JPS6159454B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for determining agglutination patterns by immunological agglutination reactions, and in particular, particle agglutination patterns for determining various blood types and detecting antibodies and antigens from agglutination patterns of blood cell particles. This relates to a determination method.

For example, as a method for determining blood type,
Publication No. 16798 discloses that a 2 to 5% suspension of test blood cells obtained by centrifugation and a specific antiserum are quantitatively dispensed into the container using a reaction container with a curved bottom in the shape of a wine cup. After stirring both, let them stand, then perform centrifugation, shake the reaction container vigorously to shake out the precipitated blood cells, and then vibrate relatively slowly to remove the aggregated components from the center of the bottom of the container. A method has been proposed in which a cohesive pattern is formed by collecting the agglomerated particles, and the agglomerated pattern is photometrically detected. That is, this method utilizes the phenomenon that aggregated and bonded particles are quickly collected in the center of the container, whereas unbonded particles are dispersed again in the liquid and do not collect in the center of the container. In this blood type determination method, after centrifugation, the reaction container is shaken vigorously to separate the precipitated blood cells from the bottom of the container.
It is used to determine the ABO blood type, which has a strong cohesive bond.

However, in the case of immunological agglutination reactions with weak binding strength, such as when determining the RH blood type or when detecting various irregular antibodies, antigens, HBs antigens, etc., the determination method described above cannot be used. is not available.
That is, if the cohesive bonding force is weak, particles such as blood cells that are once bound will separate when the reaction container is vibrated, and will not collect in the center of the reaction container. Therefore, for the detection and measurement of HBs antigen, a method has been adopted that uses a microplate equipped with a number of reaction vessels each having a conical bottom. This method uses, for example, a 10×12-well microplate to detect and measure HBs antigen using the method described below.

1 Add 1 drop (0.025 ml) of R-PHA buffer to each well of the microplate.

2. Take the sample into a diluter and dilute it twice in two series up to 10 tubes.

3. Add one drop (0.025 ml) of R-PHA buffer to the first column of the sample dilution column and one drop (0.025 ml) of R-PHA inhibition solution to the second column.

4 After shaking thoroughly for 10 seconds with a micro mixer,
Incubate at ℃ for 1 hour.

5 1 drop of R-PHAcell (0.025ml of 1% suspension)
Add to each hole.

6 Shake thoroughly for 10 seconds with a micro mixer, and
Float PHAcell uniformly.

7. Detect the aggregation pattern after leaving at room temperature for 1 hour avoiding vibration.

According to this detection method, the reaction container is left sufficiently stationary without being subjected to vibration immediately before detection, so that the precipitated aggregates are not separated, and the reaction is caused by the immunological agglutination reaction, which has a relatively weak aggregation bond. Agglomeration patterns can be obtained.

On the other hand, the applicant, in Japanese Patent Application No. 54-53370,
We have proposed a blood type determination method that can satisfactorily determine not only blood type using natural antibodies with strong aggregation strength, but also blood type using irregular antibodies with extremely weak aggregation strength. Such a blood type determination method uses, for example, a reaction container with a conical bottom, and contains blood corpuscles and a standard antiserum reagent in which the blood type is to be determined, and stirs them for a relatively short period of time ( Blood type is determined by detecting the agglutination pattern after allowing the device to stand for approximately 30 minutes. In this method, when the blood cell particles to be tested react with the antiserum reagent, the precipitated blood cell particles aggregate and bond, and are deposited thinly like snow on the conical bottom to form a uniform deposition pattern. If the blood cells do not react with the antiserum reagent, the blood cells do not aggregate and settle in a discrete manner. When they reach the conical bottom, they roll down the slope and gather in the center of the conical bottom to form an accumulation pattern. do. Therefore, the blood type can be determined by photoelectrically detecting the difference in the pattern of precipitated blood cells depending on the presence or absence of reaction with the antiserum reagent formed on the bottom of the cone.

In the various aggregation pattern determination methods described above, the problem is how to detect the aggregation pattern formed at the bottom of the reaction vessel. For example, Special Publication No. 51-16798 using a wine cup-shaped reaction container.
The method described in the publication employs a turbidity measurement method that detects the amount of light transmitted through the reaction solution.
That is, when a light beam passes through the reaction liquid, the degree of absorption changes due to blood cell particles suspended in the liquid from the liquid surface to the bottom surface, and this is measured photoelectrically. In the embodiment shown in FIG. 33 of the above-mentioned publication, light is incident from above a wine cup-shaped reaction container, and a mask having a center opening and an annular opening surrounding it is arranged below the reaction container. The light that has passed through the annular opening is made to enter the first light receiving element, and the light that has passed through the annular opening is made to enter the second light receiving element through the lens. Therefore, the amount of light that passes through the center of the reaction solution in the reaction container and enters the first light receiving element represents the turbidity of the center of the reaction solution, and the amount of light that passes through the periphery of the reaction solution and enters the first light receiving element. The amount of light incident on the second light receiving element represents the turbidity of the peripheral portion of the reaction solution. Therefore, if the amount of light passing through the center of the reaction solution decreases compared to the standard value and the amount of light passing through the periphery increases compared to the standard value, this is considered to be "agglomeration."
If the amount of light passing through the center and the periphery does not change with respect to the reference value, it can be determined that there is no aggregation. This method of detecting and determining agglomeration patterns is considered to have no problem if the distance from the agglomerate formed at the center of the bottom of the reaction vessel to the central photometric opening is small compared to the lateral spread of the aggregation pattern. It is conceivable that if the distance from the agglomerate to the central aperture becomes longer, the light incident on the periphery of the reaction liquid will be scattered by the particles and will enter the first light-receiving element through the central aperture, or vice versa. There is a drawback that the incident light is scattered by the particles, and the amount of light incident on the second light receiving element through the annular opening increases, making accurate photometry impossible. That is,
In cases where the diameter of the reaction vessel is large compared to the thickness of the bottom and it is not possible to place a mask with an opening sufficiently close to the bottom of the reaction vessel due to physical layout, or when illuminating from the bottom of the vessel, Since light needs to be received above the container, if the mask cannot be placed sufficiently close to the reaction solution, light scattered by the reaction solution and particles will reduce measurement accuracy, making accurate determination impossible. In order to eliminate this drawback, it may be possible to increase the size of the reaction vessel in order to increase the difference in turbidity, but in this case, the amount of sample increases, making it impossible to analyze a trace amount of sample. In addition, the detection optical system is quite complex, and although this is not a problem especially when the reaction vessel is large, it is difficult to make the detection optical system compact when it is necessary to use a small reaction vessel to reduce the sample amount. This makes manufacturing adjustments difficult.

Furthermore, when detecting and determining the aggregation pattern formed on the bottom surface of the reaction vessel by the above-mentioned sedimentation element, adopting the above-mentioned turbidity measurement method does not improve measurement accuracy and makes accurate determination impossible. In particular, when automatically detecting and determining a uniform deposition pattern and an accumulation pattern formed on the bottom surface of a reaction vessel by particles that settle in this way, a highly accurate detection device is required. In particular, the agglomeration pattern formed on the bottom of the reaction vessel is not necessarily clearly formed, and an intermediate deposition pattern between the uniform deposition pattern and the accumulation pattern may also be formed. It is also necessary to accurately read and judge the agglomeration pattern, including the intermediate deposition pattern.

The purpose of the present invention is to eliminate the various drawbacks of the conventional methods described above, and to be able to accurately detect and determine various aggregation patterns formed on the bottom surface of a reaction vessel.
Furthermore, the present invention aims to provide a method for determining a particle aggregation pattern that can be easily and inexpensively implemented.

The present invention provides a method for photoelectrically detecting and determining a particle aggregation pattern formed on the bottom surface by precipitation of particles in a reaction solution contained in a reaction container whose bottom surface has a slightly inclined surface. is uniformly illuminated, and an image of this bottom surface is formed by an imaging lens on the light receiving surface of a light receiving device. This method is characterized by arranging scanning-type light-receiving elements and appropriately selecting and processing image signals obtained from these light-receiving element arrays to determine an agglomeration pattern.

The present invention will be described in detail below with reference to the drawings.

FIG. 1 is a diagram showing the configuration of an example of an apparatus for carrying out the particle aggregation pattern determination method according to the present invention. The light from a light source lamp 1 is made into a parallel light beam by a commerita lens 2, and is made to uniformly enter a reaction vessel 4 via a diffuser plate 3. The bottom surface of the reaction vessel 4 has a conical shape.

FIG. 2 is an enlarged view of the bottom surface of the reaction vessel 4, showing a state in which particles are bonded and aggregated and deposited uniformly on the conical bottom surface. Such a uniform deposition pattern is obtained, for example, when performing ABO blood type determination, when a type B antiserum reagent is added to a blood cell suspension of a type A sample blood and allowed to settle naturally. be. That is, in this case, the blood cell particles that have settled due to standing still are bonded to each other at the bottom surface and are less likely to roll down the inclined surface, so that they are almost uniformly deposited on the bottom surface. If we observe this uniform deposition pattern in detail, we can see that it is quite thickly deposited at the lowest part 5A in the center, but it is deposited slightly thinner at the peripheral part 5C, and at the middle part 5B between them.
The thickness changes almost continuously.

In the present invention, the bottom surface 4A of the reaction container 4 is
The agglomerated pattern formed is imaged onto a one-dimensional light receiving device 7 by an imaging lens 6 with a deep depth of focus.
In the present invention, as this light receiving device 7, a CCD,
A one-dimensional light receiving device such as a photodiode array with a BBD, MOS type IC, or scanning circuit is used. In this example, a self-scanning one-dimensional CCD is used.
The one-dimensional CCDs are arranged so as to pass through the center of the pattern image on the bottom surface of the reaction vessel 4 formed by the imaging lens 6, as shown by the chain line in FIG.

The light receiving device 7 is driven by a drive circuit 8, and output image signals from each light receiving element are extracted in time series. This image signal is an analog signal, which is amplified by an amplifier 9, converted to a digital signal by an A/D converter 10, and stored in a memory 11. The address for this memory is supplied from the drive circuit 8. A sample number is also supplied to the memory 11 from the sample identification number detection circuit 12, and a digital quantity detected from the sample corresponding to the sample number is stored in a predetermined position. Further, an addressing circuit 13 is provided so that a designated sample number and digital information corresponding thereto are read out from the memory 11. The digital amount read out from the memory 11 is transferred to the adder circuit 14, where the outputs from each light receiving element are added in a predetermined manner, and the result of this addition is converted into an analog amount by the D/A converter 15.
The signal is supplied to a comparison circuit 16. This addition output is, for example, the sum of the outputs of the light receiving element group that receives the image of the central part 5A of the agglomerated pattern shown in FIG.
It can be the sum of the outputs of the light receiving element group that receives the image of C. The comparison circuit 16 compares these outputs, determines the agglomeration pattern based on the comparison result, and supplies the determination result to a printer or display 17. The sample number is also supplied to this printer or display 17 from the memory 11 and is also printed or displayed. In this way, the sample and the determination result can be accurately identified.

3A to 3C show the relationship between the agglomeration pattern formed on the bottom surface of the reaction vessel 4 and the light receiving element array. FIG. 3A shows an accumulation pattern in which no aggregation and bonding reaction of the particles occurs, and the particles settle and hit the bottom of the container, then roll down and accumulate in the center.
FIG. 3C shows the uniform deposition pattern shown in FIG. 2, and FIG. 3B shows an intermediate state between these, which is referred to as the intermediate deposition pattern. In this example, the one-dimensionally arranged light receiving elements are divided into 11 groups. It is preferable that the number of light receiving elements included in each group is the same. In the case of the integrated pattern shown in FIG. 3A, the light-receiving elements are in group 6 in the center, and in the intermediate stacked pattern in FIG.
In the uniform deposition pattern shown in FIG. C, a pattern image is formed on all the light receiving elements of the group 111. Therefore,
First, the outputs of the light receiving elements of the center group 6 are stored in the memory 11.
The data is read out from the source and supplied to the adder circuit 14 for addition.
Next, the outputs of the light-receiving elements of one group in the periphery, for example, group 2, are read out from the memory 11 and added. These two added outputs are converted into analog quantities by a D/A converter 15, and compared by a comparison circuit 16. At this time, the integrated pattern shown in FIG. 3A and FIG.
In the intermediate deposition pattern shown in FIG. 3C, the values of the two summed values are significantly different, whereas in the uniform deposition pattern shown in FIG. 3C, the two summed values are approximately equal. Therefore, by comparing these added values, it is possible to clearly distinguish between the integrated pattern, the intermediate deposition pattern, and the uniform deposition pattern. Next, for example, the outputs of the light receiving elements in the fourth group 4 are read out from the memory 11, added as described above and converted into analog quantities, and this added value is compared with the added value of the outputs of the light receiving elements in the sixth group 6. . In this case, in the accumulation pattern shown in FIG. 3A, the difference between both added values is large, whereas in the intermediate accumulation pattern shown in FIG. 3B, the difference between both added values is small. Deposition patterns can be accurately distinguished. By sequentially reading out the outputs of the light receiving elements from the memory 11, adding them, and comparing them, it is possible to determine not only the patterns shown in FIGS. 3A to 3C, but also various other patterns with great accuracy. .

FIG. 4 shows the configuration of an example of the comparison circuit 16. For example, the sum total of the outputs of the fourth group of light receiving elements in the intermediate portion is supplied to one input terminal 16a,
The other input terminal 16b has a second input terminal located at the periphery.
It is assumed that the sum of the outputs of the group of light receiving elements is supplied. The difference between the sums of these outputs is calculated by the first differential amplifier 1.
6c, and the difference output is sent to the second differential amplifier 16.
d to one input terminal. A reference voltage is applied to the other input terminal from a reference voltage source 16e.
This reference voltage can be adjusted to a value approximately equal to the sum of the respective outputs of the fourth group and the sixth group of light receiving elements when detecting the intermediate deposition pattern shown in FIG. 2B, for example.

As described above, the sample number and the output signal from the light receiving device 7 are temporarily stored in the memory 11, and later read out and processed as needed, so that the judgment results can be read once after processing a large number of samples. It can be printed out and displayed. In this case, necessary data can be selectively read out depending on the processing mode, and more accurate determination can be made.

The present invention is not limited to providing the memory 11 and storing digital information therein as described above, but the memory can also be omitted.
Figures 5 and 6 illustrate such an embodiment. 5 and 6, the light receiving device 7 is driven by a drive circuit 8, its output is amplified by an amplifier 9, and then converted into a digital quantity by an A/D converter 10, similar to the above embodiment. It is.

In FIG. 5, for example, the outputs of the sixth group of light-receiving elements in the center are sequentially supplied to the first adder 21 to obtain the sum, and the sum is converted into an analog quantity by the first D/A converter 22. Further, the outputs of the second group of light receiving elements in the peripheral area are added in a second adder 23, and the added output is converted into an analog quantity in a second D/A converter 24. Both of these added values are supplied to a comparison circuit 25, and the comparison result is supplied to a judgment and display circuit 26, where judgment is made and the judgment result is displayed.
Of course, it is not possible to accurately determine intermediate deposition patterns in this one-time process, so the same process is performed next on the outputs of the fourth and sixth groups of light receiving elements, as in the above example. A selection circuit 27 is provided for selecting this group of light receiving elements, which is synchronously driven by a drive circuit 8, and its output controls the operation of an A/D converter 10 having a switching function.

In the embodiment shown in FIG. 6, the A/D converter 1 is
For example, the first adder 31 adds the outputs of the sixth group of light-receiving elements in the center, and the second adder 32 adds the outputs of the second group of light-receiving elements in the periphery. The difference between these addition outputs is determined by a subtracter 33,
This difference output is supplied to the determination/display circuit 34.

As described above, according to the present invention, the agglomerated pattern formed on the bottom surface of a reaction vessel is arranged on a scanning type light receiving device using an imaging lens, and the light receiving element of this light receiving device is passed through the center of the pattern, and an image of the bottom surface is detected. By arranging them one-dimensionally over the entire area and selectively extracting and processing the outputs of these light receiving elements, not only integrated patterns and uniform deposition patterns, but also intermediate deposition patterns can be determined extremely accurately. . In particular, it is easy to increase the resolution of such a light receiving element, and thereby the determination accuracy can be further improved. In particular, sample throughput can be improved by using a memory to temporarily store the output of the light-receiving element and then selectively reading it out and processing it later. In addition, it is possible to easily and accurately identify the sample and the determination result.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the configuration of an example of an apparatus for carrying out the particle aggregation pattern determination method according to the present invention, and FIG.
The figure is also a diagram showing the configuration of the reaction vessel and the agglomeration pattern formed on its bottom surface, Figure 3A,
B and C are diagrams showing the relationship between various aggregation patterns and a light receiving device, FIG. 4 is a circuit diagram showing the configuration of an example of a comparison circuit, and FIGS. 5 and 6 are diagrams showing the particle aggregation pattern determination method according to the present invention. FIG. 3 is a block diagram showing another example of an apparatus for implementing the above. DESCRIPTION OF SYMBOLS 1...Light source, 4...Reaction container, 5A...Central part, 5B...Intermediate part, 5C...Peripheral part, 6...Imaging lens, 7...Scanning type one-dimensional light receiving device, 8...
Drive circuit, 10...A/D converter, 11...Memory, 13...Addressing circuit, 14...Addition circuit, 15...D/A converter, 16...Comparison circuit,
17... Judgment/display circuit, 27, 30... Selection circuit, 21, 23, 31, 32... Adder, 22,
24...D/A converter, 25...Comparison circuit, 2
6, 34...judgment/display circuit, 33...subtractor.

Claims (1)

[Scope of Claims] 1. In photoelectrically detecting and determining a particle aggregation pattern formed on the bottom surface by sedimentation of particles in a reaction solution contained in a reaction container whose bottom surface has a slightly inclined surface, the container The bottom surface is uniformly illuminated, and an image of the bottom surface is formed by an imaging lens on the light receiving surface of a light receiving device. A method for determining a particle aggregation pattern, characterized in that an aggregation pattern is determined by arranging scanning-type light-receiving elements, and appropriately selecting and processing image signals obtained from the array of these light-receiving elements. 2. A patent claim characterized in that the image signal obtained from the light-receiving element array is converted into a digital quantity and then stored in a memory, and then the information selectively read out from this memory is processed to determine the agglomeration pattern. The method for determining a particle aggregation pattern according to item 1.