JP2009098029A - Method and apparatus for evaluating biological reaction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique capable of precisely detecting an initiation timing of a biological reaction for a short time, by measuring a physical quantity changing in response to the degree of progression of the biological reaction. <P>SOLUTION: A method for evaluating the biological reaction is provided, which detects the initiation timing of the biological reaction by measuring the change in the prescribed physical quantity due to the biological reaction, and in which a timing is defined as the initiation timing, when an integrated value of the physical quantity exceeds a prescribed value. For example, turbidity change in a sample solution, caused by a reaction of LAL and endotoxin, is measured by the change in the light transmission of laser light, and its initiation timing is detected as the timing when an integrated value of a reduction amount of the light transmission exceeds a threshold S2. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a biological reaction evaluation method and an evaluation apparatus for measuring a change in a predetermined physical quantity due to a biological reaction and detecting a start time of the biological reaction.

  Traditionally, biological reactions have been utilized for the detection or concentration measurement of specific substances. In that case, detecting the start of the reaction by measuring a physical quantity that changes according to the degree of progress of the biological reaction, and measuring the concentration of the specific substance based on the start of the reaction Has been done.

  For example, it is known that an extract of horseshoe crab blood (LAL: Limulus amoebocyte lysate) gels when it reacts with endotoxin (lipopolysaccharide) which is a main component of the outer membrane of Gram-negative bacteria.

  Since this LAL gels by reacting with endotoxin with very high sensitivity, an extremely small amount of endotoxin can be detected. Therefore, the LAL method using this LAL as a measurement reagent is widely used as a means for detecting endotoxin.

  Specifically, in the LAL method, a LAL reagent and a specimen containing endotoxin are mixed. As a result, the reaction of the LAL reagent and endotoxin produces fine gel particles in the mixed solution, and the mixed solution gels. The presence of the gel particles depends on the scattered light intensity or the number of scattered light peaks (hereinafter collectively referred to as “light scattering intensity”), or the turbidity of the mixture due to the formation of gel particles or gelation of the mixture. Is optically measured by light transmittance. A method of optically measuring the presence of gel particles by light scattering intensity is called a light scattering particle measurement method. A method of optically measuring the turbidity of the mixed solution by the light transmittance is called a turbidimetric method. In the light scattering particle measurement method, the scattered light intensity is detected as the sum of the intensity of scattered light from each particle when the light source is local light such as a laser. In addition, there is a method in which an LED or a lamp is used as a light source, light from the light source is irradiated on the mixed solution, and the intensity of scattered light at that time is detected.

In the above case, the degree of progress of the reaction between the LAL reagent and endotoxin is measured by light scattering intensity and light transmittance. The time at which it can be determined that the reaction has started (at the time of starting the reaction) becomes shorter as the concentration of endotoxin in the sample increases, so the concentration of endotoxin is measured from the time at which it can be determined that the reaction has started.
JP 2007-097492 A JP 2004-061314 A JP-A-10-293129

  In many cases in the detection at the start of the reaction, a method has been adopted in which the reaction is started when the detected physical quantity value exceeds a preset threshold (hereinafter referred to as “threshold method”). Also called.) However, in reality, the biological reaction as described above often changes non-linearly and continuously, and it has not been easy to determine with high accuracy which time point is set as the start of the reaction.

In addition, depending on the biological reaction, if the amount of the activation factor that causes the reaction to proceed (for example, the enzyme concentration in the case of an enzyme reaction) is small, the time required for the reaction becomes long, so the value of the physical quantity exceeds the threshold value. In some cases, it takes a long time, which hinders improvement in measurement sensitivity and efficient measurement.

  The present invention has been devised in view of the above-mentioned problems, and the object of the present invention is to measure the biological reaction by measuring a physical quantity that changes according to the degree of progress of the biological reaction. It is to provide a technique capable of detecting the reaction start time in a shorter time and with higher accuracy.

  To achieve the above object, the present invention has the following features. That is, a change in a predetermined physical quantity due to a biological reaction is measured, and a time when an integrated value of the change in the physical quantity exceeds a predetermined threshold is set as a reaction start time.

More specifically, it is a biological reaction evaluation method for detecting the start of a reaction of the biological reaction by measuring a change in a predetermined physical quantity due to the biological reaction,
The biological reaction evaluation method is characterized in that a reaction start time is a time point at which an integrated value of the change in physical quantity exceeds a predetermined threshold value.

  According to this, even when the reaction activation factor in the biological reaction is small, it is possible to detect the reaction start time in a shorter time than in the case of the threshold method. Further, it is known that the correlation coefficient between the amount of the reaction activation factor and the start of the reaction is the same as in the case of the threshold method, so that highly accurate detection at the start of the reaction is possible.

  The present invention may be applied particularly in a case where the change curve of the physical quantity can be approximated to a predetermined approximate curve in which the change gradient shows an increasing tendency with time in a predetermined period after the start of measurement.

  In other words, the present invention is preferably applied to a biological reaction in which the change gradient of the physical quantity is small immediately after the start of measurement and then the change gradient increases. Such a reaction is characterized by a slow reaction rate between the start of measurement and the start of reaction. Then, when the threshold value method is used, the time from the start of measurement to the start of reaction may become extremely long, especially when there are few reaction activation factors. Therefore, if the present invention is applied to such a biological reaction, a more remarkable effect can be obtained.

  Further, the present invention may be applied to a biological reaction in which the change in the physical quantity can be approximated to a sigmoid curve. Since the sigmoid curve shows a behavior in which the change gradient is small immediately after the start of measurement and then the change gradient increases, a more remarkable effect can be obtained by applying the present invention to such a biological reaction. .

  As an example of this biological reaction, there is a reaction between the above-described extract of blood cells of horseshoe crab (LAL) and endotoxin. Examples of the physical quantity in this reaction include the light transmittance of a mixed solution of the extract of blood cells of the horseshoe crab (LAL) and a sample containing endotoxin, the scattered light intensity or scattered light of the light irradiated to the mixed solution. The number of peaks. As the physical quantity, pH, color development, fluorescence, electrical conductivity, absorbance, etc. are used in addition to light transmittance, scattered light intensity, and number of scattered light peaks according to the characteristics of each reaction.

Further, the present invention is a biological reaction evaluation apparatus for detecting a reaction start time of a predetermined biological reaction that can be approximated to a sigmoid curve,
A measurement unit for measuring a change in a predetermined physical quantity due to the biological reaction;
An integrated value calculating unit that calculates an integrated value of the change in the physical quantity based on the measured value by the measuring unit;
A reaction start detection unit for detecting the time when the integral value exceeds a predetermined threshold as the reaction start time;
A biological reaction evaluation apparatus characterized by comprising:

  According to such a biological reaction evaluation apparatus, even when the concentration of the reaction activation factor in the biological reaction is low and the reaction rate is slow, the reaction start time can be automatically set in a shorter time and with higher accuracy. Can be detected.

  The means for solving the above-described problems of the present invention can be used in combination as much as possible.

  In the present invention, it is possible to detect the start of the reaction of the biological reaction in a shorter time and with higher accuracy by measuring a physical quantity that changes according to the progress of the biological reaction. it can.

  The best mode for carrying out the present invention will be exemplarily described in detail below with reference to the drawings.

It has been found that many biological responses can approximate sigmoid curves. This sigmoid curve is a curve represented by the following mathematical formula.
f (t) = 1 / (1 + e ^{− (αt + β)} ) (1)
In Equation (1), t is time. α is a coefficient related to the activity of the reaction, and when the value of α is large, the reaction proceeds promptly. β is a coefficient related to the lag time of the reaction. When the value of β is large, the reaction moves in parallel in the time direction. In the case of a reaction in which multiple enzyme reactions are cascaded and the bottom enzyme changes the final physical quantity, the time until the bottom enzyme is activated should be treated as a change in β. is there.

  FIG. 1 is a diagram for explaining a conventional detection method at the start of a reaction when a biological reaction is approximated to a sigmoid curve. In FIG. 1, the horizontal axis represents time t, and the vertical axis represents reaction intensity (value of change in physical quantity). Here, in the order of the three curves L1, L2, and L3 (as n of Ln increases), the value of α in Equation (1) is set to be smaller. In the case where the reaction start time is detected when the reaction intensity (physical quantity value) exceeds a predetermined threshold value S1 as in the conventional case, the value of α is set as shown in FIG. As it decreases, the time required for the reaction intensity (value of change in physical quantity) to exceed the threshold value S1 becomes extremely long. As a result, in the reaction system in which α in Formula (1) is very low, the time required for detection at the start of the reaction is prolonged, which may be impractical as a reaction evaluation method.

  Therefore, in this example, the reaction intensity (value of change in physical quantity) exceeds the threshold value S1, and it is not determined that the reaction starts, but the integrated value of reaction intensity (value of change in physical quantity) sets the threshold value S2. The method of detecting the start of the reaction when exceeding (hereinafter referred to as “area method”) was adopted.

FIG. 2 is a diagram for explaining a detection method at the start of the reaction by the area method when a biological reaction is approximated to a sigmoid curve. In FIG. 2, curves L1 ′, L2 ′, and L3 ′ correspond to L1, L2, and L3 in FIG. 1, respectively.

  In FIG. 2, the value of the threshold value S2 is determined so that the reaction start time at L1 ′ coincides with the reaction start time at L1 in FIG. Then, when the area method is adopted, the time until the integrated value of the reaction intensity (value of change in physical quantity) in each curve of L2 ′ and L3 ′ exceeds the threshold value S2 in FIG. The reaction intensity (value of change in physical quantity) was clearly shortened compared to the time until the threshold value S1 was exceeded. Thus, the time required for detection can be greatly shortened by adopting the area method in this example and performing detection at the start of the reaction.

  FIG. 3 shows the logarithm of the relationship between α and T when the α value of the sigmoid function is variously changed along the horizontal axis in the threshold method and the area method according to the present embodiment, and the reaction start time T is taken along the vertical axis. It is the result plotted by. Here, both threshold value S1 and threshold value S2 are set to 0.5. As shown in FIG. 3, according to the area method, the value of T for the same α can be significantly shortened as compared with the threshold method. Further, it can be seen that the correlation coefficient between α and T is equivalent in the threshold method and the area method, and the reliability of the measurement is substantially the same in both cases.

  For example, in FIG. 3, when α = 0.1, the value of T is 100 in the threshold method, whereas it is 40.3 in the area method, and the value of T achieves a reduction rate of 60%. Yes. When α = 1, the area method is 6.3 with respect to the threshold method 10, and when α = 10, the area method is 0.8 with respect to the threshold method 1. Thus, in the area method in the present embodiment, the value of T can be significantly shortened as the value of α is smaller.

  Next, the results when the area method in the present example is used for detection at the start of the actual biological reaction will be described. Here, the area method in this example was applied to the detection at the start of the reaction in the reaction between the above-described blood crab blood extract (LAL) and endotoxin.

  Since this LAL gels by reacting with endotoxin with very high sensitivity, an extremely small amount of endotoxin can be detected. Therefore, as described above, this LAL is widely used as a measurement reagent for detecting endotoxin.

  When detecting the reaction start time in the reaction between LAL and endotoxin, the sample containing LAL and endotoxin is mixed. If it does so, a reaction liquid of LAL and endotoxin will produce | generate a micro gel particle in a liquid mixture, for example, and a liquid mixture will gelatinize. The turbidity of the admixture due to the formation of gel particles (gel aggregation) is optically measured by the change in light transmittance (turbidimetric method) to detect the start of the reaction between LAL and endotoxin. To determine the endotoxin concentration.

  FIG. 4 shows the relationship between the actual endotoxin concentration and the gel aggregation start time when the threshold method and the area method are used when the threshold values S1 and S2 are set to 0.25. Here, the horizontal axis is the endotoxin concentration, and the vertical axis is the agglutination start time and plotted in log-log. The aggregation start time corresponds to the start of the reaction in this example. As shown in FIG. 4, in both the threshold method and the area method, the relationship between the endotoxin concentration and the aggregation start time could be linearly approximated with high correlation.

As can be seen from FIG. 4, also in this case, the lower the endotoxin concentration, the larger the difference in the aggregation start time between the threshold method and the area method, and the aggregation start time when the endotoxin concentration is 0.02 pg / mL is the threshold method. Was 145 minutes, whereas the area method was 93 minutes. When the endotoxin concentration was 0.1 pg / mL, the aggregation start time was 77 minutes by the threshold method and 51 minutes by the area method. Furthermore, when the endotoxin concentration was 1 pg / mL, the aggregation start time was 31 minutes by the threshold method and 27 minutes by the area method.

  In the above description, the example in which the change in the turbidity of the mixed solution due to the progress of the reaction between LAL and endotoxin is approximated to a sigmoid curve, but this approximate curve is not limited to a sigmoid curve.

  For example, examples of approximate curves that can approximate biological responses include power functions such as allometry functions, exponential functions, logistic functions, and growth functions. Further, an increasing region of a function having a peak such as a lognormal distribution function, a Lorentz peak function, or a Voith peak function may be used as the approximate curve. These curves are suitable for the application of the present invention because they often show characteristics such that the change gradient of the reaction intensity (value of change in physical quantity) is small immediately after the start of measurement and the change gradient thereafter increases.

  Next, a second embodiment of the present invention will be described. In Example 1, an example was described in which the reaction between LAL and endotoxin was evaluated by detecting a change in turbidity due to gelation of the mixture by a change in light transmittance. In contrast, in this example, an example (light scattering particle measurement method) in which the generation and increase of gel particles due to the reaction between LAL and endotoxin is measured by changing the amount (number) of scattered light will be described.

  When LAL and a sample containing endotoxin are mixed and reacted with stirring, gel aggregation occurs in which gel particles are generated and grown in the mixed solution. In this example, the laser light is squeezed and irradiated into the mixture of the sample containing LAL and endotoxin, and the scattered light generated when the microparticles traverse the laser light passing through the mixture is detected microscopically. Then, the process of generating gel particles was observed using a laser light scattering measuring device capable of detecting individual particles. Then, the progress of the reaction between LAL and endotoxin was evaluated based on the number of gel particles detected by counting the number of peaks of scattered light.

  FIG. 5 shows the endotoxin concentration and aggregation start time when the threshold value S1 in the threshold method is 50 particles and the threshold value S2 is 50 in the area method. The graph which plotted this relationship by the logarithm is shown. Also in this case, the relationship between the endotoxin concentration and the gel aggregation start time for both the threshold method and the area method could be linearly approximated with high correlation as shown in FIG. Also in this case, the lower the endotoxin concentration, the larger the difference between the aggregation start times detected by the threshold method and the area method, and the aggregation start time at an endotoxin concentration of 0.2 pg / mL is 58 minutes by the threshold method. It became 52 minutes by law

  Next, Embodiment 3 of the present invention will be described. In this example, a reaction evaluation apparatus that detects the start of a biological reaction using the area method will be described.

  In FIG. 6, schematic structure of the reaction evaluation apparatus 1 in a present Example is shown. The reaction evaluation apparatus 1 in this embodiment is a measurement unit 2 that measures a change in light transmittance of a sample solution (for example, a mixed solution of LAL and endotoxin) in which a biological reaction in which turbidity changes with the progress of the reaction occurs. An amplifier circuit 4 for amplifying an analog signal corresponding to the light transmittance measured in the measurement unit 2, an A / D converter 6 for converting the analog signal amplified in the amplifier circuit 4 into a digital signal, and A / D conversion The digital signal converted in the device 6 is input, and the calculation unit 8 detects the reaction start time based on the area method, and the display unit 10 displays the detection result in the calculation unit 8.

  Here, the measurement unit 2 includes a laser light source 20, a light receiving element 23, an incident optical system 21 for allowing laser light emitted from the laser light source 20 to pass through the sample solution as substantially parallel light, and a laser transmitted through the sample solution. An emission optical system 22 for condensing the transmitted light on the light receiving surface of the light receiving element 23 is provided.

  For example, when the reaction evaluation apparatus 1 detects the gel aggregation start time by stirring reaction of LAL and endotoxin, a mixture containing LAL reagent and a sample containing endotoxin is introduced into the cuvette 24. Then, the magnetic stirrer bar 25 is introduced into the cuvette 24. In this state, the cuvette 24 is set in the measurement unit 2.

  Then, the operation of the stirring device 25 is started, and stirring of the mixed liquid in the cuvette 24 is continuously executed. Further, the laser light source 20 emits light, and the laser light is converted into parallel light in the incident optical system 21 and is irradiated into the mixed liquid in the cuvette 24.

  Then, in the cuvette 24, the turbidity increases with the generation and increase of the gel particles in the mixed solution, and conversely the light transmittance decreases. As a result, the amount of light that passes through the emission optical system 22 and is condensed on the light receiving element 23 is reduced.

  This change in light transmittance is amplified by the amplifier circuit 4, digitized by the A / D converter 6, and input to the arithmetic unit 8. The arithmetic unit 8 includes a ROM and a CPU that store various programs. In the arithmetic unit 8, by executing a program stored in the ROM, the input light intensity is subtracted from the initial value to obtain a difference, and the difference is integrated. Then, by executing another program, the obtained integrated value is compared with the threshold value S2 input in advance by the measurer, and when the integrated value is equal to or less than the threshold value S2, it is determined that aggregation has not started. Is done. On the other hand, when the integrated value is larger than the threshold value S2, it is determined that aggregation has started.

  The display unit 10 displays whether or not aggregation has started, and when aggregation has started, displays the aggregation start time. In this example also, the aggregation start time corresponds to the start of the reaction.

  As described above, in the reaction evaluation apparatus according to the present embodiment, an increase in turbidity due to gel aggregation in a mixed solution of a sample containing LAL and endotoxin is measured as a decrease in transmitted light of the mixed solution. .

  Then, the area of the change curve was calculated by integrating the amount of decrease in the amount of transmitted light, and it was detected that the reaction had reached the start of aggregation when this area exceeded the threshold value S2.

  According to this, the reaction start time of a biological reaction in which the light transmittance of the mixed solution changes with the progress of the reaction, such as the reaction between LAL and endotoxin, can be automatically determined by the area method. Therefore, detection at the start of reaction can be performed earlier than an apparatus using the conventional threshold method. In addition, since the calculation based on the area method can be automatically executed in the calculation unit 8, detection at the start of the reaction can be performed more easily.

  In this embodiment, the measurement unit 2 corresponds to a measurement unit. In the arithmetic unit 8, a program that subtracts the input light intensity from the initial value and integrates the difference corresponds to an integral value calculation unit. Further, in the arithmetic unit 8, the integrated value is compared with the threshold value S2, and when the integrated value is equal to or less than the threshold value S2, it is determined that aggregation has not started, and when the integrated value is greater than the threshold value S2, aggregation is started. The program that determines that the response is present corresponds to a reaction start detection unit.

  Further, in the arithmetic unit 8 in the present embodiment, detection at the start of the reaction by the area method and detection at the start of the reaction by the threshold method may be selectable at the judgment of the measurer.

  In the measurement unit 2 in this example, the degree of progress of the reaction between LAL and endotoxin was measured from the change in the light transmittance of the mixed solution, but the measurement method is not limited to this. For example, the laser light is squeezed and irradiated into a mixed solution of LAL and a sample containing endotoxin, and the peak of scattered light generated when a microparticle crosses the laser light passing through the mixed solution is detected microscopically. The progress of the reaction may be measured by counting the number of gel particles generated in the mixed solution. In this case, the arithmetic unit 8 detects that the aggregation has started by integrating the counted number of scattered light peaks and the integral value (total count number) being larger than the threshold value. Good.

  In addition, for example, by irradiating a laser beam into a mixed solution of a sample containing LAL and endotoxin, and by summing the intensity of scattered light generated when the laser light passing through the mixed solution is irradiated to each microparticle, The degree of progress of the reaction may be measured.

  That is, in this case, laser light from a laser light source is irradiated onto a minute region in the mixture, and scattered light scattered by particles passing through the minute region is detected by a light receiving element. Then, the arithmetic unit 8 calculates the sum of the intensities of the scattered light detected, and detects that aggregation has started when the value of the sum of the intensities of the scattered light becomes greater than the threshold value.

  Further, in addition to the reaction between LAL and endotoxin shown in the above examples, the present invention may be applied to human blood coagulation reaction as a similar enzyme cascade reaction. In addition, changes in various physical quantities due to enzyme reactions (for example, pH changes due to glucose gluconolactone changes due to glucose oxidase) and absorbance changes of colorimetric reagents (absorbance changes accompanying heavy metal binding to colorimetric reagents) are used. Thus, the present invention may be implemented.

It is a graph for demonstrating the detection at the time of a reaction start when a biological reaction is approximated to a sigmoid curve and the threshold method is used. It is a graph for demonstrating the detection at the time of the reaction start at the time of approximating a biological reaction to a sigmoid curve and using the area method. It is the graph which plotted the relationship between the alpha value of a sigmoid function, and the reaction start time T in the case of using the threshold method and the case of using the area method. It is the graph which calculated | required the relationship between the endotoxin density | concentration in the reaction of LAL and endotoxin at the time of using the threshold method and the area method by the turbidimetric method, and plotted by logarithm. It is the graph which calculated | required the relationship between the endotoxin density | concentration in the reaction of LAL and an endotoxin at the time of using the threshold method and the area method, and the time of agglutination of a gel by the light-scattering particle | grain measurement method, and plotted by logarithm. It is a figure which shows schematic structure of the reaction evaluation apparatus which detects automatically the reaction start time of biological reaction using the area method.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Reaction evaluation apparatus 2 ... Measurement unit 4 ... Amplifying circuit 6 ... A / D converter 8 ... Arithmetic unit 10 ... Display unit 20 ... Laser light source 21 ... Incident optical system 22 ... Exit optical system 23 ... Light receiving element 24 ... Cuvette 25 ... Magnetic stirrer bar 26 ... Stirrer

Claims (7)

A biological reaction evaluation method for detecting a start of reaction of the biological reaction by measuring a change in a predetermined physical quantity due to the biological reaction,
A biological reaction evaluation method, characterized in that a reaction start time is a time point at which an integrated value of a change in physical quantity exceeds a predetermined threshold value.   The biological reaction evaluation method according to claim 1, wherein the change in the physical quantity can be approximated to a predetermined approximate curve in which the change gradient shows an increasing tendency with time in a predetermined period after the start of measurement.   3. The biological reaction evaluation method according to claim 1, wherein the change in the physical quantity can be approximated to a sigmoid curve.   The biological reaction evaluation method according to any one of claims 1 to 3, wherein the biological reaction is a reaction between an extract of blood cells of horseshoe crab and endotoxin.   5. The biological reaction evaluation method according to claim 4, wherein the physical quantity is a light transmittance of a mixed solution of the blood cell extract of the horseshoe crab and a sample containing endotoxin.   The biological quantity according to claim 4, wherein the physical quantity is a scattered light intensity or a number of scattered light peaks of light irradiated on a mixed solution of the blood cell extract of the horseshoe crab and a sample containing endotoxin. Evaluation method of reaction. A biological reaction evaluation device for detecting a reaction start time of a predetermined biological reaction that can be approximated to a sigmoid curve,
A measurement unit for measuring a change in a predetermined physical quantity due to the biological reaction;
An integrated value calculating unit that calculates an integrated value of the change in the physical quantity based on the measured value by the measuring unit;
A reaction start detection unit for detecting the time when the integral value exceeds a predetermined threshold as the reaction start time;
An apparatus for evaluating biological reactions, comprising:

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