JP2000221195A - Prozone judging method, storage medium storing the same and automatic analyzer using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To inexpensively and easily judge whether a prozone phenomenon is generated, without using excessive expensive reagent. SOLUTION: In a prozone judging method, a reaction soln. containing an agglutinated product formed by antigen-antibody reaction is irradiated with light, and the attenuation quantity of the transmission light thereof or the intensity of the scattered light thereof is measured at a plurality of measuring points with the elapse of time to determine as substance to be examined. In this case, two arbitrary measuring points are set to an effective measurement start point Af and an effective measurement completion point Ae, the absolute value of the difference between the area A being the sum total of the time integral value of absorbancy obtained from the measured values of the adjacent measuring points, present between the measured value of the effective measurement start point Af and the measured value of the effective measurement completion point Ae, and the area B being the time integral value of absorbancy obtained by connecting the measured value of the effective measurement start point Af; the measured value of the effective measurement completing point Ae is calculated; and on the basis of the absolute value, it is judged whether a prozone phenomenon is generated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analysis method for quantifying a test substance in a sample by utilizing an antigen-antibody reaction, and an automatic analyzer using such an analysis method. The present invention relates to a prozone determination method for determining whether or not a prozone phenomenon has occurred in a reaction, and an automatic analyzer using such a prozone determination method.

[0002]

2. Description of the Related Art In recent years, clinical chemistry tests in hospitals and the like have been increasingly automated, and automation of not only biochemical tests but also serum tests is rapidly progressing.

[0003] Further, as clinical knowledge has been accumulated regarding various diseases, the existence of substances believed to be closely related to those diseases has been clarified. And those substances are, for example, alpha-fetoprotein (AFT)
Is often used clinically, for example, is used as an index (tumor marker) indicating the presence of primary liver cancer.

[0004] However, the concentration of these substances in serum is generally extremely low. Therefore, analytical measurement of these substances is performed by an immunological measurement method using an antigen-antibody reaction, for example, an immunoturbidimetric method or a latex turbidimetric method.

In these immunoassays, generally, a test substance in a sample is mixed with a reagent containing a substance capable of reacting with the test substance in the sample, and the mixed reaction solution is irradiated with light of an appropriate wavelength. The amount of the aggregated product is measured by measuring the absorbance by irradiating the sample, and the amount (concentration) of the test substance contained in the sample is determined from the obtained measured value. At this time, the intensity of the scattered light may be measured instead of the absorbance (attenuation of the transmitted light). In any case, if the amount of the test substance in the sample is within a certain range, the absorbance and the scattered light intensity are proportional to the amount of the test substance, and thus the above-described measurement method is effective.

[0006] However, in the antigen-antibody reaction,
In a state where one of the antigen or antibody is present in excess beyond a certain range defined for each reaction, the absorbance or scattered light intensity measured by the immunoassay described above is determined by the amount of the antigen or antibody. It is known that the value is lower than the value expected from, and such a phenomenon is called a prozone phenomenon. If an amount of the test substance causing this prozone phenomenon is present in the sample, the measured value obtained by the immunological assay will not match the amount of the test substance actually present. Determining whether a phenomenon has occurred is an important clinical issue.

[0007] To date, the following method has been proposed as a method for determining whether or not a prozone phenomenon has occurred in measurement using an antigen-antibody reaction. (A) a method of re-adding a reaction reagent or a sample; (b) a method of obtaining a turbidity ratio or a concentration ratio from a plurality of measured values; and (c) a reaction rate ratio from a plurality of measured values. (D) a method using a three-dimensional calibration curve of the maximum reaction rate, the reaction time until reaching the maximum reaction rate, and the concentration of the test substance from a plurality of measured values; (e) two-wavelength measurement, and the absorbance ratio thereof Is a method of making a judgment. These methods are described in, for example, the Japanese Society of Clinical Laboratory Automation, Vol. 15, No. 6, pp. 675-687 (1990).
Year) and the same volume 14 No. 3 pages 171 to 176 (1
989).

[0008]

However, in the above-mentioned method (a), all the samples are re-examined, which is one or two orders of magnitude more expensive immunological test than a general clinical biochemical test reagent. There is a problem that the cost is excessively increased because the amount of the test reagent used doubles.

In the above methods (b) to (e), it is necessary to obtain an absorbance ratio, a reaction rate ratio, or a three-dimensional calibration curve in advance prior to sample analysis. Although it is necessary to use a test substance sample with a sufficiently high concentration to perform the test, it is generally difficult to obtain and prepare such a high concentration of the test substance sample, or there is a problem that it is very costly and troublesome. is there. further,
The work of creating a calibration curve or the like must be performed every time a reagent lot changes, and there is a problem that the burden such as the cost and time required for such work becomes extremely large.

The present invention has been made in view of the above problems, and a prozone determination method capable of determining the presence or absence of a prozone phenomenon more economically and easily than conventional methods without using extra expensive reagents. And an automatic analyzer using such a prozone determination method.

[0011]

Means for Solving the Problems In order to solve the above problems, a method for determining prozone according to the present invention comprises mixing a test substance in a sample with a reagent containing a substance capable of reacting with the test substance in an antigen, Irradiating the reaction solution containing the aggregation product with light,
In an analysis method for quantifying a test substance by measuring the amount of attenuation of transmitted light or the intensity of scattered light over time at a plurality of measurement points, any two measurement points can be defined as an effective measurement start point and an effective measurement end point. And, the sum of the time integrated values of absorbance obtained from the measured values of the adjacent measurement points existing between the measured value of the effective measurement start point and the measured value of the effective measurement end point, and the effective measurement start point Determine the absolute value of the difference between the time integral value of the absorbance obtained by connecting the measured value and the measured value of the effective measurement end point, and determine the presence or absence of the prozone phenomenon based on the absolute value. I do. With such a configuration, the prozone determination method of the present invention can determine the presence or absence of the prozone phenomenon more economically and efficiently than in the past without increasing the amount of reagent used or the amount of work performed by the operator. be able to.

Preferably, in the first aspect, it is determined that the prozone phenomenon has occurred only when the absolute value is larger than a predetermined value arbitrarily determined. With such a configuration, it is possible to easily and accurately determine whether or not the prozone phenomenon has occurred.

Further, in order to solve the above-mentioned problem, the storage medium of the present invention is characterized in that a test substance in a sample is mixed with a reagent containing a substance capable of reacting with the test substance with the test substance by coagulation in a reaction solution. Forming an object; irradiating the reaction solution containing the agglutination product with light to measure the attenuation of transmitted light or the intensity of scattered light over time at a plurality of measurement points; Determining two measurement points as an effective measurement start point and an effective measurement end point; setting a criterion value for determining that a prozone phenomenon has occurred; and measuring the effective measurement start point and the effective measurement end point. Calculating an area A, which is the sum of time integrals of absorbance obtained from measured values of adjacent measuring points existing between the measured value and the measured value of the effective measurement start point and the effective measurement end point. Measurements and Calculating an area B that is a time integrated value of the absorbance obtained by tying; calculating an absolute value of a difference between the area A and the area B; comparing the determination reference value with the absolute value; And storing a program including at least a determination step of determining whether or not a prozone phenomenon has occurred. With such a configuration, the storage medium of the present invention provides a method for economically and efficiently determining whether or not a prozone phenomenon has occurred, without increasing the amount of reagents used or the amount of work performed by an operator. Can be provided.

Preferably, in the third aspect, in the determining step, if the absolute value is larger than the reference value, it is determined that a prozone phenomenon has occurred. A program for determining that no zone phenomenon has occurred is stored.
With such a configuration, it is possible to provide a method for easily and accurately determining whether or not the prozone phenomenon has occurred.

Further, in order to solve the above-mentioned problems, an automatic analyzer according to the present invention mixes a test substance in a sample with a reagent containing a substance capable of reacting with the test substance with the test substance. Irradiating the reaction solution containing light, an automatic analyzer equipped with a means and a storage medium capable of quantifying the test substance by measuring the amount of attenuation of transmitted light or the intensity of scattered light over time, An automatic analyzer, wherein the storage medium is the storage medium according to claim 3.
With such a configuration, the automatic analyzer of the present invention can more economically and efficiently determine whether or not a prozone phenomenon has occurred, without increasing the amount of reagent used or the amount of work performed by an operator. Can be.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION
A graph of a reaction curve (ie, time course) drawn by plotting measured values obtained by measuring absorbance over time in an immunological measurement method with absorbance on the vertical axis and reaction time on the horizontal axis is theoretically Is based on the finding that a test substance concentration in a sample becomes a straight line in a range where the prozone phenomenon does not occur, and does not become a straight line when the prozone phenomenon occurs. That is, when the prozone phenomenon does not occur, the slope of the reaction curve is always constant, and therefore, the slope between the measured value (A _f ) at the measurement start point and the measured value (A _e ) at the measurement end point is present. _Af- A which connects the sum of the time integrals of the absorbances obtained from the measured values of the adjacent measuring points, and the measured value ( _Af ) of the measurement start point and the measured value ( _Ae ) of the measurement end point. The technical idea that the time integral of the absorbance obtained from _e should be the same and that if there is a difference between them, it can be considered that the prozone phenomenon has occurred is the starting point of the present invention.

This will be described in detail with reference to the same time course shown in FIGS. 1A and 1B. The time course shown in FIG. 1 shows a case where there are 14 effective measurement points (observation points).

The area A shown in FIG. 1A is the measured value at the measurement start point (A _f = A ₁ ) and the measured value at the measurement end point (A _e = A
₁₄ ), the sum of the time integrals of the absorbances obtained from the measured values of the adjacent measuring points existing between the measured values, ie, the measured value of the certain measuring point (the n-th measured value; A _n ) and the adjacent measurement A time integration value of the absorbance obtained from a line segment _An - _{An + 1} connecting the measured value of the point (the (n + 1) th measured value; _{An + 1} ) (the line segment _An - _{An + 1} , the horizontal axis, and the points _An and A) _{n + 1}
Area enclosed by a vertical line drawn down on the horizontal axis from each of the above)
And can be obtained by the following equation (1).

[0019]

(Equation 1) The area B shown in FIG. 1b) is the measured value (A _f =
A ₁₎ and the measured value _(A e ₌ A ₁₄₎ and the line segment _A f _-A e _(A 1 time integral value of the absorbance obtained from _{-A 14)} connecting the measurement end point (line _A f -A _e And the horizontal axis and points A _f and A _e
Area enclosed by a vertical line drawn down on the horizontal axis from each of the above)
And can be obtained by the following equation (2).

[0020]

(Equation 2) Then, if the difference between the area A and the area B is calculated, the presence or absence of the prozone phenomenon can be determined based on whether or not the difference exists. However, in an actual measurement, there is always a measurement error within an allowable range, and a measurement error may occur. Therefore, it is impossible to make a determination based on whether or not there is a difference between the area A and the area B. It is.

However, since the measurement error and the difference (deviation) between the area A and the area B due to the measurement error are usually smaller than the deviation due to the development of the prozone phenomenon, the area A and the area The absolute value of the difference from B is determined. If the absolute value is within a certain value, it is determined that the prozone phenomenon has not occurred, and it is determined that the prozone phenomenon has occurred only when the absolute value is exceeded. Then, it is possible to make a judgment with clinically sufficient accuracy. In the present specification, the value is referred to as a criterion value. The criterion value can be appropriately set according to the type of the test substance, the analysis method to be adopted, the desired determination accuracy, and the like.

The prozone determination method of the present invention as described above is executed according to the flowchart shown in FIG.

According to FIG. 2, the processing for judging the pro zone is as follows.
First, in step S201, as a determination reference value,
The minimum value of the absolute value of the difference between the area A and the area B for determining that the prozone phenomenon has occurred is set. After confirming the setting of the determination reference value, the process proceeds to the next step S202. If the setting is not performed within the predetermined time, it is determined that the pro zone determination processing is not performed, and the process proceeds to S207 and ends.

In step S202, the time integral value of the absorbance obtained from the measured values of the adjacent measuring points existing between the measured value (A _f ) at the measuring start point and the measured value (A _e ) at the measuring end point is calculated. The area A which is the sum is calculated, and the next step S
Go to 203.

In step S203, an area B which is a time integrated value of absorbance obtained from a line segment _Af - _Ae connecting the measured value ( _Af ) at the measurement start point and the measured value ( _Ae ) at the measurement end point. Is calculated, and the process proceeds to the next step S204.

In step S204, step S2
The absolute value of the difference between the area A calculated in step S02 and the area B calculated in step S203 is calculated, and the next step S20
Go to 5.

In step S205, step S2
The determination reference value set in step S01 is compared with the absolute value calculated in step S204. If the absolute value is greater than the determination reference value, it is determined that a prozone phenomenon has occurred, and the process proceeds to step S206. Otherwise, it is determined that the prozone phenomenon has not occurred, and step S207 is performed.
To end the process.

In step S206, after performing a process indicating that a prozone phenomenon has occurred at the time of output of the measurement result, the process proceeds to step S207 and ends.

As described above, the prozone determination method of the present invention does not consume a large amount of expensive reagents and does not require any special operation from the operator, so that it is easier, more economical, and easier than before. It is possible to efficiently determine whether or not the prozone phenomenon has occurred.

Further, since the prozone determination method of the present invention does not require any special device and operation, it is applied to conventional automatic analyzers, and all immunological measurements performed using those devices are performed. Method can be applied.

Hereinafter, an example of an automatic analyzer that can be used for implementing the prozone determination method of the present invention will be schematically described with reference to FIG.

In the automatic analyzer shown in FIG. 3, a plurality of sample containers 1 each containing a sample to be inspected are provided on the upper surface of a sampler 2. The sampler 2 and other operating parts are controlled by a computer 3 via an interface 4. The sampler 2 rotates the sample container 1 below the sample dispensing probe 5 in a predetermined order. The sample dispensing probe 5 is inserted into the sample container 1, and a sample to be inspected in the sample container 1 is dispensed to the reaction container 6 by a predetermined amount by the sample pump 7 connected to the sample dispensing probe 5. The reaction container 6 into which the sample has been dispensed,
The inside of the reaction tank 9 connected to the constant temperature bath 8 is rotated to the first reagent dispensing position. Next, at the first reagent dispensing position, the reagent dispensing probe 10 is inserted into the reaction vessel 6 and the reagent pump 11 connected to the reagent dispensing probe 10 is connected.
Then, a predetermined amount of the first reagent sucked from the reagent bottle 12 is added to the reaction container 6. The reaction container 6 to which the first reagent has been added is rotated to the position of the stirring unit 13, and the stirring unit 13 stirs and mixes the contents of the reaction container 6. The reaction vessel 6 in which the contents are stirred and mixed.
Rotates rotationally to a position where the light beam from the light source 14 is blocked, and stays there for a predetermined time. The spectrophotometer 15
The absorbance of the contents in the reaction vessel 6 within this predetermined time is measured. The absorbance signal measured by the spectrophotometer 15 is input to the computer 3 via the interface 4 after passing through the analog / digital converter, and is processed according to a program stored in the computer in advance, so that the test signal in the sample is detected. Converted to the concentration of the substance. The density-converted measured value is output from the printer 17 via the interface 4 or displayed on the display 18.
Is output in an appropriate output form, such as being displayed on the screen. After the measurement, the reaction vessel 6 is rotated and moved to the position of the washing unit 19, and after the contents are discharged by the vessel washing pump 20, is rotated and moved to a predetermined position for the next analysis. . Note that commands from the operator necessary for the work are input from the input unit 16 connected to the computer 3 via the interface 4.

Next, the effect of the prozone determination method of the present invention will be described with reference to an example of the result of analyzing AFP (alpha-fetoprotein) by the reaction rate method using the above-mentioned automatic analyzer. .

FIG. 4 shows samples 4 to 4 as samples to be analyzed.
FIG. 9 shows the results of measuring the absorbance (ABS) of a sample with a known AFP content at a plurality of photometric points. FIG. 5 shows a time course created based on the measurement results.

As shown in FIG.
P is 0 ng / ml (sample 1) and 18 ng / ml, respectively.
(Sample 2), 965 ng / ml (Sample 3) and 919
ng / ml (sample 4). Absorbance was measured at a total of 33 measurement points, and the effective measurement points (effective photometry points) were the 20th to 33rd measurement points (photometry points) of the 14th meter in total.
The pro zone was determined. In the present embodiment, the judgment reference value is set to 17.

The absolute value of the difference between the area A and the area B obtained by performing various calculations from the measured values (see the lowermost part of FIG. 4) is
For sample 1 and sample 2, 0.0 and 0.2 respectively
It was confirmed that the area A and the area B were substantially equal in the concentration range where the prozone phenomenon did not occur. Also,
Test substance (AFP) for both sample 3 and sample 4
Although the concentration was within the concentration range where the prozone phenomenon occurred,
In sample 4 (concentration: 919 ng / ml), the absolute value of the difference between the area A and the area B was larger than the determination reference value, and it was accurately determined that the prozone phenomenon occurred. In the sample 3 (concentration: 965 ng / ml), the absolute value of the difference between the area A and the area B is smaller than the determination reference value. This reversal phenomenon is considered to be due to some measurement error. From this, if a measurement error is present, the absolute value of the difference between the area A and the area B becomes smaller, and it is determined that the prozone phenomenon has occurred. It has been confirmed that the technical idea of the present invention is practical only when the absolute value of the difference between A and the area B exceeds a certain value (determination reference value). From this result, it is considered that one or more different reference values for determining a normal value, an abnormal value, and / or a measurement error may be provided in a range where the absolute value is smaller than the prozone reference value. Can be

[0037]

As described above, according to the present invention,
Since the presence or absence of the occurrence of the prozone phenomenon can be determined by performing only a few calculations based on the measured values, the presence or absence of the occurrence of the prozone phenomenon can be determined with high accuracy and high reproducibility at a lower cost and easier than before. A determination method, a storage medium, and an automatic analyzer can be provided.

[Brief description of the drawings]

FIG. 1 is a time course diagram illustrating a prozone determination method according to the present invention.

FIG. 2 is a flowchart of one embodiment of a prozone determination method of the present invention.

FIG. 3 is a schematic view showing one example of the automatic analyzer of the present invention.

FIG. 4 is a diagram showing the effectiveness of one embodiment of the prozone determination method of the present invention.

FIG. 5 is a time course created based on FIG. 4;

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 sample container 2 sampler 3 computer 4 interface 5 sample dispensing probe 6 reaction container 7 sample pump 8 constant temperature bath 9 reaction tank 10 reagent dispensing probe 11 reagent pump 12 reagent bottle 13 stirring unit 14 light source 15 spectrophotometer 16 input unit 17 Printer 18 Display 19 Cleaning unit 20 Container cleaning pump

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G058 AA09 BB15 CB05 CD04 CE08 EA02 EA04 ED03 GA02 GD01 GD06 GE01 HA00

Claims (5)

[Claims] 1. A test substance in a sample is mixed with a reagent containing a substance capable of reacting with the test substance with an antigen, and a reaction solution containing the agglutination product is irradiated with light to attenuate the transmitted light. Alternatively, in an analysis method for quantifying a test substance by measuring the intensity of scattered light over time at a plurality of measurement points, any two measurement points may be used as an effective measurement start point and an effective measurement end point, The sum of the time integrals of absorbance obtained from the measured values of the adjacent measurement points existing between the measured value of the point and the measured value of the effective measurement end point, and the measured value of the effective measurement start point and the effective measurement A prozone determination method comprising: obtaining an absolute value of a difference between a time integration value of absorbance obtained by connecting a measured value of an end point to a time integral value; and determining whether or not a prozone phenomenon has occurred based on the absolute value. 2. The prozone determination method according to claim 1, wherein it is determined that the prozone phenomenon has occurred only when the absolute value is larger than a predetermined value. 3. a step of mixing a test substance in a sample with a reagent containing a substance capable of reacting with the test substance with an antigen to form an aggregated product in a reaction solution; Irradiating the liquid with light to measure the attenuation of transmitted light or the intensity of scattered light over time at a plurality of measurement points; and defining any two measurement points as an effective measurement start point and an effective measurement end point Setting a criterion value for determining that a prozone phenomenon has occurred; and measuring values of adjacent measurement points existing between the measurement value of the effective measurement start point and the measurement value of the effective measurement end point. Calculating the area A, which is the sum of the time integrated values of the absorbance obtained from the above, and the time integrated value of the absorbance obtained by connecting the measured value of the effective measurement start point and the measured value of the effective measurement end point. Calculate area B Calculating the absolute value of the difference between the area A and the area B; and determining the presence or absence of the prozone phenomenon by comparing the determination reference value with the absolute value. A storage medium storing a program including the program. 4. In the determining step, when the absolute value is larger than the determination reference value, it is determined that a prozone phenomenon has occurred, and otherwise, it is determined that no prozone phenomenon has occurred. 4. The storage medium according to claim 3, wherein a program for storing the program is stored. 5. A test substance in a sample and a reagent containing a substance capable of reacting with the test substance and the antigen are mixed, and a reaction solution containing the agglutination product is irradiated with light to attenuate the transmitted light. Alternatively, the automatic analyzer is provided with a unit capable of quantifying a test substance by measuring the intensity of scattered light over time and a storage medium, wherein the storage medium is the storage medium according to claim 3 or 4. An automatic analyzer characterized by the following.