JPH11108924A - Method and apparatus for inspecting serum protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply and rapidly measure the constitutional ratio of serum protein. SOLUTION: Serum to which 0.5 μg/ml of indocyanine green(ICG) is added is housed in a test tube 2 as a measuring sample 1 to be irradiated with pulse laser with a wavelength of 790 nm from an exciting light irradiation means 3. ICG mainly bonded to lipoprotein in serum emits fluorescence of a fluorescence life profile corresponding to the constitutional ratio of lipoprotein. This fluorescence is guided to a fluorescence detector 5 through a spectral means 4 and fluorescence life is calculated to be sent to an analyser 6. The analyser 6 calculates the constitutional ratio of lipoprotein from the calculated fluorescence life to display the same on a display device 7. By this constitution, the constitutional ratio of liporotein can be accurately inspected simply within a short time as compared with a conventional method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for measuring serum protein concentration, and more particularly to a method and apparatus for differentially measuring serum protein.

[0002]

2. Description of the Related Art In order to evaluate the presence or absence of abnormal lipid metabolism which causes factors such as heart disease and cerebrovascular disease and is related to a disease state such as diabetes, high density lipoprotein (HDL; It is diagnostically important to measure the composition ratio of serum lipoprotein subfractions such as Density Lipoprotein) and low density lipoprotein (LDL).

Conventionally, for this type of measurement, an electrophoresis method using an agarose gel or a polyacrylamide gel has been used. In this method, lipoproteins are separated according to their molecular size and their respective composition ratios are measured.

[0004] When it is necessary to perform the measurement with higher precision, a density gradient ultracentrifugation method is used. This is a method of separating and quantifying various lipoproteins by utilizing the difference in specific gravity of various lipoproteins and performing ultracentrifugation while changing the specific gravity of a solvent.

[0005]

In each of these two separation and quantification methods, a series of operations requires several hours to several tens of hours, and various operations need to be performed with great care. The abnormal test was a laborious and time-consuming test. A lipid metabolism abnormality test is indispensable for evaluating the condition of a disease such as diabetes and preventing or treating a heart disease or a cerebrovascular disease, and a method for performing such a test simply and quickly is required.

[0006] In view of the above problems, an object of the present invention is to provide a method and an apparatus which can easily and quickly measure the composition ratio of serum proteins.

[0007]

Means for Solving the Problems In order to solve the above-mentioned problems, a serum protein test method of the present invention is a method for measuring the composition ratio of various proteins contained in serum. A fluorescent reagent that binds to a protein and changes in fluorescence lifetime according to the type of the bound protein is added, and the serum, plasma or blood is irradiated with excitation light having an absorption wavelength of the fluorescent reagent, and the fluorescent reagent emits. It is characterized in that the change in the intensity of the fluorescence over time is measured and analyzed to determine the composition ratio of a predetermined protein in the serum.

It is known that the fluorescence lifetime of a fluorescent substance in a solvent generally increases as the environment around the molecule of the fluorescent substance approaches a hydrophobic environment. Since the hydrophilicity differs depending on the type of protein, when a fluorescent substance and a protein bind,
Since the hydrophobicity around the molecule of the fluorescent substance varies depending on the type of protein, the fluorescence lifetime also varies. Therefore, the temporal change of the fluorescence intensity depends on the structure of the protein. Therefore, the composition ratio of the protein can be obtained by measuring the time change of the fluorescent intensity.

Further, the serum protein testing apparatus of the present invention comprises:
A light-transmissive container containing a sample to be measured consisting of serum, plasma, or blood to which a fluorescent reagent that binds to a protein in serum and changes in fluorescence life according to the type of the bound protein is added; Excitation light irradiating means for irradiating the sample to be measured in the container with excitation light having an absorption wavelength of the fluorescent reagent,
(3) a fluorescence detection device that outputs an electric signal according to the time intensity change of the fluorescence emitted by the fluorescent reagent by the excitation light,
And (4) an analyzer for analyzing a time change of an output electric signal of the fluorescence detector to obtain a composition ratio of a predetermined protein in serum or blood.

[0010] According to this, by irradiating the sample to be measured to which the fluorescent reagent in the container is added with excitation light, the fluorescent reagent bound to the protein in the sample to be measured is unique to the protein according to the composition ratio of the protein. Emits fluorescence having a time-intensity-change waveform. The fluorescence is converted into an electric signal by a fluorescence detection device, and then analyzed by an analysis device, whereby the composition ratio of the protein in the sample to be measured can be obtained.

Here, the fluorescent reagent is preferably indocyanine green. Indocyanine green
It has the property of binding to lipoproteins and albumin in blood, and the light absorption peak wavelength in the binding state is 805 nm and the fluorescence peak wavelength is 830 nm. This is a preferred reagent because it is already widely used as a dye reagent for use, is easily available, and has no toxicity.

[0012] Preferably, the predetermined protein to be tested is a lipoprotein. This is because, as described above, it is important to examine the composition ratio of lipoprotein in blood for diagnosis of lipid metabolism abnormality. Since lipoprotein is a complex or compound of lipid and protein, it has high hydrophobicity, and thus the fluorescence lifetime of the bound fluorescent reagent is prolonged. Therefore, accurate measurement of the time change of the fluorescence intensity can be easily performed. In this case, if the fluorescent reagent is indocyanine green, indocyanine green is more preferable because it has a particularly strong affinity for lipoprotein among blood proteins.

[0013]

Embodiments of the present invention will be described below with reference to the accompanying drawings. To make it easier to understand,
In the drawings, the same components are denoted by the same reference numerals as much as possible, and the description of the overlapping components will be omitted.

FIG. 1 shows a first embodiment of the serum protein test apparatus of the present invention.
FIG. 3 is a block diagram illustrating an exemplary embodiment. Measurement sample 1
Is obtained by adding a fluorescent reagent, for example, Indocyanine green (ICG), to serum collected from the human body. ICG is a diagnostic dye reagent widely used in clinical tests such as liver function and circulatory function, and fluorescent angiography in ophthalmology, and is easily available. When this ICG is added to blood or serum, it binds to lipoproteins, albumin, globulin and the like in the blood, and its maximum absorption wavelength is from 780 nm to 805 nm in a free state.
m, and the fluorescence emission spectrum has a peak near a wavelength of 830 nm. The measurement sample 1 to which ICG is added is accommodated in a test tube 2 made of, for example, glass, which is a material that transmits at least light of 800 nm to 900 nm, which is a wavelength region including an absorption wavelength and a fluorescence wavelength region of ICG.

The excitation light source 3 irradiates the measurement sample 1 in the test tube 2 with excitation light. For example, a titanium-sapphire pulse laser can be used, and its wavelength is the same as that of ICG which is a fluorescent reagent. 7 near peak absorption wavelength
90 nm. In addition, a short pulse light source such as a semiconductor laser and an LED can be used. The spectroscopy means 4
The test tube 3 is disposed at a position not on an extension of the optical path of the excitation light from the excitation light source 3 to the test tube 2 and facing the test tube 2.
It removes unnecessary components such as excitation light reflected light contained in the fluorescence emitted from the measurement sample 1 in the inside, and a spectroscope, an interference filter, or the like can be used.

A fluorescence detector 5 is arranged on the optical path of the fluorescence transmitted through the spectroscopic means 4. A streak camera, an optical oscilloscope, or the like is used as the fluorescence detecting device 5 and converts a change in intensity of the received fluorescence with time into an electric signal. The fluorescence detector 5 is connected to the excitation light irradiating means 1 and the analyzer 6, receives a trigger signal from the excitation light irradiator 1, and outputs an output electric signal to the analyzer 6. The analysis device 6 according to a preset calibration curve,
The composition ratio of the lipoprotein is determined, and the result is output to the connected display device 7.

Next, the operation of this embodiment will be described with a specific example.

Hereinafter, an example using serum of a healthy adult male as the measurement sample 1 will be described. Add 0.
After 5 μg / ml of ICG is added and accommodated in the test tube 2, it is set at a predetermined position of the device. Then, a short pulse having a pulse width of about 6 picoseconds is irradiated at a repetition frequency of 76 MHz from a titanium-sapphire laser as the excitation light irradiation means 3. With this light pulse, a fluorescent pulse is emitted from ICG bound to each protein in the measurement sample 1. After the unnecessary wavelength component is removed by the spectroscopic means 4, the fluorescence pulse is guided to a fluorescence detection device including a streak camera, and a time-resolved fluorescence spectrum of 800 nm to 900 nm is continuously measured every 3.75 picoseconds to obtain a fluorescence pulse. A fluorescence lifetime profile, which is the time intensity change of lifetime, is required.

Here, other serum proteins such as serum albumin and globulin are present in the serum, and the binding constant of ICG to lipoprotein is two orders of magnitude higher than that of these other serum proteins. Because of the above high level, most of the ICG added to the serum becomes lipoprotein-bound. The fluorescence lifetime of unbound ICG alone is one order of magnitude shorter than the fluorescence lifetime of protein-bound ICG, and its effect can be ignored. Thus, the fluorescence lifetime profile is similar to that of lipoprotein-bound I
This reflects the configuration of the CG. That is, the composition ratio of lipoprotein can be determined from the fluorescence lifetime profile.

FIG. 2 shows an example of a fluorescence lifetime profile as a measurement result. The horizontal axis represents time, and the vertical axis represents relative fluorescence intensity when the peak fluorescence intensity is set to 1.
The solid line is the measurement result of the serum sample itself of the subject. For comparison, a saline 0.1M phosphate buffer containing 0.2 mg / ml of only LDL or HDL as a protein (P
The measurement results of BS, pH 7.4, and ICG 0.5 μg / ml) are also shown, and the dotted line is the measurement result of the LDL-only sample and the broken line is the measurement result of the HDL-only sample.

The change in the fluorescence intensity over time is represented by the following equation: When the fluorescence lifetime is τ, the fluorescence intensity at time t is represented by an exponential function exp (−t / τ)
The fluorescence lifetime of each measurement sample was 765 picoseconds. On the other hand, the fluorescence lifetime of each of the HDL and LDL samples is
They were 711 ps and 796 ps. This HDL, LDL
The lipid / protein weight ratio of each sample is 0.354 for the HDL sample and 2.78 for the LDL sample, and the lipid content per protein is about eight times higher for LDL than HDL. Therefore, LDL, which has a high lipid content and high hydrophobicity, significantly prolongs the fluorescence lifetime. From these results, it was found that the whole lipoprotein (HDL + LD
The abundance ratio of HDL in L) was 36.3%. H measured by the conventional method for the same sample
The existence ratio of DL was 37.5%, confirming the effectiveness of this measurement. The dashed line in FIG. 2 indicates HDL, LDL
7 is a result of estimating a change in fluorescence time intensity at the HDL abundance ratio of the measurement sample from the measurement result of FIG. It can be seen that the measurement result and the fluorescence lifetime change are in good agreement.

According to the present embodiment, only a predetermined reagent is added to the apparatus, the apparatus is set in the apparatus, and the apparatus is irradiated with light. Since the binding reaction of ICG with the protein is performed in a very short time, the measurement can be performed almost in real time. Therefore,
Compared with the conventional method, it is possible to measure the protein composition ratio in a simple and extremely short time.

The present inventors measured the fluorescence lifetime profile of protein-bound ICG at various protein concentrations by diluting HDL and LDL extracted from human serum with PBS. As a result, it was found that when the protein concentration was 0.2 to 2 mg / ml, the fluorescence lifetime was the same and did not depend on the protein concentration if the protein composition ratio was the same. Also,
Changing the ICG concentration in the range of 0.5-2 μg / ml had no effect on fluorescence lifetime. Therefore, it is not necessary to strictly adjust the protein concentration and ICG concentration, and the test can be easily performed, which is a preferable feature in daily clinical inspection work.

Next, a second embodiment, which is a modification of the first embodiment, will be described. FIG. 3 is a block diagram showing the second embodiment. The difference from the first embodiment shown in FIG. 2 is the difference in the configuration of the fluorescence detection device 5 that converts the fluorescence lifetime profile into an electric signal. Other components are the same as in the first embodiment, and a description thereof will be omitted.

The fluorescence detector 5 of the second embodiment comprises a photodetector 51, a constant fraction discriminator (CFD) 52, a time-voltage converter (TAC) 53,
A multi-channel analyzer (MCA) 54 is used to determine the fluorescence lifetime profile using single photon time correlation measurement. As the photodetector 51, a photoelectric conversion device such as a photomultiplier tube that outputs an electric signal in response to the incidence of a photon is used. The CFD 52 outputs a timing signal at which the signal strength reaches a certain rate regardless of the strength of the input signal. The TAC 53 outputs the difference between the rise times of the two pulse signals of the input signal and the trigger signal as a voltage signal. The MCA 54 divides the voltage value of the input signal into a plurality of regions corresponding to the voltage values, and counts the frequency of each region.

The fluorescence detecting device 5 operates as follows. The fluorescence of the measurement sample 1 is incident on the photodetector 51 after unnecessary wavelength components are removed by the spectroscopic means 4. When the amount of fluorescent light is strong in order to make a single photon state,
It is necessary to attenuate the amount of light. The photodetector 51 outputs a pulse electric signal according to the photon incidence. The generation time of this pulse corresponds to the photon incidence time. This pulse electric signal is sent to the CFD 52, and the signal waveform is adjusted so that post-processing can be performed with high accuracy. The shaped pulse signal is
A voltage signal corresponding to the difference from the trigger signal sent to the TAC 53 and sent from the excitation light irradiation means 3, that is, the time difference from the irradiation of the excitation light to the generation of the detected photon is output. The MCA 54 counts this voltage signal for each predetermined range. The photon generation frequency distribution, that is, the fluorescence lifetime profile can be obtained by repeating the photon detection of the fluorescence pulse corresponding to the continuous excitation light pulse in the same manner. This is sent to the analyzer 6 and processed in the same manner as in the first embodiment, whereby the lipoprotein composition ratio can be determined.

If the repetition period of the excitation light pulse is sufficiently short, the frequency of generation of many photons can be measured in a short time. Furthermore, this method enables highly accurate measurement even when the amount of fluorescent light is weak. Therefore, similarly to the first embodiment, it is possible to measure the composition ratio of blood protein simply and in a short time.

In the above description, an example has been described in which the fluorescence lifetime is obtained by time-resolved measurement using a pulsed light source as the excitation light source. However, the present invention is not limited to this. The fluorescence lifetime may be obtained from the change and the phase delay.

[0029]

As described above, according to the present invention, the protein composition ratio is determined by measuring the change in fluorescence time with a protein-binding fluorescent reagent without separating the protein in blood. Therefore, simple and short-time measurement is possible.

In particular, if the composition ratio of lipoproteins is measured by ICG, it can be applied to the examination of abnormal lipid metabolism, and is extremely effective in the prevention and treatment of diabetes, heart disease, cerebrovascular disease, etc. associated with abnormal lipid metabolism. It becomes something.

[Brief description of the drawings]

FIG. 1 is a block diagram illustrating a first embodiment of the present invention.

FIG. 2 is a diagram showing an example of a fluorescence lifetime profile measured in the first embodiment of the present invention.

FIG. 3 is a block diagram illustrating a second embodiment of the present invention.

[Explanation of symbols]

1 ... measurement sample, 2 ... test tube, 3 ... excitation light irradiation means,
4 ... Spectroscopic means, 5 ... Fluorescence detector, 6 ... Analyzer, 7 ...
Display device, 51 photodetector, 52 CFD, 53 TA
C, 54 ... MCA.

Claims (6)

[Claims] 1. A serum protein test method for measuring the composition ratio of various proteins contained in serum, comprising binding to proteins in serum, plasma or blood, and changing the fluorescence lifetime according to the type of the bound proteins. By adding a fluorescent reagent to the above, irradiating the serum, plasma or blood with excitation light of the excitation wavelength of the fluorescent reagent, measuring the time intensity change of the fluorescence emitted by the fluorescent reagent, and analyzing this, A serum protein test method for measuring the composition ratio of a predetermined protein in serum. 2. The method according to claim 1, wherein the fluorescent reagent is indocyanine green. 3. The serum protein testing method according to claim 1, wherein the predetermined protein is a lipoprotein. 4. A serum protein tester for measuring the composition ratio of various proteins contained in serum, wherein the fluorescence lifetime changes according to the type of protein bound to a predetermined protein in serum. Serum containing reagents,
A light-transmissive container containing a sample to be measured made of blood plasma or blood; excitation light irradiating means for irradiating the sample to be measured in the container with excitation light having an absorption wavelength of the fluorescent reagent; A fluorescence detection device that outputs an electric signal corresponding to a time intensity change of the fluorescence emitted by the fluorescent reagent, and analyzes a time change of an output electric signal of the fluorescence detection device to determine a predetermined time in the serum, plasma or blood. An analyzer for determining the protein composition ratio. 5. The serum protein testing device according to claim 4, wherein the fluorescent reagent is indocyanine green. 6. The serum protein testing device according to claim 4, wherein the predetermined protein is a lipoprotein.