JP2002055107A - Measuring method and reagent kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measuring method and a reagent kit capable of realizing higher accuracy and sensitivity by restraining influence by a zone phenomenon by improving conventional immuneoturibidimetry or immunonephelometry. SOLUTION: In this method for measuring a test substance by adding the test substance and a reactive substance for generating an insoluble high molecular complex by reacting with the test substance in a liquid phase, an adding substance having reactivity equal to the test substance is mixed with the reactive substance in the liquid phase, and is reacted in a jacking-up state covering a detecting limit with every so-called measuring apparatus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a measuring method and
It relates to a reagent kit.

[0002]

2. Description of the Related Art Various diagnostic methods have been developed for diagnosing and treating whether or not a living body is ill. Of these diagnostic methods, in particular, a specimen test using blood or urine is an essential test for diagnosing a disease in order to objectively determine the condition of a living body. Serum is most commonly used in laboratory tests, and measuring changes in components in serum is a basic test in clinical tests. Especially CRP (C
Reactive protein) is most commonly measured as a reflection of inflammatory symptoms, and immunoglobulins such as IgG, IgA and IgM, and complement components such as C3 and C4 are often measured as reflecting the state of the body. ing.

[0003] The measurement of plasma proteins such as the above-mentioned immunoglobulins and complement components was once measured by one-way immunodiffusion (SRID). However, the unified immunodiffusion method is time-consuming and difficult to measure a large number of samples simultaneously.Therefore, immunoturbidimetry or immunoturbidimetry is currently used as a method for measuring plasma protein. Most commonly used. Among them, the immunoturbidimetric method is that, when an antigen is added to a solution containing an antibody, a huge immune complex is generated by an antigen-antibody reaction, which precipitates as turbidity in the solution. Is measured by an absorptiometer, and the antigen concentration is determined by comparing with the absorbance of a standard product. On the other hand, in the immunonephelometry, an immune complex is generated by an antigen-antibody reaction, and the concentration of the antigen is determined by measuring the intensity of scattered light with respect to the irradiation light. In other words, immunoturbidimetry and immunoturbidimetry are based on the finding that the amount of insoluble immune complex generated when an antigen is added in an antibody-excess state is proportional to the amount of antigen added. A calibration curve is prepared by reacting the antibody with a predetermined concentration, and using the calibration curve, the absorbance of the immune complex obtained from the reaction between the sample and the antibody at the predetermined concentration is compared with that in the sample. Quantify the antigen concentration.

[0004] These immunoturbidimetric and immunoturbidimetric methods have higher sensitivity than the conventional one-way immunodiffusion method (in particular, immunoturbidimetric method) and can be quantified in a short period of time. It is convenient for the measurement of the plasma protein present at Enzyme-linked immunosorbent assay (EIA) and radioimmunoassay (RIA) have been used for test substances whose concentrations in plasma are low and cannot be measured by these methods. Even in the nephelometry, a device for realizing higher sensitivity has been devised.

In the immunoturbidimetry or immunoturbidimetry, a dilution test, a recovery test, and a reproducibility test are performed to confirm the performance of the measurement system. The dilution test is to prepare a dilution series of the sample and measure each diluted sample to confirm that the measured value is proportional to the dilution factor, and to assure the quantitativeness of the measurement system. In addition, the recovery test is performed by adding a known concentration of the antigen as a standard to the sample and measuring the result.The measured value obtained by subtracting the concentration of the standard from the measured value is compared with the measured value of the sample itself. And the difference between the reactivity of the sample and the reactivity of the sample.

[0006] The reproducibility test is to measure the same sample a plurality of times and check the dispersion of measured values for each measurement, and to assure the stability of the measurement system. In the immunoturbidimetry or immunoturbidimetry, the sensitivity of the measurement system is determined by measuring the absorbance or scattering intensity when an immune complex is formed by reacting a sample with an antibody, and the absorbance at 0 concentration without antigen. Or it is defined by the difference from the scattering intensity. Further, the stability of the absorbance or the scattering intensity at the concentration of the antigen contained in the sample is also a factor that affects the sensitivity. Therefore, in order to increase the sensitivity of the measurement system, it is required to increase the absorbance of the immune complex. In order to increase the absorbance, an appropriate wavelength is selected, measurement conditions such as increasing the amount of the sample and / or the antibody are set, and a surfactant or the like is added to the antiserum solution to form the immune complex. Some measures have been taken to increase turbidity.

[0007]

Generally, in an antigen-antibody reaction, in a region where the amount of antigen or antibody is excessive, since the antigen-antibody complex becomes soluble rather than insoluble, the absorbance is not proportional to the amount of antigen. The phenomenon is known. FIG. 1 shows the amount of antigen on the horizontal axis and the absorbance of the solution containing the immune complex on the vertical axis. In the region A where the amount of antigen is low and the region B where the amount of antigen is high, the absorbance is lower than the absorbance considered to be exhibited by the theoretical insoluble immune complex. In fact, in the above-mentioned dilution test, the measured value is not proportional to the dilution factor because the generation of the insoluble immune complex by the antigen-antibody reaction does not progress quantitatively in the region A (the measured value is lower than the theoretical value). ) The phenomenon is known.

In the field of clinical examinations, immunoturbidimetry and immunoturbidimetry are often performed using a dedicated or general-purpose automatic measuring device, from the dilution of a sample to the release of measurement results. Is done automatically. Therefore, when a sample is to be measured using these devices, the sample, buffer, and reaction solution are set at a predetermined position on the measuring instrument, and the instrument is switched on. It automatically dilutes, dispenses, and measures samples according to the conditions, and produces results. These measuring instruments are sold by many manufacturers, and the standards are not fixed among the measuring instruments. Therefore, the capacity of cells used for measurement, the amount of sample collected, the reaction time, and the like are not uniform. In addition, in order to increase the accuracy and stability of the measurement, it is necessary to perform the measurement under conditions most suitable for the machine. For this purpose, it is preferable to prepare a dedicated reagent for each measuring device.

However, usually, manufacturers of measuring instruments,
It is not easy for a reagent maker to prepare a different reagent for each measurement device because the manufacturer of the reagent used for the measurement device is different. Therefore, the reagent maker prepares a standard reagent, and adjusts the sample collection amount, the reagent collection amount, the measurement wavelength, and the like on the machine side to ensure the performance of the measurement system. However, as described above, since the capacity of the cell and the capacity of collecting the specimen or the reagent differ depending on the plurality of measurement devices, there is a limit to the increase in the amount of collection. In addition, increasing the amount of the sample also burdens the patient. On the other hand, when the turbidity is enhanced using a surfactant or the like, nonspecific turbidity also increases, particularly when a myeloma specimen containing an excessive amount of antigen is measured, so that sufficient performance is ensured. It was not easy, and it was difficult to eliminate the zone phenomenon.

[0010] The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to improve the conventional immunoturbidimetry or immunoturbidimetry so as to eliminate non-specific reactions and achieve higher precision and accuracy. An object of the present invention is to provide a measurement method and a reagent kit capable of realizing sensitivity.

[0011]

Means for Solving the Problems The present inventor has conducted intensive studies to solve the above-mentioned problems, and as a result, in a liquid phase, a test substance and a polymer complex which is insoluble in response to the test substance. When reacting with the reactive substance that causes the reaction, by adding an additive substance having the same reactivity as the test substance to the reactive substance in the liquid phase, there is no non-specific reaction and higher accuracy And sensitivity, and basically completed the present invention. Therefore, the measurement method according to the invention for solving the above-mentioned problem is characterized in that, in a liquid phase, a test substance and a reactive substance that reacts with the test substance to form an insoluble polymer complex are mixed by mixing the test substance and the reactive substance. A method for producing a polymer complex and measuring the test substance, wherein the liquid phase contains the test substance with respect to the reactive substance when the test substance and the reactive substance are mixed. It is characterized in that an additive substance having the same reactivity as the substance is mixed.

[0012] To this end, the following steps (a) to (c), (a) mixing the test substance and the additive substance in a liquid phase to form a mixture, and (b) the mixture Further forming the first insoluble polymer complex by adding the reactive substance to (c) irradiating the first insoluble polymer complex with light of a certain wavelength, and scattered light or transmitted light thereof And a measuring step. Further, the following steps (d) to (g),
(d) In the liquid phase, a predetermined amount of a standard substance having the same reactivity as the test substance is mixed with the additive substance to form a second mixture.
Forming a mixture, (e) further adding the reactive substance to the second mixture to form a second insoluble polymer complex, and (f) providing the second insoluble polymer complex with a predetermined wavelength. Irradiating light and measuring a scattered light or a transmitted light thereof to form a calibration curve, and (g) preparing the test substance from the scattered light or the transmitted light of the first insoluble polymer complex and the calibration curve. And a step of quantifying the concentration of the compound.

[0013] A reagent kit according to the invention for solving the above-mentioned problem comprises reacting a test substance in a liquid phase with a reactive substance which reacts with the test substance to form an insoluble polymer complex. A first solution containing an additive having the same reactivity as the test substance to the reactive substance, and a second solution containing the reactive substance. It is characterized by having.

In the above-mentioned measuring method or reagent kit, the reactive substance is preferably an antibody. In addition to the case where the reactive substance exists alone, it is preferable that the reactive substance is bound to an insoluble support or bound to a water-soluble polymer. Further, the hydrophobic substance may be improved by introducing a hydrophobic group into the reactive substance, and may be easily insolubilized by the reaction with the test substance.
Further, the test substance is preferably a plasma protein.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, embodiments of the present invention will be described with reference to the following examples, but the technical scope of the present invention is not limited by the following description. The technical scope of the present invention extends to an equivalent range. The present invention, in the liquid phase, when reacting the test substance and the reactive substance, mixing the additive substance having the same reactivity as the test substance to the reactive substance, so-called each measuring instrument It is important to react as "wearing the getter" to cover the detection limit.
The stability of the reaction generally varies depending on the absorbance to be measured, but in a low concentration range of the test substance, the measurement system becomes unstable due to low absorbance or scattering intensity. In the present invention, by adding an additive having the same reactivity as the test substance contained in the specimen, the absorbance or the scattering intensity is enhanced, and the stability of the measurement system is improved.

Examples of the "reactive substance" include an antibody, and any other substance which specifically reacts with a test substance and which forms an insoluble polymer complex by the reaction. For example, artificially multivalent ligands and receptors, enzymes and substrates may be used. Further, by introducing a hydrophobic group into the reactive substance, an insoluble polymer complex can be easily formed after the reaction with the test substance. When an antibody is selected as a reactive substance,
In addition to using the antibody solution as an antibody solution, it can be bound to an insoluble support such as latex to form a latex turbidimetric method. Further, the antibody can be used by binding to a water-soluble polymer such as dextran or ficoll, so that the antibody can be easily crosslinked with the test substance.

The "test substance" includes, for example, a plasma protein. Plasma proteins include CR as an inflammatory marker
P, α1-AG (α1 glycoprotein), haptoglobin, albumin, immunoglobulins such as IgG / IgA / IgM, C3 / C
And complement components such as 4, transferrin, haptoglobin, α2 macroglobulin, α1 antitrypsin and the like. In addition, as a “specimen” containing a test substance, urine or other bodily fluids can be measured in a similar manner in addition to serum and plasma.

As the "additive substance" having the same reactivity as the test substance with respect to the reactive substance, in the case of a plasma protein, in addition to the case where the test substance is used as a purified protein in a predetermined amount, , Or plasma or serum containing the test substance described above. Here, the "predetermined amount"
This means that the same amount of the additive substance is added to each of the unknown samples. As a result, even at the point of 0 concentration that does not contain the test substance, the measurement is performed in a state where the “predetermined amount” of the test substance is contained, and a state in which a so-called getter is worn is obtained. Therefore, the zone phenomenon in the region A where the test substance is low can be avoided.

To determine the range of the "predetermined amount",
First, it can be determined in consideration of the profile of the reaction curve of the test substance / reactive substance in the measurement system. Since the present invention is particularly effective for avoiding the zone phenomenon in the low-value area A, the maximum value MA in the low-value area A where the response curve loses linearity (shown in FIG. 1).
It is preferable to add the above test substances as “predetermined amounts”. Second, it can be determined in consideration of the range of normal values and abnormal values known in the measurement system.
In other words, when the measurement system is used, the range of the test value variation based on the range of the test value variation that can occur as a normal value or an abnormal value at the site of a general clinical test is It can be set so that the reaction curve of the active substance is included in the region showing linearity. When determining the "predetermined amount", it is preferable to select an excessively large amount of the added substance to avoid a situation where the high value region B becomes lower than the upper limit value of the range of the variation of the inspection value.

It is sufficient if the additive substance is present at the stage of reacting the test substance with the reactive substance, and it does not depend on the form (eg, powder, liquid, etc.) of the additive substance and the timing of addition. For example, when provided as the reagent kit of the present invention, it can be added in advance to the first solution.
As described above, if it is added to the first solution, it is not necessary to separately add it as an additive substance, so that the measurement operation is simplified.

When serum or plasma containing a predetermined amount of a test substance is added as an additive substance, it can be conveniently applied to all plasma protein measurement systems. It is not always necessary to use the same substance as the test substance as the additive substance, and any substance having the same reactivity as the test substance with respect to the reactive substance may be used. Alternatively, a protein obtained as a recombinant protein can be used. In particular, when the test substance is IgG, it is possible to use an F (ab ') 2 fragment as the additive substance.

Further, by preparing a calibration curve using the "standard substance", the amount of the test substance in the unknown sample can be quantitatively obtained. At that time, the same amount of the additional substance as the predetermined amount of the additional substance to be added to the sample is added to the sample for preparing the calibration curve. In addition, as the “standard substance”, generally, a predetermined amount of the test substance is often used, but the present invention is not limited to this, and has a reactivity equivalent to the test substance with respect to the reactive substance. If it is a substance, it can be used as a standard substance.

As the "liquid phase", it is preferable to use an aqueous solution, and it is usually preferable to use a buffer so that the reaction between the test substance and the reactive substance is favorably promoted. The type of the buffer is not particularly limited, and a buffer that is generally used for biochemistry can be selected from those that are suitable for the reaction system. Preferred embodiments for implementing the present invention will be described below.

[Embodiment 1] Measurement system of IgG in human serum In a first solution (hereinafter referred to as "dilution solution"), human IgG is added as an additive substance at 0.1 mg / dL to 20 mg / dL, preferably
0.5 mg / dL to 10 mg / dL, more preferably 1 mg / dL to 5 mg / dL. The second solution (hereinafter referred to as “reaction solution”) contains anti-human IgG as a reactive substance.
Adjust the antiserum to a predetermined concentration. Take 2 μL of human serum as a sample, add 250 μL of the dilution reagent, and add 100 μL of the reaction reagent after an appropriate time has elapsed. After a suitable time has elapsed, the absorbance of the reaction solution
Measure at 660 nm. The above method can provide a measurement system having good linearity when the IgG concentration in the sample is in the range of 50 mg / dL to 7500 mg / dL.

[Embodiment 2] Measurement system of IgA in human serum Human IgA was added as an additive substance in the test solution for dilution in an amount of 0.01 mg / dL or more.
3 mg / dL, preferably 0.05 mg / dL to 1.5 mg / dL, more preferably 0.1 mg / dL to 0.8 mg / dL. In the reaction solution for reaction, an anti-human IgA antiserum as a reactive substance is adjusted to a predetermined concentration. As a sample, 4 μL of human serum
, And add 250 μL of the reagent solution for dilution, and after an appropriate time, add 100 μL of the reagent solution for reaction. Further, after an appropriate time has elapsed, the absorbance of the reaction solution is measured at 600 nm. According to the above method, the IgA concentration in the sample is 10 mg /
A measurement system with good linearity can be provided in the range of dL to 1400 mg / dL.

[Embodiment 3] Measurement system of IgM in human serum Human IgM as an additive substance in the test solution for dilution was 0.01 mg / dL or more.
2 mg / dL, preferably 0.05 mg / dL to 1.0 mg / dL, more preferably 0.1 mg / dL to 0.5 mg / dL. In the reaction solution for reaction, anti-human IgM antiserum as a reactive substance is adjusted to a predetermined concentration. As a sample, 4 μL of human serum
, And add 250 μL of the reagent solution for dilution, and after an appropriate time, add 100 μL of the reagent solution for reaction. After a suitable time has elapsed, the absorbance of the reaction solution is measured at 340 nm as the main wavelength and 700 nm as the sub wavelength. The above method can provide a measurement system having good linearity when the IgM concentration in the sample is in the range of 5 mg / dL to 760 mg / dL.

[Embodiment 4] Measurement system of C3 in human serum Human C3 is added as an additive substance in the test solution for dilution in an amount of 0.01 mg / dL to 2 mg / dL.
mg / dL, preferably 0.05 mg / dL to 1.0 mg / dL, more preferably 0.1 mg / dL to 0.5 mg / dL. In the reaction solution for reaction, an anti-human C3 antiserum as a reactive substance is adjusted to a predetermined concentration. As a specimen, 4 μL of human serum is taken, 250 μL of a dilution reagent is added thereto, and after an appropriate time, 100 μL of a reaction reagent is added. Further, after an appropriate time has elapsed, the absorbance of the reaction solution is measured at 600 nm. According to the above method, the C3 concentration in the sample is 5 mg / dL to 80.
A measurement system having good linearity can be provided in the range of 0 mg / dL.

[Embodiment 5] Measurement system of C4 in human serum Human C4 as an additive substance in the test solution for dilution was 0.001 mg / dL or more.
0.2 mg / dL, preferably 0.005 mg / dL to 0.1 mg / dL, more preferably 0.01 mg / dL to 0.05 mg / dL. In the reaction solution for reaction, anti-human C4 antiserum is adjusted to a predetermined concentration as a reactive substance. As a specimen, human serum
Take 10 μL, add 250 μL of dilution reagent, and after appropriate time, add 50 μL of reaction reagent. After a suitable time has elapsed, the absorbance of the reaction solution is measured at 340 nm as the main wavelength and 700 nm as the sub wavelength. According to the above method, the C4 concentration in the sample is 1.5 mg / dL to 150 mg / dL.
A measurement system having good linearity can be provided in the range of

[0029]

[Example 1] Human IgA measurement system A healthy human serum was used as a sample, and this was used as a human IgA measurement reagent kit (trade name: TAC-2 test) by immunoturbidimetry manufactured by Institute of Medical Biology, Inc. IgA, which is a reagent kit for measuring the amount of IgA by reacting IgA in a sample with an anti-human IgA antiserum, which is a reactive substance, to form an immune complex.) The operation procedure is briefly described as follows. After adding 250 μL of the dilution reagent to 4 μL of the sample, 100 μL of the reaction reagent (including anti-human IgA antiserum) is added after an appropriate time, and before the addition of the reaction reagent (24
The difference between the measured values after the addition (50th photometric point) and after the addition (50th photometric point) was measured. Absorbance was measured at 600 nm.

The sample was diluted in advance using a dilution reagent included in the kit to prepare a dilution series of 1/1 to 1/10. The dilution linearity of the measurement system was examined using a Hitachi 7150 automatic analyzer based on the method. The measurement was performed using the reagent solution for dilution as it was according to the operation procedure of the conventional reagent kit, and 0.2% human serum as a substance added to the reagent solution for dilution in advance.
(Equivalent to about 0.2 mg / dL as human IgA).

The measurement results are as shown in Table 1, FIG. 2 and FIG. If the dilution reagent was used as it was, the absorbance deviated from the dilution line in the low antigen range with dilution,
It was measured below what was actually expected. This is Ig
It is considered that the low concentration of A caused a zone phenomenon. On the other hand, when human serum 0.2% was added to the dilution test solution, the absorbance provided by the dilution series showed a good concentration-dependent linearity, and the absorbance at each concentration was in good agreement with the theoretical value.

[0032]

[Table 1]

Example 2 Human C4 Measurement System Healthy human serum was used as a sample, and this was used as a human C4 measurement reagent kit (trade name: TAC-2 Test C4) by immunoturbidimetry manufactured by Institute of Medical Biology, Inc. This is a reagent kit for measuring the amount of C4 by reacting C4 in the sample with an anti-human C4 antiserum that is a reactive substance to generate an immune complex.) The operation procedure is briefly described as follows. After adding 250 μL of the dilution reagent to 4 μL of the sample, allow an appropriate time for the reaction reagent (including anti-human C4 antiserum) 50
μL was added, and the difference between the measured values immediately before (24th photometric point) and after (50th photometric point) the reagent solution for reaction was measured. Absorbance is 340nm as main wavelength
And was measured at 700 nm as a sub wavelength.

The sample was diluted in advance using the dilution reagent included in the kit to prepare a dilution series of 1/1 to 1/10. The dilution linearity of the measurement system was examined using a Hitachi 7150 automatic analyzer based on the method. The measurement was performed using the reagent solution for dilution as it was according to the operation procedure of the conventional reagent kit, and 0.2% human serum as a substance added to the reagent solution for dilution in advance.
(Equivalent to about 0.03 mg / dL as human C4).

The measurement results were as shown in Table 2, FIG. 4 and FIG. If the dilution reagent was used as it was, the absorbance deviated from the dilution line in the low antigen range with dilution,
It was measured below what was actually expected. This is C4
It was thought that the zone phenomenon occurred at low concentrations of. On the other hand, when human serum 0.2% was added to the dilution test solution, the absorbance provided by the dilution series showed a good concentration-dependent linearity, and the absorbance at each concentration was in good agreement with the theoretical value.

[0036]

[Table 2]

Example 3 Measurement System of Human C4 Using Myeloma Specimen In the immunoturbidimetry, a surfactant is often added to the reaction solution in order to enhance the turbidity after the reaction. When the turbidity is enhanced by the surfactant in this manner, the nonspecific reaction is also enhanced.For example, when the protein contains a high concentration such as a myeloma sample, the dilution reagent and the sample are used. Since turbidity occurs at the time of mixing, it is difficult to perform accurate quantification.

Therefore, human serum 0.2% (corresponding to about 0.2 mg / dL as human C4) was added as an additive substance to the test solution for dilution.
M myeloma serum was used as a sample, and a human C4 measuring reagent by immunoturbidimetry (described above) manufactured by Institute of Medical Biology Co., Ltd. was used for measurement with a Hitachi 7150 type automatic analyzer. The operation procedure is the same as that described in the second embodiment. Also,
The reaction reagent was added after the 24th measurement point.
The change with time of the absorbance is shown in Table 3 and FIG. Also,
The C4 concentration was measured to be 0.0 mg / dL in the conventional measurement system (a system in which human serum was not added to the dilution solution. The surfactant contained 5.5%), but the system in which human serum was added (the surfactant was used) Weight loss to 4%)
Measured 6.2 mg / dL.

[0039]

[Table 3]

As shown in Table 3 and FIG. 6, in the measurement system to which no additional substance was added (graph indicated by a square point), large turbidity (ABS = 3880) was observed when the sample and the dilution reagent were mixed (first point). ) Occurred, and the turbidity gradually decreased thereafter. Further, the addition of the reaction reagent (antiserum) increased the absorbance after the 25th photometry point again. The measured value of the C4 concentration is calculated from the difference between the absorbance before addition of antiserum (24th photometry point) and the absorbance after addition of antiserum (50th photometry point). It was considered that the myeloma specimen was quantified to a relatively low concentration because turbidity occurred due to the effect of the surfactant immediately after the dilution reagent was added, and the absorbance before the addition of antiserum was high.

On the other hand, an additive substance (human serum 0.
2%) and the amount of surfactant added was reduced to 4% (graphs indicated by triangles) in the measurement system. Absorbance (2
(4th photometric point) shows a low value, and the net reaction amount was measured as the absorbance. Therefore, it was considered that a value closer to the actual value was quantified as compared with the conventional measurement system.

Example 4 Measurement System for Human C3 In order to confirm the stability of the measurement system in the low value region, human serum was diluted to an appropriate magnification with a dilution solution (conventional measurement without adding human serum). In the system, it corresponds to an area where a zone phenomenon occurs), and a low-valued sample was prepared and measured 10 times. For the measurement, a human C3 measurement reagent kit (trade name: TAC-2 Test C3.
And an anti-human C3 antiserum, which is a reactive substance, to produce an immune complex, thereby measuring the amount of C3. ). The operation procedure is briefly described as follows. 250 μL of test solution for dilution in 4 μL of sample
After an appropriate time has elapsed, add 100 μL of the reaction solution (containing anti-human C3 antiserum). Further, after an appropriate time, the absorbance of the reaction solution was measured at 600 nm. In addition, the test solution for dilution did not contain any additional substances, and 0.2% of human serum (approximately
0.2 mg / dL).

Table 4 shows the measurement results. Comparing the measured values of C3 concentration between the case where human serum was added to the dilution test solution and the case where human serum was not added, the average value of 10 replicates was 2.81 mg / dL in the system without human serum. , CV value is 23.
It was 09%. On the other hand, in a system in which human serum was added to the dilution solution, the average value of the 10-fold measurement was 10.30 mg / dL, and the CV value was
It was 4.80%. From this fact, when human serum was not added as an additive, not only the measurement result lower than the actual value was obtained due to the zone phenomenon, but also the variation between the measurement values was large. On the other hand, when human serum was added as an additive, the insoluble polymer complex was generated proportionally by the reaction between the test substance and the reactive substance due to the avoidance of the zone phenomenon, and as a result, variation was caused. It was considered that the measurement result was small and close to the actual value.

[0044]

[Table 4]

[0045]

By using the present invention when measuring a test substance, it is possible to perform a measurement with low sensitivity and high sensitivity up to a low concentration without being affected by zone phenomena. Further, by using the present invention, accurate quantification can be performed in the measurement of a myeloma specimen without being affected by a non-specific reaction.

[Brief description of the drawings]

FIG. 1 is a schematic graph showing the relationship between test substance concentration and turbidity

FIG. 2 is a graph showing the relationship between sample concentration and turbidity when no additive is added in an IgA measurement system.

FIG. 3 is a graph showing the relationship between sample concentration and turbidity when an additive is added in an IgA measurement system.

FIG. 4 is a graph showing the relationship between sample concentration and turbidity when no additive is added in the C4 measurement system.

FIG. 5 is a graph showing the relationship between sample concentration and turbidity when an additive substance is added in the C4 measurement system.

FIG. 6 is a graph showing changes in turbidity over time when C4 is measured using a myeloma specimen.

Claims (11)

[Claims] In a liquid phase, a test substance and a reactive substance that reacts with the test substance to form an insoluble polymer complex are mixed to form the polymer complex, thereby forming the test substance. A method for measuring, wherein, in mixing the test substance and the reactive substance, an additive substance having the same reactivity as the test substance with respect to the reactive substance is mixed in the liquid phase. Measurement method characterized in that: 2. The following steps (a) to (c), (a) a step of mixing the test substance and the additive substance in a liquid phase to form a mixture, and (b) further comprising: Adding a reactive substance to form a first insoluble polymer complex, and (c) irradiating the first insoluble polymer complex with light of a predetermined wavelength and measuring scattered light or transmitted light thereof. The measurement method according to claim 1, comprising: 3. The following steps (d) to (g): (d) mixing a predetermined amount of a standard substance having the same reactivity as the test substance and the additive substance in a liquid phase; (E) adding the reactive substance to the second mixture to form a second insoluble polymer complex; (f) applying a fixed wavelength to the second insoluble polymer complex. Forming a calibration curve by irradiating the above light and measuring the scattered light or transmitted light thereof, and (g) preparing the test curve from the scattered light or transmitted light of the first insoluble polymer complex and the calibration curve. The method according to claim 2, further comprising the step of quantifying the concentration of the substance. 4. The method according to claim 1, wherein the reactive substance is an antibody. 5. The method according to claim 1, wherein the reactive substance is bound to an insoluble support. 6. The method according to claim 1, wherein the reactive substance is bound to a water-soluble polymer. 7. The method according to claim 1, wherein the test substance is a plasma protein. 8. A reagent kit for measuring a test substance by mixing a test substance and a reactive substance which reacts with the test substance to form an insoluble polymer complex in a liquid phase, A reagent kit comprising: a first solution containing an additional substance having the same reactivity as a test substance with respect to a reactive substance; and a second solution containing the reactive substance. 9. The reagent kit according to claim 8, wherein the reactive substance is an antibody. 10. The reagent kit according to claim 8, wherein the reactive substance is bound to an insoluble support. 11. The reagent kit according to claim 8, wherein the reactive substance is bound to a water-soluble polymer.