JPS58172537A - Apparatus for measuring light scattering - Google Patents

Abstract

PURPOSE:To make it possible to carry out both measurement of blood coagulation such as PT and APTT measurements and immunological measurement of a blood coagulation factor utilizing antigen-antibody reaction, by constituting the titled apparatus from one laser beam source, a liquid to be inspected irradiated by said laser beam source and one or more photodetector detecting scattered lights from the liquid to be inspected. CONSTITUTION:A light scattering intensity signal converted to an electric signal by a photodetector is treated by a signal pretreating device 6. A signal changeover device 12 is changed over so as to apply either one of output signals of photodetectors 3, 5 to the signal pretreating device 6. The signal pretreating device 6 further carries out differentiation treatment in the measurement of antigen-antibody reaction utilizing an electrically amplified width (p) and the result thereof is put in an A/D converter 7. A computer 8 subjects the information of a light scattering intensity signal converted to a digital amount by the A/D converter 7 to operation treatment to decide the point of time of blood coagulation in the case of the measurement of blood coagulation and to calculate the concn. of an antigen or an antibody in the measurement of antigen- antibody reaction. A display and printing part 9 of data treated by the computer 8 is constituted from a recorder 11 continuously observing a reaction process and a reagent portionwise pouring and automatic sample preparing mechanism 10.

Description

[Detailed description of the invention] The present invention relates to a light scattering measuring device.

More specifically, the present invention relates to a light scattering measurement device that measures blood coagulation reactions and antigen-antibody reactions as scattered light of a test liquid using one laser light source and one or more photodetectors. .

A blood coagulation test is an extremely important test for the treatment of patients with bleeding tendencies or for the follow-up management of patients undergoing anticoagulant treatment, and is also essential as a test prior to surgery.

As such a coagulation test, prothrombin time (hereinafter referred to as P
It is abbreviated as T. ) and activated partial thromboplastin time (hereinafter abbreviated as APTT).

) are well known, and each is performed as a comprehensive test of extrinsic and intrinsic coagulation mechanisms.

Furthermore, there are currently many types of automatic devices on the market that can measure PT and APTT.

The blood coagulation reaction is considered to be a complex chain reaction involving coagulation factors I to XIII, and if an abnormality is found in PT or APTT, a quantitative factor test is performed to determine which factor is deficient and to what extent. There is a need.

Conventionally, for factor quantification, a PT or APTT test is performed using a correction reagent to qualitatively determine which factor is deficient, and then P is performed using appropriate factor-deficient plasma.
The calibration relationship had to be measured by T or APTT test, which required very complicated procedures and a great deal of effort.

Furthermore, since the measurement method is indirect, the quantitative results obtained are not very accurate.

In recent years, instead of comprehensive testing of coagulation reactions such as PT or APTT, various coagulation factors have been directly tested by measuring immune activity values using antigen-antibody reactions or biological activity values using enzyme reactions with synthetic temperament. A method for quantifying is provided.

According to this method, it is possible to perform simple, highly specific, and accurate measurements; however, since it is impossible to carry out measurements using this method with conventional PT and APTT measuring devices, a new expensive device must be prepared. It was necessary and extremely uneconomical.

Conversely, the equipment that performs measurements using this method is PT, APT
This method had the disadvantage that it was impossible to carry out a very effective screening test for blood coagulation called T.

In view of the above-mentioned current situation, an object of the present invention is to
The object of the present invention is to provide a measuring device that can perform both blood coagulation measurements such as T measurement and immunological measurements such as blood coagulation factors using antigen-antibody reactions.

According to the present invention, it is possible to efficiently perform everything from screening tests to quantitative tests simply and with high precision using one device.

The present invention includes one laser light source, a test liquid irradiated by the light source, and one or more photodetectors that detect scattered light of the test liquid.

FIG. 1 shows one embodiment of the arrangement of a laser light source, a test liquid serving as a light scattering source, and a photodetector according to the present invention.

Reaction cuvette 2. Test liquid in 4. is the laser light source 1.
The sample liquid is irradiated with a constant amount of laser beam emitted from the test liquid, and the incident light beam is scattered depending on the state of the test liquid.

3. One or more photodetectors, for example two photodetectors, that measure this light scattering intensity and convert it into an electrical signal. and photodetector 5
．． is placed in a specific position relative to the reaction cuvette.

Laser light source 1. For example, the oscillation wavelength is 700 to 800 mm
A visible and near-infrared semiconductor laser is sufficient, and the reaction cuvette 2
．． For example, a tester cuvette with an inner diameter of 5 mm, and a photodetector 3. and 5. can be an ordinary silicon photo roll.

Reaction cuvette 2. Let θ be the angle between the laser optical axis passing through the center of the photodetector and the photodetector, and let θ represent the position of the photodetector. When the time-dependent change in the light scattering intensity S due to the blood coagulation reaction is recorded using the following, Figure 2 is obtained.

Also, for example, a photodetector 5 placed at a position of θ=155°. When the change in the light scattering intensity S caused by the reaction with the antigen and antibody is recorded over time using the method, Fig. 3 is obtained.

In Figure 2, which records the temporal change in the light scattering intensity S due to the blood coagulation reaction, in part a, the coagulation reaction has progressed due to the mixing of the test sample and reagent, but no fibrin precipitation has yet been observed. Part b is understood to be a state in which fibrin precipitation is actively progressing, and part c is a state in which conversion and precipitation of fibrinogen (hereinafter abbreviated as FIB) to fibrin has been completed. , it is empirically known that the time point Tc when transitioning from a to b is the time point at which fibrin precipitation begins.

FIG. 5 shows the relationship between the dilution rate of normal human plasma with saline and Tc (hereinafter abbreviated as activity curve) in PT measurement using the apparatus of the above embodiment.

That is, 100 saline diluted samples of citrated normal human plasma.
μ■Thromboplastin test solution (derived from rabbit brain) 100μ
TC was determined from the change in light scattering intensity after mixing 1 and 100 μι of a 0.02M calcium chloride aqueous solution, and the relationship with the normal human plasma sample dilution rate was plotted.

A stable activity curve was obtained even for samples with low coagulation activity, such as 10-fold dilution.

It is known to determine TC from changes in scattered light accompanying the coagulation reaction, but by using a laser with excellent intensity and monochromaticity as a light source, stable measurement with high sensitivity is possible up to the active coagulation reaction.

Furthermore, by using a scattered light measurement method using a laser light source for coagulation reaction detection, it is also possible to measure antigen-antibody reactions with the same device.

The time course of the light scattering intensity S due to the antigen-antibody reaction is shown in FIG.

That is, the light scattering intensity S starts to increase from the time when the solution containing the light source or antibody to be measured and the reagent containing the corresponding antibody or antigen are mixed, and after time TA has elapsed, the light scattering intensity S is maintained at a constant level ΔS.

FIG. 6 shows the results of a calibration curve for fibrinogen, which is plasma coagulation factor I, obtained using the apparatus of the above embodiment.

That is, anti-FIB serum (from rabbit) 12.5 times diluted solution 3
Add 60μ■ of a 61-fold dilution of standard plasma containing FIB antigen at each concentration to 00μ■, incubate at room temperature for 15 minutes, read the output of the photodetector of the above device, and calculate △S=(
The relationship between light scattering intensity S) after 15 minutes - (light scattering intensity S before the start of the reaction) and concentration was determined.

A calibration relationship sufficient for quantitative measurements was obtained.

Further, the antigen-antibody reaction can also be measured using the photodetector 3 in the above embodiment.

In this case as well, the temporal change in the light scattering intensity S as the antigen-antibody reaction in the test liquid progresses shows a pattern similar to that in FIG. 3, although there is a decrease in sensitivity.

The antigen or antibody can be quantified in the same manner as in the previous example, but as shown in Figure 4, the light scattering intensity S
The antigen or antibody can also be quantified from the time-dependent change in the rate of change (dS/dT).

For example, FIG. 7 shows the results of a calibration curve of the maximum value P of change in light scattering intensity S and fibrinogen concentration obtained using the apparatus of the above embodiment.

That is, 300μ of anti-FIB serum (rabbit) 2.5 times diluted solution
■ 21-fold dilution of standard plasma containing each concentration of FIB antigen 5
Add 0 μ■ to the photodetector of the above device 3. The output was processed by a known differential electric circuit, and the relationship between the maximum differential value P and the concentration was determined.

A stable calibration relationship sufficient for quantitative measurements was obtained.

According to this embodiment, the coagulation reaction and the antigen-antibody reaction can be measured using the same photodetector, which has the advantage of simplifying the optical system.

As described above, there has been no mention of the ability to measure blood coagulation reactions and antigen-antibody reactions with the same device using a scattered light measurement method using a laser as a light source prior to the disclosure of the technology according to the present invention.

According to the present invention, the functions of two types of devices that were conventionally required for screening tests and quantitative tests can be realized with one device.
It is clear that the effect is significant.

Furthermore, this device can measure agglutination reactions caused by antigen-antibody reactions.

In this measurement, antigen or antibody is bound to latex particles, mixed and stirred with the test sample on a flat plate, and the amount of antigen or antibody in the test sample is determined semi-quantitatively from the presence or absence of aggregation of the latex particles due to antigen-antibody reaction. A method of measuring this has been conventionally used.

In an embodiment of the device according to the invention, a photodetector 3. When the light scattering intensity of the test liquid is measured using a 100% SEM, and the background signal of the light scattered by the latex particles is electrically removed, a signal of the change in the light scattering intensity S over time is obtained depending on the presence or absence of agglutination due to antigen-antibody reaction. is obtained in a form similar to FIG.

In this case as well, a signal similar to that shown in FIG. 4 is obtained as the time-dependent change in the rate of change dS/dT of the signal, and from its maximum value P,
The concentration of antigen or antibody in the test liquid can be quantified.

For example, the device according to the present invention can easily measure fibrin degradation products (hereinafter abbreviated as FDP), which has been increasing in demand in connection with blood coagulation tests, using the method described above as a latex agglutination reaction. Can be implemented.

The fact that the device of the present invention can also be applied to methods that treat antigen-antibody reactions as agglutination reactions expands the application of the present invention.
The effect is made even more remarkable.

FIG. 8 is a block diagram of an apparatus in which the present invention is implemented as an actual light scattering measurement apparatus.

The light scattering intensity signal converted into an electric signal by the photodetector is sent to a signal preprocessor 6. will be processed.

Signal switch 12. is photodetector 3. and 5. Switch which of the output signals is input to the signal preprocessor.

Signal preprocessor5. In the measurement of the antigen-antibody reaction using an electrical amplifier or P, further differential processing is performed, and the results are sent to the A/D converter 7. Enter.

Computer 8. calculates the information of the light scattering intensity signal converted into a digital quantity by the A/D converter, determines the coagulation point in the case of blood coagulation measurement, and determines the concentration of antigen or antibody in the case of antigen-antibody reaction measurement. Calculate.

It is well known that various procedures other than the above-mentioned example have been proposed as a method for determining the blood coagulation point and calculating the concentration of antigen or antibody, and that various programs corresponding to each procedure can be created. This is not intended to limit the embodiments.

Furthermore, a display/print section 9 for displaying data processed by the computer. Recorder to continuously observe the reaction process 11. and reagent dispensing mechanism automatic specimen preparation mechanism 10. The device is configured by:

In this way, by combining the present invention and existing technology,
It is clear that an automated and simplified light scattering measuring device can be easily realized.

Although examples of the present invention have been described above, the present invention is not limited only to these examples, and can be applied to various specific examples within the scope of the present invention. .

As described above, the present invention enables the measurement of blood coagulation reactions and the measurement of antigen-antibody reactions, which were conventionally impossible with a single device, to be achieved with high precision in the same device, and improves the efficiency of the measurements. In addition to greatly contributing to rationalization,
According to this device, detailed clinical results can be obtained when conducting clinical tests, making a great contribution to the medical field.

[Brief explanation of drawings]

Fig. 1 is a diagram of the principle of the side light system according to the present invention, Fig. 2 is a temporal light scattering intensity characteristic diagram of a test liquid in a blood coagulation reaction, and Fig. 3 is a temporal light scattering intensity characteristic diagram of a test liquid in an antigen-antibody reaction. Scattered intensity characteristic diagram,
Figure 4 is a time differential characteristic diagram for the characteristic diagram in Figure 3, Figure 5 is a characteristic diagram of dilution rate and coagulation time in PT identification, and Figure 6 is the light scattering intensity of antigen concentration in FIB measurement. FIG. 7 is a calibration characteristic diagram for antigen concentration and maximum rate of change in light scattering intensity in FIB measurement, and FIG. 8 is a block diagram of a light scattering measuring device to which the present invention is applied. 1. Laser light source 2. Reaction cuvette 3. and 5 photodetector7. A/D converter8. Computer patent applicant Wako Pure Chemical Industries, Ltd.

Claims (5)

[Claims] (1) Irradiate the test liquid in the reaction container with a laser beam and measure the change in light scattering intensity of the test liquid after the reaction has started i) Measure the blood coagulation reaction of the test liquid ii) Measure the antigen-antibody reaction of the test liquid A light scattering measurement device characterized in that measurement is performed using one light source and one or more detected devices. (2) The light scattering geodetic device according to claim 1, which measures a plurality of light scattering intensities at different angles with respect to the laser optical axis. (3) The light scattering measuring device according to claim 1 or 2, wherein the blood coagulation time is measured based on a change in light scattering intensity due to the precipitation of fibuin as the blood coagulation reaction progresses. (4) The light scattering measuring device according to claim 1 or 2, wherein the measurement of the antigen-antibody reaction is based on a change in light scattering intensity due to the formation of an antigen-antibody complex. (5) The light scattering measuring device according to claim 1 or 2, wherein the antigen-antibody reaction is measured based on changes in light scattering intensity due to aggregation of insoluble carrier particles supporting the antigen or antibody.