JP2007057383A - Analyzing system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analyzing system capable of analyzing a change in the bonding state of a functional protein. <P>SOLUTION: This functional protein analyzing system (analyzing system) 1 includes a laser 21 for irradiating a detection cell 5, which houses a functional protein and a bonding substance specifically bonded to the functional protein to make them react with each other, an avalanche photodiode 31 for detecting the scattered light from the detection cell 5 and a control part 41a for calculating the index for showing a change in the bonding state of the functional protein and the bonding substance. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an analysis system, and more particularly to an analysis system for obtaining an index representing a change in a binding state between a functional protein and a binding substance that specifically binds to the functional protein.

  Conventionally, a particle size distribution measuring apparatus using a dynamic scattered light method (photon correlation method) that detects the Brownian motion of particles in a solvent from scattered light and obtains a particle size distribution based on the scattered light is known (for example, Non-Patent Document 1).

  Non-Patent Document 1 discloses a functional protein evaluation method using a particle size distribution measuring apparatus, and can measure in a wide range of particle diameters from 0.6 nm to 6000 nm. The evaluation method using the particle size distribution measuring device disclosed in Non-Patent Document 1 changes the particle size distribution before and after binding of a lectin (functional protein) and a sugar (binding substance) that specifically binds to the lectin. This is a method for evaluating whether or not a lectin is in a state of binding with a sugar by a change in the obtained particle size distribution, which is obtained by a dynamic scattering light method (photon correlation method).

Japan Society for Invention and Innovation Open Technical Report No. 2003-501687

  However, in the method for evaluating a lectin (functional protein) using the conventional particle size distribution measuring device disclosed in Non-Patent Document 1, it is only determined whether the functional protein (lectin) is bound to a binding substance (sugar). Therefore, it is difficult to know the change in the coupling state.

  The present invention has been made in order to solve the above-described problems, and an object thereof is to provide an analysis system capable of analyzing a change in the binding state of a functional protein.

Means for Solving the Problems and Effects of the Invention

  In order to achieve the above object, an analysis system according to one aspect of the present invention irradiates a detection cell for containing and reacting a functional protein and a binding substance that specifically binds to the functional protein. Based on the light source for detecting the scattered light from the detection cell, and the intensity of the scattered light detected by the detector, an index indicating a change in the binding state between the functional protein and the binding substance is obtained. A control unit.

  In the analysis system according to this aspect, as described above, the detector for detecting the scattered light from the detection cell, and the functional protein and the binding substance based on the intensity of the scattered light detected by the detector. And a control unit for obtaining an index representing a change in the binding state, so that the binding state of the functional protein can be determined based on the index representing the change in the binding state between the functional protein and the binding substance obtained by the control unit. Change can be analyzed.

  In the analysis system according to the above aspect, the index is preferably at least one index selected from the group consisting of an index reflecting the binding rate, an index reflecting the binding force, and an index reflecting the affinity. . By using an index that reflects the binding speed in this way, when the binding speed is increased, it can be determined (analyzed) that the binding between the functional protein and the binding substance is in the middle of the process, When the binding rate is constant, it can be determined (analyzed) that the binding between the functional protein and the binding substance has been completed, and therefore, based on the index reflecting the binding rate, the functional protein can be easily Changes in the binding state with the binding substance can be analyzed. In addition, if an index that reflects the binding force is used, and the binding force of the functional protein at a predetermined concentration increases under the condition that a sufficient binding substance exists, the functional protein at the predetermined concentration becomes a binding substance. Therefore, it is possible to easily analyze the change in the binding state between the functional protein and the binding substance based on the index reflecting the binding force. In addition, if an index that reflects affinity is used, it is possible to determine (analyze) which binding substance a functional protein easily binds to among multiple binding substances, so an index that reflects affinity Based on the above, it is possible to easily analyze the change in the binding state between the functional protein and the binding substance.

  In the analysis system according to the one aspect described above, preferably, the control unit includes the functional protein and the binding substance based on the intensity of scattered light detected at a plurality of points where the reaction states of the functional protein and the binding substance are different. An index representing a change in the binding state of is obtained. With this configuration, the functional protein is added to the binding substance by acquiring the intensity of scattered light (multiple scattered light intensity) detected at multiple points where the reaction state of the functional protein and the binding substance is different. When the reaction is performed, a plurality of scattered light intensities detected at different reaction times can be acquired. In addition, when the reaction is performed by adding functional protein stepwise in the presence of a sufficient binding substance, it is possible to obtain multiple scattered light intensities detected with different amounts of functional protein added. it can. In addition, the first binding substance and the functional protein are reacted to cause the first binding substance and the functional protein to dissociate the bond between the first binding substance and the functional protein and to compete with the functional protein. In the case where the reaction is performed by adding the second binding substance showing a reaction stepwise, it is possible to obtain a plurality of scattered light intensities detected with different amounts of the second binding substance added.

  In the analysis system according to the above aspect, the scattered light is preferably backscattered light. With this configuration, a highly sensitive signal can be obtained from the backscattered light, so that the detector detects the backscattered light from the detection cell, and thereby binds to the binding substance based on the backscattered light. Since an index representing a change in the binding state of a protein can be obtained, highly reliable data can be obtained.

In the analysis system according to the above aspect, the control unit preferably obtains an index based on the intensity of the scattered light and the value of the autocorrelation function, and the autocorrelation function G (Δt) is the scattered light at time t. Is defined so as to satisfy the following expression: the intensity I (t) of the light and the intensity I (t + Δt) of the scattered light after a predetermined time Δt has elapsed from the time t.
G (Δt) = <I (t) × I (t + Δt)> / <I (t)> ²

  Note that the autocorrelation function G (Δt) in the present invention is such that when the particle (functional protein) changes its position due to Brownian motion, the position of the particle changes with time from the position of the particle at a certain reference time. The degree of particle overlap as a function of time is expressed as a function of time, and since particles completely overlap at Δt = 0, the autocorrelation function G (0) = 1, and as time passes, Therefore, the autocorrelation function G (Δt) becomes a function like the above-described equation that attenuates toward zero. The inventor of the present application increases the intensity of the scattered light of the particles when optical noise is generated due to variations in the particle diameter of the functional protein, while the autocorrelation function G (Δt) is near t = 0. Considering that the value decreases in the presence of optical noise, by combining the intensity of scattered light and the value of the autocorrelation function G (Δt), it accurately represents the change in the binding state of the functional protein. I found out that the index can be obtained.

  In the analysis system for obtaining an index based on the scattered light intensity and the autocorrelation function, preferably, when the minimum time during which the detector can detect the scattered light intensity is t1, the control unit And the value of the autocorrelation function G (Δt) (0.95 × G (t1) ≦ G (Δt) ≦ 1.05 × G (t1)). With this configuration, the autocorrelation function G (Δt) in the range of 0.95 × G (t1) ≦ G (Δt) ≦ 1.05 × G (t1) has a variation in the particle diameter of the functional protein. The autocorrelation function G (Δt) in the vicinity of t = 0 that decreases mainly due to the optical noise due to the value of the autocorrelation function G (Δt) (0.95 × G (t1) ≦ G (Δt) ≦ 1.05 × G (t1)) and an index representing the change in the binding state of the functional protein more accurately by combining the intensity of scattered light rising due to optical noise Can be requested.

  In this case, preferably, the value of the autocorrelation function G (Δt) is the value G (t1) of the autocorrelation function after the minimum time t1 at which the detector can detect the intensity of the scattered light from the time t. With this configuration, the value G (t1) of the autocorrelation function when Δt = t1 is decreased mainly due to optical noise due to variations in the particle diameter of the functional protein, etc. t = 0 Since it is a nearby autocorrelation function G (Δt), the value of the autocorrelation function G (t1) after the minimum time t1 at which the detector can detect the intensity of the scattered light is combined with the intensity of the scattered light. Thus, it is possible to obtain an index representing the change in the binding state of the functional protein more accurately.

  In the analysis system for obtaining an index based on the value of the autocorrelation function G (Δt) and the intensity of scattered light, the control unit preferably calculates the intensity of the scattered light and the value of the autocorrelation function G (Δt). Based on the product, an index is obtained. With this configuration, the intensity of the scattered light increased due to the optical noise is multiplied by the value of the autocorrelation function G (Δt) decreased due to the optical noise. Of the intensity of the scattered light that has been increased due to a large amount of noise can be offset. As a result, it is possible to obtain an index representing the change in the binding state of the functional protein more accurately.

  In the analysis system for obtaining an index based on the product of the intensity of the scattered light and the value of the autocorrelation function G (Δt), preferably, the intensity of the scattered light is a plurality of times detected from a time t to a predetermined time. It is an average value of the intensity of scattered light. According to this configuration, even when the intensity of scattered light specifically increases due to optical noise due to external dust immediately after the detection time, a plurality of scattered lights for a predetermined time from the detection time. By obtaining the average value of the intensity of the light, the intensity of the scattered light that has risen specifically can be captured. As a result, by calculating the product of the autocorrelation function G (Δt) and the average value of the intensity of scattered light, an index representing the change in the binding state of the functional protein can be determined more accurately.

  In the analysis system for obtaining the index based on the product of the intensity of the scattered light and the value of the autocorrelation function G (Δt), preferably, the analysis system further includes an output unit for outputting the analysis result, and the control unit has a functionality. A graph in which the product of the intensity of scattered light and the value of the autocorrelation function G (Δt) with respect to the reaction time between the protein and the binding substance is plotted is displayed on the output unit. With this configuration, it is possible to display the transition of the product of the intensity of scattered light and the value of the autocorrelation function G (Δt) over the course of the reaction time between the functional protein and the binding substance. Based on the above, it is possible to know the binding state between the functional protein and the binding substance with respect to the reaction time.

  In the analysis system for obtaining an index based on the product of the intensity of the scattered light and the value of the autocorrelation function G (Δt), preferably, the analysis system further includes an output unit for outputting the analysis result, Plotting the product of the intensity of scattered light and the value of the autocorrelation function G (Δt) against the concentration of the functional protein when the functional protein set to multiple concentrations is mixed under the condition where the binding substance exists The graph is displayed on the output section. If comprised in this way, since the product of the intensity | strength of a scattered light and the value of autocorrelation function G ((DELTA) t) with respect to each density | concentration of functional protein can be displayed, the density | concentration of functional protein based on the graph It is possible to know the binding state between the functional protein and the binding substance.

  In the analysis system for obtaining the index based on the product of the intensity of the scattered light and the value of the autocorrelation function G (Δt), preferably, the analysis system further includes an output unit for outputting the analysis result, and the control unit has a functionality. Intensity of scattered light and autocorrelation with the concentration of inhibitory sugars when inhibitory sugars set to multiple concentrations that dissociate the binding between the functional protein and the binding substance are added under the condition where the protein and the binding substance are mixed. A graph in which the product of the value of the function G (Δt) is plotted is displayed on the output unit. If comprised in this way, the product of the intensity | strength of a scattered light and the value of autocorrelation function G ((DELTA) t) with respect to the addition amount of inhibitory sugar can be displayed. As a result, a graph showing whether the functional protein accommodated in the detection cell easily binds to the binding substance or the inhibitory sugar can be displayed. The binding state between the sex protein and the binding substance can be known.

  The analysis system according to the above aspect preferably further includes a lens unit that collects the light from the light source irradiated to the detection cell and can move the focal position of the collected light. If comprised in this way, when the functional protein and binding substance with a high density | concentration are accommodated in the detection cell, a functional protein and binding substance will be made by making the focus position of the light condensed with a lens into the light source side. The intensity of scattered light scattered by the light can be suppressed. In addition, when functional proteins and binding substances with low concentrations are contained in the detection cell, the functional protein and binding substance are set by setting the focal position of the light collected by the lens to the side opposite to the light source side. The intensity of scattered light scattered by can be increased. As a result, an appropriate scattered light intensity can be detected by the detector without depending on the concentrations of the functional protein and the binding substance accommodated in the detection cell.

  The analysis system according to the one aspect described above preferably includes an analysis device having a light source and a detector, a control unit, and a computer that is communicably connected to the analysis device. The intensity of the scattered light detected by the detector is received, and an index representing a change in the binding state between the functional protein and the binding substance is obtained based on the received intensity of the scattered light. If comprised in this way, the analysis system which can obtain | require the parameter | index showing the change of the binding state of a functional protein can be constructed | assembled with an analyzer and a computer.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 is a perspective view showing an overall configuration of a functional protein analysis system according to an embodiment of the present invention, and FIG. 2 is a block diagram of an analyzer of the functional protein analysis system according to the embodiment shown in FIG. FIG. 3-8 is a figure for demonstrating the structure of the functional protein analysis system by one Embodiment shown in FIG. First, with reference to FIGS. 1-8, the structure of the functional protein analysis system 1 by one Embodiment of this invention is demonstrated.

  The functional protein analysis system 1 according to an embodiment of the present invention has a functionality based on the scattered light intensity detected from a functional protein that performs Brownian motion in a solvent and a binding substance that specifically binds to the functional protein. This is a system for obtaining an index representing a change in the binding state between a protein and a binding substance. In the analysis system 1 according to the present embodiment, a lectin that specifically binds to a sugar chain (galactose-specific lectin CEL-IV, mannose / glucose-specific lectin ConA) is used as a functional protein, and as a binding substance. Sugar (lactose-owned polymer, glucose-owned polymer) is used. As shown in FIG. 1, the analysis system 1 according to the present embodiment includes an analysis device 2 and a personal computer (PC) 4 connected to the analysis device 2 via a USB cable 3. Note that the analyzer 2 and the personal computer 4 may be configured to be able to communicate wirelessly.

  The analysis apparatus 2 shown in FIG. 1 is accommodated in the detection cell 5 by irradiating a detection cell 5 (see FIG. 3) that contains and reacts with a measurement sample containing a functional protein and a binding substance. And a function of acquiring an autocorrelation function G2 (τ), which will be described later, obtained based on the scattered light intensity. In the present embodiment, the intensity of the scattered light is detected by using the backscattered light that is reflected in the direction opposite to the traveling direction of the laser light, which is applied to the measurement sample accommodated in the detection cell 5. is doing. As shown in FIG. 2, the analyzer 2 includes a laser 21, a plurality of mirrors 22, an attenuator 23, a movable lens 24, a measurement unit 25, a light-shielding plate 26, an absorber 27, and a condenser. An optical unit 28, an optical fiber 29, an optical fiber coupler 30, an avalanche photodiode (APD) 31, a correlator 32, a control unit 33, and a laser drive circuit 34 are provided.

  The laser 21 is provided to irradiate the detection cell 5 in which the measurement sample is accommodated with laser light. The laser beam emitted from the laser 21 is a He—Ne laser beam and has a frequency of 633 (nm). The plurality of mirrors 22 are installed at predetermined positions so that the laser light emitted from the laser 21 travels toward the detection cell 5 held by the measurement unit 25.

  The attenuator 23 has a function of adjusting the amount of laser light emitted from the laser 21. Specifically, when the measurement sample accommodated in the detection cell 5 has a high concentration or when the particle diameter of the functional protein accommodated in the measurement sample varies, the detection cell 5 is accommodated. Since the intensity of the scattered light acquired from the measurement sample is increased, the amount of attenuation by the attenuator 23 is increased to suppress the amount of laser light emitted from the laser 21. Further, when the measurement sample accommodated in the detection cell 5 has a low concentration or when the particles contained in the measurement sample are very small, the scattering obtained from the measurement sample accommodated in the detection cell 5 Since the light intensity is reduced, the amount of laser light emitted from the laser 21 is increased by decreasing the amount of attenuation by the attenuator 23.

  Here, in this embodiment, as shown in FIGS. 4 and 5, the movable lens 24 is provided for condensing the laser light emitted from the laser 21 onto the measurement sample accommodated in the detection cell 5. It has been. And this movable lens is provided so that a movement in the direction (A direction of FIG. 4 and FIG. 5) along the advancing direction of the laser beam which advances toward the detection cell 5 is provided. As a result, when a measurement sample having a high concentration is accommodated in the detection cell 5, the laser beam condensed by the movable lens 24 is moved by moving the movable lens 24 toward the laser 21 as shown in FIG. The focal position of the detection cell 5 can be set to the movable lens 24 side of the detection cell 5. As a result, it is possible to suppress the intensity of scattered backscattered light. Further, when a measurement sample having a low concentration is accommodated in the detection cell 5, as shown in FIG. 5, the laser beam condensed by the movable lens 24 is moved by moving the movable lens 24 to the detection cell 5 side. It is possible to set the focal position of the detection cell 5 to the absorber 27 (see FIG. 2) described later. As a result, it is possible to increase the intensity of the backscattered light that is scattered. Therefore, the intensity of the scattered light can be detected by the avalanche photodiode (APD) 31 without depending on the concentration of the measurement sample accommodated in the detection cell 5. The movable lens 24 also has a function of converting scattered light (backscattered light) reflected in a direction opposite to the traveling direction of the laser light into parallel light. Thereby, the scattered light obtained from the measurement sample is guided to the condensing optical unit 28 described later.

  As shown in FIGS. 1 and 2, the measurement unit 25 is provided with a cell installation unit 251 including a cell holding hole 251a for holding the detection cell 5 in which the measurement sample is accommodated. The measurement unit 25 is provided with a lid 252 (see FIG. 1) that can be opened and closed so that external light does not enter the detection cell 5 held in the cell holding hole 251a of the cell installation unit 251 during measurement. It has been. Therefore, at the time of measurement, the cell holding hole 251a holds the detection cell 5 in which the measurement sample is accommodated, and the lid 252 is closed, so that the inside of the cell installation unit 251 is in a state in which light from the outside is blocked. Become. This makes it possible to perform measurement under conditions where light from the outside is blocked. As shown in FIG. 2, a light shielding plate 26 is provided between the measurement unit 25 and the movable lens 24 so as to move in conjunction with the opening / closing operation of the lid 252 of the measurement unit 25. The light shielding plate 26 is disposed so as to block the laser light irradiated from the laser 21 from entering the detection cell 5 in a state where the lid 252 of the measuring unit 25 is opened. When the lid 252 is closed, the laser light is removed from the optical path of the laser light so as to enter the detection cell 5.

  The absorber 27 is opposed to the movable lens 24 across the measurement unit 25 holding the detection cell 5 so as to absorb the light transmitted without being reflected by the measurement sample accommodated in the detection cell 5. Is arranged. As a result, only the backscattered light scattered by the measurement sample accommodated in the detection cell 5 can be advanced in the direction toward the movable lens 24.

  The condensing optical unit 28 is provided to guide the scattered light (backscattered light) reflected and reflected on the measurement sample accommodated in the detection cell 5 to the optical fiber 29. The optical fiber 29 is provided to propagate the scattered light guided by the condensing optical unit 28 to the avalanche photodiode (APD) 31 through the optical fiber coupler 30.

  The avalanche photodiode (APD) 31 detects scattered light (analog signal) acquired by irradiating the measurement sample accommodated in the detection cell 5 with laser light, and the detected scattered light is converted into A / A The D converter 31a has a function of converting into an electrical signal (digital signal). Then, the converted electrical signal (digital signal) is stored as scattered light intensity data in a RAM 333 of the control unit 33 (to be described later) for each elapsed time and transmitted to the correlator 32 (to be described later). The avalanche photodiode 31 also has a function of amplifying the detected scattered light, and even if the scattered light detected from the measurement sample is a weak signal, the weak signal can be amplified. Is possible.

Here, in this embodiment, the correlator 32 is defined by the following formula (1) based on the scattered light intensity data for each elapsed time detected by the avalanche photodiode 31, as shown in FIGS. Output autocorrelation function G2 (τ).
G2 (τ) = <I (t) × I (t + τ)> / <I (t)> ² (1)

  The autocorrelation function G2 (τ) defined by the above equation (1) is a function using the scattered light intensity data I (t) at time t and the elapsed time τ (μsec) from time t as parameters. The autocorrelation function G2 (τ) is calculated from the position of the functional protein at a certain reference time with the change in time when the functional protein accommodated in the detection cell 5 changes its position by Brownian motion. The degree of overlap of functional proteins as a function of time is expressed as a function of time when the position of the position changes, and since the functional proteins are completely overlapped at τ = 0, the autocorrelation function G2 (0) Since the degree of overlap of functional proteins decreases with time, the autocorrelation function G2 (τ) becomes a function like the above-described equation (1) that attenuates toward 0.

  The autocorrelation function G2 (τ) is used for evaluating the size of particles that perform Brownian motion in a solvent. In a state where the functional protein and the binding substance accommodated in the detection cell 5 are not bound or in the initial stage of the binding, the functional protein and the binding substance become particles having a small molecular weight, and actively perform Brownian motion in the solvent. For this reason, the laser light irradiated to the particle having a small molecular weight that actively performs the Brownian motion is detected by the avalanche photodiode 31 as scattered light intensity data that fluctuates violently due to the Brownian motion of the particle. On the other hand, in a state where the functional protein and the binding substance accommodated in the detection cell 5 are bound or in the completion stage of the binding, the functional protein and the binding substance become particles having a large molecular weight, and slowly perform Brownian motion in the solvent. Do. For this reason, the laser light irradiated to the particle having a large molecular weight that performs the Brownian motion gently is detected by the avalanche photodiode 31 as the scattered light intensity data that gently fluctuates due to the Brownian motion of the particle. As a result, the autocorrelation function G2 (τ) relating to particles having a small molecular weight rapidly decreases (decays), and the autocorrelation function G2 (τ) relating to particles having a large molecular weight gradually decreases (decays).

  Further, the correlator 32 acquires scattered light intensity data detected by the avalanche photodiode 31 every 0.5 nsec (minimum sample time), and the autocorrelation function G2 (τ) defined by the above equation (1). Is output. In this embodiment, the value G2 (0...) Of the autocorrelation function G2 (τ) after 0.5 nsec (minimum sample time) has elapsed from the time t of the autocorrelation function G2 (τ) shown in FIGS. 5), and the value for each elapsed time is acquired.

  As shown in FIG. 2, the control unit 33 has a function of causing the laser 21 to oscillate and emit laser light by controlling the laser 21 via the laser drive circuit 34. The control unit 33 includes a ROM 331, a CPU 332, a RAM 333, and an input / output interface 334.

  The ROM 331 stores an operating system, a control program for controlling the analysis operation of the analyzer 2, and data necessary for executing the control program (such as a control signal for causing the laser 21 to oscillate). . The CPU 332 is provided to load a control program stored in the ROM 331 into the RAM 333 or directly execute it from the ROM 331. As a result, the CPU 332 can control the analysis operation of the analysis apparatus 2 by executing the control program. The data processed by the CPU 332 is transmitted to each unit of the analyzer 2 or the personal computer (PC) 4 (see FIG. 8) via the input / output interface 334. Data necessary for the processing of the CPU 332 is received from each unit of the analyzer 2 or the personal computer (PC) 4 through the input / output interface 334. For example, the control signal stored in the ROM 331 called by the CPU 332 is transmitted to the laser 21 via the input / output interface 334 and the laser drive circuit 34. Further, the CPU 332 stores the scattered light intensity data for each elapsed time detected by the avalanche photodiode 31 and the value of the autocorrelation function G2 (τ) for each elapsed time acquired by the correlator 32 in the RAM 333. Also have.

  The personal computer (PC) 4 receives the scattered light intensity data and the autocorrelation function G2 (τ) transmitted from the analyzer 2 via the USB cable 3, and receives the received scattered light intensity data and the autocorrelation function G2. Based on (τ), it is provided to obtain an index (an index reflecting the binding rate, an index reflecting the binding force, an index reflecting the affinity) indicating the change in the binding state between the functional protein and the sugar. ing. As shown in FIGS. 1 and 8, the personal computer 4 includes a main body 41, an input unit 42, and a display unit 43.

  As shown in FIG. 8, the main unit 41 includes a ROM 411, a CPU 412, a RAM 413, a hard disk 414, an input / output interface 415, an image output interface 416, a control unit 41a, and a reading device 41b. They are connected by a bus 417 so that data communication is possible.

  Here, in this embodiment, the control unit 41a and the scattered light intensity data transmitted via the input / output interface 334 (see FIG. 2) of the analyzer 2 and 0.5 (nsec) after the time t have elapsed. Based on the product of the autocorrelation function G2 (τ) and the value G2 (0.5), it has a function of obtaining an index representing a change in the binding state between the functional protein and the binding substance.

  The ROM 411 of the control unit 41a is configured by a mask ROM, PROM, EPROM, EEPROM, or the like. The CPU 412 is provided to execute an analysis application program 414 a that analyzes a functional protein installed in a hard disk 414 (described later) loaded in the RAM 413. The RAM 413 is configured by SRAM, DRAM, or the like, and is used for reading out a computer program and an analysis application program 414a stored in the ROM 411 and a hard disk 414 described later. The RAM 413 is used as a work area for the CPU 412 when executing the analysis application program 414 a installed in the hard disk 414.

  The hard disk 414 is installed with an operating system and an analysis application program 414a for analyzing a functional protein, which will be described later, and data necessary for executing the analysis application program 414a (scattered light intensity data, autocorrelation function G2 ( τ)) is stored.

Here, the analysis application program 414a installed in the hard disk 414 of the personal computer 4 will be described. The analysis application program 414a includes the average scattered light intensity data I (intensity) obtained from the scattered light intensity data for each elapsed time detected by the avalanche photodiode 31 of the analyzer 2 and the self acquired by the correlator 32. Using I2 that satisfies the following equation (2) defined by the product of the correlation function G2 (τ) and the value G2 (0.5) (intercept), the change in the binding state between the functional protein and the binding substance is It is a program that calculates the index to represent.
I2 = I × G2 (0.5) (2)

  In addition, I used in the above formula (2) is an average value of scattered light intensity data acquired from a predetermined time t (sec) to a predetermined time t + 1 (sec), and is obtained by the avalanche photodiode 31. Based on the detected scattered light intensity data for each elapsed time, it is calculated by the analysis application program 414a. Further, G2 (0.5) in the above equation (2) is a value of the autocorrelation function G2 (τ) after 0.5 nsec has elapsed from the predetermined time t shown in FIGS.

  The input / output interface 415 is, for example, a serial interface such as USB, IEEE1394, RS-235C, a parallel interface such as SCSI, IDE, IEEE1284, and an analog including a D / A converter and an A / D converter. It consists of an interface. An input unit 42 is connected to the input / output interface 415. The input / output interface 415 is connected to the input / output interface 334 of the control unit 33 of the analyzer 2 via the USB cable 3, and the scattered light intensity data detected by the analyzer 2 and the autocorrelation function G2 (τ ) Can be input to the personal computer 4. The image output interface 416 is connected to a display unit 43 configured by an LCD (Liquid Crystal Display) or a CRT, and outputs a video signal corresponding to the image data given from the CPU 412 to the display unit 43. It is configured.

  The reading device 41b is configured by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, or the like, and can read a computer program or data stored in the portable recording medium 6. Thereby, for example, the analysis application program 414a can be read from the portable recording medium 6 using the reading device 41b, and the read analysis application program 414a can be installed in the hard disk 414. The analysis application program 414a and other computer programs used in the personal computer 4 are not only provided by the portable recording medium 6, but also an external PC connected to be communicable with the personal computer 4 according to the present embodiment. It is also possible to be provided through a telecommunication line (whether wired or wireless). For example, an analysis application program 414a is stored in the hard disk of a server computer on the Internet. The personal computer 4 according to the present embodiment accesses this server computer through an electric communication line and downloads the analysis application program 414a. The downloaded analysis application program 414a can be installed in the hard disk 414. The hard disk 414 is installed with an operating system that provides a graphical user interface environment such as Windows (registered trademark) manufactured and sold by Microsoft Corporation. Note that the analysis application program 414a according to the present embodiment operates on the above operating system.

  The input unit 42 is configured so that the user can input data to the personal computer 4 by using the input unit 42.

  In the present embodiment, the display unit 43 is provided to display an image (screen) according to the video signal input from the image output interface 416. Specifically, the display unit 43 has a function of displaying a graph in which I2 defined by the above formula (2) is plotted with respect to the reaction time of the functional protein and the binding substance as an index reflecting the binding rate. Yes. In addition, the display unit 43 displays the above formula for the concentration of the functional protein when a plurality of functional proteins set at a plurality of concentrations are mixed under a condition where a binding substance sufficient as an index reflecting the binding force exists. It also has a function of displaying a graph in which I2 defined in 2) is plotted. Further, the display unit 43 displays inhibitory sugars set at a plurality of concentrations that dissociate the binding between the functional protein and the binding substance under the condition where the functional protein and the binding substance are mixed as an index reflecting the affinity. It also has a function of displaying a graph in which I2 defined by the above formula (2) is plotted with respect to the concentration of the inhibiting sugar when added.

  FIG. 9 is a flowchart showing a functional protein measurement flow by the control unit of the analyzer of the functional protein analysis system according to the embodiment shown in FIG. Next, a functional protein measurement flow by the control unit 33 of the analyzer 2 of the functional protein analysis system 1 according to the embodiment of the present invention will be described in detail with reference to FIG.

  First, when the analysis apparatus 2 is turned on, a control signal is sent from the control unit 33 of the analysis apparatus 2 to the laser drive circuit 34, the laser 21 oscillates, and laser light is emitted. When the oscillation of the laser 21 is stabilized, the control unit 33 transmits to the personal computer 4 that the measurement standby has been established, and the CPU 412 of the personal computer 4 is in the measurement standby via the image interface 416 on the display unit 43. Is displayed. Next, a functional protein and a binding substance that specifically binds to the functional protein are put into the detection cell 5 and mixed. Then, the lid 252 of the measurement unit 25 is opened, and the detection cell 5 is held in the cell of the measurement unit 25. Set in the hole 251a and close the lid 252. At this time, when the lid 252 is opened, the light shielding plate 26 blocks the optical path of the laser light, and when the lid 252 is closed, the light shielding plate 26 is removed from the optical path of the laser light, and the laser light is detected by the detection cell. 5 is irradiated. The backscattered light scattered from the detection cell 5 is received by the avalanche photodiode 31, and the avalanche photodiode 31 outputs an electrical signal for the scattered light intensity to the A / D converter 31a. Thereafter, the A / D conversion unit 31 a performs A / D conversion on the electrical signal, outputs scattered light intensity data for each time unit, and sends the data to the correlator 32.

  Next, in step S <b> 1, the CPU 332 of the control unit 33 of the analyzer 2 determines whether a measurement start signal is received from the personal computer 4. If it is determined in step S1 that the measurement start signal is not received from the personal computer 4, the above determination is repeated until the measurement start signal is received. On the other hand, if it is determined in step S1 that a measurement start signal has been received from the personal computer 4, the CPU 332 of the analyzer 2 in step S2 determines the scattered light intensity data output from the avalanche photodiode 31 and the correlator 32. The autocorrelation function G2 (τ) output from is stored in the RAM 333. Note that the scattered light intensity data and the autocorrelation function G2 (τ) stored in the RAM 333 are sequentially stored every time the reaction time between the functional protein and the binding substance elapses.

  Next, in step S3, the scattered light intensity data and the autocorrelation function G2 (τ) are transmitted to the personal computer 4 via the input / output interface 334 by the CPU 332 of the analyzer 2. In step S <b> 4, the CPU 332 of the analyzer 2 determines whether a measurement end signal is received from the personal computer 4. If it is determined in step S4 that the measurement end signal has not been received from the personal computer 4, the processes in steps S2 and S3 described above are repeated until the measurement end signal is received. On the other hand, if it is determined in step S4 that the measurement end signal has been received, in step S5, the CPU 332 of the analyzer 2 sends data to the personal computer 4 (scattered light intensity data and autocorrelation function G2 (τ)). Is stopped, and the data transmission flow process is terminated.

  FIG. 10 is a flowchart showing a display flow of the binding rate graph by the control unit of the personal computer (PC) of the functional protein analysis system according to the embodiment shown in FIG. Next, the display flow of the binding rate graph by the control unit 41a of the personal computer (PC) 4 of the functional protein analysis system 1 according to the embodiment of the present invention will be described in detail with reference to FIG.

  First, by setting the detection cell 5 in the cell holding hole 251a of the measurement unit 25, preparation for starting measurement is completed. In step S11, the CPU 412 of the control unit 41a of the personal computer 4 determines whether or not the start key displayed on the display unit 43 is turned on (clicked). If it is determined in step S11 that the start key is not turned on, the determination in step S11 is repeated until the start key is turned on. On the other hand, if it is determined in step S11 that the start key has been turned ON, a measurement start signal is transmitted to the analyzer 2 in step S12. Next, the CPU 412 of the personal computer 4 determines whether or not the data (scattered light intensity data and autocorrelation function G2 (τ)) transmitted from the analyzer 2 has been received in step S13. If it is determined in step S13 that data has not been received from the analyzer 2, the determination in step S13 is repeated until data is received. On the other hand, if it is determined in step S13 that data has been received from the analyzer 2, the scattered light intensity data and the autocorrelation function G2 (τ) transmitted from the analyzer 2 are stored in the hard disk 414 in step S14. To do. Next, in step S15, the CPU 412 determines whether or not the stop key displayed on the display unit 43 is turned on (clicked). If it is determined in step S15 that the stop key is not turned on, the processing in step S14 and the determination in step S15 are repeated until the stop key is turned on. On the other hand, if it is determined in step S15 that the stop key has been turned ON, a measurement end signal is transmitted to the analyzer 2 in step S16. Next, in step S17, the CPU 412 of the personal computer 4 receives the autocorrelation function G2 (τ) (autocorrelation function data G2 (0.5) (intercept) at 0.5 μsec), scattered light intensity data, and the like from the hard disk 414. Call. At this time, in the present embodiment, an average value I (intensity) of a plurality of scattered light intensity data detected during 1 sec from the time t at the time of detection is calculated.

  Next, in step S18, the CPU 412 is defined by the above equation (2) for each reaction time based on the calculated average scattered light intensity data I and the called autocorrelation function data G2 (0.5). I2 is obtained and stored in the hard disk 414. In step S19, the CPU 412 of the personal computer 4 calls up I2 for each reaction time stored in the hard disk 414, and creates a graph of I2 (reaction rate graph (see FIGS. 11 and 12)) with respect to the reaction time. Thereafter, in step S20, the CPU 412 displays the binding rate graph on the display unit 43 via the image output interface 416, and ends the processing of the display flow of the functional protein binding rate graph.

  Next, using I2 (= I × G2 (0.5)) acquired by the analysis application program 414a of the functional protein analysis system 1 according to the embodiment of the present invention described above, the functional protein and the binding substance are used. An experiment conducted for observing a change in the binding state with the nuclei will be described. In this experiment, as an index indicating the binding state between the functional protein and the binding substance, an index reflecting the binding rate, an index reflecting the binding force, and an index reflecting the affinity were evaluated by I2. Details will be described below.

[Experiment for obtaining an index reflecting the binding speed]
An experiment conducted for obtaining an index reflecting the coupling speed of the above-described embodiment of the present invention will be described. In this experiment, a functional protein was added to the binding substance, and a graph was prepared by plotting I2 defined by the above formula (2) until a predetermined time elapsed from the time when the functional protein was added. The binding rates of the functional protein and the binding substance according to 1 and 2 and Comparative Examples 1 and 2 were evaluated. In Examples 1 and 2 and Comparative Examples 1 and 2, buffers composed of 10 mM Tris-HCl, 150 mM NaCl, and 20 mM CaCl ₂ (pH 7.5) were used, respectively.

Example 1
1 μmol of galactose-specific lectin CEL-IV was added to 10 pmol of lactose-owned polymer and glucose-owned polymer, and a graph (see FIG. 11) in which I2 defined by the above formula (2) was plotted every 15 sec was created. The data is displayed on the display unit 43 of the personal computer (PC) 4 (see FIG. 1).

(Example 2)
1 μmol of mannose / glucose-specific lectin ConA was added to 10 pmol of lactose-owned polymer and glucose-owned polymer, and a graph (see FIG. 12) in which I2 defined by the above formula (2) was plotted every 15 sec was created. The data is displayed on the display unit 43 of the personal computer (PC) 4 (see FIG. 1).

(Comparative Example 1)
A graph was prepared by adding 1 μmol of galactose-specific lectin CEL-IV to 10 pmol of lactose-owned polymer and glucose-owned polymer, and plotting the average scattered light intensity data I (intensity) obtained by the control unit 41a every 15 sec interval. The result is shown in FIG.

(Comparative Example 2)
1 μmol of mannose-glucose specific lectin ConA was added to 10 pmol of lactose-owned polymer and glucose-owned polymer, and a graph was prepared by plotting average scattered light intensity data I (intensity) obtained by the control unit 41a every 15 sec interval. The result is shown in FIG.

  As shown in FIG. 11 above, the galactose-specific lectin CEL-IV added to the lactose-owned polymer and glucose-owned polymer according to Example 1 was found to have an increased I2 for the lactose-owned polymer. On the other hand, the galactose-specific lectin CEL-IV was found to have no increase in I2 for the glucose-owned polymer. Thus, the galactose-specific lectin CEL-IV showed a specific increase in I2 only in the lactose-owned polymer, and showed an almost constant value after showing an increase in I2 over about 2 min immediately after addition. . Thus, it is considered that the galactose-specific lectin CEL-IV and the lactose-owned polymer were bound to each other for about 2 minutes immediately after the addition, and then the binding between the galactose-specific lectin CEL-IV and the lactose-owned polymer was completed.

  As shown in FIG. 12, the mannose glucose-specific lectin ConA added to the lactose-owned polymer and glucose-owned polymer according to Example 2 was found to have an increased I2 for the glucose-owned polymer. On the other hand, the mannose glucose-specific lectin ConA was found to have no increase in I2 for the lactose-owned polymer. Thus, in mannose glucose-specific lectin ConA, a specific increase in I2 was observed only in the glucose-owned polymer, and an increase in I2 was observed from about 1 min to about 3 min after addition. Thus, it is considered that the mannose / glucose-specific lectin ConA and the glucose-owning polymer were bound within about 1 min to about 3 min after the addition, and then the binding between the mannose / glucose-specific lectin ConA and the glucose-owning polymer was completed. . Further, when the transition of I2 of the galactose-specific lectin CEL-IV with respect to the lactose-owned polymer shown in FIG. 11 and the transition of I2 of the mannose glucose-specific lectin ConA with respect to the glucose-owned polymer shown in FIG. I2 of the galactose-specific lectin CEL-IV for the owned polymer is increased immediately after addition, whereas I2 of the mannose glucose-specific lectin ConA for the glucose-owned polymer is increased from about 1 min after the addition. Was observed. It is considered that this indicates that the speed of starting binding differs depending on the type of functional protein (galactose-specific lectin CEL-IV, mannose-glucose-specific lectin ConA).

  15 shows an autocorrelation function G2 between a mannose glucose-specific lectin ConA and a galactose-specific lectin CEL-IV displayed on the display unit of the functional protein analysis system according to the embodiment shown in FIG. It is the graph which plotted value G2 (0.5) of (tau) for every elapsed time. Referring to FIG. 15, it can be seen that the value of G2 (0.5) of mannose glucose-specific lectin ConA and galactose-specific lectin CEL-IV decreases with time. If the functional protein and the binding substance coexist, a chemical equilibrium occurs between the functional protein and the binding substance. For this reason, when the functional protein and the binding substance are in an equilibrium state, the scattered light intensity data detected by the avalanche photodiode 31 due to the competition reaction (binding reaction and dissociation reaction) between the functional protein and the binding substance is competitive. It is considered that the value of the autocorrelation function G2 (τ) is detected with the elapsed time as it is detected in a state including optical noise due to the reaction.

  As shown in FIG. 13 above, the galactose-specific lectin CEL-IV added to the lactose-owned polymer and glucose-owned polymer according to Comparative Example 1 increased I (average scattered light intensity data) for the lactose-owned polymer. I found out. On the other hand, it was found that the galactose-specific lectin CEL-IV did not increase I (average scattered light intensity data) for the glucose-owned polymer. As a result, in the galactose-specific lectin CEL-IV, a specific increase in I (average scattered light intensity data) was observed only in the lactose-owned polymer. However, in the transition of I (average scattered light intensity data) of the galactose-specific lectin CEL-IV with respect to the lactose-owned polymer shown in FIG. 13, there is an optical detected with the above-described competitive reaction (binding reaction and dissociation reaction). Noise is not taken into account. On the other hand, in Examples 1 and 2 shown in FIG. 11, the optical noise detected with the competition reaction is taken into account, so that the reaction between the galactose-specific lectin CEL-IV and the lactose-owned polymer is accurately performed. It is considered possible to analyze.

  As shown in FIG. 14 above, the mannose glucose-specific lectin ConA added to the lactose-owned polymer and glucose-owned polymer according to Comparative Example 2 increased I (average scattered light intensity data) for the glucose-owned polymer. I found out. On the other hand, it was found that the mannose glucose-specific lectin ConA did not increase I (average scattered light intensity data) for the lactose-owned polymer. As a result, in the mannose glucose-specific lectin ConA, a specific increase in I (average scattered light intensity data) was observed only in the glucose-owned polymer. However, the transition of I (average scattered light intensity data) of the mannose glucose-specific lectin ConA with respect to the glucose-containing polymer increases from about 1 min to about 10 min, then decreases for about 1 min, and increases after about 11 min. Thus, a portion where I (average scattered light intensity data) is stable could not be confirmed. For this reason, it could not be judged whether or not the reaction between the mannose / glucose-specific lectin ConA and the glucose-containing polymer was completed. This is because the above-described competitive reaction (binding reaction and dissociation reaction) frequently occurred, so that the optical noise accompanying the competitive reaction appeared in the transition of I (average scattered light intensity data), and the mannose glucose-specific lectin ConA This is probably because I (average scattered light intensity data) is not stable even when the reaction between the glucose and the glucose-containing polymer is completed.

  Thus, Examples 1 and 2 contain binding substances (lactose-owned polymer, glucose-owned polymer) that specifically bind to functional proteins (galactose-specific lectin CEL-IV, mannose-glucose-specific lectin ConA). After the functional protein (galactose-specific lectin CEL-IV, mannose / glucose-specific lectin ConA) is added to the detection cell 5, the avalanche photodiode 31 detects the scattered light from the detection cell 5 every predetermined time. By doing so, the control unit 41a of the personal computer 4 can obtain an index that reflects the binding speed between the functional protein and the binding substance based on the intensity of the scattered light detected by the avalanche photodiode 31.

[Experiment for obtaining an index that reflects binding strength]
An experiment conducted for obtaining an index reflecting the binding force of the above-described embodiment of the present invention will be described. In this experiment, the galactose-specific lectin CEL-IV was added step by step to the lactose-owned polymer, and the function according to Example 3 was created by creating a graph plotting I2 defined by the above formula (2). The binding force between the sex protein and the binding substance was evaluated. In Example 3, a buffer solution consisting of 10 mM Tris-HCl, 150 mM NaCl, and 20 mM CaCl ₂ (pH 7.5) was used.

(Example 3)
Add galactose-specific lectin CEL-IV with a minimum amount of 10 pmol (10, 20, 40, 80, 160, 320, 640, 1280, 2560, 3840 (pmol)) to 10 pmol of lactose-owned polymer. Then, measurement was made 5 minutes after the addition, and a graph (see FIG. 16) in which I2 defined by the above formula (2) was plotted was created and displayed on the display unit 43 of the personal computer (PC) 4 (see FIG. 1). did. In the experiment for obtaining the index reflecting the binding rate, as described above, the binding between the galactose-specific lectin CEL-IV and the lactose-owned polymer was completed within about 2 min immediately after the addition, It was measured 5 minutes after the addition when the reaction was progressing.

  Referring to FIG. 16 above, in the presence of sufficient sugar (10 pmol lactose-owned polymer), an increase in I2 was observed when about 320 pmol to about 640 pmol of galactose specific lectin CEL-IV was added. Thus, in the presence of sufficient sugar, galactose-specific lectin CEL-IV at a concentration of about 320 pmol to about 640 pmol is considered to actively bind to sugar (lactose-owning polymer).

  Thus, in Example 3, the functional protein (galactose-specific lectin CEL) was contained in the detection cell 5 containing a binding substance (lactose-owning polymer) that specifically binds to the functional protein (galactose-specific lectin CEL-IV). -IV) is added stepwise, the scattered light detected by the avalanche photodiode 31 is detected by the avalanche photodiode 31 by detecting the scattered light from the detection cell 5 for each added amount of the functional protein. An index reflecting the binding force between the functional protein and the binding substance can be obtained by the control unit 41a of the personal computer 4 based on the strength of the.

[Experiment to find an index that reflects affinity]
An experiment conducted for obtaining an index reflecting the affinity of the above-described embodiment of the present invention will be described. In this experiment, in the state where the binding substance is added to the functional protein, inhibitory sugars (mannose and glucose) that dissociate the binding between the binding substance and the functional protein are added step by step. The affinity between the functional protein according to Example 4 and the binding substance was evaluated by preparing a graph plotting the defined I2. In Example 4, a buffer solution consisting of 10 mM Tris-HCl, 150 mM NaCl, and 20 mM CaCl ₂ (pH 7.5) was used.

Example 4
Into the reaction solution in which 1 μmol of mannose-glucose specific lectin ConA is added to 10 pmol of glucose-containing polymer and allowed to stand for 10 minutes or more, each of the inhibitory sugars (mannose and glucose) having a minimum amount of 0.1 μmol (double amount) ( 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8 (μmol)) was added and measured 5 minutes after the addition. A graph (see FIG. 17) plotting I2 defined in 2) was created and displayed on the display unit 43 of the personal computer (PC) 4 (see FIG. 1). In the experiment for obtaining the index reflecting the binding rate, as described above, the binding between the mannose / glucose specific lectin ConA and the glucose-containing polymer was completed within about 3 min immediately after the addition. It was measured 5 minutes after the addition when the reaction was progressing.

  Referring to FIG. 17, a decrease in I2 was observed for mannose / glucose-specific lectin ConA with respect to a small amount (about 0 μmol to about 3 μmol) of an inhibiting sugar (mannose). Therefore, if a decrease in I2 is observed with a small amount of inhibitory sugar, the functional protein is considered to have a high affinity with the inhibitory sugar. Therefore, the mannose / glucose-specific lectin ConA of this embodiment is It is thought that it shows high affinity. That is, when a small amount of mannose of about 0 μmol to about 3 μmol is added, it is considered that mannose / glucose-specific lectin ConA and mannose begin to bind.

  As described above, Example 4 contains a binding substance (glucose-owned polymer) that specifically binds to a functional protein (mannose / glucose-specific lectin ConA) and a functional protein (mannose / glucose-specific lectin ConA). After the inhibitory sugar (mannose) that dissociates the binding between the binding substance and the functional protein is added stepwise to the detection cell 5, the scattered light from the detection cell 5 for each added amount of inhibitory sugar is avalanche-photographed. By detecting with the diode 31, the control unit 41a of the personal computer 4 can obtain an index reflecting the affinity between the functional protein and the binding substance based on the intensity of the scattered light detected by the avalanche photodiode 31. Is possible.

  In the present embodiment, as described above, based on the scattered light intensity data detected by the avalanche photodiode 31 and the autocorrelation function G2 (τ) calculated by the correlator 32, the functional protein and the binding substance By installing in the hard disk 414 an analysis application program 414a for obtaining an index I2 representing a change in the binding state of the protein, the change in the binding state of the functional protein is analyzed based on I2 obtained by the analysis application program 414a. can do.

  In the present embodiment, if I2 obtained by the product of the scattered light intensity data and the value G2 (0.5) of the autocorrelation function G2 (τ) is used, the coupling shown in FIG. 11 and FIG. When the speed is increased, it can be judged (analyzed) that the binding of the functional protein (galactose-specific lectin CEL-IV) and the binding substance (lactose-owning polymer) is in the middle of the process, When the binding rate is constant, it can be judged (analyzed) that the binding between the functional protein (galactose-specific lectin CEL-IV) and the binding substance (lactose-owning polymer) has been completed. Thus, the change in the binding state between the functional protein and the binding substance can be easily analyzed. In addition, from the graph shown in FIG. 16, the functional protein (galactose-specific lectin CEL-IV) at a concentration of about 320 pmol to about 640 pmol under conditions where a sufficient (10 mol) binding substance (lactose-owned polymer) is present. When I2 increases, it can be judged (analyzed) that the functional protein at that concentration is actively bound to the binding substance, so that based on I2, the functional protein and the binding substance can be easily Changes in binding state can be analyzed. In addition, from the graph shown in FIG. 17, it is determined which functional sugar (mannose / glucose-specific lectin ConA) easily binds to which of the two inhibitory sugars (mannose and glucose) (mannose). Since (analysis) can be performed, the change in the binding state between the functional protein and the binding substance (inhibiting sugar) can be easily analyzed based on I2.

  In the present embodiment, the correlator 32 acquires the autocorrelation function value G2 (0.5) at τ = 0.5 nsec, so that the autocorrelation function value G2 (0. 5) is an autocorrelation function G2 (τ) in the vicinity of τ = 0, which decreases as a main cause of optical noise due to variations in the particle diameter of the functional protein accommodated in the detection cell 5, and therefore avalanche. By combining the value G2 (0.5) of the autocorrelation function after the minimum time (minimum sample time) 0.5 nsec in which the photodiode 31 can detect the scattered light intensity and the average scattered light intensity data I, An index that accurately represents the change in the binding state of the functional protein can be obtained.

  In the present embodiment, the analysis application program 414a installed in the hard disk 414 of the personal computer 4 is based on the product of the average scattered light intensity data I and the autocorrelation function value G2 (0.5). By obtaining I2, the intensity of the scattered light increased due to the optical noise is multiplied by the value G2 (0.5) of the autocorrelation function decreased due to the optical noise to obtain the optical value. Of the intensity of the scattered light increased due to the static noise can be offset. As a result, it is possible to obtain an index representing the change in the binding state of the functional protein more accurately.

  In the present embodiment, by calculating I2 using average scattered light intensity data I that is an average value of a plurality of scattered light intensities detected between time t (sec) and time t + 1 (sec), Even when the intensity of the scattered light specifically increases due to optical noise due to dust from the outside immediately after the detection time, the average value of the intensity of the plurality of scattered lights for 1 min from the detection time is obtained. The intensity of the scattered light that has risen specifically can be captured. As a result, by obtaining the product of the value G2 (0.5) of the autocorrelation function and the average scattered light intensity data I, it is possible to obtain an index representing the change in the binding state of the functional protein more accurately.

  The embodiments and examples disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present invention is shown not by the above description of the embodiments and examples but by the scope of claims for patent, and includes all modifications within the meaning and scope equivalent to the scope of claims for patent.

  For example, in the above embodiment, the control unit 41a of the personal computer 4 uses the scattered light intensity data detected by the avalanche photodiode 31 and the autocorrelation function G2 (τ) calculated by the correlator 32 to combine them. Although the example which calculates | requires the parameter | index which reflects speed | velocity | rate, the parameter | index reflecting a binding force, and the parameter | index reflecting an affinity was shown, this invention is not restricted to this, The parameter | index reflecting a binding velocity and the parameter | index reflecting a binding force And any one of the indices reflecting affinity may be obtained.

  In the above embodiment, an example is shown in which I2 calculated by the product of the scattered light intensity data detected by the avalanche photodiode 31 and the autocorrelation function G2 (τ) calculated by the correlator 32 is used as an index. However, the present invention is not limited to this, and an index that is calculated based on the scattered light intensity data and the autocorrelation function G2 (τ) by a method other than the product of the scattered light intensity data and the autocorrelation function G2 (τ) is used as an index. It may be used as

  Further, in the above embodiment, I2 calculated by the product of the scattered light intensity data detected by the avalanche photodiode 31 and the value G2 (0.5) of the autocorrelation function G2 (τ) calculated by the correlator 32. However, the present invention is not limited to this, and an index calculated based on scattered light intensity data and a value calculated from other than the autocorrelation function G2 (τ) may be used as the index. Good.

  In the above embodiment, by connecting a personal computer (PC) to the analyzer through a USB cable (communication line), the scattered light intensity data detected by the avalanche photodiode of the analyzer and the correlator are calculated. An example is shown in which the autocorrelation function G2 (τ) is transmitted and the CPU of the personal computer obtains I2 based on the received scattered light intensity data and the autocorrelation function G2 (τ). Not limited to this, the analysis apparatus may obtain I2 based on the scattered light intensity data and the autocorrelation function G2 (τ).

It is the perspective view which showed the whole structure of the functional protein analysis system by one Embodiment of this invention. It is a block diagram of the analyzer of the functional protein analysis system by one Embodiment shown in FIG. It is a perspective view of the detection cell used for the functional protein analysis system by one Embodiment shown in FIG. It is the schematic diagram which showed the movable lens of the analyzer of the functional protein analysis system by one Embodiment shown in FIG. It is the schematic diagram which showed the movable lens of the analyzer of the functional protein analysis system by one Embodiment shown in FIG. It is the graph which showed the autocorrelation function about ConA output by the correlator of the analyzer of the functional protein analysis system by one Embodiment shown in FIG. It is the graph which showed the autocorrelation function about CEL-IV output by the correlator of the analyzer of the functional protein analysis system by one Embodiment shown in FIG. FIG. 2 is a block diagram of a computer of the functional protein analysis system according to the embodiment shown in FIG. 1. It is the flowchart which showed the measurement flow of the functional protein by the control part of the analyzer of the functional protein analysis system by one Embodiment shown in FIG. It is the flowchart which showed the display flow of the binding rate graph by the control part of the personal computer of the functional protein analysis system by one Embodiment shown in FIG. 1 is a graph according to Example 1 in which I2 is plotted against the reaction time of a glucose- and lactose-containing polymer with a galactose-specific lectin CEL-IV. FIG. 6 is a graph according to Example 2 in which I2 is plotted against the reaction time of glucose-owned polymer and lactose-owned polymer with mannose glucose-specific lectin ConA. It is the graph by the comparative example 1 by which I was plotted with respect to the reaction time of the lactose possession polymer and the lactose possession polymer, and the galactose specific lectin CEL-IV. It is the graph by the comparative example 2 by which I was plotted with respect to the reaction time of a lactose possession polymer and a glucose possession polymer, and mannose glucose specific lectin ConA. It is the graph which plotted the value G2 (0.5) of the autocorrelation function G2 ((tau)) in the predetermined time t of mannose glucose specific lectin ConA and galactose specific lectin CEL-IV for every elapsed time. FIG. 4 is a graph according to Example 3 in which I2 is plotted when galactose-specific lectin CEL-IV is added under conditions where sufficient lactose-owned polymer is present. It is the graph by Example 4 which plotted I2 at the time of adding various concentration inhibitory sugar (glucose, mannose) with respect to mannose glucose specific lectin ConA.

Explanation of symbols

1 Functional protein analysis system (analysis system)
2 analyzer 4 computer 5 detection cell 21 light source (laser)
24 Movable lens (lens)
31 Avalanche photodiode (detector)
32 Correlator 41a Control unit 43 Display unit (output unit)

Claims (14)

A light source for irradiating a detection cell for containing and reacting a functional protein and a binding substance that specifically binds to the functional protein; and
A detector for detecting scattered light from the detection cell;
An analysis system comprising: a control unit for obtaining an index representing a change in a binding state between the functional protein and the binding substance based on the intensity of the scattered light detected by the detector.   The analysis system according to claim 1, wherein the index is at least one index selected from the group consisting of an index reflecting a binding rate, an index reflecting a binding force, and an index reflecting affinity.   The control unit represents a change in the binding state between the functional protein and the binding substance based on the intensity of scattered light detected at a plurality of points where the reaction state between the functional protein and the binding substance is different. The analysis system according to claim 1 or 2, wherein an index is obtained.   The analysis system according to claim 1, wherein the scattered light is backscattered light. The control unit obtains the index based on the intensity of the scattered light and the value of the autocorrelation function,
The autocorrelation function G (Δt) satisfies the following expression by the scattered light intensity I (t) at time t and the scattered light intensity I (t + Δt) after a predetermined time Δt has elapsed from the time t. The analysis system according to any one of claims 1 to 4, wherein
G (Δt) = <I (t) × I (t + Δt)> / <I (t)> ² When the minimum time during which the detector can detect the intensity of the scattered light is t1,
The controller is based on the intensity of the scattered light and the value of the autocorrelation function G (Δt) (0.95 × G (t1) ≦ G (Δt) ≦ 1.05 × G (t1)). The analysis system according to claim 5, wherein the index is obtained.   The value of the autocorrelation function G (Δt) is an autocorrelation function value G (t1) after a minimum time t1 at which the detector can detect the intensity of scattered light from the time t. The analysis system described.   The analysis system according to claim 6 or 7, wherein the control unit obtains the index based on a product of an intensity of the scattered light and a value of the autocorrelation function G (Δt).   The analysis system according to claim 8, wherein the intensity of the scattered light is an average value of the intensity of a plurality of scattered lights detected during a predetermined time from the time t. An output unit for outputting the analysis result is further provided,
The control unit displays, on the output unit, a graph in which a product of the intensity of the scattered light and the value of the autocorrelation function G (Δt) with respect to a reaction time between the functional protein and the binding substance is plotted. Item 10. The analysis system according to Item 8 or 9. An output unit for outputting the analysis result is further provided,
The control unit is configured such that the intensity of the scattered light and the autocorrelation function with respect to the concentration of the functional protein when the functional protein set to a plurality of concentrations is mixed under a condition where a sufficient binding substance is present. The analysis system according to claim 8 or 9, wherein a graph in which a product of a value of G (Δt) is plotted is displayed on the output unit. An output unit for outputting the analysis result is further provided,
The control unit is configured to add the inhibitory sugar set to a plurality of concentrations that dissociate the binding between the functional protein and the binding substance under a condition where the functional protein and the binding substance are mixed. The analysis system according to claim 8 or 9, wherein a graph in which a product of the intensity of the scattered light and the value of the autocorrelation function G (Δt) with respect to an inhibitory sugar concentration is plotted is displayed on the output unit.   13. The lens unit according to claim 1, further comprising a lens unit that collects the light from the light source irradiated on the detection cell and that can move a focal position of the collected light. Analysis system. An analyzer having the light source and the detector;
A computer having the control unit and connected to the analyzer to be able to communicate;
The control unit of the computer receives the intensity of the scattered light detected by the detector, and changes the binding state between the functional protein and the binding substance based on the received intensity of the scattered light. The analysis system according to claim 1, wherein an index to be expressed is obtained.

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