JPS6222066A - Latex agglutination reaction measuring instrument - Google Patents

Abstract

PURPOSE:To obtain a latex agglutination reaction instrument which permits quantitative measurement with good measurement efficiency, has excellent operability and can inexpensively be produced by measuring the change, with time, of the quantity of transmitted light of the light irradiated on plural sample cuvettes each contg. a mixture composed of a sample and latex reagent after correcting the difference in the detection sensitivity. CONSTITUTION:The light from a light source 2 passes through the plural cuvettes 1 and is detected by a photoelectric detector 4 when the mixtures composed of the sample and latex reagent are put into the plural cuvettes 1 and the measurement is started by a switch mechanism 4 and a computer 8. The signal for the quantity of the light transmitted through the cuvettes is inputted via a multiplexer 5 to a sensitivity correcting circuit 19 consisting of a ratio calculating circuit 6 and a digital to analog converter 9, by which the detection sensitivity of the quantity of each transmitted light is corrected. The corrected quantity is inputted via an analog-to-digital converter 9 to a computer 8. The microcomputer 8 calculates the change, with time, of the quantity of the transmitted light via the signal from a timer 10 and stores the data thereof into a memory 11 and delivers the data to a display device 12, a printer 14 and a communication device.

Description

Detailed Description of the Invention "Industrial Application Field" The present invention provides a latex agglutination reaction measuring device that enables quantitative determination by objective measurement, has good measurement efficiency, has excellent operability, and can be manufactured at low cost. It is related to.

"Prior art and its problems" A method for measuring an immune reaction using latex agglutination reaction includes, for example, F[lP, CRP, RA, A
A known method for measuring blood proteins such as SLO is to visually determine the state of aggregation on a glass plate, but while this method is simple, it is only semi-quantitative and requires dilution. There were problems such as being complicated and causing individual differences in judgment.

On the other hand, there are also known methods and devices that detect the state of aggregation as a change in optical absorbance; for example,
1575, JP 54-108883, JP 54-108895, JP 57-149
However, the above method described in Japanese Patent Application Laid-Open No. 57-183848 has the advantage that quantitative measurement is performed repeatedly by objective measurement, but on the other hand, it uses a single light source and a detector. , the measurement efficiency can be reduced to 1” fJ by manual operation.
″″Then, the optical system of the specimen is exposed.
.

This has the disadvantage that the operation becomes complicated because it has to be inserted frequently. To overcome these shortcomings and improve efficiency and ease of operation, automated machines that use a single light source and detector in a time-sharing manner are commercially available, but they are very expensive. There was a problem.

The present invention solves the above-mentioned problems, and provides a latex agglutination reaction measuring device that enables quantitative determination by objective measurement, has good measurement efficiency, has excellent operability, and can be manufactured at low cost. With the goal.

"Summary of the Invention" The reaction time course when measuring the latex agglutination reaction as a change in the amount of transmitted light is as shown in Figure 3. However, when multiple samples are measured simultaneously using different optical systems, differences in sensitivity may occur. Therefore, there is no compatibility between I(t).

In order to correct the difference in sensitivity, for example, the ratio R of the subsequent transmitted light amount r(t) to the transmitted light amount IO at the initial stage of the reaction.
(t) = [I(t)/Iol becomes.

+Yu KJ Takuji J↓ r Small Pre-Kugilta 1 Independently instruct the start of measurement of the amount of transmitted light of the Yushima small cage.For example, start the measurement of each sample by a switch mechanism, and in conjunction with the switch, each If the reaction times of the samples are counted in parallel using a timer and the R(t) of each sample at each time is determined simultaneously, it is possible to measure the temporal change in the amount of transmitted light of multiple samples separately, which is very efficient. We have discovered that it is possible to construct a device with excellent operability, and have arrived at the present invention.

That is, the present invention provides means for holding a plurality of sample cuvettes containing a mixture (test sample liquid) of a sample and a latex reagent that reacts with an antigen or antibody in the sample to cause an agglutination reaction, and means for irradiating each light beam and detecting the amount of transmitted light of each separately; means for correcting the difference in sensitivity in detecting the amount of transmitted light; and means for instructing the start of measurement of the amount of transmitted light of the test sample liquid. , and a timer that separately measures the elapsed time for each of the sample cuvettes from the start of measurement of the amount of transmitted light of the sample liquid to be tested, and simultaneously measures changes over time in the amount of transmitted light of a plurality of samples in parallel. Specifically, the present invention is characterized by the fact that conventional methods have only considered the use of a single light source and detector, but have not considered parallel measurement using multiple optical systems. On the other hand, it is characterized in that it enables parallel measurements with multiple optical systems by performing sensitivity correction between different optical systems.

That is, in the basic configuration block diagram of the present invention shown in FIG. 8, there are a sample cuvette 1, a light source 2 that irradiates the sample cuvette, and a means 3 for detecting the amount of transmitted light that has passed through the sample cuvette 1. Multiple optical systems consisting of
A means 20 for holding the optical system is provided. and,
The difference in sensitivity of the optical system is corrected by means 21 for correcting the difference in sensitivity of the optical system.
The measurement of the amount of transmitted light is started by the means 4 for instructing the start of measurement of the amount of transmitted light, and the elapsed time from the start of measurement of the amount of transmitted light of the test sample liquid is determined by the timer 22.
The time is measured by

"Embodiments" Next, preferred embodiments of the present invention will be described with reference to the drawings.

FIG. 1 is a block diagram of a measuring device according to the present invention, which includes a plurality of sample cuvettes 1 containing a mixture of a sample and a latex reagent (test sample liquid), a plurality of light sources 2, and a corresponding plurality of a photoelectric detector 3 that irradiates a sample cuvette 1 with a light beam and separately detects a plurality of transmitted light quantities, and a multiplexer 5 that sequentially switches and transmits a plurality of transmitted light quantities I(t) detected by the photoelectric detector 3 one after another. , a sensitivity correction circuit 19 that corrects sensitivity differences between multiple detections, an A/D converter 7 that converts the corrected results into digital values, and a a switch mechanism 4 for instructing the start of measuring the amount of transmitted light of each sample liquid to be tested; and a timer 10 for measuring the elapsed time from the start of measurement of the amount of transmitted light for each sample liquid in conjunction with the instruction of the switch mechanism; and a computer 8 and a memory 11, which control the data processing of the digitalized changes in the transmitted light amount and the operation of the entire apparatus, and simultaneously measure and process the temporal changes in the transmitted light amount of multiple samples. Note that means for holding the cuvette 1 are omitted in the figure.

As the light source 2, a light emitting element such as an LED or a tungsten lamp can be used. Furthermore, the light source 2 does not need to be arranged in multiple stages, and the irradiation light may be guided from a single light source to each sample cuvette using an optical fiber.

As the photoelectric detector 3, a light receiving element such as a photodiode or a photocell that generates an electric signal corresponding to the amount of transmitted light can be used. Similar to the light source, the photodetector may also guide the transmitted light of each sample cuvette through an optical fiber to a single photodetector.

In the above embodiment, the sensitivity correction circuit 19 includes a ratio calculation circuit 6, a DIA converter for setting Io at the initial stage of one reaction, or an input/& converter 8 for setting the amount of transmitted light of the pseudo test sample. Consists of. The sensitivity correction circuit 19 includes a means for storing the amount of transmitted light at the initial stage of the reaction of the test sample liquid, and a means for calculating the ratio of the amount of transmitted light thereafter to the amount of transmitted light at the initial stage, or Any other means may be used as long as it is a means for determining the ratio between the blank value of the light amount and the amount of transmitted light of the test sample liquid or the logarithm of the ratio. For example,
It can also be replaced by a program operation using the computer 8 and memory 11.

The sensitivity correction circuit has a means for storing the amount of transmitted light at the initial stage of the reaction of the test sample solution, and a means for calculating the ratio of the amount of transmitted light after that to the amount of transmitted light at the initial stage. This method is preferable because the detection sensitivity can be corrected only by measuring the amount of transmitted light relative to the object, the detection sensitivity is very good, and the operability is excellent.

As a sensitivity correction circuit, the method of calculating the ratio of the blank value of the amount of transmitted light of the pseudo test sample liquid to the amount of transmitted light of the test sample liquid, or the logarithm of the ratio, is based on the amount of transmitted light for one test sample liquid. In addition to measurement, it is necessary to measure a pseudo test sample (the same system of reaction mixture, but not containing antigen or antibody) for each detection optical system, which makes the operation somewhat complicated.

The amount of transmitted light in the initial stage may be the amount of transmitted light at any specific time within a certain period of time in the initial stage, the maximum value, the minimum value, or the average value. Good too.

When setting the maximum value, it is effective when air bubbles are mixed in when the reagent and sample are mixed; when setting the minimum value, it is effective when it takes time for the mixture of reagent and sample to become uniform. In addition, when using the average value, it is effective when AC noise is superimposed on the signal due to fine particles, etc., or when AC noise is generated due to the vibration of relatively large particles. In addition, if air bubbles are mixed in as described above when mixing the reagent and specimen, or if it takes time for the mixture of the reagent and specimen to become uniform, if the time period in which the correct l is obtained is known in advance, it is possible to It is preferable to use the amount of transmitted light at a specific point in time.

The switch mechanism 4 for instructing the start of measurement of the amount of transmitted light of the test sample liquid is a means for instructing a plurality of cuvettes independently in terms of time or position. It may be linked to the measurement operation. Here, 1-hour independent means that measurement can be started at any independent timing for two or more cuvettes. For example, one switch can be used to adjust the operator's procedure By pressing the buttons appropriately at the same time, it means that measurement will be started for each of the multiple cuvettes. Also, positional independence means, for example, providing two or more switches corresponding to two or more cuvettes and operating each one independently.
This means that measurements can be started independently for two or more cuvettes.

In order to fully exhibit the functions of the measuring device of the present invention having the above configuration, in the above embodiment, a data numerical display device 12 (LED, CRT display, etc.), a data numerical display control switch 13, a data printing Printer 1
4. A communication device 15 for communicating with an external combination computer and a display device 18 for displaying the measurement status of each sample cuvette are provided.

FIG. 2 shows a perspective view of a specifically configured measuring device of the present invention, in which 12' is a switch for displaying data of the measured sample, 13' is a switch for switching between a plurality of display data, and 16 is a switch for displaying data of a measured sample. A holder for holding a sample cuvette, 1
7 is a pipettor 1! 8 is a measurement status display device. Here, as an example, an example is shown in which te books are held simultaneously.

The pipettor 17 is for dispensing the sample or latex reagent, and the operation of this pipetter 17 is controlled by the switch 4.
When the sample and latex reagent are dispensed and mixed by the dispensing operation of the pipetter 17, the photometry operation starts and the timer IO starts. In conjunction with this timer 10, the measurement status display device 118 lights up to notify which sample cuvette is undergoing measurement operation.

"Effect" Next, an example in which latex agglutination reaction was measured using the measuring device of the present invention will be shown.

The test was conducted using the following reagents and samples and by the following measurement operations.

Reagent: A latex reagent made by sensitizing polystyrene latex with FDP with an average particle size of 0.085 g m and an antibody titer of 5.
0JLg Ag/ml anti-FDP solution (rabbit origin) sample: Take a standard FDP sample 50IL1 diluted to an appropriate concentration into a test tube cuvette with an inner diameter of 6 layers, and add anti-FDP to this.
Add 200 pL of the solution, mix, and heat at 37° C. for 30 minutes to use as a sample.

Measurement operation: Dispense the latex reagent into the 250 IL sample using a 200 pl pipettor with a switch, and start measurement at the same time as the dispensing. The amount of transmitted light of the sample is measured one after another using the device of the present invention that can perform simultaneous measurements at 18 locations, and the subsequent amount of transmitted light I(t) is calculated with respect to the initial transmitted light amount value Io.
The ratio R(t) was obtained from the computer of the measuring device.

However, R(t) in this test was calculated using the following formula.

R(t)=It/Io The results were as follows.

(1) Compatibility of measured values due to correction The difference between Io and I(t) (however, t-2 minutes or 3 minutes) in each measurement optical system is defined as Δl, and the comparison with R(t) is shown in the following table. 1: t = 2 minutes Table 2: t = 3 minutes In either case, since the sensitivity of each optical system is different, Δ■ is not compatible, but the value of R(t) according to the present invention is Since the difference in sensitivity has been corrected, it is a compatible amount between different optical systems. (2) R( t), the calibration relationship with FDP concentration was measured. The results are shown in Figure 5.

(3,) Reproducibility R(t) measured with each optical system for two types of samples in the cases of t = 2 minutes and t = 3 minutes is converted into a concentration value using the calibration curve in Figure 5. Converted. The results are shown in Tables 3 and 4 below.

, Ru/, Senkin ≦) '1 Daisoku..., r Table 3: t-2 minutes Table 4: When t = 3 minutes and t = 2 minutes, for the 48 samples measured in (1) to (3) above , it takes only a short time to get results from measurement operations! It takes about 2 minutes, and according to the present invention, a large amount of specimen can be measured quickly with a simple operation.

"Effects of the Invention" As described above, according to the present invention, it is possible to start measuring multiple samples one after another at any timing, and by the time the photometry holder is full of sample cubes, the first Now that the sample measurement has been completed, by using it again,
A large number of samples can be measured quickly and efficiently without any idle time.

Moreover, since it has a simple configuration, such a large-volume sample processing apparatus can be manufactured at low cost, and each sample can be operated independently at any timing, so it has excellent operability.

[Brief explanation of the drawing]

Fig. 1 is a block diagram showing an embodiment of the present invention, Fig. 2 is a perspective view of the measuring device of the present invention, and Fig. 3 is a graph showing the reaction time course when the latex agglutination reaction is measured as a change in the amount of transmitted light. , Fig. 4 is a graph showing the relationship between transmitted light l ratio and reaction time, and Fig. 5 is a graph showing the relationship between transmitted light l ratio and reaction time.
FIG. 6 is a graph showing the measurement of the calibration relationship with the DP concentration, and is a basic block diagram of the present invention. In the figure. 1...Sample cuvette, 2...Light source, 3...Photoelectric detector, 4...Switch mechanism for instructing the start of transmitted light measurement, 5...Multiplexer, 7...A/D converter ,
8... Computer, lO... Timer, 11...
Memory, 16...Holder, 17...Pipetter-1
18... Sensitivity 1 degree correction circuit Patent applicant Wako Pure Chemical Industries, Ltd. "Figure 2 Figure 3 Figure 1 (t) Time (min,) Figure 4 -Bu'i (min,) Figure 5 FDP concentration (, ug/rr+z) Figure 6 Procedural amendment July 7, 1985 1, Indication of the incident 1985 Patent application No. 162462 λ Name of the invention Latex agglutination reaction measuring device Contact information 7): L03-270-85714, Date of amendment order. 5. Detailed description of the invention in the specification subject to amendment. 6. Contents of amendment (1) "Measurement efficiency is also well"
is corrected as ``high measurement efficiency.'' (2) The statement "On the other hand, there was an optical problem..." from line 7 on page 4 to line 2 on page 5 of the specification has been deleted, and the following is replaced in its place: Insert the text. ``On the other hand, there are many known methods and devices that detect the state of aggregation as a change in optical absorbance.
rch, Biochem, Biophys, Dango, 338-355 (1955), ■Croatica
Chemiea Actj Sat 2, 457-466 (19
70), ■Specification of British Patent No. 989,617. ■Special Publication No. 58-11575, ■Japanese Patent Publication No. 54-10
Publication No. 8693, ■Japanese Unexamined Patent Publication No. 54-108695,
JP 57-149951, ■ JP 57-1
It is described in Publication No. 63848 and the like. That is, for example, ■
Hours and Minutes "Publication No. 11575/1983 describes the near-infrared rays when a mixture of insoluble carrier particles carrying antibodies or antigens or a suspension containing them and the basic medium of the subject is placed in the reference side cell of a spectrophotometer. By controlling the intensity of the transmitted light in the outer region, a reaction between a suspension containing insoluble carrier particles carrying antibodies or antigens in the sample-side absorption cell and an analyte containing the antigen and/or antibody is generated. When the mixture is added, the intensity of transmitted light in the near-infrared region is I, and the formula A = log IO/
The absorbance A is determined from r and the turbidity is measured. Conventionally, this method of determining the state of aggregation as a change in absorbance has been carried out using a reference example and a pair of sample-side absorption cells, as in the above example, in order to cover for the instability of the light source and detector. However, although the above method has the advantage of allowing highly accurate quantification through objective measurement, it requires manual operation to measure changes in the absorbance of multiple samples because it is a measuring device for each sample. The sample cell must be repeatedly moved in and out of the optical system while strictly controlling the reaction time. However, this is very complicated and extremely difficult. Particularly in dynamic measurements in which absorbance is measured at two or more points in the course of a reaction, it is practically impossible to take in and take out a large number of samples and perform parallel measurements. Usually, a method is adopted in which one sample monopolizes the optical system until the end of the measurement, which results in extremely low measurement efficiency. In order to eliminate the trouble of repeatedly putting the sample cell in and out of the optical system, it is possible to incorporate multiple absorbance measurement systems as described above, but the reference side must also be built in at the same time, which would complicate the structure and increase manufacturing costs. Even if it were possible, it would be expensive, and the number of built-in absorbance measurement systems would also be limited. On the other hand, there are commercially available machines that are equipped with a single light source and detector and can automatically exchange multiple sample cells, but these have the drawback of being very expensive. In addition, regardless of the method, blanking the absorbance of the sample using the basic medium of the specimen does not guarantee correct blank absorbance measurement if the actual specimen has coloration or turbidity. (3) "This invention" written on page 5, line 3 of the specification is amended to read "this invention is". (4) “Efficiency” stated on page 5, line 4 of the specification is replaced with “
"It's highly efficient," he corrected. (5) "Difference in sensitivity" stated on page 5, line 14 of the specification is corrected to "However, difference in sensitivity and sample blank." (6) "For example" written on page 5, line 14 of the specification is deleted. (7) “This R(t)” written from line 17 to line 18 on page 5 of the specification is changed to “R(t) in a biochemical reaction system.”
(t) is”. (8) "Naru." written on page 5, line 19 of the specification is amended to "It turns out to be naru." (9) “The inventors are” stated on page 5, line 20 of the specification.
be amended to read, "Furthermore, the inventors of the present invention, etc.""By correcting sensitivity between different optical systems," stated in αQ Mei Specification, Volume 7, Volume 4.
is corrected as "by devising and implementing a method for correcting the sensitivity between different optical systems." Next to "Characteristics" written on page 7, line 6 of the alpha specification, insert a new line and insert "Furthermore, this method also makes it possible to correct sample blanks." (6) "Of the optical system" stated on page 7, line 12 of the specification is corrected to "between the optical systems." (c) "Multiple detections" stated on page 8, line 8 of the specification is replaced with "
Detection between multiple optical systems is corrected. "It is also possible" written on page 10, line 1 of the θmine specification.
Next, insert "Also, the timer 10 can be replaced by one basic timer and a program operation using the computer 8 and memory 11." (g) "One" written on page 10, line 5 of the specification is amended to "one". αe Akira Specification 1 is amended to read "one". cL Description on page 10, line 13 of the specification A “(reaction mixture)”
amend it to "(for example, a reaction mixture"). Qll Specification 1 Ni 1 Amend it to "one". Amend "one by one" written on page 12, line 1 of the alpha specification to "one by one". ``The ``switch'' described on page 12, line 15 of the Kan specification is amended to read ``numeric display device.'' The above procedural amendment dated October 20, 1986

Claims (9)

[Claims] (1) [1] A means for holding a plurality of sample cuvettes containing a mixture (test sample liquid) of the sample and a latex reagent that reacts with the antigen or antibody in the sample to cause an agglutination reaction; [2] means for irradiating each of the sample cuvettes with a light beam and separately detecting the amount of transmitted light for each; [3] means for correcting the difference in sensitivity in detecting the amount of transmitted light for each; a means for instructing the start of measurement of the amount of transmitted light; and [5] a timer for separately measuring the elapsed time from the start of measurement of the amount of transmitted light of the test sample liquid for each of the sample cuvettes; A latex agglutination reaction measuring device characterized by measuring changes over time in the amount of transmitted light. (2) the means for correcting the sensitivity difference in detecting the amount of transmitted light is a means for storing the amount of transmitted light at an initial stage of reaction of the test sample liquid;
2. The measuring device according to claim 1, further comprising means for determining a ratio of a subsequent amount of transmitted light to the amount of transmitted light at the initial stage. (3) The means for correcting the sensitivity difference in detecting the amount of transmitted light includes means for calculating the ratio of the blank value of the amount of transmitted light of the pseudo test sample liquid and the amount of transmitted light of the test sample liquid, or the logarithm of the ratio. A measuring device according to claim 1, characterized in that: (4) Claims 1 to 3, wherein the means for instructing the start of measuring the amount of transmitted light of the test sample liquid is a means for instructing each of the plurality of cuvettes independently in time or position. The measuring device according to any one of Items. (5) Any one of claims 1 to 3, wherein the means for instructing the start of measuring the amount of transmitted light of the test sample liquid is linked to the dispensing operation of a sample or latex reagent dispenser. The measuring device according to item 1. (6) The measuring device according to claim 2, wherein the amount of transmitted light in the initial stage is the amount of transmitted light at any specific time within a certain period of time in the initial stage. (7) The measuring device according to claim 2, wherein the amount of transmitted light in the initial stage is the maximum value of the amount of transmitted light within a certain period of time in the initial stage. (8) The measuring device according to claim 2, wherein the amount of transmitted light in the initial stage is an average value of the amount of transmitted light within a certain period of time in the initial stage. (9) The measuring device according to claim 2, wherein the amount of transmitted light in the initial stage is the minimum value of the amount of transmitted light within a certain period of time in the initial stage.