JPS6114459B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method and a measuring device for effectively measuring suspended solids in a suspension, and in particular, the present invention relates to a method and a measuring device for effectively measuring suspended solids in a suspension. The present invention relates to a method and apparatus for determining the amount or concentration of an immunochemical complex by measuring a liquid sample existing as an immunochemical complex by nephelometry.

In general, the dimensions of the immunochemical complexes formed as a result of antigen-antibody reactions are compared with the dimensions of the various component molecules originally contained in the serum sample, antiserum solution, buffer solution, etc. that make up the reaction solution. It's obviously big.
Therefore, the light scattering ability of the reaction solution increases as a result of the antigen-antibody reaction, so it can be used for nephelometric analysis.

However, dust particles or air bubbles may be accidentally mixed into the reaction solution, and the scattered light intensity value obtained by turbidimetry may not necessarily be due to the generated immunochemical complex. It is often not limited. Particularly when measuring antigens that are present in extremely small amounts in serum or those that are out of the proper mixing ratio, the amount of immunochemical complexes produced is small, and these dust particles and air bubbles are Contamination gives fatal measurement errors.

However, the size of dust particles and bubbles is determined not only by comparison with the various component molecules originally contained in the serum sample, antiserum solution, buffer solution, etc. that make up the reaction solution, but also by the size of the particles formed as a result of antigen-antibody reactions. Although it is larger than an immunochemical complex and has a greater light scattering ability, the various component molecules mentioned above and the immunochemical complex are mixedly integrated, and their light scattering is almost the same no matter where they are taken. Dust particles and air bubbles that accidentally enter the sample have a low concentration relative to serum samples, etc., and their scattering is perturbed.

Focusing on this point, we irradiated the antigen-antibody reaction solution contained in a test tube with a laser beam, and observed the scattered light generated by this laser beam using a photodetector for a certain period of time. A technique that eliminates fluctuations in the scattered light intensity as being caused by mixed dust particles and uses the minimum scattered light intensity obtained during the observation period as the measured value (for example, Japanese Patent Application No. 154687-1982)
(Refer to Japanese Unexamined Patent Publication No. 51-90883)). This means that even if dust particles accidentally enter the laser beam and emit spurious scattered light that crosses the irradiated laser beam, it will not be added to the measured value, and the measured value will not be added to the measured value. This is assumed to be determined by the uniform scattered light.

However, if dust particles or air bubbles are suspended in a small space irradiated by the laser beam and are not removed from the space during the observation period, errors will obviously occur. Laser beams are used to minimize the probability of such phenomena occurring, and making the laser beam narrower is effective, but it is impossible to reduce the probability determined by the dust particles themselves. be.
Therefore, dust particles that do not move due to the Brownian motion of the solution molecules and may remain in the space irradiated by the laser beam cause extreme measurement errors in the above method.

The present inventors believe that the immunochemical complexes generated as a result of antigen-antibody reactions are sufficiently uniformly dispersed in the concept of turbidimetry, and that the intensity of scattered light is uniform in any part of the reaction solution. Therefore, by forcing the reaction solution to flow across the irradiated light beam in a constant state, the space occupied by the dust particles or bubble particles can be reduced to a very short period of time passing across the light beam. The present invention was completed by noting that a uniform scattered light output can be obtained except for time. That is, by distinguishing between this uniform scattered light output and the instantaneous pulse-like output caused by dust particles or air bubbles, it is possible to obtain nephelometric measurements that can truly avoid the effects of dust particles or air bubbles. It is something that can be obtained.

Therefore, the first objective of the present invention is to measure the amount of immunochemical complexes without the influence of dust particles or air bubbles, and furthermore, it is considered difficult to measure them because they exist in extremely small amounts. The aim is to enable the analysis of many serum-specific proteins. A secondary object of the invention is to produce a very inexpensive device that does not require the employment of special light sources such as laser beams.

Hereinafter, the present invention will be explained in detail with reference to the drawings. FIG. 1 is an explanatory diagram showing the measurement principle of the present invention, showing a fluidized cell body 3, a suspension supply means for forcefully flowing a reaction liquid 41, which is a suspension _4, into the fluidized cell body 3, and the fluidized cell body 3. 3, and a means for measuring and displaying the amount of light scattered by the reaction solution ₄₁ .

First, the light emitted from the light source lamp 1 is made into a constant light velocity by the condensing lens 2, and irradiates the reaction liquid ₄₁ in the fluidized cell body 3, causing the reaction liquid in the fluidized cell body 3 to become a suspension. Scattered light is emitted while the liquid ₄₁ is flowing. The reaction liquid ₄₁ is forced to flow through the fluid cell body 3 at a constant speed by the suction action of a suspension supply means, for example, a peristaltic pump 5, and is then forced into a drain tank 6.
is discharged. On the other hand, the scattered light is observed and recorded over time by a photodetector 7, a signal amplifier 8, and a recorder 9.
Provide data as shown in FIG. In this case, the light source lamp 1 may be a tungsten lamp or the like, and any light source system that emits a stable light flux with a constant amount of light may be used without using the condenser lens 2. The photodetector 7 may be a photocell tube or other light source. Any type of device can be used as long as it converts into an electrical signal. The type and shape of the fluid cell body 3 does not matter as long as it is a fluid type used for nephelometric analysis, but it is also possible to use a fluid cell body ₃₁ with an annular outlet that is excellent in air bubble discharge as shown in FIG. can.

Curve A shown by a solid line in FIG. 2 is an example of a scattered light output curve recorded using the reaction solution ₄₁ using the above apparatus, where the horizontal axis represents time t and the vertical axis represents scattered light output _VE . In the figure, the base of the curve is almost a horizontal straight line (this DC level is V _E1 ), and several pulses P ₁ , P ₂ , etc. protrude from this base.
...is recognized. These pulses P ₁ , P ₂ . . . are clearly caused by the passage of dust particles or air bubbles, and the DC level at the base is caused by the immunochemical complex and the reaction product particles in the reaction solution ₄₁ . It represents the strength of uniform scattering determined by the amounts of various component molecules that are smaller than the complex. This can be demonstrated by preparing multiple samples containing graded amounts of this immunochemical complex and recording the scattered light output curve for each sample in the same manner. The height _of the pulses P ₁ , P ₂ .

Curve B indicated by a dotted line in the figure is one of the scattered light output curves measured and recorded in the same manner for a certain pre-reaction solution ₄₂ .
This is an example. Pre-reaction liquids used in the case of antigen-antibody reactions include serum samples, antiserum (reagents), and buffer solutions. It contains various minute component molecules that are suspended without being involved in the process. These minute scattering particles do not change before and after the reaction, except for the antigen and antibody, and the scattered light based on them is included as the scattered light component of the reaction solution. Therefore, for each of the pre-reaction solutions 4 ₂ , 4 ₂ ′......, the DC levels of scattered light output V _E2 , V _E2 ′... were measured under the same conditions as for the reaction solution 4 ₁ , and
It is necessary to subtract each from the scattered light output V _E1 due to the reaction solution. In this case, for zero point correction, a buffer solution or distilled water containing almost no light scattering substance is made to flow through the fluidized cell body 3, and the scattered light output curve C at that time is determined, and the horizontal level of the curve C is If the axis is taken as the axis, errors due to device characteristics or stray light can be eliminated. Each solution 4 ₁ , 4
When the concentration of ₂ , 4 ₂ '...... is low, there is additivity between the scattered light output curves, and in the case of Fig. 2, V _E1 and V
The difference in _E2 , V _Ea , will represent the amount or concentration of the immunochemical complex.

As described above, in the above method proposed by the present invention, the scattered light output curves before and after the reaction are recorded on the recorder 9, the DC level shown at the base of both curves is read, and the Based on the created calibration curve, it is possible to accurately measure the amount or concentration of the immunochemical complex.

Furthermore, the present invention not only proposes the above-mentioned method concept, but also provides a more effective and rapid measuring method and a device based on the method, and in particular, the structure of the fluid cell body ₃₁ and the signal processing circuit A. This data processing method has an unprecedented feature.

First, the fluid cell body ₃₁ is characterized by an annular outflow passage 19 for smoothly discharging air bubbles, and a stepped common hole is provided in the axial direction of the columnar body 10.
1 and the light guide 12, and the outflow path 20
It has been established. The flow tube 11 has both ends open and one end constricted to connect the connecting end 13 to the suction nozzle 23.
The glass tube is housed in the flow tube storage hole 14 on the large diameter side of the stepped common hole, and is connected to a leak-preventing packing 16 interposed between the inner circumferential stepped portion 15 and the open end 11a and the storage hole opening. Pressing tool 1 screwed onto portion 14a
It is fixed in the main body 10 by 7. The above flow tube 1
1 should have as small a volume as possible to prevent the flow of the test liquid from becoming turbulent and to enable measurement even with a small amount of sample. Further, it is desirable that the tube has a thin cylindrical shape so that the sample liquid flows smoothly and water droplets and dust particles do not accumulate. The light guide 12 is made of a solid cylindrical glass rod, both ends and the cylindrical surface of which are optically polished, and are inserted and fixed into the light guide hole 18 on the small diameter side of the stepped common hole. In this case, the flow tube 11 and the light guide 12
The light guide tip 12a is preferably coaxial with the flow tube opening end 11a, and the light guide tip 12a projects inward from the flow tube opening end 11a.

A hole provided by a tapered inner circumferential step 15' is present between the flow tube storage hole 14 and the light guide hole 18, and the hole and the light guide 12 form an annular outflow path 19. An outflow path 20 is formed outward from the annular outflow path 19 and is connected to an outflow pipe 22 via a connector 21 . Connector 21
, the outflow pipe 22 , the flow pipe end 13 and the reaction liquid 4
It is connected to the suction nozzle 23 through which it is fed by removable connection sleeves 24 and 25 to prevent leakage.

A through hole 26 for attaching the photodetector 7 is provided on the side wall of the columnar body 10 on the side of the flow tube storage hole, and a through hole 26 is provided for attaching the photodetector 7.
A liquid level detector 27 can also be provided downstream of the flow from the outlet pipe 22, for example, near the connection part of the outflow pipe 22. The liquid level detector 27 includes, for example, a light emitter 27a and a light receiver 27.
A start switch 30, which will be described later, detects the difference in the amount of transmitted light or the amount of reflected light before and after the reaction liquid 4 passes through the outflow pipe 22. It emits a signal.

As a result, the reaction liquid ₄₁ enters the connecting end 13 of the flow tube 11 through the suction nozzle 23 due to the suction action of the peristaltic pump 5, filling the flow tube 11 and forming an annular outflow path 19 around the light guide 12 and an outflow path. 20
The water then flows towards the outflow pipe 22. The annular outflow channel 19 plays an important role here. That is, the reaction liquid ₄₁ often contains air bubbles in addition to dust particles, and the air bubbles that flow into the flow cell 3 cause serious trouble not only when measuring scattered light but also during colorimetric analysis. The reason for this is that if there is a bent part in the outflow path, a layer will be formed in the liquid flow at that part, and the mixed air bubbles will stagnate and will not flow out. In particular, if bubbles stagnate in the space occupied by the light beam, extreme measurement errors will occur. The annular outflow path 19 described above serves to eliminate the possibility of air bubbles stagnating in the space occupied by the light beam, and the air bubbles that are mixed in will quickly pass through any part around the protruding end of the light guide 12. It is carried out to the outflow path 20.

Furthermore, in the fluid cell body ₃₁ , the light emitted from the light source lamp is introduced into the fluid tube 11 through the light guide 12, so there is no need for a large entrance window to take in the light. tube 11
The volume of can be minimized. In addition, there is no need to narrow down the luminous flux, and sufficient light energy can be easily irradiated without using special methods such as using a laser beam. Such structural features can also be used in fluorescence analysis, and it is clear that the present invention can be implemented as a measuring instrument for both nephelometry and fluorescence analysis. This introduced light irradiates the reaction liquid ₄₁ along the flow axis direction, that is, the cylindrical axis direction of the flow tube 11. Therefore, light scattering by the reaction liquid occurs throughout the flow tube 11, but
The scattered light observed by the photodetector 7 is limited to the portion emitted from the space L circled by the dotted line.
In addition, as the fluid cell body 3, the above fluid cell body 3 ₁
Of course, other materials can also be used.

Next, as shown in FIG. 4, the signal processing circuit A receives a scattered light output signal E 1 obtained by amplifying the electrical signal continuously emitted by the photodetector 7 with the signal amplifier 8, and receives the scattered light output signal E ₁ at fixed time intervals. an A-D converter 29 which samples the digital signal D into a digital signal and inputs the digital signal D to an arithmetic unit at the same periodic intervals; After writing and recording to memory at different addresses and all data has been recorded,
The display means 34 extracts a predetermined number of data from the smallest value out of all the data, calculates the average value thereof, and displays the data.
Arithmetic device 33 and A-D converter 2
The clock generator 32 generates clock signals C ₁ and C ₂ to the computer 9 and the arithmetic unit 33.
The arithmetic unit 33 is a microcomputer 3.
3 ₁ , TTL logic circuit, etc. are used, and as the display means 34, a printer or digital display device 3 is used.
4 ₁ is used. Note that the arithmetic unit 33 may be provided with a zero compensation device 28 to set the value on the display means 34 to zero when distilled water or the like is caused to flow through the fluid cell body 3, thereby eliminating the influence of stray light, etc. .

The measurement and signal processing are started by the operator pressing the start switch 30 after the flow tube 11 is first filled with the flow of the reaction liquid. Actually, it is not necessary to observe the entire length of the reaction liquid ₄₁ , so the flow tube 11
It is also possible to issue the start signal _S1 at the same time the liquid flow reaches the liquid level detector 27 provided further back in the flow. From then on, the given reaction liquid flows one after another, and the photodetector 7 continues to observe the light scattering components distributed in the flow direction of the reaction liquid ₄₁ and emit electrical signals until it runs out. .
As mentioned above, the distribution of this light scattering component is uniform for immunochemical complexes and dissolved substances smaller in size, and is uniform for dust particles and air bubbles.

Next, the input switch 31 is closed by the start signal S1 issued from the start switch ₃₀ ,
The scattered light output signal E ₁ is input to an A-D converter 29 which receives a clock signal C ₁ provided by a clock generator 32.
The scattered signal E ₁ at regular time intervals according to the period of
is sampled to form a digital signal D, and the digital signal D is transferred to an arithmetic unit 33, for example, a microcomputer ₃₃₁ , at the same periodic intervals.

The series of operations of the microcomputer ₃₃₁ is also started by the start signal S2 issued by the start switch ₃₀ , and is periodically controlled by the clock signal _C2 provided by the clock generator 32. microcomputer 33
The data regarding the digitally quantified scattered light output signal _E1 , which is successively transferred to the microcomputer 331, is stored in a memory built into the microcomputer ₃₃₁ , and data _processing is performed in the manner described later. The number of data stored in the memory is determined by the observation time of the reaction solution _{41 ,} that is, the time from _{t S} to t _E in FIG. 2, and the period of the clock 32. Since it is not necessary to coincide with the leading and trailing ends of the flow of the reaction liquid ₄₁ , it is actually desirable to set t _S and t _E within a range that takes safety into account. For example, the amount of reaction liquid is 2 ml, the time required for the entire amount to pass through space L is 20 seconds, and the measurement is started 5 seconds after the leading edge of the flow of reaction liquid passes through space L, and 0.2
It is possible to record 50 pieces of data at second intervals and finish the measurement with a 5 second flow margin. This means that the reaction solution ₄₁ is divided into 50 sections for measurement.

Now, assuming that 50 pieces of data are recorded as in this example, the microcomputer ₃₃₁ processes these data as follows.

If you look at the recorded data in terms of ranking, there is no regularity and the numerical values are randomly arranged. This is due to the random distribution of local signal fluctuations caused by dust particles and air bubbles.

After the data recording is completed, the microcomputer ₃₃₁ first rearranges each of these pieces of data starting from the smallest value, then adds a predetermined number of pieces of data in order from the smallest value of the rearranged 50 pieces of data, and calculates the corresponding data. This added value is divided by the number of pieces of added data to obtain an average value and output to the digital display device ₃₄₁ or display means 34 such as a printer.

This arithmetic processing method is extremely superior in the following points. In other words, if you look at the data group that has been recorded and sorted in order of size, in order of decreasing numerical value,
Although the number is indefinite, the data of several numbers from the beginning show extremely consistent values. This is because the reaction liquid is a homogeneous phase without dust particles or air bubbles being mixed in at least some of the 50 sections of the reaction solution mentioned above. This is because it is extremely rare for all 50 compartments to be contaminated with foreign substances in a carefully prepared reaction solution. The further you go to the back row, the more the numbers fluctuate. It is clear that the preceding approximately coincident portion of this data sequence represents a number corresponding to the base of the curve shown in FIG.

The microcomputer ₃₃₁ selects only the data group corresponding to this base, calculates and outputs the average value limited to these data groups, but in reality, fluctuations, noise, and
Due to slight unevenness in the distribution of immunochemical complexes in the reaction solution, even data derived from the basal region may not exhibit completely consistent values, and it is difficult to determine the number of data to be limited and selected by experiment in advance. I'll decide.

For example, if it is specified that 25 pieces of data are selected in ascending order from 50 pieces of data and their average value is calculated, even if the data from the 21st data onwards contains data with the addition of pseudo scattering signals due to dust particles. ,
The average value contains only 20% of the net error,
This will display a more or less accurate value. Furthermore, in this way, there is no risk of receiving an accidental measured value that is even smaller than the true value due to fluctuations inherent in the device, as would be the case when the minimum value is used as the representative value.

In exactly the same manner as the above method, the scattered light output intensity of the pre-reaction solution, that is, in the case of antigen-antibody reaction, the serum sample, antiserum solution, and buffer solution, is measured, and each of them is subtracted from the scattered light output intensity of the reaction solution. The scattered light output intensity based on the reaction product particles (immunochemical complex) is determined, and its amount or concentration is determined from a calibration curve determined in advance.

As described above, the present invention continuously or intermittently measures the amount of scattered light obtained by irradiating a certain amount of light flux onto a suspension such as a reaction liquid or a pre-reaction liquid that is forced to flow through a fluidized cell body. A method and device for measuring the amount or concentration of light scattering substances in a liquid by determining a series of steady and small measured values. Allows accidental dust particles and air bubbles to pass through quickly. Therefore, the scattered light caused by these dust particles and air bubbles becomes pulsed and is excluded from the measurement results, making it possible to obtain accurate measurement results of suspended solids. This makes it possible to analyze many serum-specific proteins that were thought to be difficult to measure because they existed in extremely small amounts. Furthermore, although only a small number of samples are required for measurement, a large number of spot measurements can be carried out, and measurement errors caused by fluctuations and noise inherent in the device, as well as slight unevenness in the distribution of suspended solids, can be kept to a low level. Since there is no need to use a special light source, the structure of the device is easy to handle, and there is no risk of reading errors.

[Brief explanation of the drawing]

FIG. 1 is a schematic explanatory diagram showing the measurement principle of the present invention,
FIG. 2 is a scattered light output curve diagram obtained as a result of the measurement method shown in FIG. 1, FIG. 3 is a longitudinal cross-sectional view of a flow cell body with an annular outflow channel according to the present invention, and FIG. 4 is a measurement according to the present invention. FIG. 2 is a block diagram of the device. DESCRIPTION OF SYMBOLS 1... Light source, 3... Fluid cell body, 3 ₁ ... Fluid cell body with annular outflow path, 4 ₁ ... Reaction liquid, 7...
Photodetector, 8...Signal amplifier, 9...Recorder, 1
1...Flow tube, 12...Light guide, 29...
A-D converter, 30...Start switch, 32
... clock generator, 33 ₁ ... microcomputer, 34 ₁ ... digital display device.

Claims (1)

[Claims] 1. A suspension that is forced to flow through a pipe is irradiated with a constant amount of light, and constant scattered light is generated by suspended substances that are uniformly dispersed in the suspension. A photodetector continuously observes the large but fluctuating scattered light caused by dust particles or air bubbles that are larger than these suspended substances and accidentally mixed into the suspension, and determines whether the scattered light is caused by the dust particles or air bubbles. Scattered light output signals containing pulses are input at fixed time intervals and converted into digital signals, and the digital signals are once recorded as data in an arithmetic unit, and then a predetermined percentage of all data is extracted from the smallest value. A method for measuring light scattering substances in a solution that measures the amount or concentration of suspended substances by extracting them and calculating their average value. 2 Suspension is a reaction solution that uniformly disperses reaction product particles and various component molecules smaller than the particles, and a pre-reaction solution that uniformly disperses various component molecules. Claim 1, which measures the amount or concentration of reaction product particles, which are moderately large suspended solids, by determining
2. Method for measuring light scattering substances in a solution as described in Section 1. 3 Suspension supply means for forcing the suspension 4 to flow into the fluid cell 3, means for irradiating the suspension 4 with a constant amount of light, and detecting light scattered by the suspension 4 to generate an electrical signal. A photodetector 7 converts the electrical signal into a signal, a signal amplifier 8 amplifies the electrical signal, and receives the scattered light output signal E from the amplifier 8, samples it at regular time intervals, converts it into a digital signal, and converts the signal into a digital signal. A solution in a solution characterized by comprising a signal processing circuit A that once records data based on the signal D, then selects data with a small value and calculates the average value thereof, and a display means 34 that shows the calculation result. Light scattering substance measuring device. 4 The fluid cell body 3 is provided with a stepped common hole in the axial direction of the columnar body 10, and the large diameter side of the stepped common hole is used as a flow tube storage hole 14, and one end is constricted into the hole 14 to form a suction nozzle. The flow pipe 11 with the connecting end 13 to the light guide 12 is housed and fixed by the pressure fitting 17, and the small diameter side of the stepped common hole is used as the light guide hole 18.
is inserted and fixed, and an annular outflow path 19 is formed between the holes 14 and 18 by the hole provided by the tapered inner circumferential step 15' and the light guide 12, and from the annular outflow path 19, an annular outflow path 19 is formed. A light scattering substance in a solution according to claim ₃ , which is a flow cell body 31 with an annular outflow path, which has an outflow path 20 and a photodetector through hole 26 on the side surface of the flow tube storage hole 14. measuring device. 5 The signal processing circuit A receives the scattered light output signal E from the signal amplifier 8 and outputs the clock generator 3.
2, an A-D converter 29 samples the clock signal C1 at fixed time intervals according to the period of the clock signal C1 given by _C1 , and inputs the digital signal D to the arithmetic unit 33 at the same periodic intervals; The digital signal D is periodically controlled by the clock signal _C2 provided by the 32, and the digital signal D is converted into data and recorded, and a predetermined number of data from the smallest value is extracted from all the recorded data, and the average value is calculated. 4. The light scattering substance measuring device in a solution according to claim 3, which comprises a computing device 33 for calculating.