JPS6116016B2 - - 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 reaction container whose bottom surface is curved in the shape of a wine cup is used, and a 2 to 5% suspension of test blood cells obtained by centrifugation and a specific antiserum are quantitatively dispensed into the container. After stirring both, let stand,
Next, centrifugation is performed, and the reaction container is violently vibrated to shake out the precipitated blood cells, and then vibrated relatively slowly to collect the agglomerated components in the center of the bottom of the container to form an agglutination pattern. A method of photometrically detecting this has been proposed. That is,
This method utilizes the phenomenon that coagulated and bonded particles are quickly collected in the center of the container, whereas unbonded particles are redispersed in the solution and do not collect in the center of the container. In this blood type determination method, the reaction vessel is violently shaken after centrifugation to separate the precipitated blood cells from the bottom of the vessel, and is therefore 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 detection and measurement of HBs antigen,
A method using a microplate equipped with a large number of reaction vessels each having a conical bottom surface has been adopted. This method uses, for example, a 10×12-well microplate to detect and measure HBs antigen using the method described below.

(1) Add one drop (0.025ml) 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 solution to the first column of the sample dilution series 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-PHA cell (0.025ml of 1% suspension)
Add to each hole.

(6) Shake thoroughly for 10 seconds with a micro mixer, and
- Uniformly suspend PHA cells.

(7) Detect the aggregation pattern after leaving for 1 hour at room temperature 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 proposed a blood type determination method that can satisfactorily determine not only blood type using natural antibodies with strong agglutinating force, but also blood type using irregular antibodies with extremely weak aggregating bonding force. 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 to determine the blood type in this reaction container, stirs the blood, and processes the blood for a relatively short period of time. The blood type is determined by detecting the agglutination pattern after the device is allowed to stand for about 30 minutes. In this method, when the blood cells to be tested react with the antiserum reagent, the precipitated blood cells aggregate and are deposited thinly like snow on the bottom of the conical shape, forming a uniform deposition pattern. If they do not react with the reagent, the blood cell particles do not aggregate, but settle in a discrete manner. When they reach the conical bottom, they roll down the slope, and gather at the center of the conical bottom to form an accumulation pattern. Therefore, the blood type can be determined by optically 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 lies in 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 periphery does not change with respect to the standard value, it is determined that there is no aggregation.
It can be determined that 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. The incident light is scattered by the particles, and the amount of light incident on the second light receiving element through the annular aperture increases,
The drawback is that accurate photometry cannot be performed. In other words, when 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 close enough to the bottom of the reaction vessel due to physical layout, Since the light needs to be illuminated and received above the container, if the mask cannot be placed sufficiently close to the reaction solution, light scattered by particles in the reaction solution will reduce photometry accuracy, leading to inaccurate determinations. It becomes possible. 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, which is not a problem especially when the reaction vessel is large, but when it is necessary to use a small reaction vessel to reduce the sample amount, it is necessary to make the detection optical system small. This makes manufacturing and adjustment difficult.

Furthermore, when detecting and determining the agglomeration pattern formed on the bottom surface of the reaction vessel by the above-mentioned sedimented particles,
If the turbidity measurement method described above is adopted, the measurement accuracy cannot be improved and accurate determination cannot be made. 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 surface of the reaction vessel is not always clearly formed, and a pattern intermediate between a uniform deposition pattern and an accumulation pattern may also be formed. It is also necessary to accurately read and judge the agglomeration pattern, including the pattern. Furthermore, when detecting aggregation patterns photoelectrically, it is necessary to take into account scratches formed on the bottom of individual reaction vessels, dust attached to the bottom, variations in transmittance of individual reaction vessels, and variations in the concentration of individual reaction solutions. , fluctuations in the light source, uneven illumination of the light source, and other various factors are mixed in, and these become background noise. Such background noise may make accurate determination difficult. Additionally, in order to reduce the effects of such background noise, it is necessary to clearly form aggregation patterns, but this requires a sufficiently long standing time, which reduces processing efficiency. At the same time, it is necessary to increase the amount of sample, which makes analysis of ultra-trace amounts difficult.

The purpose of the present invention is to eliminate the various drawbacks of the above-mentioned conventional methods, eliminate the influence of the background noise mentioned above, and accurately detect and judge the agglomeration pattern formed on the bottom surface of a reaction vessel. Moreover, it is an object of the present invention to provide a method for determining a particle aggregation pattern that can be carried out easily and inexpensively, and can quickly obtain determination results for a small amount of sample.

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. The image of the bottom surface is focused on the light-receiving surface of the light-receiving device by an imaging lens, and the image of the bottom surface is detected by the light-receiving device before an agglomeration pattern is formed on the surface of the reaction vessel. The output is memorized as background noise, and then the image of the bottom surface is detected by a light receiving device during or after the formation of the agglomerated pattern, the memorized background noise is subtracted from the output, and this subtraction output is obtained. This method is characterized in that the agglomeration pattern is determined by processing.

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 collimator 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 is made into a conical shape. When performing an immunological analysis using a particle agglutination reaction, for example, a predetermined amount of a hemocyte suspension of sample blood is dispensed into the reaction container 4, and then a predetermined amount of an antiserum reagent is dispensed, and after stirring, It is necessary to allow the blood cell particles to settle in a substantially stationary state and to detect the aggregation pattern formed on the bottom of the container to make a determination.

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 A 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 on the bottom surface and are less likely to roll down the inclined surface, so that they are almost uniformly deposited on the bottom surface. When this uniform deposition pattern is observed in detail, it is found that it is quite thickly deposited at the bottom part 5A in the center, whereas it is deposited slightly thinner at the peripheral part 5C, and in 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 in the area is detected by being imaged on the light receiving device 7 by the imaging lens 6 with a deep depth of focus, but in this example, the difference in brightness between the center 5A and the peripheral area 5C For this purpose, as shown in FIG. 3, the light receiving device 7 is provided with a first light receiving element 7A located at the center and a second light receiving element 7B located at the periphery. After amplifying the outputs of these light receiving elements 7A and 7B with amplifiers 8A and 8B, respectively, an A/D converter 9
is converted into a digital quantity and stored in the memory 10. In the present invention, the above-described detection is performed immediately after dispensing the sample and reagent into the reaction vessel 4, that is, before an aggregation pattern is formed on the bottom surface 4A, and the output obtained from the light receiving device 7 at that time is stored in the memory 10. to be memorized. In this way, the output detected before the formation of the agglomeration pattern takes into account factors such as scratches on the bottom of the reaction vessel 4, attached dust, variations in transmittance, variations in the concentration of the reaction solution, variations in the light source 1, and uneven illumination. This is called background noise. Due to the presence of such background noise, the agglomerated pattern cannot be detected accurately, and detection must wait until the agglomerated pattern is completely formed, resulting in a disadvantage of low processing capacity.

In the present invention, in order to eliminate such drawbacks, as shown in FIG. Lamp 1', collimator lens 2', diffuser plate 3',
An imaging lens 6' and a light receiving device 7' are provided. The reaction container 4 is moved from the first photometric position to the second photometric position as shown by arrow A. The two outputs of the second photodetector 7' are connected to amplifiers 8A' and 8A', respectively.
B' to one input terminal of the first and second differential amplifiers 11A and 11B. The other input terminal of these differential amplifiers is supplied with a signal that reads out the digital quantity stored in the position designated by the addressing circuit 12 and converts it into an analog quantity by the D/A converter 13. This analog signal represents the background noise in the center and periphery as described above.
First and second differential amplifiers 11A and 11B
At this time, this background noise component is removed from the output of the second light receiving device 7', and therefore only a signal truly representing the agglomerated pattern is output.
These outputs are supplied to the third differential amplifier 14,
Find the difference between the center and the periphery of the agglomeration pattern.
Furthermore, this differential output is supplied to one input terminal of the fourth differential amplifier 15, and a reference voltage adjusted to a predetermined value from the reference voltage source 16 is applied to the other input terminal. As described above with reference to FIG.
The differential output supplied from the fourth differential amplifier 15 is smaller than the reference value, and the output of the fourth differential amplifier 15 is, for example, 0 volts. On the other hand, in an accumulation pattern in which no aggregation-bonding reaction occurs and the sedimented particles roll on the bottom of the reaction vessel and accumulate in the center, the brightness of the peripheral area is brighter than that of the center, so the third difference is dynamic amplifier 14
The difference output from the fourth differential amplifier 15 becomes larger than the reference value, and the output of the fourth differential amplifier 15 becomes large, for example, +15V. By supplying the output obtained from the fourth differential amplifier 15 to the judgment display circuit 17 in this way, the agglomeration pattern formed on the bottom surface 4A of the reaction vessel is judged, and the result can be printed out or displayed. can do.

As described above, in the present invention, background noise is detected in advance before the aggregation bonding reaction progresses, this is memorized, and the background noise is detected during or after the aggregation bonding progresses and the aggregation pattern is formed. Since the aggregation pattern is determined by processing the signal after the background noise has been removed from the detected signal, it is possible to make accurate determinations that are not affected by background noise, and the aggregation pattern is completely formed. Since the determination can be made even before the process is performed, processing efficiency is significantly improved. Another advantage is that only trace amounts of samples and reagents are needed.

The present invention is not limited to the above-mentioned example, and can be modified and changed in many ways. For example, in the above-mentioned example, two photometric sections were provided, but of course, only one photometric section could be provided, thereby allowing the test liquid to be photometrically measured twice. In this case, the influence of variations in the first and second photometric sections is completely eliminated. Also,
In the above example, photometry was performed twice, but it is also possible to perform photometry more times. Furthermore, various modifications can be made to the configuration of the signal processing circuit.
For example, in the example shown in FIG. 1, the output of the third differential amplifier 14 is compared with one reference value, but it can also be compared with a plurality of reference values at different levels. Further, in the example described above, the memory 10 is a digital memory, and the output of the first light receiving device 7 is converted into a digital quantity by the A/D converter 9 and then stored, but an analog memory can also be used. /D converter 9
And the D/A converter 13 can be omitted.

FIG. 4 shows another example of the signal processing circuit, and the same parts as those shown in FIG. 1 are denoted by the same reference numerals. In this example, the difference between the outputs of the first light receiving device 7 is stored in advance, and this is subtracted from the difference between the outputs of the second light receiving device 7'. For this purpose, the outputs from the first and second light receiving elements 7A and 7B of the first light receiving device 7 are input to an amplifier 8.
After amplification by A and 8B, the first differential amplifier 20
and find the difference between them. This difference is converted into a digital amount by the A/D converter 9 and stored in the digital memory 10. On the other hand, the first light receiving device 7'
The outputs from the second light-receiving elements 7A' and 7B' are amplified by amplifiers 8A' and 8B', respectively, and then supplied to the second differential amplifier 21. is supplied to one input terminal of the At this time, the other input terminal is supplied with the difference signal read from the address position of the memory 10 designated by the address designating circuit 12, and the difference between these two difference signals is determined. The differential output of the third differential amplifier 22 is compared with the reference value from the reference voltage source 16 in the fourth differential amplifier 15 as in FIG. 1, and the comparison result is supplied to the judgment/display circuit 17.

Furthermore, in the example described above, a reaction vessel with a conical bottom was used, but a single-sided roof type, a gable roof type,
Reaction vessels having bottoms of various shapes, such as pyramid shapes, can also be used.

Furthermore, the structure of the light receiving device can be modified in various ways, for example, as shown in FIG.
The light-receiving elements 30A and 30B may be arranged in the center and the periphery, or a large number of light-receiving elements 31A to 31J may be arranged in a distributed manner as shown in FIG. It is also possible to arrange such linear solid-state imaging devices 32 in the diametrical direction, or to arrange three light receiving elements 33A to 33C concentrically as shown in FIG. 5D.

[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 of the present invention, FIG. 2 is a diagram showing the reaction vessel and the aggregation pattern formed on its bottom surface, and FIG. FIG. 4 is a diagram showing a modification of the apparatus for carrying out the method of the present invention; FIGS. 5A, B, C, and D are plan views showing modifications of the light receiving device. be. 1, 1'... Light source lamp, 2, 2'... Collimator lens, 3, 3'... Diffusion plate, 4... Reaction container, 6,
6'...Imaging lens, 7,7'...Light receiving device, 10...Memory, 11A, 11B, 14, 15, 20, 2
1, 22... Differential amplifier, 16... Reference voltage source, 17
...Judgment display circuit.

Claims (1)

[Claims] 1. In order to photoelectrically detect and judge the particle aggregation pattern formed on the bottom surface by the settling of particles in a reaction solution contained in a reaction container with a slightly inclined surface on the bottom surface, the bottom surface of the container is uniformly illuminated. Then, the image of the bottom surface is focused on the light receiving surface of the light receiving device by an imaging lens, and the image of the bottom surface is detected by the light receiving device before the agglomeration pattern is formed on the bottom surface of the reaction vessel, and its output is back-up. The ground noise is stored as ground noise, and then the image of the bottom surface is detected by a light receiving device during or after the formation of the agglomerated pattern, the memorized background noise is subtracted from the output, and this subtraction output is processed. A method for determining a particle aggregation pattern, characterized in that the aggregation pattern is determined by