JP2763614B2 - Method for determining antigen or antibody concentration by immunoagglutination - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for determining that an antigen or antibody in a specimen is at a high concentration by immunoaggregation.

[Conventional technology]

As a device for measuring the concentration of an antigen (or antibody) contained in a sample, an insoluble carrier sensitized with an antibody (or antigen) that specifically reacts with the target antigen (or antibody) is mixed into the sample. Thus, there is an immunoagglutination measuring device which causes an antigen-antibody reaction and measures the degree of aggregation of the carrier by means of a particle counting means (JP-A-60-119163).

The degree of aggregation A is calculated from the number of aggregated particles (polymer) P and the number of unaggregated particles (monomers) M, for example, by A = P / (M + P).
Converted to density.

FIG. 4 shows the elapsed time T from the mixing of the sample and the reagent on the horizontal axis.
The vertical axis shows the aggregation growth curve with the aggregation degree A taken.
The curves f ₁ , f ₂ , and f ₃ correspond to high-, medium-, and low-concentration samples, respectively, and the progress of the reaction varies depending on the concentration of the sample (that is, the antigen concentration). Further, the cohesion degree A does not simply increase with respect to the elapsed time T, and when the time elapses too much, a phenomenon is seen in which the cohesion degree decreases. Also, the time during which the degree of agglomeration decreases is faster as the concentration is higher.

FIG. 5 shows the relationship between the sample concentration C and the agglutination degree A when the elapsed times are T ₁ ′ and T ₂ ′. Curves f ₄ and f ₅ correspond to elapsed times T ₁ ′ and T ₂ ′, respectively. Cohesion degree A
Does not simply increase with respect to the concentration C. When the amount of the antigen is too large, a phenomenon in which the degree of aggregation A decreases (prozone phenomenon) is observed. For example, suppose that a certain sample is measured at time T ₂ ′ and the degree of aggregation a ₃ is obtained. However, this cohesion degree
For a _3, the concentration C ₂ is present in a concentration C ₁ and an excess region in the normal range, it is not possible immediately to identify the concentration of cohesion.

Then, a method described in Japanese Patent Application Laid-Open No. 61-280568 was devised. In this method, the degree of aggregation is determined not only at time T ₂ ′ but also at time T ₁ ′. If aggregation degree at time T ₁ 'is a _2, the antigen concentration is C _2, if the aggregation degree is a ₁ it can be seen that the antigen concentration of C _1.

However, the peak of the curve f ₅ reaches usually several 10%, the peak of the curve f ₄ does not reach only a few percent, hardly occurs a difference in cohesion measurement of time T ₁ '. That is, there is also a problem of reproducibility, and only an agglomeration degree with poor reliability can be obtained. For this reason, it is difficult to specify the concentration by the above method.

If the measurement is performed by extending the time T ₁ ′, that is, by approaching the time T ₂ ′, the peak of the curve f ₄ also increases, so that the difference in the degree of agglomeration is likely to occur, and the measurement accuracy also increases. enter a low measurement density side, closer to the similar shape of the curve f _5. For this reason, it becomes difficult to specify the concentration. Then, in the vicinity of the peak of the curve f ₅ and foremost, with respect to the concentration C,
Since the change in the cohesion degree A is extremely small, it is difficult to perform the concentration conversion, and the result is uncertain.

As described above, it is not possible to guarantee the accuracy of the measurement result over the entire concentration region. Therefore, when the concentration of the antigen to be measured is extremely high, it is necessary to detect this as a high concentration region and issue an alarm.

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

(A) A method in which the cohesion degree A ₁ ′ at the time T ₁ ′ is used as a monitor, and when this value exceeds a certain value, the region is determined to be a high concentration region. As shown in FIG. 6, cohesion shown by curve f ₄
When A ₁ ′ is equal to or larger than a ₄ , it is determined that the region is a high concentration region. The region indicated by oblique lines is that region.

(B) A method of determining a high density region when αA ₁ ′ + A ₂ ′ becomes a certain value or more instead of A ₁ ′ in (A).
A ₂ ′ is the degree of aggregation at time T ₂ ′. α is a constant.

[Problems to be solved by the invention]

In the case of the method (a), as described above, since the accuracy of the cohesion degree is not obtained, the judgment region is likely to vary. In other words, even though it is not a high-density area, it is determined to be a high-density area,
Despite being a high-density area, it may not be determined to be a high-density area. In the high density by prozone phenomenon may cohesion is less than a _4, erroneous determination may occur.

In the case of the method (b), an erroneous determination occurs similarly to the case of the method (a).

Conventionally, when the measurement is repeatedly performed, if the concentration of the previous sample is extremely high, it may affect the measurement result of the next sample, thereby causing a measurement error.

An object of the present invention is to provide a method for determining the concentration of an antigen or an antibody by immunoaggregation, which can correctly determine a high concentration region even in a measurement system in which a prozone phenomenon occurs.

An object of the invention of claim (2) is to provide a method for determining the concentration of an antigen or an antibody by immunoaggregation, which can appropriately determine that the concentration of a previous sample is high enough to affect the next measurement result. .

[Means for solving the problem]

The concentration determination method of the present invention comprises mixing and reacting a sample having an antigen or antibody to be measured with a reagent containing an antibody or an antigen-insoluble carrier sensitized with the antigen or antibody. Accordingly, the antigen or antibody in the specimen to an insoluble carrier to agglomerate with each other as a medium, sample and reagent different predetermined time T ₁ and to each other from the time of mixing of the
When T ₂ has elapsed, the cohesion degrees A ₁ and A ₂ at each elapsed time are measured, and the cohesion degrees A ₁ and A ₂ are plotted on a two-dimensional basis as a set of data. If it is within a predetermined area, it is determined that it is in a high concentration area exceeding the measurement range.

The above-mentioned region is given in a form that bisects the aggregation characteristic curve with the analyte concentration as a parameter drawn on the two-dimensional plane.

According to the invention of claim (2), in the case of claim (1), if the point where the cohesion degrees A ₁ and A ₂ are plotted two-dimensionally as a set of data is within a predetermined region, the point is remarkably increased. It is determined that the warning area has a high concentration.

(Operation)

Samples such as human serum contain various kinds of substances such as antigens and antibodies. The reagent of the immunoagglutination measurement device contains an antibody or an insoluble carrier sensitized with an antigen that specifically reacts with a specific antigen or antibody. Thus, by mixing the sample and the reagent, an antigen-antibody reaction occurs, and the insoluble carrier mutually aggregates via the antigen or antibody in the sample. The degree of aggregation varies depending on the amount of antigen or antibody to be measured contained during detection. Therefore, the amount of the target antigen or antibody is measured by measuring the degree of aggregation and further converting the degree of aggregation.

In the present invention, a set of agglomeration degrees A ₁ and A ₂ is obtained by measuring the degree of agglomeration with a time lag. Both do not make the same change in concentration, so T ₁
Taking cohesion A ₁ and T ₂ cohesion A ₂ measurement of the measuring in two axes, according to the concentration change, it is possible to draw a cohesive characteristic curve on a two-dimensional. Therefore, if the area is determined so as to bisect the aggregation characteristic curve, the area can be divided into a high density area and a non-high density area based on the density.

The area can be defined as an area that satisfies, for example, any of the following inequalities (a) to (c).

The method according to claim (2) is applicable to a case where the preceding sample has an extremely high concentration and the measurement result of the next sample may be affected, for example, when the following inequalities (b) and (d) are satisfied. Can be issued assuming that the determination condition is satisfied.

〔Example〕

One embodiment of the present invention will be described with reference to FIGS. First, the immunoagglutination measuring device used in this embodiment will be described.

An apparatus using the CIA method (Counting Immunoassay method) is known as an apparatus for measuring a small amount of protein in a sample with high sensitivity and rapidity. This is a device that measures the degree of agglutination associated with latex agglutination using an antigen-antibody reaction by a particle counting method and quantifies the antigen.
A-100 (trade name, manufactured by Toa Medical Electronics Co., Ltd.) is one example. In this embodiment, this immunoagglutination measuring device is used.

In this device, a sample (containing the antigen to be measured) and a reagent (containing latex particles to which an antibody specifically reacting with the antigen to be measured in the sample is included) are mixed. As a result, a reaction occurs between the antigen and the antibody, and the latex particles begin to aggregate with each other, for example, two, three,..., Using the antigen in the sample as a medium, and gradually aggregates are formed. After reacting for a predetermined time, a mixture of the sample and the reagent is introduced into the flow cell. A sheath flow is formed in the flow cell, and a laser beam is applied to a tiny stream of particles. The number of unagglomerated latex particles and the number of aggregated latex particles are determined by measuring and discriminating the scattered light emitted from each particle, and the degree of aggregation is calculated from the numbers of both. The determined degree of aggregation is converted to an antigen concentration by a calibration curve. As described above, the target antigen in the sample is quantified. The method of determining the degree of aggregation by the above method is described in detail in JP-A-60-119163 and JP-A-60-243565.

In this example, the degree of agglomeration A was determined by the number of unagglomerated latex particles.
From M, the number of aggregated latex particles P, A = P (M + P) is defined and quantified. In addition, the degree of aggregation is measured twice in one measurement. After time T ₁ from the mixing of sample and reagent (e.g., about 30 seconds) performs a first round of measurements (T ₁ measurement), the second measurement after a time T ₂ (e.g. after about 15 minutes) (T ₂ measurements)
I do. In this way, by performing the measurement twice with a time lag, a set of data on the cohesion degrees A ₁ and A ₂ can be obtained.
It is checked whether or not the cohesion degrees A ₁ and A ₂ satisfy predetermined conditions, and it is determined whether or not the cohesion degrees are in a high concentration region. This will be described in an easy-to-understand manner.

The obtained set of data A ₁ and A ₂ is plotted on two dimensions with the two axes being A ₁ and A _2, and it is determined whether or not the data is in the high concentration area. The high-concentration region is, for example, an over-range region outside the measurement range. A ₁ and A ₂ are T ₁ measurement and T ₁ respectively.
It is the degree of aggregation in ₂ measurements. FIG. 1 is a diagram for judging the over-range which the aggregation degree A _1, A ₂ and biaxial, f ₆
Shows the aggregation characteristic curve when α-fetoprotein (AFP) was measured. Aggregation curve f ₆ measures the degree of aggregation with varying concentration can be obtained by plotting the resultant cohesion. Numbers were appended to each point of the first drawing of the curve f ₆ is AFP concentration, unit is [ng / ml]. The degree of agglomeration is expressed as a percentage.

The area for over range determination
It is determined to f ₆ to the original. In other words, the aggregation characteristic curve f ₆ starts near the origin and draws the curve shown in FIG. 1 in the case of AFP as the concentration increases. of course,
If the measurement object changes, the shape of the curve also changes. Over-range region is taken as the encompasses curved section of the subsequent point corresponding to the concentration Cx with the upper curve f ₆ (i.e. higher concentrations), and does not include a curved portion of the previous i.e. low concentrations that point region . By thus bisecting the curve f _6, the concentration Cx more analytes can be determined to be in the over-range region.

In the case of AFP, as an example, the following (a) to
An area satisfying any one of the conditions (c) could be set as an overrange area. In FIG. 1, it is shown by hatching. Curves a to c in FIG. 1 correspond to equations (a) to (c), respectively.
Here is a function with the inequality sign equal to:

(A) A ₂ −A ₂ (C ₆ )> − α [A ₁ −A ₁ (C ₆ )] (b) A ₁ > [A ₁ (C ₀ ) + A ₁ (C ₁ ) + A ₁ (C ₂ )] / 3 + β (c) A ₂ > A ₂ (Cx) where α and β are positive constants, and in the case of AFP, α = 2
0 and β = 1.0 were good.

Note that A ₁ (C ₀ ), A ₁ (C ₁ ), A ₁ (C ₂ ), A ₁ (C ₆ ), and T ₁ measurements of the calibrators with concentrations C ₀ , C ₁ , C ₂ , and C ₆ respectively. a degree of aggregation, a ₂ (C ₆₎ is the degree of aggregation in the T ₂ measured concentration C _6. A ₂ (Cx) is the degree of aggregation in the T ₂ measurement of the concentration Cx, and is a calibration curve determined by measuring calibrators at the concentrations C ₀ , C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , and C ₆ Required from.

FIG. 2 is a calibration curve diagram. The concentrations C _{0 to} C ₆ are specifically
0,2,8,32,128,512,2048 [ng / ml]. Cx is 1000 [ng / ml] here. That is, 1000 [ng / ml] or more is set as the overrange region.

The highest concentration of 2 million [ng / ml] shown in FIG. 1 is a sample having an extremely high concentration that does not normally appear. β is set to such a value that the high-concentration sample can be distinguished from the sample of extremely low concentration (C _{0 to} C ₂ ) with a margin. If α is too small, a sample having a concentration higher than the concentration Cx tends to be determined to be in the overrange region due to data variation. If α is too large, a sample having a concentration lower than the concentration Cx tends to be determined to be outside the overrange region. As described above, the area for the overrange determination must be determined in consideration of the occurrence of data variation.

In the above, the determination method has been described for the case of AFP. However, in the case of other measurement substances such as carcinoembryonic antigen (CEF), ferritin (FRN), β2-microglobulin (B2M), etc. Can be determined and determined. Investigating, CEA, FRN, B2M, like AFP,
It has been found that the overrange region can be determined under the conditions (a), (b), and (c) described above. However, it is necessary to select appropriate values for α and β so as to match each item.

In the above formula, C _n (n is an integer of 3 or more) can be used instead of C ₆ .

Next, another embodiment will be described. In this example, the concentration
A higher density Cy is set than Cx, and a region having the density Cy or higher is set as another determination region. Here, Cy is set to about 200,000 [ng / ml], and a carry-over alarm is issued for a sample having a concentration equal to or higher than this. The carryover alarm is an alarm that is issued when there is a possibility that the measurement result of the next sample may be affected when the concentration of the previous sample is extremely high.

As shown in FIG. 3, the determination conditions will be described. A region satisfying both of the following conditions (b) and (d) is defined as a carry-over warning region. In FIG. 3, the range indicated by the cross hatching is the carry-over warning area. A curve d in FIG. 3 shows the following equation (d) and a function obtained by converting an inequality sign into an equal sign.

(B) A ₁ > [A ₁ (C ₀ ) + A ₁ (C ₁ ) + 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 invention's effect〕

In the concentration determination method of the present invention, in the first measurement,
The degree of aggregation is measured twice at different times, and the two degrees of aggregation take different values for each concentration. Therefore, by plotting the two values as one set on a two-dimensional basis, even if the prozone phenomenon occurs, it is possible to determine satisfactorily whether or not the values are in the target density region regardless of the prozone phenomenon. For this reason, an uncertain measurement result is not issued, and the inspection accuracy can be improved.

According to the density determination method of claim (2), when the plotted point is in a predetermined high density area different from the density area, it is determined that the plotted point is in the alarm area. It can be seen that the measurement result is high and may affect the measurement result of the next sample, and the test accuracy can be further improved.

[Brief description of the drawings]

FIG. 1 is an explanatory view of an agglutination characteristic curve showing an AFP overrange region in one embodiment of the present invention, FIG. 2 is a calibration curve diagram thereof, and FIG. 3 is a diagram showing an AFP carryover warning region in another embodiment. FIG. 4 is an explanatory diagram of the aggregation characteristic curve shown, FIG. 4 is an explanatory diagram showing the relationship between elapsed time and the degree of aggregation in the conventional example, and FIG.
FIG. 6 and FIG. 6 are explanatory diagrams each showing the relationship between the antigen-antibody and the degree of aggregation. a to c: curves indicating determination conditions, f _6: aggregation property curves

Continuation of front page (58) Field surveyed (Int.Cl. ⁶ , DB name) G01N 33/543

Claims (2)

(57) [Claims] 1. A sample having an antigen or antibody to be measured is mixed with a reagent containing an insoluble carrier sensitized with an antibody or antigen specifically reacting with the antigen or antibody.
By reacting the antigen or antibody in the specimen to an insoluble carrier to agglomerate with each other as the intermediary, when sample and reagent different predetermined time from the mixing of T ₁ and T ₂ has elapsed, at each elapsed time The cohesion degrees A ₁ and A ₂ are measured, and the cohesion degrees A ₁ and A ₂ are plotted on a two-dimensional basis as a set of data.If the plotted point is within a predetermined area, the measurement range is set. A method for determining the concentration of an antigen or an antibody by immunoaggregation, which comprises determining that the concentration is in an extremely high concentration region. 2. If a point obtained by plotting the cohesion degrees A ₁ and A ₂ on a two-dimensional basis as a set of data is within a predetermined region, it is determined that the region is an alarm region having a remarkably high concentration. Item (1): A method for determining the concentration of an antigen or an antibody by immunoagglutination.