JPH095324A - Fluorescent-material-labeled antiserum and igg fraction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure an antibody to an antigen containing a coloring matter which is not substantially fluorescent by giving fluorescence to the antibody by mixing the coloring matter in the antibody. SOLUTION: When an antiserum containing an antibody to an antigen containing a coloring matter which is not substantially fluorescent and its IgG fraction are separated and the coloring matter is mixed in the antiserum or IgG fraction, fluorescence appears in the molecular of the coloring matter or the fluorescence of the molecular increases. When the fluorescence from the mixture is measured, the coloring matter can be detected even when its amount is very small. When an anti-MG antibody is used, it is indicated that the MG labeling the antibody is useful as a fluorescent label. When the antibody is labeled with a malachite green coloring matter, the amount of the background fluorescence of a sample can be reduced effectively and, therefore, high- sensitivity measurement becomes possible as compared with other coloring matters.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antiserum, an antibody, and an IgG fraction for a dye which is not substantially fluorescent as an antigen, and which becomes fluorescent when mixed with the dye, It relates to antibodies and IgG fractions.

[0002]

2. Description of the Related Art A spectroscopic analysis method has been widely used as a method for analyzing trace substances in the course of changes in in-vivo metabolites and pollutants in the environment. In particular, absorption spectrum analysis and fluorescence spectrum analysis methods are widely used because of simple measurement methods.

Although the absorption spectrum analysis method has lower detection sensitivity as compared with the fluorescence analysis method described later, it has been widely used because many substances to be measured have optical absorption in the UV-visible region.

That is, the absorbance (Abso
rbance) and the concentration c of the substance to be measured follow the formula below.

Absorbance = log (I ₀ / I) = εcl I ₀ : Intensity of incident light, I: Intensity of light passing through the sample ε: Absorbance coefficient of the substance to be measured, l: Optical path length What is obtained by measuring the absorbance is the ratio of incident light to transmitted light, which does not change even when the light source intensity or the sensitivity of the detector is increased.

On the other hand, if the substance to be measured is fluorescent, a fluorescence analysis method can be used. In this case, the amount of light emitted by the substance when irradiated with excitation light is measured, and therefore the light source is used. The sensitivity of the measurement can be improved by increasing the sensitivity of the detector or increasing the sensitivity of the detector.

That is, between the intensity F of the light emitted as fluorescence, the concentration c of the fluorescent substance, and the excitation light intensity I ₀ , F = ΦI ₀ (1-10 ⁻ ε ^cl ) I ₀ : The intensity of the incident light , Ε: Absorbance coefficient of the substance to be measured, l: Optical path length, Φ: Quantum yield of fluorescence, which is proportional to the intensity of incident light.

Further, when the substance to be measured is contained in a mixture sample consisting of multiple components, the absorption spectrum of impurities often overlaps with the absorption spectrum of the substance to be analyzed in the measurement of the absorbance, and thus It is difficult to selectively measure only the absorbance.

On the other hand, even when the substance to be measured is contained in a mixture sample consisting of multiple components, non-fluorescent contaminants can be ignored in the fluorescence analysis method, and fluorescent contaminants can be ignored. Even when they coexist, it is possible to selectively measure only the substance to be analyzed by selecting the excitation wavelength and the fluorescence wavelength.

Therefore, the fluorescence analysis method is superior to the absorption analysis method in terms of detection sensitivity and measurable property of the mixture.

Therefore, conventionally, an antibody for a fluorescent dye has been prepared as an analysis technique which utilizes the advantage of high sensitivity measurement of the fluorescence analysis method and the high selectivity in the antigen-antibody reaction. These antibodies react with the dye to reduce or increase the fluorescence intensity of the dye.

Therefore, specific quantitative analysis of these dyes has been carried out by using these changes in fluorescence intensity.

However, the above-mentioned high-sensitivity and high-selective fluorescence analysis technique utilizing the antigen-antibody reaction has an essential limitation that it cannot be used when the analyte is not substantially fluorescent.

On the other hand, it is known that a dye that is not substantially fluorescent under normal measurement conditions can be measured for fluorescence under special conditions, that is, it becomes fluorescent.

For example, malachite green is not normally fluorescent, but it is known that it becomes fluorescent in glycerin. It is explained that the structure of the dye molecule is fixed by the influence of the solvent and the like, and as a result, the dye molecule becomes fluorescent.

Further, there are many fluorescent substances in the living body, and the fluorescence (self-luminescence) becomes a background during the fluorescence measurement of the biological sample, which can reduce the detection sensitivity. Even in the case of fluorescence immunoassay using a titer plate, the fluorescence emitted by the plastic material of the plate also becomes a background, which can reduce the detection sensitivity. there were.

[0017]

In view of the above points, the present invention specifically binds to a dye that is not substantially fluorescent under ordinary measurement conditions, and the bond fixes the molecular structure of the dye molecule. The purpose is to find an antibody capable of increasing or expressing fluorescence. Further, the present invention provides a fluorescent dye which enables highly sensitive measurement as compared with other dyes due to extremely low background fluorescence.

[0018]

The antiserum according to the present invention comprises:
It comprises an antibody against an antigen having a non-fluorescent dye, characterized in that the mixture with the substantially non-fluorescent dye is fluorescent.

That is, the present invention is an antiserum containing an antibody against an antigen having a dye that is substantially non-fluorescent, wherein the antiserum or a mixture of the antibody and the dye has fluorescence. It is related to antiserum.

Furthermore, the present invention provides an IgG fraction in antiserum containing an antibody against an antigen having a dye which is not substantially fluorescent, wherein the mixture of the IgG fraction and the dye has fluorescence. It relates to the characteristic IgG fraction.

[0021] The present invention also relates to an antiserum, wherein the antigen is formed by adding the dye to an immunological substance.

The present invention also relates to an IgG fraction characterized in that the antigen is formed by adding the dye to an immunological substance.

Further, in the present invention, the immunological substance is bovine serum albumin, human serum albumin, ovalbumin, bovine gamma globulin, equine serum globulin, human gamma globulin, sheep gamma globulin, bovine thyroglobulin, porcine thyroglobulin, hemocyanin. And at least one selected from the group consisting of synthetic polypeptides.

In the present invention, the immunizing substance is bovine serum albumin, human serum albumin, rabbit serum ovalbumin, bovine gamma globulin, horse serum globulin, human gamma globulin, sheep gamma globulin,
It relates to an IgG fraction characterized by being selected from at least one selected from the group consisting of bovine thyroglobulin, porcine thyroglobulin, hemocyanin and synthetic polypeptides.

Furthermore, the present invention relates to an antiserum, wherein the dye has a triphenylmethane structure.

The present invention also relates to an IgG fraction, wherein the dye has a triphenylmethane structure.

The present invention also relates to an antiserum characterized in that the dye having a triphenylmethane structure is malachite green.

Further, the present invention relates to an antiserum, wherein the dye has a diphenylmethane structure.

The present invention also relates to an IgG fraction, wherein the dye has a diphenylmethane structure.

The present invention also relates to an antiserum characterized in that the dye having the diphenylmethane structure is auramine O.

The present invention further relates to an IgG fraction, wherein the dye having the diphenylmethane structure is auramine O.

The present invention will be described in detail below.

(Dye Not Substantially Fluorescent) The dye that can be used in the present invention is particularly a dye that does not substantially show fluorescence in an ordinary solvent such as water, or one that shows weak fluorescence but is weak. Not limited.

That is, in the present invention, "not substantially fluorescent" means that no fluorescence spectrum is shown under normal measurement conditions, or only extremely weak fluorescence is shown, and the fluorescence spectrum analysis is substantially carried out by a commercially available device or the like. What is said to be impossible (Taiji Nishikawa et al., "Fluorescence phosphorescence analysis method",
Kyoritsu Shuppan, 30 pages, 1984).

In the present invention, when the fluorescence quantum yield is 1% or less under a specific condition, it is a dye which does not exhibit substantially fluorescence as referred to herein, and the fluorescence quantum yield is 10% under a specific condition.
^In the case of less than ² %, it means a dye which does not show substantially fluorescence.

However, when extremely weak fluorescence can be detected by special measuring conditions, measuring apparatus, etc., the dye which is not substantially fluorescent in the present invention becomes fluorescent by the treatment according to the present invention, and fluorescence analysis is performed. Means that the fluorescence quantum yield of the dye is extremely small under normal measurement conditions (for example, 0.01% or less), but it significantly changes due to interaction with an antibody by the treatment according to the present invention ( (For example, 1% or more).

Further, the dyes usable in the present invention are
Those which are fluorescent (fluorescence quantum yield is larger than 1%) in a common solvent such as water are also included.

In this case, it means that the fluorescence intensity is further increased by the treatment according to the present invention, which means that it is possible to obtain an analytical sensitivity higher than the sensitivity obtained by ordinary fluorescence analysis. Here, an increase in fluorescence intensity means that the fluorescence quantum yield changes and increases.

In the present invention, it is desirable that the above-mentioned increase in fluorescence quantum yield is at least 10 times or more.
00 times, more preferably 1000 times or more (more preferably 10 times)
000 times or more) is desirable.

For example, regarding malachite green,
An increase in fluorescence quantum yield of about 1000 times or more can be obtained.

The dye which can be used in the present invention does not necessarily have absorption in the visible region (having an absorption maximum at 350 nm or more), but one having an absorption in the ultraviolet region (having an absorption maximum at 350 nm or less), Alternatively, the absorption band may extend to the visible part.

The molecular structure of the dye that can be used in the present invention is not particularly limited, and those containing various chromophores (chromophores) are possible.

Particularly, those containing a chromophore having a stable pH at around neutral pH are desirable.

For example, a dye having a triphenylmethane skeleton in its molecular structure is particularly preferable (for example, "Dye Handbook" edited by Kodansha Scientific Co., Ltd., Shin Okawara).
(See), and malachite green, which is a pigment having a triphenylmethane skeleton in the molecular structure, are particularly preferably used.

A dye having a diphenylmethane skeleton (for example, see "Dye Handbook" edited by Kodansha Scientific Co., Ltd., Shin Okawara) can also be preferably used. As the diphenylmethane dye, for example, an auramine dye can be preferably used. It is particularly preferable to use auramine O.

(Immunity substance) The immunity substance usable in the present invention is not particularly limited.

Generally usable, for example, albumin (bovine serum, human serum), rabbit ovalbumin, serum globulin (bovine gamma globulin, horse serum globulin, human gamma globulin, sheep gamma globulin), thyroglobulin (bovine thyroglobulin). , Porcine thyroglobulin), hemocyanin, synthetic polypeptides and the like can be preferably used.

(Preparation of Antigen Having Dye Which Is Not Substantially Fluorescent) In the present invention, a method for preparing an antigen having a dye which is not substantially fluorescent is not particularly limited.

For example, it is possible to separate the fraction of the antigen having the dye by agitating and mixing the immunological substance and a dye that is not substantially fluorescent for a predetermined time and using gel filtration chromatography.

Further, if necessary, it can be purified by using a purification means utilizing an immunoreaction (Kouichiro Kawashima (translated): "Introduction to Immunoassay", Nanzandou, p. 70, 1987).

The concentration of the obtained antigen having the dye can be quantified by, for example, the Lowry method.

(Preparation of antiserum) The antibody of the present invention can be preferably prepared in the immunization step.

The immunized animal is not particularly limited, but rabbits and guinea pigs can be preferably used.

The immunoadjuvant usable in the present invention is not particularly limited, but general Freund's incomplete adjuvant, aluminum adjuvant and the like can be preferably used.

The immunoinjection method usable in the present invention is not particularly limited, but subcutaneous injection, intraperitoneal injection and the like can be preferably used, for example, in guinea pigs.

In the present invention, the confirmation and collection of the antiserum can be carried out by carrying out a booster immunization, if necessary, and carrying out a test collection to examine the antibody titer.

In the present invention, there is no particular limitation on the separation of the collected antiserum, and it is possible to separate the serum by a general method, for example, after coagulating the voted blood and then centrifuging. The specific antibody activity of the obtained antiserum with respect to the antibody dye can be preferably measured by enzyme immunoreaction or the like ("Measuring method using biological activity", Maruzen, Shin-basic biochemistry experimental method 6, page 98).

(IgG Fraction) In the present invention, the method for purifying the IgG fraction from the antiserum is not particularly limited. A salting-out method, a gel filtration method, an ion exchange chromatography method and the like can be preferably used, and a protein A method is particularly preferably used. Furthermore, the obtained IgG fraction can also be concentrated by a centrifugation method and adjusted to a predetermined concentration.

(Fluorescence spectrum measuring method) In the present invention, the above antiserum or antibody is mixed with a non-fluorescent dye to make the mixed solution fluorescent, and various fluorescence spectra of the resulting mixed solution are measured. It can be measured by the method (fluorescence / phosphorescence analysis method, Nishikawa / Hiramoto, Kyoritsu Shuppan, Chapter 2, pages 49-99).

In particular, it is possible to quantify only the fluorescence from the dye with high sensitivity without the interference of coexisting substances, by measuring the fluorescence difference spectrum from the control mixture.

The fluorescence quantum yield expressed in the present invention is
Depends on the dye, immunity substance, immunized animal, immunization process, separation and purification means, etc.

Further, according to the present invention, it is possible to quantitatively analyze the dye by utilizing the expressed fluorescence. The limit of quantitative analysis depends on the above quantum yield.

(Triphenylmethane Dye) For example, for malachite green, it is possible to compare the absorbance sensitivity with the measurement sensitivity by fluorescence detection as follows.

Malachite green was dissolved in PBS and 4
Prepare a dilution series in the concentration range of ˜4000 nM.

The absorbance at 617 nm of these solutions was measured (Hitachi 557 type dual wavelength spectrophotometer, optical path length 1 cm quartz cell, control PBS) (Table 1) to prepare a concentration-absorbance curve (FIG. 6).

Table 1 Concentration (nM) 4 12 40 120 400 1200 4000 Absorbance (617 nm) 0 0 0.002 0.009 0.036 0.132 0.464 As shown in the figure, there is a linear correlation between concentration and absorbance in the range of 40 to 4000 nM. Was obtained, but it could not be measured at 12 nM or less.

On the other hand, the IgG of the present invention derived from guinea pig 1
A fixed amount of the fraction (final concentration 0.2 μM) was added to prepare a dilution series (PBS solution) of malachite green in which the dye concentration was adjusted to the range of 0 to 12 nM.

The fluorescence of these solutions was measured (Hitachi fluorescence spectrophotometer 850 type, excitation side optical path length 1 cm quartz cell, excitation wavelength 617 nm, emission wavelength 650 nm, both excitation light and emission bandpass width 5 nm, photomultiplier tube Gain is Hi
gh, time constant 1.5 seconds, fluorescence integration time 30 seconds) (Table 2), and a concentration-fluorescence intensity curve is prepared (FIG. 5).

The fluorescence intensity was shown by the output value (arbitrary relative value) of the fluorescence spectrophotometer.

Table 2 Concentration (nM) 0 0.04 0.12 0.4 1.2 4 12 Fluorescence intensity (650 nm) 0.026 0.026 0.034 0.066 0.18 0.487 1.22 As shown in the figure, concentration-fluorescence intensity was linear in the range of 0.12 nM to 12 nM. A strong correlation is obtained, indicating that quantitative analysis is possible in this concentration range.

Therefore, 12 nM cannot be detected by the absorbance measurement, but 12 nM can be detected by the fluorescence detection using the antibody of the present invention.
Furthermore, it is possible to detect with 0.12 nM, which is a 2-digit lower concentration, with high quantification and high sensitivity (300 times the sensitivity by absorbance).

(Diphenylmethane Dye) Furthermore, when auramine O (AO), which is a diphenylmethane dye, is used, the same effect as that of rhodamine is exhibited. That is, in the absorption spectrum of AO, anti-MG-KLH
The IgG mixture shows a decrease in absorption intensity and a shift to longer wavelengths (Fig. 7).

Auramine O is anti-MG-KLH I
Fluorescence is developed by mixing with gG (FIG. 8).
The fluorescence intensity is estimated to be about malachite green.

The above results indicate that the anti-MG-KL according to the present invention was used.
It is shown that H IgG can recognize and bind at least the diphenylmethane group of the dye molecule.

(Measurement based on low background fluorescence using malachite green) Many fluorescent substances exist in the living body. For example, 47 for retinol (vitamin A)
At around 0 nm (Biochemistry Encyclopedia Tokyo Kagaku Dojin, p. 1373), riboflavin emits fluorescence with a maximum near 536 nm (p. 1,350). These fluorescences (autofluorescence) serve as backgrounds when measuring the fluorescence of a biological sample and can reduce the detection sensitivity.

In the case of fluorescence immunoassay using a titer plate, the fluorescence emitted by the plastic material of the plate also becomes the background, and the detection sensitivity can be reduced.

In this case, if the excitation wavelength and the fluorescence wavelength can be set to the shorter wavelength side or the longer wavelength side from the excitation and fluorescence wavelengths of these background fluorescence, the influence of the background fluorescence can be effectively reduced. it can.

For this purpose, a fluorescent dye having excitation and fluorescence wavelengths separated from the wavelength of autofluorescence is useful as the fluorescent dye. For example, malachite green used in the present invention is one of the suitable pigments.

In fact, malachite green has been shown to be a very effective dye in fluorescent immunoassays for serum components, as explained below. That is, it is possible to label the antibody against the test substance with malachite green (hereinafter referred to as MG) by a covalent bond, react the anti-MG antibody with the antibody, and observe the fluorescence spectrum emitted. That is, when an anti-MG antibody is used, MG labeled with the antibody is shown to be useful as a fluorescent label. At this time, the background fluorescence of the sample (various background fluorescence of the titer plate, etc.)
By comparing the case of observing with the excitation wavelength of G and the case of exciting with a shorter wavelength, the case of labeling with MG shows only extremely low background fluorescence, which is more sensitive than other dyes. It becomes possible to measure.

[0080]

According to the present invention, an antiserum containing an antibody against an antigen having a dye which is not substantially fluorescent, and I
The gG fraction was separated, and these separated antisera or IgG were separated.
By mixing the fraction with the dye, the dye molecule develops or increases fluorescence. Measuring the fluorescence from this mixture allows microanalysis of the dyes. The use of anti-MG antibody indicates that MG labeled with the antibody is useful as a fluorescent label.

Furthermore, in the case of labeling with a malachite green dye, it is possible to effectively reduce the background fluorescence amount of the sample, and therefore, it becomes possible to perform a highly sensitive measurement as compared with other dyes.

[0082]

Embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

(1) Preparation of Malachite Green Labeled Hemocyanin and Malachite Green Labeled Bovine Serum Albumin Hemocyanin (Keyhole Lympet Hemocyanin, Calbioch)
EM (manufactured by em) (KLH) was dissolved in 10 ml of a 0.5 M carbonate buffer (pH 9.5) in a 10 ml Erlenmeyer flask. To this, 6.5 mg of malachite green isothiocyanate (MGITC, manufactured by Molecular Probe Co.) was added, and the mixture was stirred at 4 ° C. for 24 hours with protection from light.

The above reaction solution was subjected to gel filtration chromatography (equilibrated with phosphate buffered saline (PBS)).
Bio-Rad Econo-Pac 10DG), unreacted M
GITC and MG labeled KLH (hereinafter MG-KLH) were separated.

The protein concentration of the obtained MG-labeled KLH fraction was 220 μg / ml (Lowry method).

10 ml of 9.5 mg bovine serum albumin
0.5 M carbonate buffer (pH 9.5) in an Erlenmeyer flask
It was dissolved in 10 ml. Malachite green isothiocyanate (MGITC, Molecular Probe)
6.5 mg was added, and the mixture was shielded from light and stirred at 4 ° C all day and night.

The above reaction solution was subjected to gel filtration chromatography (equilibrated with phosphate buffered saline (PBS)).
Bio-Rad Econo-Pac 10DG), unreacted M
GITC and MG labeled BSA (hereinafter referred to as MG-BSA) were separated.

The protein concentration of the obtained MG-labeled BSA fraction was 237 μg / ml (Lowry method).

(2) Immunization of experimental animals (i) Initial immunization 1.0 ml of the MG-KLH solution prepared in (1) was diluted with 1.0 ml of physiological saline to prepare an antigen solution. 0.2
Using a syringe (5 ml) with a 2 μm filter,
RAS under aseptic conditions (Ribi Adjuvant System, Ribi)
Injected into vials. Then, the vial was shaken vigorously for 2 minutes to prepare an emulsion of the antigen solution and the adjuvant.

The obtained emulsion was guinea pig (Ha
rtley, female, SPF, n = 3), under anesthesia (neptal, dose 8 mg / guinea pig), 0.5 ml per animal was administered (0.1 ml × 4 places subcutaneously in the back of the back, 0 intraperitoneally). .1 ml).

(Ii) Booster Immunization Two booster immunizations were performed at an interval of 3 weeks from the first immunization using the same amount of antigen and adjuvant as in the first immunization.

(3) Measurement of antiserum body titer (i) Timing of antibody titer determination was determined by the following preliminary test.

Two weeks after the first booster immunization, 0.5 ml of blood was respectively collected from the immunized guinea pigs by heart blood sampling.
Was partially sampled, the antibody titer was measured, and it was confirmed that a sufficient increase in the antibody titer was observed 2 weeks after the booster immunization.

As a result, whole blood was collected 2 weeks after the second booster immunization, and then the final antibody titer was measured in the same manner.

(Ii) Preparation of antiserum The collected blood was left in an incubator at 37 ° C. for 1 hour to promote the formation of blood clots. Then, the mixture was left overnight at 4 ° C. to contract the blood clot, and then the antiserum was separated by centrifugation.

The specific antibody activity of the obtained antiserum against malachite green was confirmed by the enzyme immunoassay method as follows.

(Iii) Enzyme immunoassay MG-KLH solution (220 μg / ml as KLH) 1
50 μl of 50 mM carbonate buffer (pH 9.6) 15 ml
96-well immunotiter plate (made by Greiner, ELISA-PLATE F-f)
0.1 ml was dispensed into each well. This was left overnight at 4 ° C. to adsorb MG-KLH on the inner wall of the titer plate.

A washing solution (containing phosphate buffered saline (PBS) and 0.05% Tween 20) (0.1 ml / well) was added to the immunotiter plate, and the solution was discharged. This washing step was repeated three times. Next, the blocking solution (1
% Gelatin PBS) was added at 0.1 ml / well, and the mixture was left at 4 ° C. overnight.

Then, the cells were washed again with the washing solution in the same manner, and diluted solution of antiserum (100 to 6400-fold dilution system) and guinea pig serum not immunized as a control (not diluted).
0.1 ml was added to each well. This titer plate was allowed to stand at room temperature for 6 hours to react the antigen with the antibody.

[0100] After that, washing with the above washing solution was performed, and horseradis
h peroxidase-labeled goat anti-guinea pig IgG antibody solution (final concentration = 16 μg / ml, Cappel) 0.1 ml /
The wells were added and left overnight at 4 ° C. to react the antiserum-derived IgG with the enzyme antibody.

After that, the reaction substrate solution (ABTS Peroxidase Substrate System, Kirkegaad &
0.1 ml of Perry Laboratories) was added to each well. This was heated at 37 ° C. for 10 minutes to carry out an enzymatic reaction, and then a reaction stop solution (1% SDS aqueous solution) was added. This immunotiter plate is a plate reader (Bio
-Radn, model 3550), set to 405n
The absorbance at m was measured.

FIG. 1 shows an example of measuring the antibody titer of whole blood collected one week after the second booster immunization.
It has been shown to show significant antibody activity as compared to the control up to a 3200-fold dilution.

Further, it showed antibody activity against malachite green labeled bovine serum albumin (MG-BSA),
Since it showed no activity against bovine serum albumin (BSA), it is shown that this antiserum specifically recognizes malachite green.

(4) Purification of IgG fraction The IgG fraction was purified from the obtained antiserum using protein A. Protein A fixed column (Protein A column kit for antibody purification Ampure PAKit, Am
ersham) was used to obtain about 4 ml of IgG fraction from 1 ml of this antiserum.

The obtained IgG fraction was placed in a cartridge for sample pretreatment (ULTRACENT-10 for centrifugal concentration manufactured by Tosoh Corp.) in an amount of 1 to 1.5 ml at a temperature of 4 ° C. and 5000 rpm.
It was centrifuged at m for 30 to 60 minutes. By repeating this, about 4 ml was finally concentrated to 0.7 ml.

(5) Reaction of Antibodies with Malachite Green Bovine gamma globulin was used as a standard sample to quantify the protein concentration of the obtained concentrated IgG fraction (Bio-Rad protein assay, manufactured by Bio-Rad).

The results are as follows.

Guinea pig serum total protein concentration IgG fraction. 1 150 mg / ml 6.15 mg / ml 2 146 mg / ml 7.83 mg / ml According to this result the antiserum was diluted so that its protein concentration was equal to the IgG fraction. This dilution and IgG
Prepare a 100-fold to 6400-fold diluted solution of each fraction,
MG-KLH and MG-BSA were adsorbed to each well of a 96-well immunotiter plate, and 0.1 ml was added to each well.
The mixture was left overnight at ℃ to carry out an antigen-antibody reaction. After that, enzyme immunoreaction was performed in the same procedure as in the measurement of antibody titer, and the antiserum and I
The antibody activities of the gG fractions were compared. The result is shown in FIG. This shows that the IgG fraction showed a significantly higher activity than the antiserum, and that the specific activity was increased by purifying the IgG fraction.

(6) Absorption spectrum The concentration of the protein and the dye was adjusted so that the above-mentioned protein concentration was 2 μM and MG was 4 μM, and guinea pig γ-globulin was used as a control (blank) against anti-MG-KLH IgG. IgG) and the above IgG fraction and MG
The absorption spectrum of the mixture was measured (Fig. 3).

As shown in FIG. 3, MG and blank IgG
The absorption spectrum of the mixed solution of No change in the absorption maximum wavelength and the spectrum of only MG, but the mixed solution of the IgG fraction having anti-MG-KLH activity and MG has an absorption maximum wavelength of 6 to 6 as compared with MG alone. It can be seen that the wavelength is shifted to the long wavelength side by 7 nm.

It is considered that this is because the anti-MG-KLH specifically bound to MG to form a conjugate, whereby the electronic state of the dye was changed and the absorption maximum wavelength was shifted.

(7) Fluorescence spectrum Further, the fluorescence spectrum of the above mixture was measured (Fig. 4,
HITACHI 850 fluorescence spectrophotometer (manufactured by Hitachi), excitation wavelength 617 nm, fluorescence wavelength 630 to 800 nm, bandpass 5 nm (excitation side, fluorescence side), scan 60 nm /
Min, time constant 0.5 seconds, photomultiplier tube gain LOW).

FIG. 4 shows the fluorescence spectrum of the mixture of IgG fraction having anti-MG-KLH activity and MG (excitation light 617n
m, fluorescence 630-800 nm) from which the fluorescence spectrum of the mixture of blank IgG and MG is subtracted. Taking the difference spectrum causes problems in fluorescence analysis such as fluorescence of the cell itself and Raman scattering of the solvent. It becomes possible to remove noise and obtain a pure fluorescence spectrum of the sample.

The difference spectrum shows that anti-MG-KLH I
It is clearly shown that the MG of the gG conjugate is fluorescent. That is, it is shown that non-fluorescent MG becomes fluorescent by specifically forming a conjugate with this antibody.

Therefore, it becomes possible to perform a specific and quantitative fluorescence analysis of MG that has become fluorescent by the above-mentioned treatment.

(8) Reduction of background fluorescence by using malachite green As described below, an antibody against a test substance was labeled with malachite green (hereinafter referred to as MG) by a covalent bond, and the antibody was treated with anti-M.
The G antibody was reacted and the emitted fluorescence spectrum was observed, which showed that the background fluorescence was reduced by using the anti-MG antibody, and thus the MG labeled with the antibody was extremely useful as a fluorescent label. .

A sample in which mouse serum components were bound to a titer plate was prepared, and the background fluorescence amount and spectrum of this sample were observed at the MG excitation wavelength.
Compared to the case of excitation at a shorter wavelength.

The procedure and the result will be described in detail below.

(A) M as a fluorescent label for fluorescent immunoassay
G (i) Labeling of MG with rabbit anti-mouse IgG antibody 1.05 mg of rabbit anti-mouse IgG antibody (manufactured by Wako) was added to 0.5 ml of phosphate buffered saline (20 mM, pH 7.3, hereinafter referred to as PBS). After dissolution, 9.5 ml of carbonate buffer solution (0.5 M, pH 9.0) was added and stirred.
To this solution, malachite green thiocyanate (M
40 μl of dimethyl sulfoxide (DMSO) in which 0.3 mg of GITC, manufactured by MPInc) was dissolved to prepare a reaction mixture. Protect this from light and stir overnight at 4 ° C to remove M
GITC was covalently attached to IgG and labeled.

The reaction mixture was applied to a desalting column (10DG, manufactured by Bio-Rad) equilibrated with PBS, and P
Gel filtration chromatography was performed using BS as an eluent to separate and remove unreacted MGITC to obtain a fraction of MG-labeled rabbit anti-mouse IgG antibody (hereinafter referred to as MG-labeled antibody).

900 μl of PBS was added to 100 μl of this fraction to prepare a 10-fold diluted solution of the same fraction, and its absorption spectrum was measured. The results are shown in FIG. PBS as a control
Was used. It was confirmed that the antibody was labeled with MG by the maximum absorption being observed around 620 nm.

(Ii) Reaction between MG-labeled antibody and anti-MG antibody and fluorescence spectrum To 100 μl of the above-mentioned MG-labeled antibody fraction, 5 μl of anti-MG antibody fraction and 895 μl of PBS were added, and the mixture was stirred at room temperature for 1 hour to give MG. The antigen-antibody reaction of the anti-MG antibody was fully equilibrated.

Next, the fluorescence spectrum of this solution was measured with a spectrofluorometer (HITACHI 850 type). The excitation wavelength is 620 nm and the emission wavelength is 630 to 750 nm.
Excitation side bandpass 5.0 nm, emission side bandpass 5.0
nm, scan speed 60 nm / min, and photomultiplier tube gain was high.

The results are shown in FIG. 10 (a). 650-6
Fluorescence with a maximum between 60 nm was observed. This confirms that the MG labeled with the antibody became fluorescent. Furthermore, (b) of FIG. 10 is a fluorescence spectrum when the MG-labeled antibody was reacted with a blank antibody (antibody that does not bind to MG), but it shows almost no fluorescence. This showed that the reaction between the MG-labeled antibody and the anti-MG antibody was specific.

(B) Comparison of background fluorescence amount of serum (i) Preparation of serum Blood was collected from a mouse (BALB / C, male, adult) and left at room temperature for 30 minutes to coagulate the blood. This is left overnight at 4 ° C to separate the blood clots formed by blood coagulation,
Serum was collected.

(Ii) Binding of serum components to titer plate Mouse serum was added to a plastic titer plate (gre
50 μl was placed in each well of Iner (96 wells) and left at room temperature for 6 hours to bind serum components to the titer plate. Thereafter, each well of the same titer plate was washed with a buffer for washing (phosphate buffered saline, 0.5% Twe.
(including en20) to remove unbound serum components.

(Iii) Measurement of background fluorescence amount The titer plate prepared in (B) was set in the cell holder of a spectrofluorometer (HITACHI 850 type). At that time, the bottom of the titer plate was bent at 45 degrees to both the excitation light and the detector (Fig. 11). With this arrangement, the excitation light reflected on the bottom surface of the titer plate was prevented from directly entering the detector. In addition, an extra portion of the titer plate that interferes with irradiation of excitation light and fluorescence measurement was cut in advance. The background fluorescence observed here is a combination of fluorescence derived from both the serum component and the titer plate material.

The measurement conditions are the following two types.

FIG. 12A: Excitation wavelength 440 nm, fluorescence wavelength 450 to 600 nm, and FIG. 12B: Excitation wavelength 620 nm, fluorescence wavelength 630.
˜780 nm and (a), CCJV (9-
(2-carboxy-2-cyanovinyl) j
ulolidine) fluorescence measurement conditions,
Fluorescein, a fluorescent dye generally used in fluorescence immunoassay, is measured under the following conditions (excitation maximum wavelength = 483.5 nm, fluorescence maximum wavelength 525 nm, biochemical encyclopedia, Tokyo Kagaku Dojin, 10
It is also close to page 91). Measurement result, (b)
In the case of, it is clear that the background fluorescence amount is remarkably reduced as compared with the case of exciting at 440 nm (a).

From the above results, it is concluded that the use of MG for fluorescence measurement has an advantage that the background level can be extremely reduced and high-sensitivity measurement can be performed as compared with the case of using CCJV or fluorescein. it can.

[0131]

INDUSTRIAL APPLICABILITY As described above in detail, according to the present invention, an antibody having a dye that is substantially non-fluorescent as an antigen is prepared, and by mixing this antibody with the dye, the fluorescence is substantially reduced. It is possible to make non-active dyes fluorescent,
Enables trace fluorescence analysis of the dye. Further, by using MG, the background level can be extremely reduced and high-sensitivity measurement can be performed, as compared with the case of using other dyes such as CCJV and fluorescein.

[Brief description of drawings]

FIG. 1 is a diagram showing an antibody titer of antimalachite green labeled hemocyanin serum according to the present invention.

FIG. 2 is a diagram showing a comparison of antibody activities of an antiserum according to the present invention and IgG fractions.

FIG. 3 is a diagram showing an absorption spectrum of a mixed solution of an IgG fraction from an anti-malachite green labeled hemocyanin serum according to the present invention and malachite green.

FIG. 4 is a view showing a fluorescence difference spectrum of a mixed solution of an IgG fraction from an antimalachite-green labeled hemocyanin serum according to the present invention and malachite-green.

FIG. 5 is a diagram showing a concentration-fluorescence intensity curve of malachite green.

FIG. 6 is a diagram showing a concentration-absorbance curve of malachite green.

FIG. 7 is a diagram showing an absorption spectrum of a mixed solution of an IgG fraction from an anti-malachite-green labeled hemocyanin serum and auramine O according to the present invention.

FIG. 8 is a diagram showing a fluorescence difference spectrum of a mixed solution of an IgG fraction from an anti-malachite-green labeled hemocyanin serum and auramine O according to the present invention.

FIG. 9 is a diagram showing an absorption spectrum of MG-labeled mouse IgG according to the present invention.

FIG. 10 is a diagram showing a fluorescence spectrum of an MG-labeled rabbit anti-mouse IgG antibody, (a) showing the anti-MG according to the present invention.
It is a figure which shows the fluorescence spectrum at the time of making an antibody react, and (b) is a figure which shows the fluorescence spectrum at the time of making a blank react.

FIG. 11 is a view showing the arrangement of titer plates in the spectrophotometer according to the present invention.

FIG. 12 is a diagram showing back ground fluorescence of a titer plate to which mouse serum components are bound, (a) is a diagram measured with excitation light of 440 nm and a fluorescence wavelength of 450 to 600 nm, and (b) is excitation. Light 620nm, fluorescence wavelength 6
It is a figure measured at 30 to 780 nm.

Claims (14)

[Claims] 1. An antiserum comprising an antibody against an antigen having a dye that is substantially non-fluorescent, wherein the antiserum or a mixture of the antibody and the dye has fluorescence. 2. An IgG fraction in antiserum containing an antibody against an antigen having a dye that is not substantially fluorescent, wherein the mixture of the IgG fraction and the dye has fluorescence. IgG fraction. 3. The antiserum according to claim 1, wherein the antigen is formed by adding the dye to an immunological substance. 4. The Ig according to claim 2, wherein the antigen is formed by adding the dye to an immunological substance.
G fraction. 5. The immunological substance is bovine serum albumin, human serum albumin, rabbit serum ovalbumin,
4. The antibody according to claim 3, wherein at least one is selected from the group consisting of bovine gamma globulin, horse serum globulin, human gamma globulin, sheep gamma globulin, bovine thyroglobulin, porcine thyroglobulin, hemocyanin, and synthetic polypeptide. serum. 6. The immunological substance is bovine serum albumin, human serum albumin, rabbit serum ovalbumin,
The IgG according to claim 4, wherein at least one is selected from the group consisting of bovine gamma globulin, horse serum globulin, human gamma globulin, sheep gamma globulin, bovine thyroglobulin, porcine thyroglobulin, hemocyanin, and synthetic polypeptide. Fractions. 7. The antiserum according to claim 1, wherein the dye has a triphenylmethane structure. 8. The I according to claim 2, wherein the dye has a triphenylmethane structure.
gG fraction. 9. The antiserum according to claim 7, wherein the pigment having a triphenylmethane structure is malachite green. 10. The IgG fraction according to claim 8, wherein the dye having a triphenylmethane structure is malachite green. 11. The antiserum according to claim 1 or 3, wherein the dye has a diphenylmethane structure. 12. The I according to claim 2, wherein the dye has a diphenylmethane structure.
gG fraction. 13. The antiserum according to claim 11, wherein the dye having a diphenylmethane structure is auramine O. 14. The IgG fraction according to claim 12, wherein the dye having a diphenylmethane structure is auramine O.