at a fixed position and a fixed angle. However, in an automatic analyzer in which the light source and the light source move, it is difficult to always keep the light source and the light source in a fixed positional relationship, and the light source often shifts slightly depending on the light source. The displacement between the two affects the fluorescence intensity. In addition, the distortion and the slight difference in the wall thickness of the velvet surface during the velvet molding also affect the measurement results. In the case of the above-described method of measuring the fluorescence polarization, since the measurement result is calculated based on the ratio of the fluorescence intensities, there are few problems caused by the above-mentioned huette. However, the components to be measured that can be analyzed based on the fluorescence polarization method are limited to low molecules in terms of molecular weight, and are not suitable for measuring large molecular components, which are highly required in clinical analysis. In addition, in the above-described top / top photometry method, since the measurement is performed while moving the covert in order to improve the accuracy of data, a fluctuation occurs in the measurement liquid level. The fluctuation of the liquid level causes a problem that the accuracy of data is actually reduced, and also has a problem that it is difficult to apply the data to a conventional automatic analyzer. That is, the conventional direct fluorescence measurement method of performing normal fluorescence analysis by a light measurement method of a type in which light crosses a filter is difficult to apply to an automatic analyzer because of the above-described problems. An object of the present invention is to provide a fluorescence analysis method capable of reducing a measurement error due to a difference between cuvettes when measuring a fluorescence intensity based on a fluorescent substance generated in each cuvette. (Means for solving the problem) The present invention is a fluorescence analysis method for generating a fluorescent substance according to the concentration of a measurement target substance in a sample in a cuvette, and measuring fluorescence generated by irradiation with excitation light, The fluorescent substrate used to generate the fluorescent substance is irradiated with excitation light for each cuvette containing a reaction solution coexisting with the fluorescent substance, and the fluorescence intensity and the fluorescent substrate at a wavelength for the fluorescent substance Is measured for each cuvette, and the measured fluorescence intensity corresponding to the fluorescent substance is corrected for each cuvette using the measured fluorescence intensity corresponding to the fluorescent substrate. [Action] According to the above method, a certain amount of a measurement auxiliary substance different from the measurement target substance is included in the measurement solution and analyzed, and the measured value of the measurement target substance is corrected based on the measurement result. This eliminates the need for extra measurement time for correction and cleaning of the kit. At this time, the measurement auxiliary substance and the measurement wavelength thereof are selected such that the fluorescence intensity is not affected by the difference in the concentration of the measurement target substance and shows a constant value. Now, for example, assume that the substance to be measured is A, the auxiliary substance for measurement is B, and the fluorescence intensity of A in the state of being put in an ideal non-scratched cover is F ₁ , and the fluorescence intensity of B is F ₂ . Also the fluorescence intensity of the A and B were measured in cuvette that further normal use and F ₃ and F _4, respectively. At this time, since there is an equal concentration and an equal amount of B in both the cubes, F ₂ = F ₄ should be satisfied. For this reason, F ₃ becomes F ₃ × F ₂ / F ₄ after correcting the error due to the packet. As described above, by measuring the fluorescence intensity of B in a manner similar to that of A for one of the kits, it is possible to correct the kitt difference and perform direct fluorescence measurement with high accuracy. [Example] In a preferred embodiment according to the present invention, together with the substance to be measured, a substance different from the substance to be measured contained in the measurement solution in a certain amount is simultaneously measured, and the former is determined based on the measurement result of the latter. Correct the measurement result. The generated fluorescent substance, which is the substance to be measured, is irradiated with the excitation light in the coexistence state of the measurement auxiliary substance as a component for correction. When the component for correction is a substrate for the reaction, the fluorescence intensity of the reaction product can be corrected by the fluorescence intensity of the substrate that is the reaction component.
Great effect. Hereinafter, an embodiment of the fluorescence analysis method according to the present invention will be described. First, add 2 ml each to 20 different kibet
A ^0-7 M FITC (Fluorescein isothiocyanate) solution was dispensed, and the fluorescence intensity was measured. Excitation was measured at 480 nm and fluorescence was measured at 530 nm. Further, a sample with no scratches or distortion was fixed to a sample holder of a fluorometer, and the same sample as above was repeatedly measured. The measurement results are shown in Tables 1 and 2. Table 1 shows the measured values in a kibet without scratches and distortion. Table 2 shows the measured values for the different cubes. For each of the above measurements, a popular fluorometer in which the excitation light and the fluorescence respectively passed through the wall of the cube and the excitation light incidence direction and the fluorescence emission direction were perpendicular was used. As is evident from the above tables, the measurement results vary greatly due to differences in the cubes, indicating that correction of the cube difference is necessary for direct fluorescence photometry. Next, the sample containing the drug to be measured, theophylline
50μ, antibody solution 500μ, enzyme solution (β-
(Galactosidase) 500μ, 50μm of umbelliferone / theophylline conjugate labeled with a substrate derivative (galactose) and 400μ of Bieine buffer (pH 8.5) at 37 ° C.
The reaction was performed for 20 minutes. The theophylline substrate derivative-labeled umbelliferone theophylline conjugate in this sample competes with the antibody to react. Then, the fluorescence intensity of the fluorescent substance produced as a result of hydrolysis of the substrate derivative-labeled umbelliferone-theophylline conjugate that could not bind to the antibody by the catalytic action of the enzyme (β-galactosidase) was measured. Theophylline concentration was determined by fitting to a previously prepared standard curve. For the measurement, an excitation wavelength of 400 nm and a fluorescence wavelength of 450 nm were used. The measurement reproducibility of the fluorescence intensity at this time is shown in Table 3 below. Although not directly involved in the reaction at the time of the reaction, Texas Red (1 mg / 5 ml solution) was added to the reaction solution in 10 μl portions, and the reaction was carried out at 37 ° C. for 20 minutes as described above. For the measurement, the first photometry is performed at 400 nm for excitation and 450 nm for fluorescence to measure the theophylline concentration. Next, excitation 590 nm for the same reaction solution,
A second photometry was performed at 630 nm fluorescence. At this time, the first result was corrected based on one of the second results, and the reproducibility of the measurement was obtained. The results are shown in Table 4 below. From the above results, it can be seen that the measurement reproducibility was significantly improved by correcting the difference of the Cubeto using the Texas Red measurement value. However, in this method, substances that do not participate in the reaction of the sample must be added to the cube. Next, 50 μl of a sample containing α-phytoprotein (AFP) as a substance to be measured, glass beads to which an anti-AFP antibody is bound, and alkaline phosphatase (ALP) -labeled anti-AFP
Antibody solution 50μ, 10mM Tris-HCl buffer (pH7.4) 100μ
Was added and reacted at 37 ° C. for 20 minutes. Thereafter, the glass beads were sufficiently washed with a buffer. The glass beads were transferred to a kubet, and 10 mM 50 μm of umbelliferone phosphate (MUP), which is an auxiliary substance for measurement and a substrate,
Add M diethanolamine buffer (pH 9.0) and add
Allowed to react for minutes. Excitation 375 nm, fluorescence for umbelliferone (MUB) generated as a result of MUP enzyme reaction
450 nm was used. Since the concentration of MUB to be measured changes according to the concentration of AFP contained in the sample, AFP in the sample can be quantified based on the fluorescence intensity of MUB. On the other hand, since MUP is a substrate, it is always added in a fixed amount to the entire reaction.Moreover, the enzymatic reaction proceeds in a large excess region of the substrate, and the generated MUB is expressed in terms of concentration.
A trace three orders of magnitude lower than UP. Therefore, the MUP amount in the reaction solution can be considered to be constant regardless of the degree of progress of the enzyme reaction. MUP is a fluorescent substrate and shows a different fluorescence spectrum from the product MUB. Also, by appropriately selecting the photometric wavelength of excitation and fluorescence, the MUP measurement value shows a constant value regardless of the MUB concentration in the reaction solution. Therefore, the reproducibility measurement of MUB photometry was performed using the MUP measurement value for correction. For the measurement of MUP as this measurement auxiliary substance,
Excitation 356 nm and fluorescence 410 nm were used. The results are shown in Table 5 below. As described above, when MUB is corrected based on the measured value of MUP, measurement reproducibility is significantly improved. First
The figure shows the MUP fluorescence spectrum for different MUB concentrations. [Effects of the Invention] According to the present invention, although there is no need to add an extra substance unrelated to the reaction of the sample, the difference between cuvettes in which the sample is subjected to the reaction treatment (the scratches and dirt on the cuvette). Difference) can be corrected, so that the measurement by the direct fluorescence photometry method using the automatic analyzer can be put to practical use.

[Brief description of the drawings] FIG. 1 is a graph showing the fluorescence spectrum of MUP.

   ────────────────────────────────────────────────── ─── Continuation of front page    (56) References JP-A-62-105036 (JP, A)                 JP-A-62-59842 (JP, A)                 JP-A-63-177038 (JP, A)                 JP-A-62-12837 (JP, A)                 Japanese Patent Laid-Open No. Sho 50-106993 (JP, A)                 Tokiko Sho 59-30215 (JP, B1)                 Anal. Chem. (1982) Vo               l. 54, No. 4P. 634-637                 Anal. Chem. (1977) Vo               l. 49, no. 4P. 667-668

Claims (1)

(57) [Claims] In a fluorescence analysis method for generating a fluorescent substance according to the concentration of a substance to be measured in a sample in a cuvette and measuring fluorescence generated by irradiation with excitation light, the fluorescent substrate used for generating the fluorescent substance However, the excitation light is irradiated for each cuvette containing the reaction solution coexisting with the fluorescent substance, and the cuvette changes the fluorescence intensity at the wavelength corresponding to the fluorescent substance and the fluorescence intensity at the wavelength corresponding to the fluorescent substrate. A fluorescence analysis method comprising: measuring the fluorescence intensity for each cuvette; and correcting the fluorescence intensity measurement value corresponding to the fluorescent substance using the fluorescence intensity measurement value corresponding to the fluorescent substrate for each cuvette.