JPH03110469A - Method for deciding concentration of antigen or antibody by immune agglutination - Google Patents

Abstract

PURPOSE:To allow good decision even when a prozone is generated by deciding a high concn. region based on the two-dimensional agglutination characteristic curve plotted according to the degree of agglutination upon lapse of the different time after a specimen and a regent are mixed. CONSTITUTION:The agglutination characteristic curve f6 is determined when the degrees of agglutination A1, A2 upon lapse of the different time T1, T2 after mixing of the specimen and the reagent for each of the calibrators of the concns. C1, C2... are measured and a pair of the degrees of the agglutination are two-dimensionally plotted. The good decision is made and the accuracy of the inspection is enhanced without erroneous decision even in the case of the generation of the prozone phenomenon to decrease the degrees of the agglitination when the concn. increases to the prescribed value or above if the overrange region of the concn. Cx or above is designated as the high-concn. region. The region based on the conditions of equation may also be designated as the high-concn. region by determining constant alpha, beta, etc. for every specimen based on the curve f6. A2(Cx):the degree of agglutination for the concn. Cx at the time T2.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for determining the high concentration of an antigen or antibody in a specimen by immunoagglutination.

[Conventional technology]

As a device for measuring the concentration of antigen (or antibody) contained in a sample, an insoluble carrier sensitized with an antibody (or anti-R) that specifically reacts with the target antigen (or antibody) is mixed into the sample. There is an immunoagglutination measuring device that generates an antigen-antibody reaction by doing so, and measures the degree of aggregation of the carrier using a particle counting means (Japanese Patent Laid-Open Publication No. 1983-1963).

The agglomerated variable is calculated, for example, from the number of aggregated particles (polymer) P1 and the number of unagglomerated particles (monomer) M as A=P/(M+P), and further, this aggregated variable is calculated using a predetermined relational expression:
Converted to concentration.

In Figure 4, the horizontal axis is the elapsed time T from the mixing of the sample and reagent;
The graph shows an agglomerative growth curve with agglomerative eccentricity plotted on the vertical axis.

Curve f1. ft and f= correspond to high concentration, medium concentration, and low concentration of the analyte, respectively, and the reaction progress rate differs depending on the analyte concentration (that is, antigen concentration). In addition, the degree of aggregation A does not simply increase with respect to the elapsed time T, but a phenomenon in which the degree of aggregation decreases as time passes is observed. In addition, the time when the degree of aggregation decreases is the time when the concentration becomes high.

FIG. 5 shows the relationship between the concentration C of the specimen and the agglutination variable when the elapsed time is T, 'l T,'.

Curve f1. fa are the elapsed times 7, +, T, and
+ is supported. The degree of aggregation A does not simply increase with respect to the concentration C. When the amount of antigen is too large, a phenomenon in which the agglutination rate decreases (prozone phenomenon) is observed. for example,
Time T for a certain specimen! It is assumed that the agglomeration degree a3 is obtained by measuring the agglomeration degree a3. However, for this degree of aggregation a1, there is a concentration C8 in the normal range and a concentration CI in the excessive range, so the concentration cannot be immediately determined from the degree of agglutination.

Therefore, a method described in Japanese Patent Application Laid-Open No. 81-280568 was devised. In this method, the degree of cohesion is determined not only at time T,' but also at time T1'. It can be seen that if the degree of agglutination at time 7.1 is at, the antigen concentration is C1, and if the degree of agglutination is al, the anti-R concentration is C3.

But curves! The peak of , usually reaches several 10%, but
The peak of curve f4 reaches only a few percent, and at time T
+' measurement hardly causes a difference in the degree of aggregation. In other words, there are problems with reproducibility, and only unreliable agglomeration degrees can be obtained. For this reason, it is difficult to specify the concentration using the above method.

If the time 7.1 is extended, that is, the measurement is made closer to time T,1, the peak of the song If will also be higher, making it easier for differences in aggregation to occur and improving measurement accuracy, but this time the peak position will be It shifts to the low measured concentration side and becomes close to a similar shape to the curve f. This makes it difficult to determine the concentration. and,
Above all, the curve f.

Near the peak of , the change in the degree of aggregation A with respect to the concentration C is extremely small, so it is difficult to convert the concentration and the results are uncertain.

As described above, the accuracy of the measurement results cannot be guaranteed for the entire concentration range, so if the antigen concentration to be measured is extremely high, it is necessary to detect this as a high concentration range and issue an alarm.

Conventional methods for determining high concentration include the following methods (a) and (a).

(b) A method of using the degree of aggregation A1' at time T1° as a monitor and determining a high concentration region when this value exceeds a certain value. As shown in FIG. 6, when the degree of aggregation A1° shown by curve f4 is 84 or more, it is determined that the region is in a high concentration region. The area indicated by diagonal lines is the area.

(b) Instead of Alo in (a) above, αA, '+A4
A method of determining a high concentration area when ' exceeds a certain value. At' is time T! is the cohesion degree at ′. α is a constant.

[Problem to be solved by the invention]

In the case of method (a), as described above, the accuracy of the degree of cohesion is not achieved, so variations tend to occur in the determination area. In other words,
The area may be determined to be a high concentration area even though it is not a high concentration area, or may not be determined to be a high concentration area even though it is a high concentration area. Furthermore, due to the prozone phenomenon, the degree of aggregation may become smaller than a4 at high concentrations, resulting in erroneous determination.

In the case of method (b), as in the case of method (b),
Misjudgment occurs.

Furthermore, in the past, when measurements were repeatedly carried out, if the previous sample had an extremely high concentration of 1E, this could affect the measurement results of the next sample, resulting in measurement errors.

An object of the present invention is to provide a method for determining the concentration of an antigen or antibody using immunoagglutination, which allows accurate determination of high concentration regions even in a measurement system in which the prozone phenomenon occurs.

The object of the invention as claimed in claim (3) is to provide a method for determining the concentration of an antigen or antibody by immunoagglutination, which can appropriately determine whether the concentration of a previous specimen is high enough to affect the next measurement result. .

[Means to solve the problem]

The concentration determination method of the present invention involves mixing and reacting a specimen having an antigen or antibody to be measured with a reagent containing an antibody that specifically reacts with the antigen or antibody or an insoluble carrier sensitized with the antigen. When the insoluble carriers are mutually agglutinated by the antigen or antibody in the sample as a medium, and when predetermined times T and T, which are different from each other, have elapsed since the time when the sample and reagent were mixed, the degree of agglutination A at each elapsed time is determined. , , At was measured, and the aggregation degree A5. It is characterized in that At is plotted on a two-dimensional plane as a set of data, and if the plotted point is within a predetermined area, it is determined that it is in a high concentration area.

The above region is given in the form of bisecting the agglutination characteristic curve drawn on the two dimensions and using the analyte concentration as a parameter.

In the invention of claim (2), the above-mentioned area is divided into the following (al~(
Each inequality of C1 % expression%)) () (However, α and β are positive constants, and n is an integer of 3 or more.^1(CO),^+(Ct>, At(Ct),^l
(C,) are the degrees of aggregation during measurement at time T of samples with concentrations CI+Ct, ct, c, respectively, and 8, (C,
) is the degree of aggregation when measuring concentration C, at time T. As (Cx) is the degree of aggregation when measuring the concentration Cx at time T. ) is an area that satisfies one of the conditions shown in

The invention of claim (3) provides that the point where the agglomeration degrees A, , A, are plotted on a two-dimensional plane as − set of data is the following (b
l, (dl inequality % formula % ()] (where β, γ, and δ are constants) A set of data is determined to be a warning area on two dimensions.

[For production]

A specimen such as human serum contains various substances such as antigens and antibodies. The reagent for an immunoagglutination measuring device contains an insoluble carrier sensitized with an antibody or antigen that specifically reacts with a certain antigen or antibody. Therefore, by mixing the specimen and the reagent, an antigen-antibody reaction occurs, and the insoluble carriers aggregate with each other via the antigen or antibody in the specimen. The degree of agglutination changes depending on the type of antigen or antibody to be measured contained in the specimen. Therefore,
By measuring this degree of agglutination and further converting the degree of agglutination, the amount of the target antigen or antibody can be determined.

By the way, in this invention, one set of aggregation degrees AI+AI is obtained by measuring the aggregation degrees at different times. Since both do not change in the same way with respect to concentration, T
, the measured aggregation degree A, and T, the measured aggregation degree A, are taken as two axes, and an agglutination characteristic curve can be drawn on two dimensions according to the concentration change.

Therefore, if a region is determined to be a part of this agglomeration characteristic curve, it is possible to divide the region into a high-concentration region and a low-concentration region based on the concentration.

As shown in claim (2), the area includes the (al~
(It can be defined as an area that satisfies any of the conditions shown in the inequality C1.

In the method of claim (3), when the concentration of the previous sample is extremely high and there is a possibility that it will affect the measurement results of the next sample, the judgment condition for issuing an alarm is determined by the inequality (bl, (dl)). The condition is to satisfy.

〔Example〕

An embodiment of the present invention will be described with reference to FIGS. 1 and 2. First, the immunoagglutination measuring device used in this example will be explained.

The CIA method (Counting Immunoas
There is one using the say method). This is a device that measures the degree of agglutination accompanying a latex agglutination reaction using an antigen-antibody reaction using a particle counting method, and quantifies the antigen. (manufactured by Medical Electronics Co., Ltd.) is an example. In this example, this immunoagglutination measuring device is used.

With this device, the sample (containing the antigen to be measured)
By mixing the reagent (containing latex particles bound with antibodies that specifically react with the antigen to be measured in the sample), the antigen-antibody causes a reaction, and the latex particles react with the antigen in the sample. For example, two, three,
As such, they begin to coagulate with each other, and agglomerates are gradually formed.

After reacting for a predetermined period of time, the mixed solution of the sample and reagent is introduced into the flow cell. A sheath flow is formed in the flow cell, and a trickle of particles is irradiated with laser light. By measuring and discriminating the scattered light emitted from each particle, the number of unagglomerated latex particles and the number of aggregated latex particles are determined, and the degree of aggregation is calculated from both numbers. The determined degree of agglutination is converted into antigen concentration using a calibration curve. As described above, the target antigen in the specimen is quantified. The method for determining the degree of agglomeration using the above method is described in Japanese Patent Application Laid-Open No. 1963/1963 and Japanese Patent Application Laid-Open No. 60-24358.
Learn more about Publication No. 5.

In this example, the number of agglomerated latex particles is M
, from the number P of agglomerated latex particles, A=P/(M0P) is defined and quantified. Furthermore, the degree of aggregation is measured twice in one measurement. After time T1 from mixing the sample and reagent (
For example, after about 30 seconds), the first measurement (T, measurement) is performed, and after a time T (for example, after about 15 minutes), the second measurement (T is measured) is performed. In this way, by shifting the time 2
By performing measurements twice, a set of data on the degree of aggregation A, , A, is obtained. It is checked whether the degree of aggregation A,, A, satisfies a predetermined condition, and it is determined whether or not it is in a high concentration region. Let me explain this in an easy-to-understand manner.

The obtained set of data A, , A, represents two wheels ^,.

A is plotted on a two-dimensional plane, and it is determined whether or not it is in a high concentration region. The high concentration region is, for example, an overrange region that is outside the measurement range.

Note that AI and At are T, measurement, and T, aggregation degree in measurement, respectively. Figure 1 is a diagram for determining overrange using the cohesion degree AI and At as two axes, and f is α
- Shows the aggregation characteristic curve when measuring fetoprotein (AFP). The aggregation characteristic curve f is obtained by measuring the degree of aggregation while changing the concentration and plotting the obtained degree of aggregation. The numbers attached to each point of the curve f in FIG. 1 are AFP concentrations, and the unit is (ng/ml).

Furthermore, the degree of aggregation is expressed as a percentage.

The area for overrange judgment is this cohesive characteristic curve f
, can be determined based on. In other words, the aggregation characteristic curve f starts near the origin, and as the concentration increases, in the case of AFP, it draws the curve shown in FIG. 1. Of course, if the object to be measured changes, the shape of the curve will also change. The overrange region is an area that includes a curve portion after a point corresponding to a certain concentration Cx on the curve T6 (that is, a higher concentration), but does not include a curve portion before that point, that is, a lower concentration. By dividing the curve f in this way,
A specimen with a concentration equal to or higher than Cx can be determined to be in the overrange region.

In the case of AFP, for example, the following +al~(C
An area that satisfies either condition 1 can be defined as an overrange area. In FIG. 1, it is shown by diagonal lines. Curves a-C in FIG. 1 each represent a function of the formula +al~(C1 with the inequality sign being an equal sign.

fal At-At(Cm) > -a [A1-^
, (CI)) (blAI>[^+ (Co) 1^1 (C
I)+Al(C1>1 /3+β(C1＾□〉＾BaCx
) However, α and β are positive constants, and in the case of AFP, α=
20, β=1.0 was good.

In addition, ^+(Co), AI(CI), AI(Cri, ^1
(Ca) is the degree of agglomeration in the T measurement of the calibrators at concentrations c, , c, , c, , c, respectively, and 8 g (C-
) is T of the concentration C6, the degree of aggregation in the measurement. Man.

(Cx) is T of the concentration Cx, the degree of aggregation in the measurement,
Concentration C@+ Cat Cat Cat Cat CaI
It is obtained from the calibration curve determined by measuring the calibrator of C.

? f! Figure 2 is a calibration curve diagram. Concentrations 00 to C1 are specifically 0, 2, 8, 32, 128, 512°2048
[n g/mf), Cx is here 1000
(ng/mA'). In other words, 1000 [n
g/ml] or more is defined as an overrange region.

The highest concentration shown in Figure 1, 2 million [ng/mj7
) is a sample with an extremely high concentration that would not normally occur. β converts this high concentration sample to an extremely low concentration (C・~Ct)
The values are set such that the samples can be easily distinguished from each other. If α is too small, a sample with a higher concentration than the concentration Cx will be likely to be determined to be in the overrange region due to data variations. If α is too large, a sample with a lower concentration than the concentration Cx will be more likely to be determined to be outside the overrange region. In this way, the area for overrange determination must be determined on the premise that data variation will occur, taking this into consideration.

The determination method for AFP has been explained above, but other measurement substances such as carcinoembryonic antigen (CEF), ferritin (FRN), β2-microglobulin (8
2M) etc., determine the overrange area in the same way,
can be determined.

Upon investigation, I found that CE, FRN, and 82M can also determine overrange areas under the conditions of +a+, (bl, (C1) described above, similar to AFP.However, α.

It is necessary to choose an appropriate value for β to suit each item.

Next, other embodiments will be described. In this example, the concentration C
A density cy higher than x is set, and an area having a density higher than cy is set as another determination area. Here, Cy is approximately 200,000 [n
g/rrl), and a carryover alarm is issued for samples with a concentration above this concentration. The carryover alarm is an alarm that is issued when the previous sample has an extremely high concentration and there is a possibility that the measurement results of the next sample will be affected.

As shown in Fig. 3, to explain the judgment conditions, the area that satisfies both the following conditions (bl, (dl) is the carryover warning area. The curve d in Fig. 3 represents a function obtained by changing the inequality sign of dl to an equality sign.

(bl ^, > (AI (On) + ^ + (CI) 10 people + (ci)) /3 + β (di At-δ〈γ[^,
−[A, (C,)+^+(C,)10A, (CI))
/3+β, where β, γ, and δ are constants.

〔Effect of the invention〕

In the concentration determination method of the present invention, the degree of aggregation is measured twice at different times in the first measurement, and these two degrees of aggregation take different values depending on the concentration. Therefore, by plotting both values as a set on a two-dimensional plane, even if a prozone phenomenon occurs, it is possible to satisfactorily determine whether or not the concentration is in the target concentration range. Therefore, uncertain measurement results are no longer produced, and inspection accuracy can be improved.

In the concentration determination method of claim 31, when the plotted point is in a predetermined high concentration region different from the concentration region, it is determined that the point is in the alarm region, so that the concentration of the previous sample is extremely high. It becomes clear that there is a possibility that the measurement result of the next sample may be affected, and the test accuracy can be further improved.

[Brief explanation of drawings]

Fig. 1 is an explanatory diagram of an agglutination characteristic curve showing the overrange region of AFP in one embodiment of the present invention, Fig. 2 is its calibration curve, and Fig. 3 shows the carryover warning range of AFP in another embodiment. FIG. 4 is an explanatory diagram showing the relationship between elapsed time and degree of aggregation in the conventional example,
FIGS. 5 and 6 are explanatory diagrams showing the relationship between the antigen and antibody and the degree of agglutination, respectively. a-C...Curve showing determination conditions, f6...Agglomeration characteristic curve (Fig.

Claims (3)

[Claims] (1) By mixing and reacting a sample containing an antigen or antibody to be measured with a reagent containing an antibody that specifically reacts with the antigen or antibody or an insoluble carrier sensitized with the antigen, When the insoluble carriers are mutually aggregated through antigen or antibody, and different predetermined times T_1 and T_2 have elapsed since the mixing of the sample and reagent, the degree of agglutination A_1 and A_ at each elapsed time is determined.
2 is measured, the agglomeration degrees A_1 and A_2 are plotted on a two-dimensional plane as a set of data, and if the plotted point is within a predetermined area, it is determined that it is in a high concentration area. A method for determining the concentration of an antigen or antibody by immunoagglutination. (2) The predetermined area is one of the following (a) to (c)
Each inequality (a) A_2-A_2(C_n)>-α[A_1-A_
1(C_n)](b)A_1>[A_1(C_0)+A
_1(C_1)+A_1(C_2)]/3+β(c)A
_2>A_2(Cx) (However, α and β are positive constants, and n is an integer of 3 or more. A_1(C_0), A_1(C_1), A_1(C_
2), A_1(C_n) are the concentrations C_0, C_1, and
A_2(C_n) is the aggregation degree of the sample C_2 and C_n measured at time T_1, and A_2(C_n) is the degree of aggregation at time T_1 of the concentration C_n.
This is the degree of aggregation during measurement in No. 2. A_2(Cx) is the degree of aggregation when measuring the concentration Cx at time T_2. ) Claim (1) is an area that satisfies any of the conditions shown in
The method for determining the concentration of an antigen or antibody by immunoagglutination as described above. (3) The point where the agglomeration degrees A_1 and A_2 are plotted on a two-dimensional plane as a set of data is the inequality (b) A_1>[A_1(C_0)+A_1(C_1) of the following (b) and (d).
+A_1(C_2)]/3+β(d)A_2−δ<γ[
A_1-[A_1(C_0)+A_1(C_1)+A_
1(C_2)]/3+β] (where β, γ, and δ are constants) The antigen or antibody produced by immune aggregation according to claim (2), which is determined to be in the alarm region when both conditions are satisfied. concentration determination method.