JP3025096B2 - Fluorescence analysis - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a method for irradiating a fluorescent substance with a plurality of types of laser light having a wavelength longer than the maximum excitation wavelength under basic conditions, whereby the fluorescent substance is excited and absorbed by multiphoton absorption. The present invention relates to a method for performing fluorescence analysis with high sensitivity using a small device by utilizing emission of fluorescence having a shorter wavelength than light.

[0002]

2. Description of the Related Art
In order to analyze a fluorescent biological sample or a biological sample labeled with a fluorescent dye by a fluorescent analysis method, a fluorescent substance, particularly fluorescein, a coumarin derivative, or the like is used for 300 to 600 nm
Was irradiated as excitation light, and the resulting fluorescence was measured and analyzed. However, in the case of such a conventional method, since the wavelength difference between the excitation light and the fluorescence is generally only 20 to 90 nm, when performing the fluorescence analysis, a part of the fluorescence is also added to the cut filter for absorbing and removing excess excitation light. Due to absorption loss, it was difficult to sufficiently increase the analytical sensitivity.

On the other hand, an excitation light source for fluorescence analysis preferably has a large output and is monochromatic light, and it is naturally desirable that the excitation light source be small in size in order to facilitate downsizing of the analyzer. A light source that satisfies these requirements is a semiconductor laser, but no semiconductor laser can be used as an excitation light source in the 300 to 600 nm region.

[0004]

Means for Solving the Problems The present inventor has disclosed in Japanese Patent Application No. Hei.
In 287858, a method for exciting a fluorescent substance with a laser beam having a wavelength (2λ ± 100 (mm)) about twice the maximum excitation wavelength under basic conditions to measure fluorescence has been found. The present invention relates to a method for measuring fluorescence emission by so-called multiphoton absorption, in which a plurality of photons having energy smaller than the maximum excitation wavelength are absorbed and one photon is emitted.

That is, under basic conditions, a conventional substance which emits fluorescence by excitation light of 300 to 600 nm has a wavelength longer than the maximum excitation wavelength as long as it is a set of wavelengths satisfying the following formula and It can absorb and excite light. Since the fluorescent substance at that time is considered to pass through the vibronic state generated by the interaction between the electronic state and the vibration state as an intermediate state, the fluorescent substance has the same wavelength and the same intensity as the fluorescent light emitted when excited at 300 to 600 nm. Can emit fluorescent light.

As a result, a fluorescent substance can be excited with high efficiency by a commercially available semiconductor laser having a long wavelength oscillation, so that the size of the analyzer can be reduced. In addition, the inventors have found that high sensitivity can be realized because the absorption loss of fluorescence by the filter can be suppressed low.

The maximum excitation wavelength (λ nm) described in the present invention is an excitation wavelength at which the intensity of fluorescence emitted by one-photon absorption under basic conditions becomes maximum. The maximum excitation wavelength mainly corresponds to the maximum absorption wavelength of the fluorescent substance under basic conditions.
Further, the absorption of a plurality of photons means the absorption of two or more photons, and more preferably the absorption of two photons (two-photon absorption).
It is.

In the present invention, only one type of wavelength can be used as the excitation wavelength, provided that the mathematical formulas and are satisfied. in this case,

[0009]

(Equation 3)

[0010]

(Equation 4)

Must be satisfied. The method of the present invention can be used for the following fluorescence analysis.

(1) A fluorescent substance to be measured is excited under a basic condition by a plurality of laser beams having a wavelength longer than the maximum excitation wavelength of the fluorescent substance which satisfies the formula and the fluorescence is measured. Fluorescence analysis.

(2) An immunological substance is immobilized on the surface of an optical fiber, and (a) a substance exhibiting the same immune reaction as the substance to be measured and the substance to be measured labeled with a fluorescent substance is applied to the immunological substance on the surface. After a competitive reaction, or (b) immunoreacting the substance to be measured with the immune substance on the surface and then reacting a substance immunoreactive with the substance to be measured labeled with a fluorescent substance, A fluorescence immunoassay method characterized in that the fluorescence is measured by exciting with a plurality of laser beams having a wavelength longer than the maximum excitation wavelength of the fluorescent substance that satisfies the formula and the above.

(3) Immobilizing an immunological substance on the surface of an optical fiber; (a) Competitively reacting the immunological substance on the surface with a substance to be measured and a substance showing the same immune reaction as the substance to be measured to which biotin is bound Or (b) immunoreacting the immunological substance on the surface with the analyte and then reacting the immunoreactive substance with the analyte to which biotin is bound, and then avidin labeled with a fluorescent substance is reacted with A fluorescent immunoassay method comprising reacting, under basic conditions, exciting with a plurality of laser beams having a wavelength longer than the maximum excitation wavelength of the fluorescent substance satisfying the mathematical formula and measuring the fluorescence.

Examples of the fluorescent substance include base-soluble fluorescein, coumarin derivatives, rhodamine B, dansyl derivatives and the like. The basic condition is desirably pH 8 to 13. If the pH is higher than this, the fluorescent substance may be hydrolyzed, which is not preferable.

The method (1) comprises measuring a fluorescence by exciting a fluorescent substance to be measured under basic conditions with a plurality of laser beams having a wavelength longer than the maximum excitation wavelength of the fluorescent substance satisfying the formula and (See Example 1). The fluorescent substance to be measured may be a fluorescently labeled substance to be measured. In this case, as a method of fluorescently labeling the substance to be measured, there are a method of directly binding the fluorescent substance and a method of binding via a substance that specifically binds to the substance to be measured or avidin-biotin.

The method (2) is roughly classified into a competitive method (a) and a sandwich method (b).

In the competitive method (a), a substance (for example, an antigen) which shows the same immunoreactivity as the substance labeled with a fluorescent substance soluble in a base having a known concentration and a substance to be measured are mixed. An optical fiber on which an immunological substance (for example, an antibody) is immobilized is immersed in this solution to react competitively. In the competition method, the higher the concentration of the analyte, the lower the amount of binding of the analyte labeled with the fluorescent substance to the optical fiber, and thus the lower the fluorescence intensity (see Examples 2, 4, and 6).

In the sandwich method (b), an optical fiber on which an immunological substance (for example, an antibody) is immobilized is immersed in a solution of a substance to be measured (for example, an antigen) and reacted (antigen-antibody reaction), and then this optical fiber is dissolved in a base. And immersed in a solution of a substance (for example, an antibody) immunoreactive with the substance to be measured labeled with a fluorescent substance. In the sandwich method, the substance to be measured is sandwiched between the immunological substance on the optical fiber and the substance immunoreactive with the substance to be measured labeled with a fluorescent substance. Therefore, in this method, as the concentration of the analyte increases, the amount of the substance that immunoreacts with the analyte labeled with the fluorescent substance binds to the optical fiber, and the fluorescence intensity increases (Examples 3, 5, 7).

When the concentration of the substance to be measured is measured by the method (2), it is necessary to increase the binding amount of the fluorescent substance per molecule of the immunological substance (antigen or antibody) in order to improve the measurement sensitivity. Therefore, a substance exhibiting the same immune reaction as the substance to be measured or a substance immunoreactive with the substance to be measured is bound to biotin, and the biotin is bound to avidin labeled with a fluorescent substance, or a plurality of reactive groups. It is preferable that avidin labeled with a fluorescent substance soluble in a base is bound to the plurality of reactive groups via biotin.

In these methods, since a large number of avidin labeled with a fluorescent substance is bound to a substance exhibiting the same immunoreactivity as the substance to be measured or a substance immunoreactive with the substance to be measured, the same substance as the substance to be measured is used. It is possible to increase the amount of the fluorescent substance bound per molecule of the substance exhibiting an immune reaction or the substance immunoreacting with the substance to be measured. As a result, it is useful to dramatically improve the detection sensitivity. Avidin and biotin can be replaced by a set of compounds having similar effects. For example, antibody-protein A and the like.

Examples of the substance having a plurality of reactive groups include:
Polypeptides such as polylysine, chitosan, polygalactosamine and polyneuraminic acid, or aminoglycans are used, and chitosan is particularly preferred. 200 to 100,000 reactive active groups per molecule, preferably 4000 to 100
Desirably, there are 5,000.

The method (3) is suitable when an unstable fluorescent substance is used, and also includes a competitive method (a) and a sandwich method (b). Utilizing the specific binding of avidin and biotin, a substance to be measured in which biotin is directly bound or in which a substance having a plurality of reactive groups with biotin bound to a plurality of reactive groups is bound A substance exhibiting the same immune reaction as described above or a substance immunoreacting with the substance to be measured is first subjected to an immunoreaction on an optical fiber, and then avidin labeled with a fluorescent substance is bound thereto. This is because a certain fluorescent substance is susceptible to hydrolysis or oxidation in an aqueous solution, so that if the label is performed immediately before the measurement, the decrease in the fluorescent substance can be suppressed to a low level.

In the competitive method (a), a substance to be measured and a substance exhibiting the same immunoreactivity as the substance to which biotin is bound are mixed, and an optical fiber on which the immunological substance is immobilized is immersed in this solution. Immune reaction. Next, when avidin labeled with a fluorescent substance is reacted, a substance exhibiting the same immunoreactivity as the substance to be measured is labeled with the fluorescent substance via biotin-avidin. In the competition method, the higher the concentration of the analyte, the lower the amount of binding of the analyte labeled with the fluorescent substance to the optical fiber, and thus the lower the fluorescence intensity (see Examples 8 and 10).

In the sandwich method (b), an optical fiber on which an immunological substance is immobilized is immersed in a substance to be measured solution to cause an immunoreaction, and the immunological substance on the optical fiber and the substance to be measured are bound. Next, this optical fiber is immersed in a solution of a substance immunoreactive with the substance to be measured to which biotin is bound. The immunological substance on the optical fiber and the substance immunoreactive with the biotin-bound substance to be measured are bound in a state where the substance to be measured is sandwiched. Next, when this optical fiber is immersed in a solution of avidin labeled with a fluorescent substance soluble in a base, a substance immunoreacting with the substance to be measured via biotin-avidin is labeled with the fluorescent substance. In the sandwich method, the higher the concentration of the substance to be measured, the larger the amount of a substance immunoreactive with the substance to be measured labeled with a fluorescent substance, which binds to the optical fiber, so that the fluorescence intensity increases (see Examples 9 and 11).

Biotin is bound to a substance having the same immunoreactivity as the analyte or a substance immunoreactive with the analyte via the substance having a plurality of reactive groups described in the method (2). Is desirable. Avidin and biotin can be replaced by a set of compounds having similar effects.

In the method (2) and the method (3), a substance exhibiting the same immune reaction as the substance to be measured or a substance immunoreactive with the substance to be measured binds to a substance having a plurality of reactive groups, and the plurality of reactions are performed. An active group to which avidin labeled with a fluorescent substance is bound via biotin can be produced by the following method. That is, after reacting biotin with most of the active groups of the substance having a plurality of reactive groups, the biotin is reacted with a substance exhibiting the same immune reaction as the substance to be measured or a substance immunoreactive with the substance to be measured, and then biotin is reacted. It is produced by binding to avidin labeled with a fluorescent substance.

In the analysis methods (2) and (3) of the present invention, an apparatus as shown in FIGS. 1 and 3 can be used. These devices include a small light source (such as a semiconductor laser) (4), an optical fiber (1) for transmitting excitation light or fluorescence, a detection unit (4) in which an immunological substance is immobilized, and fluorescence generated by the detection unit. And a detector (7) for measuring.

As shown in FIG. 2, the detection unit uses the competitive method (a)
In the case of, an immunological substance is immobilized on the surface of the detection section, to which a substance exhibiting the same immunological reaction as the substance to be measured labeled with a fluorescent substance soluble in a base or a substance to be measured by the immune reaction is bound. ing. On the other hand, in the case of the sandwich method (b), the substance to be measured is bound to the immunological substance immobilized on the surface of the detection unit, and furthermore, the substance to be immunoreacted with the substance to be measured labeled with a fluorescent substance soluble in a base is bound. .

The optical fiber is usually made of a resin, which is a copolymer of a monomer such as methyl acrylate, ethyl acrylate, methyl methacrylate and a monomer such as styrene, because resin is cheaper and easier to use. Optical fibers are used. To immobilize the immunological substance on the surface of the resin optical fiber, a formyl group is introduced as a reactive group, and the immunological substance is covalently bonded.

[0031]

According to the present invention, since fluorescence analysis can be performed using a semiconductor laser having a wavelength longer than 600 nm, the size of the entire apparatus can be reduced. Further, since the absorption loss of fluorescence by the filter is small, high sensitivity can be realized.

[0032]

Embodiments of the present invention will be described below, but the present invention is not limited to these embodiments and can be applied in a wide range.

Example 1 (Detection of aflatoxin B ₁ present in stored peanuts) (1) One peanut sample was ground in 3 ml of methanol, and the supernatant was recovered by centrifugation. Was filtered with a membrane filter. (2) The filtrate of (1) was irradiated with semiconductor laser light at 810 nm, 1130 nm and 1550 nm, and the fluorescence at 425 nm was measured. Aflatoxin B ₁ : Ex. 362 nm Em. 425 nm 27624 = 12346 + 8826 + 6452 (cm ^-1 ) (3) Aflatoxin B ₁ was contained in 11 of 6000 samples.
Was observed.

Example 2 (Measurement of Human Pancreatic Amylase Antibody by Competition Method) (1) In 1 ml of 20 mM borate buffer (pH 7.5),
2 mg of a goat-derived human pancreatic amylase antibody and 4 mg of 7-hydroxycoumarin-3-carboxylic acid (umbelliferone) were dissolved. An aqueous carbodiimide (CHM
C) 10 mg was added, and the mixture was allowed to stand at room temperature overnight under light shielding, then separated and purified by anion exchange chromatography, and a human pancreatic amylase antibody labeled with umbelliferone (UA.
b) A solution was obtained. (2) 10 mg of NiSO ₄ was dissolved in 0.5 ml of water, and then 2.5 ml of ethanol was added. 30 white precipitates formed
The supernatant obtained by centrifugation at 00 rpm was used as a Ni-ethanol solution. To 0.4 ml of a 50 mM KOH ethanol solution,
0.1 ml of the Ni-ethanol solution was added, and 50
A 50% glutaraldehyde solution (50 μl) was added to prepare a reaction solution. (3) Diameter 1 mm mainly composed of polymethyl methacrylate
The cross section of the optical fiber is immersed in the reaction solution of (2), and
The mixture was reacted at 0 ° C. for 10 minutes to introduce a formyl group on the surface of the optical fiber. This was washed with 20 mM hydrochloric acid and water. (4) The optical fiber of (3) is immersed in a solution of 2 mg of human pancreatic amylase solution in 1 ml of a phosphate buffered saline (PBS) solution, left at 4 ° C. overnight, and then the optical fiber is taken out and washed with water. Then, 1% NaBH ₄ aqueous solution
After immersion for 5 minutes and further washing with water and a PBS solution, a human pancreatic amylase-immobilized sensor was prepared. (5) 20 μl of the serum sample (containing a human-derived human pancreatic amylase antibody) to be measured was added to the UA. b. The sensor part of (4) was immersed in a solution obtained by mixing 20 μl of the solution, and allowed to stand at room temperature for 30 minutes to allow the antibody to be measured and the labeled antibody to react with the immobilized antigen competitively. Washed with PBS solution. (6) The sensor portion of (5) was immersed in a 1% sodium carbonate solution, and 670 nm and 9 nm were measured using the apparatus shown in FIG.
The semiconductor laser light of 16 nm was irradiated, and the fluorescence of 450 nm was measured. 7-hydroxycoumarin-3-carboxylic acid Ex. 3
87 nm Ex. 450 nm 25840 = 14925 + 10915 (cm ^-1 ) (387 nm) (670 nm) (916 nm) (7) Using a human pancreatic amylase antibody solution of known concentration as a standard, the antibody was detected up to 30 ng / ml in serum samples. As a comparative example, when the same measurement was performed using a device using a nitrogen laser, the detection limit was 40 ng / ml.
Also, the device became large and expensive.

Example 3 (Measurement of Human Pancreatic Amylase Antibody by Sandwich Method) (1) In the same manner as in (1) of Example 2, goat-derived human pancreatic amylase antibody labeled with umbelliferone (U-
A. b. ) A solution was obtained. (2) A human pancreatic amylase-immobilized sensor was prepared in the same manner as in (2) to (4) of Example 2. (3) The sensor part of (2) is immersed in 20 μl of a serum sample to be measured (containing a human-derived human pancreatic amylase antibody) and left at room temperature for 30 minutes to allow the antibody to be measured to react with the immobilized antigen. Then, the plate was washed with a PBS solution containing Tween 20. (4) The sensor part of (3) is replaced with the U-
A. b. The sample was immersed in the solution, left at room temperature for 20 minutes, and reacted with the labeled antibody using the antibody to be measured as an antigen. (5) When human pancreatic amylase antibody was measured in serum according to (6)-(7) of Example 2, the antibody was found to be 20 ng.
/ ml could be detected.

Example 4 (Measurement of hCG by Competition Method) (1) 100 μg of human chorionic gonadotropin (hCG) and biotin 2 in 1 ml of 20 mM borate buffer (pH 8)
mg of water-soluble carbodiimide (CHMC) 10 mg
Was added and left overnight at 4 ° C. to cause a reaction, followed by separation and purification by gel filtration to obtain a biotinylated hCG solution. (2) 1 mg of avidin and 0.2 ml of triethylamine were dissolved in 1 ml of ethanol. Next, 1.8 mg of fluorescein isothiocyanate (fluorescein) was dissolved and reacted at room temperature for 6 hours under light shielding. The solvent of the reaction solution was removed under reduced pressure, and the residue was subjected to 50 mM acetate buffer (pH 4.5) 1
suspended in ml. This was separated and purified by a gel filtration method to obtain avidin modified with fluorescein. (3) mixing this with the biotinylated hCG of (1),
HC labeled with fluorescein + avidin + biotin
A G (f-hCG) solution was obtained. (4) hCG by the same method as in (2) to (4) of Example 2.
An antibody-immobilized sensor was prepared. (5) The f-hCG solution of (3) is added to 10 μl of the hCG solution to be measured at each concentration, the sensor portion of (4) is immersed, and left at room temperature for 30 minutes, and the h
CG and f-hCG were competitively reacted with the immobilized hCG antibody. Thereafter, the sensor portion was washed with a PBS solution containing Tween 20. (6) The sensor portion of (5) was immersed in a 1% sodium carbonate solution, and 780 nm and 136 nm were measured using the apparatus shown in FIG.
The semiconductor laser light of 2 nm was irradiated, and the fluorescence of 518 nm was measured. Fluorescein Ex. 496 nm Ex. 518 nm 20161 = 12821 + 7340 (cm ^-1 ) (496 nm) (780 nm) (1362 nm) By this analysis method, hCG in the measurement object could be detected up to 1.2 ng / ml. As a comparative example, when the same measurement was performed using an apparatus using an argon laser, the detection limit was 1.2.
ng / ml, but the equipment was very large and expensive.

Example 5 (Measurement of hCG by Sandwich Method) (1) An hCG antibody solution derived from biotinylated sheep was obtained in the same manner as in (1) of Example 4. (2) Fluorescein-modified avidin was obtained in the same manner as in (2) and (3) of Example 4, and this was mixed with the biotinylated hCG antibody of (1), and fluorescein + avidin + Sheep hC labeled with biotin
G antibody was obtained. (3) A human-derived hCG antibody-immobilized sensor was prepared in the same manner as in (3) and (4) of Example 2. (4) The sensor portion of (3) was immersed in 10 μl of the hCG solution to be measured at each concentration, allowed to stand at room temperature for 30 minutes, and reacted with the immobilized hCG antibody using hCG as an antigen. Thereafter, the sensor portion was washed with a PBS solution containing Tween 20. (5) Next, the sensor part was immersed in the labeled hGC antibody solution of the above (2), and the labeled antibody was reacted with the hGC to be measured as an antigen. (6) When the same measurement as (6) of Example 4 was performed,
The detection limit was 1 ng / ml and the device could be made smaller.

Example 6 (Measurement of hGC by Competition Method Using Chitosan) (1) 4 mg of Na ₂ CO ₃ and 10 g of biotin were added to 100 μl of water.
A solution in which mg was dissolved was mixed with a 1.8 μM solution of chitosan (the number of amino groups was 4000 on average per molecule). Next, 100 mg of water-soluble carbodiimide (CHMC) was added thereto.
Was added thereto, and the mixture was reacted overnight at room temperature with stirring. Then, the reaction was stopped by adding a few drops of acetic acid. Then 0.2g / ml
Adding a Na ₂ CO ₃ and mixtures 4ml of 0.1 g / ml of NaCl, precipitated biotinylated chitosan (b-c).
After collecting the precipitate by centrifugation,
g / ml Na ₂ CO ₃ and 0.3 g / ml NaCl
Washed twice. The precipitate was suspended in 2 ml of 10 mM potassium phosphate buffer (pH 7), and further suspended in 500 ml of the same buffer.
Was dialyzed overnight at 4 ° C. to obtain a bc suspension. (2) hCG1 to be measured is added to the bc suspension of (1).
Of water-soluble carbodiimide (CHM
C) 10 mg was added and reacted at 4 ° C. for 6 hours. After the completion of the reaction, the mixture was dialyzed overnight against a 10 mM potassium phosphate buffer (pH 7), and unreacted substances were removed using an anion exchange column to obtain hCG-bound bc. (3) Dansyl-modified avidin was obtained in the same manner as in (2) of Example 4, except that dansyl chloride was used instead of fluorescein isothiocyanate. This was mixed with the hCG-bound bc of (2) above, and the hC labeled with dansyl + avidin + biotin + chitosan was mixed.
G (labeled antigen) was obtained. (4) hCG was obtained in the same manner as in (2) to (4) of the embodiment.
An antibody-immobilized sensor was prepared. (5) In the same manner as in (5) to (7) of Example 4, the hCG to be measured and the labeled antigen of (3) were converted to the hCG of (4).
It was made to react competitively with the CG antibody immobilized sensor. (6) Using the apparatus shown in FIG. 1, the semiconductor laser light was irradiated at 1020 nm, and the fluorescence at 510 nm was measured. Dansyl chloride Ex. 340 nm Ex. 510 nm 29412 = 9804 (cm ^-1 ) x 3 (340 nm) (1020 nm) As a result, the detection limit of hGC by this measurement is 0.7 ng.
/ ml.

Example 7 (Measurement of hCG by a sandwich method using chitosan) (1) Biotinylated chitosan (bc) was obtained in the same manner as in (1) of Example 6. Next, a goat-derived hCG antibody was prepared using the goat-derived hCG antibody in the same manner as in Example 6, (2).
Bc bound to the antibody was obtained. (2) An avidin modified with dansyl was obtained in the same manner as in (3) of Example 6, mixed with bc bound with the hCG antibody of (1), and labeled with dansyl + avidin + biotin + chitosan. The obtained hCG antibody (labeled antibody) was obtained. (3) The hCG to be measured was reacted with the immobilized hCG antibody in the same manner as in (3) and (4) of Example 5, and then the labeled antibody of (2) was reacted. (4) 810 nm, 942 nm and 15
The semiconductor laser light of 50 nm was irradiated, and the fluorescence of 510 nm was measured. Dansyl chloride Ex. 340 nm Em. 510 nm 29412 = 12346 + 10614 + 6452 (cm ^-1 ) (340 nm) (810 nm) (942 nm) (1550 nm) As a result, the detection limit of hCG by this measurement is 0.3 ng.
/ ml. As a comparative example, when the same measurement was performed using a nitrogen laser, the detection limit of hCG was 0.3 ng.
/ ml, but the equipment was very large and expensive.

Example 8 (Measurement of Calcitonin by Competition Method) (1) Except that calcitonin was used instead of hCG,
A biotinylated calcitonin solution was obtained in the same manner as in (4) of Example 4. (2) An avidin (ra) solution modified with rhodamine B isothiocyanate in the same manner as in (2) to (3) of Example 4, except that rhodamine B isothiocyanate was used instead of fluorescein isothiocyanate I got (3) A goat-derived human calcitonin antibody-immobilized sensor was prepared in the same manner as in (2) to (4) of Example 2. (4) 5 μl of the biotinylated calcitonin solution of the above (1) is added to 10 μl of the calcitonin solution to be measured at each concentration, and the sensor portion of the above (3) is immersed in this solution and left at room temperature for 20 minutes to immobilize the solution. The calcitonin to be measured and biotinylated calcitonin were reacted with the calcitonin antibody. After that, the sensor part was replaced with Tween 20-containing PB
Washed with S solution. (5) Next, the sensor portion of (4) was immersed in the ra solution of (2) for 5 minutes to react avidin with biotin. Thereafter, the sensor portion was washed with a PBS solution containing Tween 20. (6) Then, a 50 mM Tris-HCl buffer (pH 9.
5) and 780 nm and 1 nm using the device shown in FIG.
The semiconductor laser light of 550 nm was irradiated, and the fluorescence of 627 nm was measured. Rhodamine B Ex. 520 nm Em. 627 nm 19231 = 12821 + 6452-42 (cm ^-1 ) (520 nm) (780 nm) (1550 nm) The detection limit of calcitonin by this analysis method is 1.3 ng / m.
l.

Example 9 (Measurement of Calcitonin by Sandwich Method) (1) A method similar to (1) of Example 4 was used except that a rabbit-derived human calcitonin antibody was used instead of hCG.
A human calcitonin antibody derived from a biotinylated rabbit was obtained. (2) Avidin (r-a) modified with rhodamine B was obtained in the same manner as in (2) of Example 4, except that rhodamine B isothiocyanate was used instead of fluorescein isothiocyanate. (3) A goat-derived human calcitonin antibody-immobilized sensor was prepared in the same manner as in (2) to (4) of Example 2. (4) The sensor part of (3) was immersed in 10 μl of the calcitonin solution to be measured at each concentration, and allowed to stand at room temperature for 30 minutes to react the calcitonin to be measured with the immobilized calcitonin antibody. After that, Tween 2
Washed with PBS solution containing 0. Further, the sample was immersed in the above-mentioned (1) human calcitonin antibody solution derived from a biotinylated rabbit for 20 minutes to react the calcitonin (antigen) to be measured with the biotinylated human calcitonin antibody. (5) The sensor part of (4) is replaced with (5) of Example 8.
In the same manner as described above, dipping in the ra solution of (2) above,
Avidin was reacted with biotin. (6) This solution was irradiated with the same semiconductor laser beam as in (6) of Example 8, and the fluorescence at 627 nm was measured. As a result, the detection limit was 1 ng / ml.

Example 10 (Measurement of Calcitonin by Competition Method Using Polylysine) (1) Except that polylysine was used instead of chitosan,
A biotinylated polylysine (b-p1) suspension was obtained in the same manner as in Example 6, (1). (2) Calcitonin 50 was added to the bp1 suspension of the above (1).
μg, and add water-soluble carbodiimide (CHMC)
10 mg was added and reacted at 4 ° C. for 6 hours. After completion of the reaction, the mixture was dialyzed overnight against a 10 mM potassium phosphate buffer (pH 7), and unreacted substances were removed using an ion exchange column to obtain calcitonin-bound bp1. (3) Avidin (na) modified with NBD was obtained in the same manner as in (2) of Example 4, except that nitrobenzoxadiazol chloride (NBD) was used instead of fluorescein isothiocyanate. Was. (4) A goat-derived human calcitonin antibody-immobilized sensor was obtained in the same manner as in Example 8, (3). (5) bp1 suspension 5 in which calcitonin of (2) was bound to 10 μl of the calcitonin solution to be measured at each concentration
Then, the sensor portion of (4) was immersed in this solution, and allowed to stand at room temperature for 30 minutes. The calcitonin to be measured and the calcitonin of (2) were bound to the immobilized calcitonin antibody. p1 was allowed to react competitively. (6) In the same manner as in (5) of Example 8, the sensor portion of (5) was immersed in the na solution of (3) to bind avidin to biotin. (7) Using the apparatus shown in FIG. 3, 689 nm and 1550 nm
Was irradiated, and the fluorescence at 540 nm was measured. NBD Ex. 475 nm Em. 540 nm 21053 = 14601 + 6452 (cm ^-1 ) (475 nm) (689 nm) (1550 nm) The detection limit of calcitonin by this analysis method is 0.7 ng / ml.
Met.

Example 11 (Measurement of calcitonin by a sandwich method using polylysine) (1) A bp1 suspension was obtained in the same manner as in Example 10, (1). Next, using a rabbit-derived human calcitonin antibody, b-p1 bound with a rabbit-derived human calcitonin antibody was obtained in the same manner as in Example 10, (2). (2) In the same manner as in (3) and (4) of Example 10, n
-A and a goat-derived human calcitonin antibody-immobilized sensor were prepared. (3) The sensor part of (2) was immersed in 10 μl of the calcitonin solution to be measured at each concentration, and allowed to stand at room temperature for 30 minutes to allow the calcitonin to react with the immobilized calcitonin antibody. Thereafter, the plate is washed with a PBS solution containing Tween 20 and further immersed in the bp1 suspension to which the human calcitonin antibody of the above (1) is bound for 20 minutes. p1 was reacted. (4) In the same manner as in (5) of Example 8, the sensor portion of (3) was immersed in the na solution of (2) to bind avidin to biotin. (5) Using the apparatus shown in FIG. 3, 689 nm and 1550 nm
Was irradiated, and the fluorescence at 540 nm was measured. As a result, the detection limit of calcitonin was 0.4 ng / ml.
Met.

[Brief description of the drawings]

FIG. 1 shows a fluorescence measurement system using three semiconductor lasers.

FIG. 2 shows a fluorescence detection unit in the above device.

FIG. 3 shows a fluorescence measurement system using two semiconductor lasers.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 Optical fiber 2 Semiconductor laser 3 Guide rail for optical axis alignment 4 Sensor chip 5 Sample injection cell 6 Filter 7 Detector 8 Half mirror 9 Immune substance 10 Substance to be measured 11 Same immunity as the substance to be measured labeled with fluorescent substance Substances that show a reaction 12 Substances that immunoreact with the substance to be measured labeled with a fluorescent substance

Claims (6)

(57) [Claims] 1. A method for producing a fluorescent substance under basic conditions,
A fluorescence analysis method comprising: exciting with a plurality of laser beams having a wavelength longer than the maximum excitation wavelength of the fluorescent substance satisfying the following formula and measuring fluorescence. (Equation 1) (Equation 2) 2. An immunological substance is immobilized on the surface of an optical fiber, and (a) competition is made between the immunological substance on the surface and the substance exhibiting the same immune reaction as the substance to be measured and the substance to be measured labeled with a fluorescent substance. Or (b) immunoreacting the substance to be measured with the immunological substance on the surface, and then reacting the substance to be immunoreacted with the substance to be measured labeled with a fluorescent substance. A fluorescence immunoassay method comprising: exciting with a plurality of laser beams having a wavelength longer than the maximum excitation wavelength of the fluorescent substance satisfying the formula of claim 1 and measuring the fluorescence. 3. A substance exhibiting the same immunoreactivity as the substance to be measured or a substance immunoreactive with the substance to be measured is bound to biotin, and avidin labeled with a fluorescent substance is bound to the biotin. 3. The fluorescent immunoassay according to 2. 4. A substance exhibiting the same immunoreactivity as the substance to be measured or a substance immunoreactive with the substance to be measured binds to a substance having a plurality of reactive groups, and the plurality of reactive groups are mediated by biotin. The fluorescent immunoassay according to claim 2, wherein avidin labeled with a fluorescent substance is bound thereto. 5. An immunological substance is immobilized on the surface of an optical fiber, and (a) a substance exhibiting the same immune reaction as the substance to be measured and the substance to be measured to which biotin is bound is competitively reacted with the immunological substance on the surface. Or (b) immunoreacting the immunological substance on the surface with the analyte and then reacting the immunoreactive substance with the analyte bound to biotin, and then reacting avidin labeled with a fluorescent substance. 2. A fluorescence immunoassay method, wherein the fluorescence is measured under basic conditions by exciting with a plurality of laser beams having a wavelength longer than the maximum excitation wavelength of the fluorescent substance that satisfies the formula of claim 1 and the fluorescence. 6. A substance exhibiting the same immunoreactivity as the substance to be measured or a substance immunoreactive with the substance to be measured binds to a substance having a plurality of reactive groups, and biotin binds to the plurality of reactive groups. The fluorescent immunoassay according to claim 5, which comprises: