JP2007171213A - Fluorescence immunity measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly sensitive real-time fluorescence immunity measuring method. <P>SOLUTION: This fluorescence immunity measuring method for a specimen of the present invention comprises (1) a process for adding a specimen pigment complex with a substantially nonfluorescent pigment coupled to the specimen, and an anti-specimen pigment complex antibody that is antibody for the specimen pigment complex, and capable of recognizing both the specimen and the pigment respectively as epitopes, into a solution containing the specimen of an unknown amount, (2) a process for measuring a change of a fluorescent intensity in the pigment, based on an antigen-antibody reaction of the specimen pigment complex with the anti-specimen pigment complex antibody, under the presence and absence of the specimen, and (3) a process for determining quantitatively the amount of the specimen, by comparing the change of the fluorescent intensity measured under the presence of the specimen in the process (2), with the the change of the fluorescent intensity measured under the absence of the specimen in the process (2), and by investigating the fluorescent intensity reduced by the antigen-antibody reaction of the specimen with the anti-specimen pigment complex antibody. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a fluorescence immunoassay method.

  Conventionally, an immunoassay method (FIA) using fluorescence is known as a high-sensitivity analysis method for trace substances in the course of changes in metabolites in the living body and pollutants in the environment. Such a method is generally based on the measurement of the fluorescence intensity of a fluorescent dye labeled for detection based on an antigen-antibody reaction.

  Therefore, the conventional fluorescence immunoassay method using an antigen-antibody reaction has a problem that since the dye is fluorescent, the background fluorescence due to non-specific adsorption or the like is large and the detection sensitivity is reduced. Furthermore, there is a problem that it is difficult to measure in real time.

JP-A-9-5324

  The present invention uses a dye that is not substantially fluorescent under normal measurement conditions, and makes the dye fluorescent by suppressing intramolecular movement of the dye molecule based on an antigen-antibody reaction. It is an object of the present invention to provide a method capable of suppressing the influence of various background fluorescence from the system and enabling high-sensitivity and real-time fluorescence immunoassay.

  In order to solve the above problems, an immunoassay method according to the present invention is a specific antibody developed by the present inventor (Japanese Patent Laid-Open No. 9-5324), and is a dye that is not substantially fluorescent to a subject. An antibody using an analyte dye complex having an antigen as an antigen is used.

  That is, the immunoassay method according to the present invention has the general concept shown in FIG. 1 and the competition method as an example of the embodiment schematically shown in FIG. An immune reaction is caused, and as a result, a dye that is not substantially fluorescent becomes fluorescent by interacting with the antibody, and the amount of the antigen present in the sample can be quantified.

  That is, the present invention relates to a fluorescence immunoassay method for a subject, comprising (1) preparing a subject dye complex by binding a substantially non-fluorescent dye to the subject; and (2) the subject. A step of preparing an anti-analyte dye complex antibody having an analyte dye complex as an antigen, and (3) an antigen antibody with the analyte dye complex against the anti-analyte dye complex antibody in the presence of the analyte And a step of measuring a change in the fluorescence intensity of the dye based on the reaction.

  The fluorescent immunoassay method described above includes: 1) an analyte dye complex in which a dye that is not substantially fluorescent to the analyte is bound to a solution containing an unknown amount of the analyte; and an antibody against the analyte dye complex A step of adding an anti-analyte dye complex antibody that recognizes both the analyte and the dye as epitopes, and 2) the analyte dye in the presence and absence of the analyte Measuring a change in fluorescence intensity of the dye based on an antigen-antibody reaction between the complex and the anti-analyte dye complex antibody, and 3) the fluorescence measured in the presence of the analyte in 2) By comparing the change in intensity with the change in fluorescence intensity measured in the absence of the analyte, and examining the fluorescence intensity reduced by the antigen-antibody reaction between the analyte and the anti-analyte dye complex antibody Quantify the amount of the analyte It is preferable to provide a step.

  Further, the fluorescence immunoassay method described above includes 1) adding the anti-analyte dye complex antibody to a solution containing an unknown amount of the analyte, and then adding the analyte dye complex, and 2) Measuring the change in fluorescence intensity of the dye based on the antigen-antibody reaction between the analyte dye complex and the anti-analyte dye complex antibody in the presence and absence of the analyte; and 3) the 2 ), The change in the fluorescence intensity measured in the presence of the analyte and the change in the fluorescence intensity measured in the absence of the analyte are compared, and the analyte and the anti-analyte dye complex And a step of quantifying the amount of the analyte by examining the fluorescence intensity reduced by the antigen-antibody reaction with the antibody.

  Further, the above-described fluorescence immunoassay method comprises a competitive reaction by an antigen-antibody reaction between the analyte and the analyte dye complex with respect to an antigen-antibody reaction between the analyte dye complex and the anti-analyte dye complex antibody. It is more preferable to further include a step of monitoring in real time using the fluorescence intensity as an index.

  The present invention also provides the fluorescence immunoassay method described above, wherein the anti-analyte dye complex antibody is an IgG fraction.

  Further, the present invention provides the fluorescence immunoassay method described above, wherein the subject is insulin.

  Furthermore, the present invention provides the fluorescence immunoassay method described above, wherein the dye has a triphenylmethane structure.

  The present invention also provides the fluorescence immunoassay method described above, wherein the dye having a triphenylmethane structure is malachite green.

  The present invention also provides the fluorescence immunoassay method described above, wherein the dye has a diphenylmethane structure.

  Furthermore, the present invention provides the above-described fluorescence immunoassay method, wherein the dye having a diphenylmethane structure is auramine O.

  According to the present invention, it is possible to produce an antibody using a substance bound with a dye that is not substantially fluorescent as an antigen, and to make the dye that is not substantially fluorescent by mixing this antibody with the antigen. It becomes possible, and trace fluorescence analysis of the antigen becomes possible. In addition, the background level can be extremely reduced, and highly sensitive measurement is possible. Furthermore, real-time measurement is possible. That is, when the label is always active (the label activity is independent of the antigen-antibody reaction), the antibody causes non-specific adsorption on an immunotiter plate or the like and becomes a background. On the other hand, according to the present invention, even if either antibody or MG-labeled antigen causes some nonspecific adsorption, fluorescence does not occur unless both react, and that alone does not become a background.

  Furthermore, living organisms are immune-tolerant to their own constituent substances and cannot produce antibodies, and in the case of different types of animals with respect to specific substances (for example, insulin), the amino acid sequence is In almost the same case, no animal can be used to produce antibodies. On the other hand, in the present invention, since the antigen is an artificial substance in which MG is labeled on the measurement target, an antibody can be easily generated. Therefore, since it is difficult to produce antibodies conventionally, it is possible to measure even substances for which immunoassay is impossible or extremely difficult.

  Hereinafter, the present invention will be described in detail.

(Dye that is not substantially fluorescent)
The dye that can be used in the present invention is not particularly limited as long as it does not substantially exhibit fluorescence in a normal solvent such as water, or exhibits fluorescence but is weak. That is, in the present invention, substantially non-fluorescent means that it does not show a fluorescence spectrum under normal measurement conditions, or shows only very weak fluorescence, and it is substantially impossible to perform fluorescence spectroscopic analysis with a commercially available apparatus or the like. (Yasuharu Nishikawa et al., “Fluorescence phosphorescence analysis”, Kyoritsu Shuppan, 30 pages, 1984).

In the present invention, in the case of fluorescence quantum yield less than 1% in specific conditions, a dye which does not exhibit substantial fluorescent here, fluorescence quantum yield than ^{2% 10-2} under certain conditions In the case of, it means a dye that does not substantially exhibit fluorescence.

  However, in the case where extremely weak fluorescence can be detected by special measurement conditions, a measuring device, etc., the dye that is not substantially fluorescent in the present invention becomes fluorescent by the treatment according to the present invention, and fluorescence analysis is possible. This means that the fluorescence quantum yield of the dye is extremely small (for example, 0.01% or less) under normal measurement conditions, but greatly changes by interacting with the antibody by the treatment according to the present invention (for example, 1%). )).

  Furthermore, the dyes that can be used in the present invention include those that are fluorescent in a common solvent such as water (fluorescence quantum yield is greater than 1%).

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

  In the present invention, the increase in the fluorescence quantum yield is preferably at least 10 times or more, but is preferably increased by 100 times, more preferably 1000 times or more (more preferably 10,000 times or more).

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

  The dye that can be used in the present invention is not necessarily required to have absorption in the visible part (having an absorption maximum at 350 nm or more), but has absorption in the ultraviolet part (having an absorption maximum at 350 nm or less), or an absorption band. May extend to the visible region.

  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.

  In particular, those containing a stable chromophore near neutral pH are desirable.

  For example, a dye having a triphenylmethane skeleton in the molecular structure is particularly preferable (see, for example, “Dye Handbook” edited by Kodansha Scientific, Nobu Okawara), and a malachite that is a dye having a triphenylmethane skeleton in the molecular structure. Green or the like is particularly preferably used.

  In addition, a dye having a diphenylmethane skeleton (for example, see “Dye Handbook” edited by Kodansha Scientific Co., Ltd., Nobu Okawara). As the diphenylmethane dye, for example, an auramine dye can be preferably used. In particular, use of auramine O is preferable.

(Subject)
There is no particular limitation on the analyte that can be measured using the method according to the present invention. That is, it is only necessary that the dye according to the present invention can be bound and the obtained dye analyte complex can be an immunizing antigen in the antigen-antibody reaction according to the present invention.

  In this case, if necessary, the antigenicity can be improved by subjecting the dye analyte complex to a specific chemical modification. For example, bovine serum albumin, human serum albumin, rabbit serum albumin, ovalbumin, bovine gamma globulin, horse serum globulin, human gamma globulin, sheep gamma globulin, bovine thyroglobulin, butyroglobulin, hemocyanin, synthetic peptide and the like.

  More specifically, what can be a subject in the present invention includes, for example, various physiologically active substances (peptide hormones, enzymes, various antibodies), and pathogens (bacteria, viruses). In the present invention, insulin is particularly preferable among them.

  Furthermore, since the chemical structure (for example, amino acid sequence) of the analyte is very close to the present invention, an antibody having a sufficient difference when used as an antigen as it is cannot be obtained. Even when it cannot be used, it can be preferably used. That is, even when the chemical structure is similar, in the present invention, a substance further bound with a specific fluorescent dye is used as an antigen. Due to this structure, the obtained antibody has sufficient antigen recognizability. It will have.

(Analyte dye complex preparation method: a method of binding a substantially non-fluorescent dye to an analyte)
In the present invention, the method for producing an antigen having a dye that is not substantially fluorescent (hereinafter referred to as analyte dye complex or dye analyte complex) is not particularly limited.

  For example, it is possible to stir and mix the analyte and a dye that is not substantially fluorescent for a predetermined time, and to separate the fraction of the antigen having the dye using gel filtration chromatography.

  Further, if necessary, for example, it is possible to purify by using a purification means utilizing an immune reaction (for example, the Biochemical Society of Japan, continued chemistry experiment course, Volume 5 Immunobiochemical Research Method (Tokyo Kagaku Dojin) (See pages 25-31, 1.3 Methods for Specific Purification of Antibody).

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

(Anti-analyte dye complex antibody: antibody production method)
Preparation of the antibody used in the present invention (hereinafter referred to as anti-analyte dye complex antibody) can be preferably prepared using a normal immunization process.

Although it does not restrict | limit especially as an immunized animal, A rabbit and a guinea pig can be used conveniently.

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

  There is no particular limitation on the immunoinjection method that can be used in the present invention, but for example, in guinea pigs, subcutaneous injection, intraperitoneal injection, and the like can be suitably used.

  In the present invention, antiserum production can be confirmed and collected by performing additional immunization if necessary, collecting a test and examining the antibody titer.

  In the present invention, separation of the collected antiserum is not particularly limited, and serum can be separated by a general method, for example, after clotting the determined blood, and by centrifugation. The specific antibody activity against the antigenic dye of the obtained antiserum can be preferably measured by enzyme immune reaction or the like (for example, the Biochemical Society of Japan, continued chemical experiment course, Volume 5 Immunobiochemical Research Method (Tokyo Kagaku Dojin), 62 -See page 65, 1.4.6 Enzyme Immunoassay).

(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 can be particularly preferably used. Furthermore, the obtained IgG fraction can also be concentrated by ultrafiltration by centrifugation and can be prepared to a predetermined concentration.

(Fluorescence spectrum measurement method)
In the present invention, by mixing the antibody and a non-fluorescent dye, the mixed solution becomes fluorescent, and the fluorescence spectrum of the obtained mixed solution can be measured by various measuring methods (fluorescence and fluorescence). (Phosphorescence analysis, Nishikawa, Hiramoto, Kyoritsu Shuppan, Chapter 2, 49-99).

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

  The fluorescence quantum yield expressed in the present invention depends on incidental factors in dye production, analyte, and antibody production.

  Furthermore, according to the present invention, it is possible to quantitatively analyze a subject using the expressed fluorescence. The limit of quantitative analysis depends on the quantum yield.

(Subject measurement method)
Various specific embodiments of the fluorescence immunoassay method according to the present invention are possible as shown below, and can be appropriately selected depending on the purpose of the measurement. One preferred embodiment for detecting an analyte in the present invention is a competitive method as schematically shown in FIG. In this method, even when the analyte exists as a mixture in the sample, it can be measured with high sensitivity (due to low background fluorescence) and in real time. That is, an analyte dye complex obtained by binding a dye to an analyte is prepared, and an antibody (including both polyclonal and monoclonal antibodies) is prepared according to a conventional method using the complex as an antigen. Based on the competitive reaction between the analyte dye complex and the analyte with respect to the analyte dye complex antibody, the concentration of the analyte can be measured. At this time, it is possible to monitor the change in fluorescence obtained over time in real time under the condition that either concentration is excessive.

  More specifically, an example in which insulin is used as a subject will be described below. However, it is not limited to insulin.

(1) Malachite green (MG) is selected as a pigment, and MG-ins are covalently labeled insulin as MG-Ins, and insulin that is not labeled with MG is simply insulin (Ins). Furthermore, an antibody using MG-Ins as an antigen is referred to as an anti-Mg-Ins antibody. Such antibodies are usually obtained by known methods.

  The MG-Ins obtained here becomes fluorescent by the antigen-antibody reaction with the anti-Mg-Ins antibody (Japanese Patent Laid-Open No. 9-5324). That is, it is considered that the vibration and rotation in the MG molecule was suppressed by the binding with the antibody, and the non-radiative transition from the excited state to the ground state was suppressed. Therefore, it becomes possible to monitor in real time by the fluorescence change of MG which generates such an antigen-antibody reaction process.

(2) This antigen-antibody reaction is suppressed by the presence of a competitive antigen-antibody reaction by insulin coexisting in the measurement system, and the degree of suppression depends on the amount of insulin coexisting.

  Therefore, under the condition that the anti-Mg-Ins antibody and MG-Ins are in a constant amount, it is possible to quantify insulin as the analyte in the measurement system from the degree of this suppression.

(3) Several methods are possible to quantitatively evaluate the process or degree of this suppression. Specifically, as the insulin concentration in the measurement system increases, the increase in fluorescence from the MG becomes dull, and the slope of the curve showing the time change of the increase in MG fluorescence (Fig. 2, Slope1 and Slope2) decreases. The time to reach a certain fluorescence intensity is delayed (FIG. 2, t1 and t2), and the fluorescence intensity after a certain time (FIG. 2, t3) decreases.

  A calibration curve can be created from the correlation between the numerical value and the insulin concentration. Specifically, for example, the correlation between the fluorescence intensity 5 minutes after the start of the reaction and the insulin concentration can be obtained.

(4) The order of the antigen-antibody reaction is not particularly limited. For example, it is possible to add a certain amount of MG-Ins after the reaction of insulin and the antibody is first reacted. The reason is that MG-Ins, which is the original sequence, is expected to bind to the antibody more strongly than insulin. Therefore, in this case, the change in fluorescence intensity over time is measured in real time by the change in fluorescence amount over time as the insulin that is in equilibrium with the antibody is driven out and replaced by MG-Ins (Fig. 3).

  Furthermore, as another aspect of the present invention, an antigen antibody between the obtained dye analyte complex and the antibody according to the present invention is first subjected to a pretreatment for binding the dye to the analyte in the sample. There is a method for directly measuring the concentration of an analyte based on the reaction. This method can be used particularly when the analyte in the sample is present in high purity.

  According to the present invention, the dye is based on a competitive antigen-antibody reaction between the complex and the analyte against an antibody having an analyte having a dye that is not substantially fluorescent (dye analyte complex) as an antigen. Becomes fluorescent, and by measuring the change in fluorescence, the analyte can be quantified in real time.

  Examples of the present invention will be described in detail below, but the present invention is not limited to such examples.

(1) Preparation of Malachite Green Labeled Insulin (MG-Ins) 10.0 mg insulin (Zn free, porcine, manufactured by Biomedical Technologies Inc.) was dissolved in 5 ml carbonate buffer (0.5 M, pH 9.0). To this solution, 1.5 mg of malachite green isothiocyanate (MGITC, manufactured by Molecular Probe Inc.) dissolved in 0.1 ml of acetone was added, and the mixture was stirred at 4 ° C. for 24 hours.

Next, 3 ml of the above reaction solution was applied to a gel filtration column (Econo-PalcODG, manufactured by Bio-Rad) equilibrated with phosphate buffered saline (PBS), and 4 ml of PBS was added from the top of the force ram. The gel was subjected to gel filtration chromatography to remove unreacted MGITC and to separate the MG-Ins fraction.

(2) Immunization of MG-Ins
2-1. Initial Immunization 50 μl of the obtained MG-Ins fraction was diluted with 2.1 ml of physiological saline to obtain an antigen solution. This was aseptically injected into a vial of an adjuvant (RibiAdjuvant System, manufactured by Ribi) using a syringe (5 ml) with a 0.22 μm filter. The vial was then shaken vigorously for 2 minutes to prepare an emulsion of antigen solution and adjuvant. RAS vials were handled according to the attached instruction manual. The obtained emulsion was administered to guinea pigs (Hartley, female, SPF, n = 4) under anesthesia (Nembutal, dose: 8 mg / kg), 0.5 ml per animal (subcutaneously 0.1 ml X 4 sites, abdominal cavity) In 0.1ml).

2-2. Booster immunization Booster immunization was performed twice at 3-4 week intervals from the initial immunization. Antigen amount and adjuvant were the same as for the first immunization.

(3) Measurement of anti-MG-1ns serum anti-body value The specific antibody activity of the antiserum obtained above against MG-Ins was confirmed by enzyme immunoassay as follows.

3-1. Timing of antibody titer measurement
Two weeks after the first boost, a small amount of blood was collected from the immunized guinea pig and the antibody titer was measured. Blood collection was performed by incising the auricular vein with a scalpel and collecting the leaked blood with a hematocrit measuring capillary. One end of this capillary was sealed with seal-ease (Becton Dickinson and Company), this was set in a centrifuge (KOKUSANMODELH-103RS domestic centrifuge) and centrifuged at 3000 rpm for 10 minutes at 4 ° C. to separate the serum.

  As a result of measuring the antibody titer of this serum by the method described later, it was confirmed that a sufficient increase in antibody titer was observed 2 weeks after the booster immunization. Based on this result, whole blood was collected 2 weeks after the second booster, and the final antibody titer was measured in the same manner.

3-2. Preparation of antiserum The whole blood collected from guinea pigs was taken into a sterilized test tube and allowed to stand at 37 ° C for 1 hour in an incubator to promote clot formation. Next, after leaving overnight at 4 ° C. to clot the clot, it was set in a centrifuge (KOKUSAN MODEL H-103RS manufactured by Kokusan Centrifuge Co., Ltd.) and centrifuged at 3000 rpm, 4 ° C. for 10 minutes to separate the antiserum. .

3-3. Enzyme immunoassay
Dissolve 70 μl of the gel filtration fraction of MG-Ins in 7 ml of 50 mM carbonate buffer (pH 9.6), and dispense 0.1 ml into each well of a plastic 96-well immunotiter plate (greiner, ELISA-PLATE). did. This was left overnight at 4 ° C. to adsorb MG-Ins to the inner wall of the titer plate.

  After 0.1 ml of washing solution (PBS containing 0.05% Tween 20) was added to each well of the immunotiter plate, the well was discharged. This washing process was repeated three times. Next, 0.1 ml of blocking solution (PBS containing 1% gelatin) was added to each well and left at 4 ° C. overnight.

  Thereafter, the well was washed again in the same manner, and 0.1 ml of a diluted antiserum solution (100 to 12, 800 times) in PBS or immunized guinea pig serum (not diluted) was added to each well as a control. This titer plate was allowed to stand at room temperature for 10 hours to react the antigen and the antibody.

  After washing with the washing solution in the same manner, 0.1 ml of horseradish peroxidase-labeled goat anti-guinea pig IgG antibody solution (final concentration was 16 μg / ml, manufactured by Cappe1) was added to each well and allowed to stand overnight at 4 ° C. And an enzyme antibody were reacted.

  Thereafter, the substrate was washed in the same manner with a washing solution, and 0.1 ml of a reaction substrate solution (ABTS Peroxidase Substrate System, manufactured by Kirkegaard & Perry Laboratories) was added to each well according to the instruction manual for the reagent. This was placed in a 37 ° C. incubator for 45 minutes to carry out the enzyme reaction, and then 0.1 ml of a reaction stop solution (1% SDS aqueous solution) was added to each well. This immunotiter plate was set on a plate reader (Bio-Rad, model 3550), and the absorbance at 405 nm was measured.

3-4. Results of antibody titer measurement
The antibody titer of the antiserum obtained by collecting all blood one week after the second boost was measured. The result of the example is shown in FIG. This antiserum showed significant antibody activity compared to the control up to 3200-fold dilution.

(4) Purification of IgG fraction From the antiserum obtained above, the IgG fraction was purified using protein A. Protein A is a protein produced by Staphylococcus aureus and is known to specifically bind to Fc fragments of IgG such as humans, mice, and rabbits (referenced by Kazutomo Imabori, supervised by Tamio Yamakawa, `` Biochemical Dictionary '', p1107, Tokyo Chemical Doujin (1984)). Using a protein A-immobilized column (Protein A column kit for antibody purification, Ampure PA Kit, manufactured by Amersham) according to the instruction manual, IgG fraction of about 4 ml was obtained from 1 ml of the antiserum.

  This IgG fraction was concentrated by centrifugation. A sample pretreatment cartridge centrifugal concentration ULTRACENT-10 (manufactured by Tosoh Corporation) was charged with 1 to 1.5 ml of IgG fraction and centrifuged at 5000 rpm (x3000G) for 30 to 60 minutes under cooling at 4 ° C. This was repeated and 4 ml of Protein A strength ram fraction was concentrated to 0.7 ml.

  Protein quantification of this concentrated IgG fraction was performed using a Bio-Rad protein assay (Bio-Rad) according to the instruction manual. Bovine gamma globulin was used as a standard sample. As a result, the protein concentration of the concentrated IgG fraction obtained from each guinea pig was 34.5 to 42.4 μM.

(5) Fluorescence of MG with anti-MG-Ins antibody (IgG) The concentrated IgG fraction derived from anti-MG-Ins serum was reacted with MG and MG-InS, and the fluorescence spectrum of MG was examined. The fluorescence spectrum is measured using a Hitachi 850 fluorescence spectrophotometer (manufactured by Hitachi), excitation wavelength 620 nm, fluorescence wavelength 630 to 750 nm, bandpass 5 nm (both excitation and emission sides), scan speed 60 nm / min, time constant It was measured with a photomultiplier tube gain High for 2 seconds. The sample was placed in a quartz cell for fluorescence measurement with an optical path length of 1 cm on the excitation light side and measured.

5-1. Reaction and fluorescence spectrum of anti-MG-Ins antibody (IgG) and MG
A mixed solution of anti-MG-Ins antibody (IgG) and MG was prepared using PBS. Final concentrations were adjusted to 4 μM for IgG and 4 μM for MG. The concentration of MG was set so as not to cause concentration quenching. In addition, since the IgG fraction includes those that do not react with MG, it is meaningless to set the concentration ratio of IgG and MG strictly, and it was arbitrarily set. As a control, a blank IgG fraction purified from guinea pig gamma globulin (INTER-CELLTECHNOLOGIES, INC.) By protein A ram was used.

  The fluorescence spectrum of this mixed solution is shown in FIG. This is obtained by subtracting the fluorescence spectrum of the mixture solution of blank IgG and MG from the fluorescence spectrum of the mixture solution of anti-MG-Ins antibody (IgG) and MG. The reaction between blank IgG and MG gives all the backgrounds that are not necessary for specific antigen-antibody reaction (fluorescence of the cell itself, Raman scattering of the solvent, fluorescence due to non-specific reaction of antibody and MG, etc.). The fluorescence spectrum is still obtained by subtracting this, indicating that the anti-MG-Ins antibody (IgG) converted MG to fluorescence due to its specific antibody activity.

5-2. Reaction of anti-MG-Ins antibody (IgG) and MG-Ins Using PBS, a mixed solution of anti-MG-Ins antibody (IgG) and MG-Ins was prepared. Final concentrations were adjusted to 4 μM for IgG and 0.67 μM for MG-Ins. The concentration of MG-Ins was calculated from MG ε ₆₂₀ = 1.5 × 10 ⁵ M ^−l cm ⁻¹ (literature DGJay, Pro. Natl. Acad. Sci. USA, Vol. 85 pp5454-5458 (1988)). The basis for setting this concentration is the same as in the case of the reaction between the antibody (IgG) and MG.

  As a control blank IgG, a blank IgG fraction purified from a guinea pig gamma globulin (INTER-CELL TECHNOLOGIES, INC.) By protein A force ram was used.

  The fluorescence spectrum of this liquid mixture is shown in FIG. This is obtained by subtracting the fluorescence spectrum of the mixture solution of blank IgG and MG-Ins from the fluorescence spectrum of the mixture solution of anti-MG-Ins antibody (IgG) and MG-Ins. From this difference spectrum, it can be seen that the anti-MG-Ins antibody (IgG) converted MG labeled with insulin into fluorescence due to its specific antibody activity.

5-3. Reaction of anti-insulin antibody and MG-Ins
(i) The results obtained in 5-2. show that the antibody can recognize both insulin and MG as epitopes. On the other hand, an antibody that recognizes only insulin cannot effectively convert MG to fluorescence.

  In fact, some anti-insulin antibodies have an epitope around the MG labeling site on MG-Ins, and these antibodies recognize MG-Ins even though they are weak, and as a result, suppress free rotation within the MG molecule. It is also possible to make MG fluorescent. However, such an antigen-antibody reaction is weaker than that of anti-MG-Ins antibody, and since MG cannot be accurately recognized, it is considered that the efficiency of making MG fluorescent is low. In order to confirm the above prediction, the fluorescence spectrum generated by the antigen-antibody reaction between the anti-insulin antibody and MG-Ins was measured.

  (ii) First, an IgG fraction containing anti-porcine insulin IgG (hereinafter referred to as anti-insulin IgG) was purified from anti-porcine insulin antiserum (manufactured by HYCOR BIOMEDICAL INC.) using protein A ram. The IgG concentration was determined by Bio-Rad protein assay (manufactured by Bio-Rad) in the same manner as described above, and was 8.78 μM.

  (iii) Next, this IgG fraction was reacted with MG-Ins, and the fluorescence spectrum was measured. As controls, blank IgG and anti-MG-Ins antibody (IgG) were used. The final concentration of each component was adjusted to 0.5 μM for MG-Ins, blank IgG and anti-MG-Ins antibody, and to 0.5, 2.0, and 8.0 μM for anti-insulin IgG.

  The reason for setting a plurality of anti-insulin IgG concentrations is as follows. In other words, anti-insulin antibody is expected to require a larger amount of antibody than anti-MG-Ins antibody (IgG) to detect it, since it has little activity to make MG fluorescent. This is because. Since it is impossible to predict in advance how much is sufficient, a plurality of concentrations were set.

  (iv) The results are shown in FIG. The activity of anti-insulin IgG to fluoresce MG on MG-Ins was less than or equal to blank IgG at the same IgG concentrations of 0.5 and 2.0 μM, and no significant difference was observed.

  The anti-insulin IgG concentration is 8.0 μM for the first time, showing slightly higher activity than the blank IgG, but it is significantly weaker than the positive control anti-MG-Ins antibody (IgG).

  From this result, it was shown that the antibody according to the present invention needs to be able to recognize both MG and Ins of MG-Ins as an antigen as epitopes.

(6) Determination of insulin by competitive antigen-antibody reaction of anti-MG-Ins antibody (IgG) and insulin
(i) The degree of competitive reaction between a fixed amount of MG-Ins and a variable amount of insulin against a certain amount of anti-MG-Ins antibody (IgG) was measured by the time change of fluorescence emitted by MG-Ins. From this experimental result, the correlation (calibration curve) between the fluorescence intensity after a certain period of insulin addition and the added insulin concentration was determined.

  (ii) The fluorescence measurement was performed as follows.

  First, 18 μl of PBS solution of anti-MG-Ins antibody (IgG) and 0 to 900 μl of PBS solution of insulin were mixed in a quartz cell for fluorescence measurement (optical path length 1 cm), and PBS was added to prepare a solution volume of 987 μl.

  This cell was set in a cell holder of a fluorescence spectrophotometer connected to a constant temperature bath (endocal refrigerated circulating bath RTE-5, manufactured by NESLAB Instruments, Inc.), and the temperature was maintained at 35 ° C. Further, the measurement sample was always stirred using a magnetic stirrer bar so that the antigen-antibody reaction proceeded promptly and uniformly. This state was left for 10 minutes and pre-incubated. Next, 13 μl of MG-Ins in PBS was instantaneously added into the cell using a microsyringe.

  (iii) Recording of the fluorescence spectrophotometer was started before the addition, and changes in fluorescence intensity before and after the addition of MG-Ins were measured in real time. The final concentration of each component was 0.25 μM for IgG, 0-29.8 μM for insulin, and 0.078 μM for MG-Ins. The fluorescence measurement conditions were an excitation wavelength of 620 nm, an emission wavelength of 650 nm, a bandpass filter of 5 nm (both excitation side and fluorescence side), a time constant of 2 seconds, and a photomultiplier tube gain of High.

  An example of the temporal change in fluorescence intensity is shown in FIG. The vertical axis is fluorescence intensity, the horizontal axis is time, the upper curve is without insulin, and the lower is when insulin is 7.44 μM. This result shows that the fluorescence intensity after the same time decreases as the insulin concentration increases.

  Further, FIG. 9 shows the relationship between the fluorescence intensity 5 minutes after addition of MG-Ins and the insulin concentration. In this figure, the value obtained by subtracting the fluorescence intensity (background) immediately before MG-Ins addition from the fluorescence intensity after 5 minutes is plotted for each insulin concentration (n = 1). As is apparent from this figure, the fluorescence intensity after 5 minutes monotonously decreased as the insulin concentration of the measurement sample increased. Using this relationship as a calibration curve, the amount of insulin in the unknown sample can be estimated.

It is a figure which shows typically the fluorescence immunoassay method which concerns on this invention. It is a figure which shows the method to evaluate quantitatively the degree of suppression by the anti-MG-Ins antibody and the reaction with MG-Ins. It is a figure which shows the principle of the method of quantifying insulin using the competitive reaction of MG-Ins and insulin with respect to anti-MG-Ins antibody based on this invention. It is a figure which shows the antibody value of the antiserum one week after the 2nd boost. It is a figure which shows the fluorescence spectrum (difference spectrum) when MG reacts with an anti-MG-Ins antibody (IgG). It is a figure which shows the fluorescence spectrum (difference spectrum) of MG when MG-Ins couple | bonds with the anti-MG-Ins antibody (IgG). It is a figure which shows a fluorescence spectrum when MG-Ins couple | bonds with an anti-insulin antibody (IgG). Here, (1) is anti-MG-Ins antibody 0.5 μM, (2) is blank IgG 0.5 μM, (3) is anti-insulin IgG 0.5 μM, (4) is anti-insulin IgG 2.0 μM, (5) is anti-insulin. The case with IgG 8.0 μM is shown. It is a figure which shows the time change of the fluorescence intensity in the case of competitive reaction of MG-Ins and insulin with respect to an anti-MG-Ins antibody. It is a figure which shows the relationship between the fluorescence intensity 5 minutes after MG-Ins addition, and an insulin concentration.

Claims (9)

A fluorescent immunoassay method for a subject,
1) an analyte dye complex in which a dye that is not substantially fluorescent to the analyte is bound to a solution containing an unknown amount of the analyte, and an antibody against the analyte dye complex, the analyte and Adding an anti-analyte dye complex antibody that recognizes both of the dyes as epitopes, and
2) measuring a change in fluorescence intensity of the dye based on an antigen-antibody reaction between the analyte dye complex and the anti-analyte dye complex antibody in the presence and absence of the analyte;
3) In 2), the change in the fluorescence intensity measured in the presence of the subject is compared with the change in the fluorescence intensity measured in the absence of the subject. Quantifying the amount of the analyte by examining the fluorescence intensity reduced by the antigen-antibody reaction with the analyte dye complex antibody;
A fluorescence immunoassay method comprising: A fluorescent immunoassay method for a subject,
1) adding the anti-analyte dye complex antibody to a solution containing an unknown amount of the analyte, and then adding the analyte dye complex;
2) measuring a change in fluorescence intensity of the dye based on an antigen-antibody reaction between the analyte dye complex and the anti-analyte dye complex antibody in the presence and absence of the analyte;
3) In 2), the change in the fluorescence intensity measured in the presence of the subject is compared with the change in the fluorescence intensity measured in the absence of the subject. Quantifying the amount of the analyte by examining the fluorescence intensity reduced by the antigen-antibody reaction with the analyte dye complex antibody;
A fluorescence immunoassay method comprising:   The competitive reaction by the antigen-antibody reaction between the analyte and the analyte dye complex to the antigen-antibody reaction between the analyte dye complex and the anti-analyte dye complex antibody is monitored in real time using the fluorescence intensity as an index. The fluorescence immunoassay method according to claim 1 or 2, further comprising a step. The fluorescence immunoassay method according to any one of claims 1 to 3, wherein the anti-analyte dye complex antibody is an IgG fraction. The fluorescence immunoassay method according to any one of claims 1 to 4, wherein the subject is insulin. The fluorescent immunoassay method according to claim 1, wherein the dye is a dye having a triphenylmethane structure. The fluorescence immunoassay method according to claim 6, wherein the dye having a triphenylmethane structure is malachite green. The fluorescent immunoassay method according to claim 1, wherein the dye is a dye having a diphenylmethane structure.   The fluorescence immunoassay method according to claim 8, wherein the dye having a diphenylmethane structure is auramine O.

Images