JPH0712816A - Bio-substance measuring method utilizing specific bonding reaction - Google Patents

Abstract

PURPOSE:To determine an object to be measured with high sensitivity in a short time by determining the object based on the secular change of the detected intensity of light emission or fluorescence. CONSTITUTION:By catching the specific bonding reaction of a sample of a known concentration through an enzyme-substrate reaction, the detected intensity of generated luminescence (fluorescence) is continuously measured for a prescribed period of time. A characteristic curve prepared based on the measured results and indicating the relation between the measuring time and detected intensity, namely, the time characteristic is approximated to a straight line by using the method of least squares. The inclination of the straight line represents the secular change of the detected intensity and depends upon the quantity of a bio-substance contained in the sample. Therefore, by using a plurality of samples of known concentrations, a calibration curve representing the relation between the inclination of the time characteristic and concentration of a substance to be measured is prepared. Then, by receiving 12 the light emitted from a sample contained in a reaction container 11 and measuring 14 the inclination of the time characteristic of the sample containing the substance by an unknown amount, the substance to be measured in the sample is determined 15 from the calibration curve. Therefore, the substance can be determined without waiting for the saturation of detected intensity nor being affected by background noise caused by the stray light, etc., from the measuring instrument.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a measurement reaction utilizing a specific binding reaction of a living body such as an immune reaction.

[0002]

2. Description of the Related Art Conventionally, in order to quantify an antigen or an antibody, an enzyme which uses an enzyme activity as a label to trace an antigen-antibody reaction between the antibody or the antigen and a substance to be measured and quantifies the antigen or the antibody based on the result. Immunoassays are performed primarily in the medical or biotechnology fields. As one of the enzyme immunoassay methods, there is a method utilizing bioluminescence, chemiluminescence, or fluorescence. This is performed by reacting an enzyme-labeled antigen or antibody with a substrate, detecting luminescence or fluorescence generated by this enzyme reaction with a light receiver, and measuring the intensity thereof.

The intensity of luminescence or fluorescence generated by the reaction of the above-mentioned enzyme and substrate is measured by, for example, a photon counting method. In this case, the intensity of luminescence or fluorescence at a certain time point or the intensity of luminescence or fluorescence within a specific time range is measured.

[0004]

In the above-described enzyme immunoassay, when a substance that emits light in an extremely short time such as luminol or acridinium ester is used as a substrate that emits light or fluorescence, the measurement is also extremely difficult. Finish in a short time. However, when a peak of luminescence or fluorescence appears in a short time, it is difficult to operate unless a measuring device having a high responsiveness is configured, which is an obstacle to automation. So, among the commonly used substrates,
For example, as in the case of a dioxetane derivative or a luminol / p-iodophenol system, the luminescence or fluorescence reaction may proceed slowly. For example, a dioxetane derivative is shown in FIG.
As shown in, the emission intensity continues to increase slowly until about 20 minutes after the start of the enzyme-substrate reaction. In FIG. 9, the concentration of alkaline phosphatase is 10 ⁻⁴ unit / ml.

When such a substrate having a slow luminescence or fluorescence reaction is used, the problem of missing the peak value of luminescence or fluorescence is solved, but it is extremely difficult to carry out the measurement in a short time. Because the intensity of luminescence or fluorescence is not sufficient in the short time after the initiation of the enzyme-substrate reaction, and measurement is difficult,
This is because it takes a long time to obtain sufficient strength for measurement. In addition, the lower the concentration of the substance to be measured, the more unclear the emission or fluorescence peaks, which may even delay the discovery of the peak portion.

Further, in the conventional photon counting method, when background noise is generated due to stray light or the like in the measuring device, the background noise is included in the counting result and the base rises, making it difficult to perform high-sensitivity measurement.

In the above explanation, the problem in the enzyme immunoassay was explained, but the similar problem is the specific binding of the living body such as the reaction by the complementary DNA strand or the reaction between the DNA binding strand and the DNA. It is also recognized in the reaction. The present invention has been made in view of such points,
Provided is a method for measuring a biological substance using a specific binding reaction, which can quantify a substance to be measured in a short time and with high sensitivity.

[0008]

According to the present invention, a substance to be measured and a specific reaction substance specifically bound to the substance to be measured and labeled with an enzyme are reacted, and then the substance to be measured and the specific substance are reacted. A method for measuring a biological substance, which comprises quantifying a substance to be measured by detecting luminescence or fluorescence generated by reacting a reaction substance with a substrate that emits luminescence or fluorescence when reacting with the enzyme, the detection intensity of luminescence or fluorescence. There is provided a method for measuring a biological substance using a specific binding reaction, which comprises quantifying the substance to be measured based on the amount of change with time.

The method for measuring a biological substance of the present invention will be described in more detail below. Here, the specific binding reaction means, for example, an antigen-antibody reaction, a reaction by a complementary DNA chain (DNA
-Including RNA binding).

The substance to be measured is an antigen or an antibody when the specific binding reaction is an antigen-antibody reaction, and is bound to a carrier as in a sandwich assay in a solid-phase enzyme-linked immunosorbent assay (ELISA method). It may be an antibody bound to an antigen. Examples of such substances to be measured include various pathogenic bacteria, viruses, hormones, allergens, blood group antigens, tissue group antigens, antigens such as amino acids, or antibodies thereof.

The enzyme used for labeling is not particularly limited as long as it is usually used as a labeling enzyme. For example, alkaline phosphatase, horseradish peroxidase and β-galactosidase can be preferably used.

The substrate that reacts with the enzyme to generate luminescence or fluorescence is selected according to the enzyme used for labeling. For example, when alkaline phosphatase or β-galactosidase is used as the labeling enzyme, a dioxetane derivative is preferably used as the substrate.

In the method for measuring a biological substance of the present invention, the quantification of the biological substance is performed based on the amount of change over time in the detected intensity of luminescence or fluorescence. That is, the detection intensity of luminescence or fluorescence is continuously measured for a predetermined time after the enzyme substrate reaction is started and a predetermined time has elapsed. From this measurement result, the amount of change in the detected intensity over time is obtained.

For example, a sample having a known concentration is prepared, a specific binding reaction in this sample is captured by an enzyme substrate reaction, and the detection intensity of luminescence or fluorescence generated by the enzyme substrate reaction is continuously measured as described above. Based on the obtained measurement results, create a characteristic line showing the relationship between measurement time and detection intensity (hereinafter also referred to as time characteristic), apply the least squares method to this characteristic line, and make this characteristic line a straight line. To approximate. The slope of this line indicates the amount of change in the detected intensity over time,
It depends on the state of the specific binding reaction in the sample solution, that is, the amount of the biological substance in the sample solution. Therefore, using a plurality of samples of known concentration, create a calibration curve consisting of the relationship between the slope of the time characteristics and the concentration of the substance to be measured, and the slope of the time characteristic of the sample containing an unknown amount of the substance to be measured. Can be measured and the substance to be measured in the sample can be quantified from the calibration curve.

The calibration curve does not consist of the relationship between the concentration of the substance to be measured and the amount of change in the detection intensity over time. Instead, a calibration curve was prepared using samples containing enzymes at different concentrations, and It may be based on the relationship with the amount of change in detection intensity over time. Since the amount of the enzyme always has a certain correlation with the amount of the substance to be measured to which the specific reaction substance labeled with the enzyme binds, the amount of the substance to be measured can be determined from the amount of the enzyme.

[0016]

According to the method for measuring a biological substance using the specific binding reaction of the present invention, the amount of change over time in the detection intensity of luminescence or fluorescence is obtained. The amount of change with time depends on the amount of the substance to be measured, and an unknown amount of the substance to be measured is determined based on the relationship between the amount of the known substance to be measured and the amount of change with time. This allows
The substance to be measured can be quantified without waiting for the detection intensity of luminescence or fluorescence due to the enzyme substrate reaction to reach saturation. Further, since the amount of change in the detected intensity of luminescence or fluorescence over time does not include the base included in the detected intensity, it is less affected by background noise generated by stray light or the like in the photon counting measurement device.

[0017]

The method for measuring a biological substance of the present invention will be described in detail below. FIG. 1 is a block diagram showing an example of a chemiluminescence measuring apparatus used in the method for measuring a biological substance according to this embodiment.
Reference numeral 11 in the drawing accommodates a sample solution inside and allows bioluminescence, chemiluminescence, or fluorescence generated by a luminescence or fluorescence reaction to be transmitted to the outside. As the reaction vessel 11, for example, a glass tube, a plastic tube such as polystyrene, or a microtiter plate can be used.

Light or fluorescence emitted from the reaction container 11 is received by the light receiving element 12. The signal obtained by the light receiving element 12 is amplified by the amplifier 13, then photon-counted by the photon counter 14, and the counting result is processed by the data processor 15. As the data processing device 15, a device that can perform arithmetic processing using a computer such as a computer can be used.

In this example, a dioxetane derivative (AMPPD; 3- (4-methoxyspiro [1,2-dioxyethane-3,2'-tricyclo [3,3,1,1 ^3,7 ]] was used as a luminescent substrate. Decan] -4-yl) phenyl phosphate disodium; Tropix "Elisa
Light Test Kit ") was used.

The reagents used in this example were prepared as assay buffers: 0.1 M diethanolamine, 1 mM magnesium chloride, 0.02% sodium azide; PH 9.5. Substrate solution is 10% Emerald, 0.4 mM AMP
PD was used. Moreover, as a washing buffer, 0.00
Tris-HCl buffered saline (PH7.6) containing 5% Tween 20 was used.

In the method for measuring a biological substance according to this example, a two-step sandwich sassay by the solid phase method was performed.
First, as shown in FIG. 2A, the reaction container 11 (Nun
2 μg / ml of anti-human HBs antibody 21 (Nippon Special Immune), which reacts specifically with HBs antigen 22 as a substance to be measured, on the inner wall of the well of a microplate manufactured by c.
0 μl was used to solidify by overnight. After completion of the solid phase immobilization, the solid phased surface of the reaction vessel 11 was blocked with 3% bovine albumin for the purpose of preventing nonspecific binding of the antigen.

Next, as shown in FIG. 2 (B), 100 μl of the sample was added to the reaction vessel 11 and the primary reaction was carried out at room temperature for 30 minutes. Here, as the sample, a sample was used in which a predetermined concentration of HBs antigen (Meiji Milk Industry) was dissolved in the above-mentioned washing buffer. As a result, as shown in FIG. 2 (C), the HBs antigen 22 in the sample was treated with anti-human HBs.
It bound to antibody 21.

After the completion of the primary reaction, the reaction solution was removed and the reaction vessel 11 was washed with a washing buffer solution. After washing, an enzyme-labeled anti-HBs antibody solution (manufactured by Olympus Optical Co., Ltd.) was added to the reaction vessel 11 and a secondary reaction was carried out for 30 minutes (antigen-antibody reaction).
Carried out. As a result, as shown in FIG. 2D, the secondary antibody 24 labeled with the enzyme 23 bound to the HBs antigen 22 bound to the anti-human HBs antibody 21. Enzyme 23
Is alkaline phosphatase.

After the completion of the secondary reaction, the reaction solution was removed and the reaction vessel 11 was washed again with the washing buffer. Figure 2
As shown in (E), after washing, an AMPPD solution having a concentration of 17 mg / ml and an emerald solution having a concentration of 0.1% were added, and then an enzyme substrate reaction was performed at room temperature.

After a lapse of a predetermined time after the initiation of the enzyme substrate reaction, the luminescence intensity was measured using the above-described chemiluminescence measuring device. The emission intensity was measured in units of 1 second, and the number of photons counted in 1 second was used as a measurement value, and the measurement was continuously performed for a predetermined time.

FIG. 3 shows HBs with an antigen concentration of 10 ng / ml.
The relationship between the luminescence intensity due to the antigen-antibody reaction and the time when the luminescence intensity of the sample containing the antigen is measured as described above is shown. The horizontal axis of FIG. 3 shows the time from the start of the measurement of the emission intensity, and the vertical axis shows the emission intensity (unit: the number of counts per second). Here, the measurement of the luminescence intensity was started after 17 minutes had elapsed since the start of the enzyme substrate reaction, and was performed for 60 seconds in units of 1 second.

As is clear from FIG. 3, when an antigen-antibody reaction occurs, chemiluminescence occurs due to the enzyme substrate reaction with the enzyme labeled with the above-mentioned labeled antibody. This emission intensity increases with time. Further, the tendency that the luminescence intensity increases shows the dependence on the antigen concentration, and increases as the antigen concentration increases.

Next, different concentrations of alkaline phosphatase (TOYOBO) dissolved in the above-mentioned washing buffer were prepared as samples, and after 17 minutes had elapsed since the initiation of the enzyme-substrate reaction according to the procedure described above. Get 60 points of data. The slope of the characteristic line when the relationship between the luminescence intensity at each enzyme concentration and time (time characteristic) was represented by a graph was obtained by the method of least squares.

A calibration curve as shown in FIG. 4 is obtained with the vertical axis representing the slope of the time characteristic obtained from each of these samples and the horizontal axis representing the concentration of alkaline phosphatase (unit: unit / ml). It was

The concentration of HBs antigen in the sample can be quantified based on the calibration curve shown in FIG. That is, the luminescence intensity of a sample containing an unknown amount of HBs antigen was measured under the same conditions as when the calibration curve was created, and the alkaline phosphatase corresponding to the measured value was measured from the corresponding calibration curve.
It is possible to determine the concentration of zase and to determine the concentration of HBs antigen corresponding thereto.

According to the method for measuring a biological substance of this example,
A calibration curve was prepared based on the slope of the time characteristics of alkaline phosphatase concentration and luminescence intensity, and HBs in the sample
Seeking the concentration of antigen. Since the amount of change in emission intensity over time does not include the base included in emission intensity, even if the base rises due to background noise due to stray light or the like, it is hardly affected by it and prevents the S / N ratio from decreasing. be able to.

In FIG. 5, H of various concentrations is obtained in the same manner as described above.
For the sample containing the Bs antigen, measurement was continuously performed for 30 seconds from 30 seconds to 60 seconds after the start of the enzyme substrate reaction, and the slope of the time characteristic was determined by the least squares method based on the obtained data. The results obtained by approximation are shown below. In this case, the number of measurement points was set at 30 points every one second, and the results of two samples for each antigen concentration were plotted. As comparative data, similarly, values obtained by averaging the luminescence intensities after 30 seconds to 60 seconds were plotted for each antigen concentration are shown in FIG. As is clear from comparison between FIG. 5 and FIG. 6, if the luminescence intensity is plotted on the vertical axis, the measurement sensitivity becomes only about 1 ng / ml because the quantitative measurement becomes difficult as the antigen concentration becomes lower (see FIG. 6). ). On the other hand, according to the slope of the time characteristic based on the method of least squares, a result corresponding to the antigen concentration can be obtained, so that the quantitative measurement is already possible even when the enzyme-substrate reaction starts very early. (Fig. 5
reference).

In FIG. 7, the sandwich assay was performed in the same manner as described above, and 60 seconds after starting the enzyme substrate reaction, 12
This is a result obtained by continuously measuring for 60 seconds until 0 second, and approximating the slope of the time characteristic by the least square method based on the obtained data. The number of measurement points is 60 points. Also,
Similarly, as comparative data, values obtained by averaging the luminescence intensities every 1 second after 1 minute to 2 minutes were plotted for each antigen concentration to obtain FIG. As is clear from comparing FIGS. 7 and 8,
Just plotting the emission intensity on the vertical axis, the lower the antigen concentration becomes, the more difficult the quantitative measurement becomes (FIG. 8).
reference). On the other hand, according to the slope of the time characteristic based on the method of least squares, the result according to the antigen concentration is obtained with better correlation, and the measurement sensitivity is 10 ^-2 to 10 ^-1 ng.
This suggests that ultrasensitive quantitative measurement of / ml is possible (see FIG. 7).

In the above-described embodiment, the light emission intensity is measured at a unit of 1 point / second, but the present invention is not limited to this, and it may be 0.1 second / point or 2 points / point. Seconds are fine. That is, 1 second within 10 seconds to 2 minutes, preferably 30 seconds to 60 seconds after starting the measurement.
It is advisable to use measured values obtained at 0 to 100 points, preferably 30 to 60 points. The measurable range may be from the beginning of the progress of the luminescence or fluorescence reaction to the saturation of the reaction, in this example, 30 seconds to 17 minutes after the start of the reaction, but it is practically the least squares method. It suffices to provide an effective range in which each plot is closer to a straight line, and select a measurement start time from which measurement can be performed as quickly as possible.

Further, in this embodiment, the antibody was immobilized on the inner wall of the reaction vessel, but it is also possible to measure the antibody by immobilizing the antigen. Further, the antigen or antibody is not limited to be immobilized on the inner wall of the reaction container, and the antigen or antibody is immobilized on the fine particles such as magnetic beads, glass beads or polystyrene beads, and the sandwich assay is performed in the same manner as described above. You can also do it. Further, as the reaction container, slides, columns, filters, tubes and the like having various forms may be used. Further, the luminescence or fluorescence substrate, enzyme, washing buffer, reaction temperature, pH, dilution concentration, etc. used are various known techniques (for example,
Bioluminescence and chemiluminescence, Imai, 1989), the same effect as the present invention can be obtained even if the change is made within the measurement range. Further, it was confirmed that luminol and p-iodophenol were used as luminescent substrates, and the measurement could be performed quickly and with high sensitivity by the same principle.

[0036]

According to the method for measuring a biological substance using the specific binding reaction of the present invention, the amount of the substance to be measured is determined based on the amount of change over time in the detection intensity of luminescence or fluorescence. The substance to be measured can be quantified without waiting for the detection intensity of luminescence or fluorescence due to the reaction of 1 to reach saturation. In addition, since the base included in the detected intensity is removed, the amount of change over time in the detected intensity of luminescence or fluorescence is less affected by background noise.
As a result, the substance to be measured can be quantified with high sensitivity and in a short time. Further, since it is not necessary to find the peak of luminescence or fluorescence, the measurement can be carried out easily and surely.

[Brief description of drawings]

FIG. 1 is a block diagram showing an example of a chemiluminescence measuring apparatus used in a method for measuring a biological substance according to this embodiment.

2 (A) to (E) are process diagrams showing respective steps of a sandwich assay in the method for measuring a biological substance in the present example.

FIG. 3 is a characteristic diagram showing the relationship between the emission intensity of a sample and time in the method for measuring a biological substance according to this example.

FIG. 4 is a characteristic diagram showing a calibration curve based on the relationship between the concentration of alkaline phosphatase and the slope of the time characteristic in the method for measuring a biological substance of this example.

FIG. 5 is a diagram showing the results of measuring the time characteristics of samples containing various concentrations of HBs antigens measured 30 seconds to 60 seconds after the initiation of the enzyme substrate reaction according to the method for measuring biological substances of the present example.

FIG. 6 shows various concentrations of HBs according to the same method as in FIG.
The figure which plotted the value which averaged the luminescence intensity measured for 30 to 60 seconds after starting an enzyme substrate reaction about the sample containing an antigen for every antigen concentration.

FIG. 7 is a diagram showing the results of measuring the time characteristics measured for 60 seconds to 120 seconds after the start of the enzyme substrate reaction for samples containing various concentrations of HBs antigen according to the method for measuring a biological substance of this example.

FIG. 8 shows HBs of various concentrations according to the same method as in FIG.
The figure which plotted the value which averaged the luminescence intensity for every 1 second after 1 to 2 minutes after starting an enzyme substrate reaction about the sample containing an antigen for every antigen concentration.

FIG. 9 is a characteristic diagram showing the relationship between luminescence intensity and measurement time in a method for measuring a biological substance using a specific binding reaction.

[Explanation of symbols]

11 ... Reaction vessel, 12 ... Light receiving element, 13 ... Amplifier, 14
... Photon counter, 15 ... Data processing device.

Claims (1)

[Claims] 1. After reacting a substance to be measured with a specific reaction substance which is specifically bound to the substance to be measured and labeled with an enzyme, the substance to be measured and the specific reaction substance are reacted with the enzyme. A method for measuring a biological substance, which comprises quantifying a substance to be measured by detecting luminescence or fluorescence produced by reacting a substrate exhibiting luminescence or fluorescence, comprising the steps of: A method for measuring a biological substance using a specific binding reaction, which comprises quantifying a substance to be measured.