JPH07191033A - Immunoassay method using liposome - Google Patents

Abstract

PURPOSE:To surely estimate the concentration of carboxyfluorescein (CF) in a short time by simultaneously measuring the fluorescence intensity ratio and transmittance of a CF fluorescent pigment and deciding the concentration of the pigment based on both measured values. CONSTITUTION:An argon laser 10 is used as a light source and a laser beam from the laser 10 is made incident on a cell cuvet 18 containing an injected specimen through an ND filter 14 and aperture 16. A transmittance measuring means is positioned on the opposite side of the light source with respect to the cell 18 in a linear positional relation and a fluorescence intensity measuring means is positioned in a perpendicular positional relation with respect to the light source section. The fluorescence from a CF fluorescent pigment contained in the specimen is condensed by means of a condenser 32 through a pinhole 30 and made incident on a photosensor 40 after the light other than a fluorescent component is removed through a filter 34. The fluorescence of the pigment is measured on the basis of the intensity of excited light intensity detected by means of a monitor 23. When such an optical system constitution is used, the transmittance and fluorescence intensity of the specimen can be measured simultaneously. The conversion of the fluorescence intensity measured value into the fluorescence intensity ratio is performed by using the intensity of liposome destroyed by a surface-active agent and the intensity of the liposome itself.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunoassay method using liposomes. More specifically, the present invention relates to a liposome immunoassay method capable of reliably determining a carboxyfluorescein fluorescent dye concentration in a short time in an immunoassay method using a liposome using a carboxyfluorescein fluorescent dye as a fluorophore.

[0002]

2. Description of the Related Art Immunoassays based on the antigen-antibody reaction are extremely superior in terms of specificity and sensitivity.
It is widely used in various fields such as medical biology research and clinical examination. Based on this antigen-antibody reaction, methods such as radioimmunoassay (RIA) using a radioisotope and enzyme immunoassay (EIA) using an enzyme have been developed in order to perform microquantification of a sample.

However, although the RIA is very excellent in terms of sensitivity, it uses a radioisotope as a labeling substance, so a qualified person who is the first-class radiation handling supervisor is required.
Measurement can be performed only within a specific radioactivity management facility,
Furthermore, there is a problem that a special facility is required for the treatment of radioactive isotope waste.

Since EIA uses an enzyme as a labeling substance, it does not cause a problem like RIA, but it has a problem that an unreacted labeling substance has to be separated, which makes the measurement very troublesome.

In addition to these methods, an immunoassay method (LILA) using liposomes has recently been proposed. This L
Unlike conventional RIA and EIA, ILA has the advantages that it is a homogeneous system that does not require separation of unreacted labeling substance, and that it can be rapidly measured.

The liposome is prepared by suspending an amphipathic complex lipid having both a hydrophilic group and a hydrophobic group in the same molecule (for example, lecithin, cholesterol, phosphatidic acid) in a buffer solution at a certain temperature or higher. It is an endoplasmic reticulum composed of a prepared lipid bilayer. A protein antigen or a specific antibody is covalently bonded to this liposome membrane using a bifunctional crosslinking agent. Carboxyfluorescein (CF) is encapsulated as a fluorescent label in the liposome. CF is a fluorescein dye, has high water solubility, and is easily encapsulated in liposomes. When this is reacted with a specific antibody or an antigen, an antigen-antibody complex is formed on the surface of the liposome membrane. At this time, if complement is present, it is activated and a membrane attack complex is formed. It binds to the liposome membrane to increase the permeability of the lipid bilayer, and CF in the liposome flows out into the external solvent. When CF is encapsulated in a liposome at a high concentration, it does not fluoresce due to self-quenching, but it begins to fluoresce when it flows out of the liposome and is diluted with an external solvent. The amount of antigen and the amount of antibody can be determined by measuring this fluorescence intensity.

[0007]

As shown in FIG. 3, CF has a proportional relationship between the fluorescence intensity and the concentration as the molar concentration increases up to a certain concentration (C _P ), but it exceeds C _P. In the region of extremely high concentration, on the contrary, it exhibits the characteristic that the fluorescence concentration decreases. Therefore, the concentration of CF in the fluorescence intensity I _fA is C
Two kinds of values, ₁ and C ₂ , are obtained, and it is impossible to determine which concentration is correct.

Therefore, conventionally, in the inspection method for estimating the concentration from the fluorescence intensity of CF, the concentration region of CF is limited or the measurement object is diluted in several ways, and the concentration is determined from the characteristics before and after. A roundabout and redundant method such as estimation was adopted.

Therefore, an object of the present invention is to provide a novel fluorescence detection method capable of reliably estimating the CF concentration in a short time without using a conventional roundabout method.

[0010]

In order to solve the above-mentioned problems, the present invention encloses a carboxyfluorescein fluorescent dye in a liposome and covalently bonds a protein antigen or a specific antibody to the liposome membrane to form a liposome membrane surface. An antigen-antibody complex is formed, and a complement is allowed to act on the complex to form a membrane attack complex. The membrane attack complex destroys the liposome membrane, and the carboxyfluorescein fluorescent dye encapsulated in the liposome is transferred to the liposome. In an immunoassay using a liposome, which comprises measuring the amount of antigen and the amount of antibody by measuring the fluorescence intensity of the released carboxyfluorescein fluorescent dye, the fluorescence intensity ratio of the carboxyfluorescein fluorescent dye The transmittance was also measured at the same time, and the carboxy was measured based on the both measured values. Providing immunoassay using liposomes and determining the concentration of Ruoresein fluorescent dye.

[0011]

As described above, according to the method of the present invention, the transmittance is simultaneously measured together with the fluorescence intensity ratio of the carboxyfluorescein fluorescent dye. The transmittance of the carboxyfluorescein fluorescent dye is inversely proportional to the concentration. That is, the transmittance decreases as the concentration of the carboxyfluorescein fluorescent dye increases. Therefore, by creating a calibration curve of the fluorescence intensity ratio and the transmittance for the concentration of a known carboxyfluorescein fluorescent dye, the fluorescence intensity ratio and the transmittance measurement value of the obtained carboxyfluorescein fluorescent dye of the unknown sample are measured by the calibration curve. The concentration of the carboxyfluorescein fluorescent dye in the sample can be reliably determined in a short time by applying

In this way, the concentration of the carboxyfluorescein fluorescent dye is reliably determined in a short time, and as a result, the amount of antigen and the amount of antibody can be reliably estimated in a short time. It can also be very useful in promoting basic physics and chemistry research.

The term "fluorescence intensity ratio" used in this specification means that the total CF encapsulated in liposomes is
It means the fluorescence intensity with respect to the amount. The term “transmittance” means the ratio of the amount of light transmitted without being scattered by the substance in the cell to the amount of irradiation light.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in more detail below with reference to the drawings.

The immunoassay method itself using liposomes is known. For example, “Pharmaceutical Factory”, Vol. 7, No. 5
(1987) 421-425, "Biological Sciences", V
ol. 38, No. 5 (Oct. 1987) 498-
Page 500 and "Clinical Examination", Vol. 34, No. 7
(July 1990) pp. 868-871.

FIG. 1 is a schematic perspective view of an example of an optical measurement system that can be used to carry out the method of the present invention. As shown, the light source is an argon laser 10 having a wavelength of 488 nm. Flash xenon can be used as the light source. The beam emitted from the argon laser 10 passes through the beam expander, passes through the aperture 16 from the ND filter 14, and is incident on the quartz cell cuvette 18 into which the sample is injected. The transmittance measuring means is arranged in a linear positional relationship with the light source with the cell 18 interposed therebetween, and the fluorescence intensity measuring means is arranged so as to be orthogonal to the light source. The transmittance measuring means is composed of, for example, a filter (λ = 488 nm bandpass filter) 20 and an output monitor 22. On the other hand, the fluorescence emitted from the carboxyfluorescein fluorescent dye (CF) in the sample of the cell 18 passes through the pinhole 30 and is condensed by the condensers (concentrators) 32 and 32.
Light other than the fluorescent component is removed by the filter (λ = 514 nm) 34, the fluorescent light having a wavelength of 514 nm is collected by the condenser lens 36, and is made incident on the photosensor 40 through the field stop 38. The fluctuation of the output of the laser excitation light also affects the measured values of the fluorescence intensity ratio and the transmittance. Therefore, a monitor 23 for measuring the excitation light output immediately before entering the cell 18 and a half mirror 24 for making the excitation light incident on this monitor are provided. The excitation light intensity detected by the monitor 23 is used as a reference. By using the optical system having such a configuration, the transmittance and fluorescence intensity of the sample can be measured at the same time. In order to convert the obtained measured value of the fluorescence intensity into the form of the fluorescence intensity ratio, the fluorescence intensity obtained by destroying the liposome with a surfactant is set to 100%,
Each measurement value may be converted with the fluorescence intensity of the liposome itself being 0%.

FIG. 2 is a standard calibration curve showing the relationship between the fluorescence intensity ratio and the transmittance at 488 nm with respect to the antigen concentration (mg / ml) prepared by using the optical measurement system shown in FIG.
There are three types of immunoassays that utilize the complement-dependent membrane damage reaction: the release method, the competition method, and the sandwich method.
The calibration curve of the present invention is prepared using the sandwich method. The sandwich method consists of activating complement by sandwiching an antigen with an antibody on the surface of a liposome and a free antibody (secondary antibody).

As an example of preparing a calibration curve in the sandwich method, a C-reactive protein (hereinafter referred to as “CRP”) contained in serum and known to increase in content during inflammation will be described below. The method for creating the calibration curve is shown below. The liposome used is a mixture of several phospholipids such as cholesterol and dipalmitoylphosphatidylcholine. CF is encapsulated in this to prepare multilamellar liposomes. The primary antibody is an anti-human CRP mouse monoclonal antibody, which is treated with periodic acid to create a binding group and then bound to a liposome. The liposome to which the primary antibody is bound is diluted to an appropriate concentration with 10 mM TES buffer solution containing an absorbent for stabilizing the reaction, and this is used as a liposome reagent. The buffer also has a function of preventing spontaneous disintegration of liposomes. The standard serum for measuring fluorescence intensity used at each fixed point for preparing a calibration curve is prepared by diluting the purified CRP antigen in several stages with the above buffer solution so that the concentration of each fixed point is obtained. After preparing the above, the method for obtaining the calibration curve is as follows. A fixed amount of the above-mentioned standard serum was mixed with the liposome reagent, and the mixture was allowed to react at 37 ° C. near human body temperature for 1 hour. A secondary antibody (for example, anti-human CRP rabbit antibody) solution and a complement (for example, guinea pig serum) solution are added to this mixed solution in a fixed amount, and the mixture is further reacted for 1 hour to stabilize the reaction. The fluorescence intensity and the transmittance are measured to prepare a calibration curve as shown in FIG.

For example, when the measured transmittance of the sample is 78% and the fluorescence intensity ratio is 45%, the CRP in the sample is shown in FIG.
The concentration can be determined to be 0.1 mg / ml. When the CRP concentration is determined only from the fluorescence intensity ratio as in the conventional case, the CRP concentration corresponding to the fluorescence intensity ratio of 45% is 0.1 mg /
Both 4 mg / ml were obtained in addition to ml and it was not possible to immediately determine which was the correct concentration. The transmittance curve is CR
Since there is an exponential decay with increasing P concentration, there is only one intersection of CRP concentrations corresponding to a given transmittance.
Therefore, even if the CRP concentration corresponding to the measured value of the transmittance of the sample and the measured value of the fluorescence intensity ratio do not completely match, the measurement of the fluorescence intensity ratio closest to the CRP concentration corresponding to the measured value of the transmittance is performed. CRP of sample with CRP concentration corresponding to the value
It can also be determined to be the concentration.

Thus, by simultaneously measuring the measured values of the transmittance of the sample, it is possible to reliably determine the true antigen concentration corresponding to the measured value of the fluorescence intensity ratio in a short time based on the measured values of the transmittance. You can Therefore, as in the conventional measuring method for estimating the concentration from the fluorescence intensity of CF, the concentration region is limited, or the measurement target is diluted in several ways and the concentration is estimated from the front and back characteristics, which is redundant and redundant. All operations are unnecessary.

Hereinafter, the effectiveness of the method of the present invention will be illustrated by the following specific examples.

Example A specific example of antibody measurement using the complement-dependent membrane damage reaction will be described. Reagents and methods used were the same as those described above. ， CR as a sample provided by the hospital
The P-positive serum was diluted 100-fold with a 10 mM TES buffer solution containing an absorbent, and the fluorescence intensity of the sample was measured to obtain the CRP concentration in each sample. The CRP measurement of the same sample as above was carried out by the TIA method which has been conventionally used and which optically detects the precipitation reaction and the agglutination reaction during the highly reliable antigen-antibody reaction, and the correlation between the two methods was investigated. As a result, the correlation shown in FIG. 4 was obtained, and the correlation coefficient was as good as 0.98, confirming the reliability of this method.

[0023]

As described above, according to the method of the present invention, the transmittance is simultaneously measured together with the fluorescence intensity ratio of the carboxyfluorescein fluorescent dye. Apply a calibration curve of fluorescence intensity ratio and transmittance for known carboxyfluorescein fluorescent dye concentrations, and apply the measured fluorescence intensity ratio and transmittance of carboxyfluorescein fluorescent dye of an unknown sample to the calibration curve. As a result, the concentration of the carboxyfluorescein fluorescent dye in the sample can be reliably determined in a short time. In this way, the concentration of the carboxyfluorescein fluorescent dye is reliably determined in a short time, and as a result, the amount of antigen and the amount of antibody can be reliably estimated in a short time. It can also be very useful in promoting research.

By simultaneously measuring the transmittance of the sample together with the fluorescence intensity ratio, the true CF concentration corresponding to the measured value of the fluorescence intensity ratio can be reliably determined in a short time based on the measured transmittance value. be able to. Therefore, the conventional C
There is no need for a roundabout and redundant operation such as limiting the concentration region, or diluting the measurement object several times and estimating the concentration from the front and back, as in the measurement method that estimates the concentration from the fluorescence intensity of F. become.

[Brief description of drawings]

FIG. 1 is a schematic perspective view of an example of an optical measurement system that can be used to carry out the method of the present invention.

FIG. 2 is a diagram showing a calibration curve of fluorescence intensity ratio and transmittance created by using the apparatus of FIG.

FIG. 3 is a diagram of a characteristic curve showing a relationship between carboxyfluorescein fluorescent dye concentration and fluorescence intensity converted into an output voltage value.

FIG. 4 is a characteristic diagram showing a correlation between a CRP concentration measurement value obtained by the method of the present invention and a CRP concentration measurement value obtained by a conventional TIA method.

[Explanation of symbols]

 10 Light Source 12 Beam Expander 14 ND Filter 16 Aperture 18 Cell 20 Filter 22 Output Monitor 30 Pinhole 32 Condenser 34 Filter 36 Condenser Lens 38 Field Stop 40 Photo Sensor

Claims (2)

[Claims] 1. Encapsulating a carboxyfluorescein fluorescent dye in a liposome, covalently binding a protein antigen or a specific antibody to the liposome membrane to form an antigen-antibody complex on the surface of the liposome membrane, and allowing the complement to act on the complex. To form a membrane attack complex, and the membrane attack complex destroys the liposome membrane to release the carboxyfluorescein fluorescent dye encapsulated in the liposome to the outside of the liposome, and the fluorescence intensity of the released carboxyfluorescein fluorescent dye. In an immunoassay using a liposome, which consists of measuring the amount of antigen and the amount of antibody by measuring, the transmittance is also measured at the same time as the fluorescence intensity ratio of the carboxyfluorescein fluorescent dye, and based on the both measured values obtained Characterized by determining the concentration of carboxyfluorescein fluorescent dye Immunoassay using liposomes. 2. A calibration curve of the fluorescence intensity ratio and the transmittance is prepared in advance for a known concentration of carboxyfluorescein fluorescent dye, and the measured values of the fluorescence intensity ratio and the transmittance measured for an unknown sample are used as the calibration curve. The method of claim 1 comprising determining the concentration of the carboxyfluorescein fluorescent dye in the unknown analyte by applying