JP4092374B2 - Affinity substance measurement method - Google Patents

Description

  The present invention relates to a method and an apparatus for measuring an affinity substance utilizing agglutination reaction of carrier particles.

  As a method for detecting or measuring the presence of an affinity substance, for example, an enzyme immunoassay or a radioimmunoassay has been conventionally used. In either method, the affinity substance is bound to its binding partner, and the affinity substance is measured based on the binding level of both. These methods are highly sensitive and accurate. However, the reagent is unstable because an enzyme or a radioisotope is used as a label. In addition, when using radioisotopes, there are restrictions on storage and preservation, so detailed consideration and technology are required for measurement. Therefore, a simpler measuring method has been demanded. Moreover, since these methods require a relatively long time for measurement, it is difficult to cope with an urgent inspection. Under these circumstances, highly sensitive and rapid measurement methods have been actively studied.

  Since 1970, an analytical method for measuring the binding between an affinity substance and a binding partner using the aggregation of carrier particles as an indicator has been put into practical use. This method enables quantitative analysis by optically measuring the degree of aggregation of carrier particles. For example, a method of optically measuring immunological particle agglutination using latex particles as carrier particles is called latex agglutination turbidimetry. The reaction temperature in these analytical methods is generally in the range of 37 to 45 ° C., and the specific agglutination reaction proceeds by stirring with a stirring blade or the like. At this time, the time required for measurement (reaction) is about 10 to 20 minutes, which is quicker than the enzyme immunoassay or radioimmunoassay. On the other hand, it is said that measurement sensitivity or measurement range is inferior to enzyme immunoassay.

The particle size distribution measurement method in the latex agglutination method is also known (Non-Patent Document 1 / Cambiaso et al., J. Immunol. Methods 18, 33, 1977, Non-Patent Document 2 / Shigetaka Matsuzawa, Chemistry and Industry, Vol. 36, No. 4) No., 198 3 ). The latex agglutination turbidimetry measures the light transmittance of the particle suspension, whereas the particle size distribution measurement measures the state and number of dispersed individual particles. Ca in the beam Biaso et al reported, was reacted for 20 minutes the reagent and the antigen which an antibody is bound to a latex particle size 0.8μm at 37 ° C.. The number of particles after the reaction was counted, and the antigen was measured based on the level of decrease in the number of particles due to aggregation. The number of particles was measured with a counter based on the principle of laser scattered light.

  On the other hand, Matsuzawa et al. Reacted a reagent in which an antibody was bound to latex particles having a particle diameter of 1 μm with an antigen for 6 hours. After the reaction, the antigen was measured by measuring the average particle volume by the electric resistance method. However, only the PAMIA system (Sysmex Corporation) using the laser scattering method by the sheath flow has been put into practical use and has spread. In PAMIA, latex particles having a particle diameter of 0.78 μm are used. After reaction at 45 ° C. for 15 minutes, latex particles are counted and immunoassay is performed. PAMIA has higher sensitivity than latex agglutination turbidimetry, but is said to be less sensitive than high sensitivity immunoassays such as radioimmunoassay (RIA) and enzyme immunoassay (EIA).

  In latex agglutination turbidimetry, latex particles having a particle diameter of 0.05 to 0.6 μm are generally used. In the case of the latex agglomeration particle size distribution analysis method, such a small particle is easily affected by a measurement interfering substance. For example, lipids, proteins, blood cell components, etc. coexist in body fluids such as blood and urine. These coexisting substances are difficult to distinguish from the carrier particles. Thus, relatively large particles have been used to avoid the effects of these measurement interfering substances, which may not allow correct counting of carrier particles. On the other hand, when the particle diameter is about 1 μm as in Matsuzawa et al., Agglutination reaction is difficult to occur, so latex particles of about 0.8 μm have been used. Also, the aperture (pore) used by Matsuzawa et al. To measure the average particle volume is 30 μm. This size is susceptible to aperture clogging. However, it is impossible to detect particles of 0.8 to 1 μm with an aperture having a larger diameter.

  Furthermore, in order to promote the aggregation of carrier particles based on the binding between the affinity substance and the binding partner, and to facilitate the detection of the formed aggregate, a method of applying an AC voltage to the reaction system is known (special feature). Kaihei 7-83928 / Patent Document 1). This method is a method for detecting or measuring the presence of an affinity substance by agglomeration of carrier particles, and an AC voltage is applied to the reaction system so that an electric field strength of 5 to 50 V / mm is obtained in the presence of a salt of 10 mM or more. It is characterized by applying to.

  When the carrier particles holding the binding partner are placed in an electric field, they line up along the electric field (pearl chaining). Thereafter, when the electric field is stopped, the carrier particles arranged side by side are redispersed. If an affinity substance is present during pearl chain formation, the binding partner binds to the affinity substance. As a result, even after the electric field is stopped, the carrier particles do not redisperse, and the presence of a pearl chained carrier is still observed. The measurement method utilizes this phenomenon. That is, in the electric field, the reaction of the affinity substance is promoted. If the carrier particles are redispersed after the electric field is stopped, the reaction product can be detected.

Japanese Patent Laid-Open No. 7-83928 Cambiaso et al., J. Immunol. Methods 18, 33, 1977 Matsuzawa, Shigetaka, Chemistry and Industry, Vol.36, No.4, 1983

In the method for measuring an affinity substance using application of an electric field, the reaction of the affinity substance occurs during pearl chain formation of carrier particles holding a binding partner. Carrier particles aggregated by the reaction of both are detected or measured as an indicator of binding. Therefore, if the reaction between the two can be promoted, the reaction efficiency may be improved. Alternatively, it would be useful if a method for clarifying the factors that inhibit the reaction and removing the influence is provided.
An object of the present invention is to provide a method for promoting the binding of carrier particles in a method of aggregating carrier particles by binding an affinity substance and a binding partner. Or the subject of this invention is providing the method for suppressing the influence of the inhibition factor of both coupling | bonding.

  In order for the binding partner held by the pearl chained carrier particles to efficiently bind to the affinity substance, it is necessary to provide a condition in which as much affinity substance as possible contacts the binding partner. In other words, it can be said that the condition where there are many affinity substances that miss the opportunity of contact with the binding partner is a condition with low reaction efficiency. Under such conditions, in the measurement method in which the binding between the two is detected by aggregation of carrier particles, improvement in measurement sensitivity may be hindered. The present inventors have clarified a method for improving the efficiency of binding between an affinity substance and a binding partner, and completed the present invention.

In addition, the temperature of the reaction solution to which a voltage is applied rises due to Joule heat. The present inventors analyzed the influence of the temperature of the reaction solution on the reaction between the affinity substance and the binding partner. As a result, it was confirmed that an increase in the temperature of the reaction solution may inhibit the binding of the two. Then, it has been clarified that suitable reaction conditions can be realized by controlling the temperature conditions of the reaction solution to which an electric field is applied, and the present invention has been completed. That is, the present invention provides the following measurement method, measurement apparatus, or method for promoting aggregation of carrier particles holding a binding partner by an affinity substance.
[1] A method for measuring an affinity substance, comprising the following steps.
(1) Incubating a reaction liquid in which carrier particles bound with a binding partner having binding activity with a measurement target affinity substance and a measurement target affinity substance are mixed,
(2) a step of applying a voltage pulse to the reaction solution in step (1),
(3) After step (2), aggregates of carrier particles formed by binding with the affinity substance to be measured, and carriers that did not bind to the affinity substance to be measured and did not form an aggregate Counting either or both of the particles, and
(4) After the step (3), the step of determining the level of the substance to be measured based on either or both of the aggregate formation level and the level of the carrier particles that did not form the aggregate [2] step The method according to [1], wherein (1) is a step of incubating the reaction solution at 37 to 90 ° C.
[3] The method according to [2], wherein step (1) is a step of incubating the reaction solution at 40 to 90 ° C.
[4] The method according to [1], wherein the reaction solution contains a water-soluble polymer.
[5] The method according to [1], wherein the viscosity of the reaction solution in step (2) is adjusted to 0.8 to 3 mPas.
[6] The method for measuring an affinity substance according to [1], wherein the step (2) is performed at 0 to 20 ° C.
[7] The method for measuring an affinity substance according to [6], wherein the step (2) is performed at 0 to 10 ° C.
[8] The method according to [1], wherein either or both of the aggregate and the carrier particles that did not form the aggregate are counted using the three-dimensional information as an index.
[9] The method according to [1], wherein the binding between the affinity substance and the binding partner is binding by an antigen-antibody reaction.
[10] The method according to [9], wherein the affinity substance is an antigen and the binding partner is an antibody or a fragment containing an antigen-binding region thereof.
[11] The method according to [9], wherein the affinity substance is an antibody or a fragment containing an antigen-binding region thereof, and the binding partner is a fragment containing an antigen or an antigenic determinant thereof.
[12] The method according to [1], wherein the voltage pulse is an AC voltage pulse.
[13] A method for measuring an affinity substance, comprising the following steps.
(1 ′) a step of incubating a reaction liquid containing at least a binding partner having binding activity with a measurement target affinity substance and a measurement target affinity substance before or after mixing with the agglutination reagent component. The carrier particles are aggregated by an agglutinating reagent, and the aggregation is inhibited by an affinity substance to be measured.
(2 ′) applying a voltage pulse to the reaction solution in step (1 ′) in the presence of the agglutinating reagent component;
(3 ′) After step (2 ′), any of the aggregates of the carrier particles formed by binding to the agglutinating reagent and the carrier particles whose aggregation is inhibited by binding to the affinity substance to be measured or Counting both, and
(4) After the step (3 ′), the step of determining the level of the substance to be measured based on either or both of the aggregate formation level and the carrier particle level that did not form the aggregate [14] The method according to [13], wherein in step (1 ′), after the reaction solution is incubated, an agglutinating reagent is mixed before step (2 ′).
[15] The method according to [13], further comprising a step of further incubating the agglutinating reagent after mixing and before the step (2 ′).
[16] The method according to [13], wherein in the step (1 ′), the reaction solution is incubated in the presence of an agglutinating reagent, and then the step (2 ′) is performed.
[17] An apparatus for aggregating carrier particles, comprising carrier particles bound with a binding partner having binding activity with a specific substance, and means for applying a voltage pulse to a reaction liquid containing the specific substance. An apparatus having means for heating the temperature of the liquid to 37 ° C to 90 ° C.
[18] A method for aggregating carrier particles, comprising a step of applying a voltage pulse to a reaction liquid containing a carrier particle bound to a binding agent having a binding activity with a particular substance and the particular substance. The method is characterized in that the temperature of the reaction solution is 0 ° C. to 20 ° C. during application of.
[19] The method according to [18], wherein the binding between the binding partner and the specific substance is an antigen-antibody reaction.
[20] The method according to [18], wherein the voltage pulse is an AC voltage pulse.
[21] The method according to [18], wherein a water-soluble polymer is added to the reaction solution.
[22] The method according to [18], wherein the viscosity of the reaction solution is adjusted to 0.8 to 3 mPas.
[23] The method according to [18], comprising a step of incubating the carrier particles and the specific substance at 37 ° C. to 90 ° C. before applying a voltage pulse.
[24] An apparatus for aggregating carrier particles, comprising carrier particles bound to a binding partner having binding activity with a specific substance, and means for applying a voltage pulse to a reaction solution containing the specific substance. An apparatus having means for setting the temperature of the reaction solution to 0 ° C. to 20 ° C. during application of.
[25] An affinity substance or an agglutinating reagent of the carrier particle, which includes the following elements and binds a carrier particle bound with a binding partner having a binding activity to the affinity substance to be measured and the affinity substance to be measured. A measuring device for measuring the aggregation by the index.
a: space for holding the reaction solution,
b: means for incubating the temperature of the reaction solution at 37 ° C. to 90 ° C.
c: means for applying a voltage pulse to the reaction solution,
d: means for setting the temperature of the reaction solution to 0 ° C. to 20 ° C. when a pulse voltage is applied, and
e: Means for counting either or both of carrier particles and agglomerates of carrier particles contained in the reaction solution

  According to the present invention, there is provided a method for promoting the binding reaction of the carrier particles holding the binding partner with the affinity substance in the reaction solution to which an electric field is applied, or a method for suppressing the influence of the factor inhibiting the reaction. sponsored. There are not many reports showing suitable reaction conditions in the measurement of affinity substances using the aggregation of carrier particles as an indicator. According to the present invention, for example, an increase in sensitivity of a measurement method using an immunological binding reaction using carrier particle aggregation as an index, or a reduction in reaction time can be realized. The present invention contributes to the optimization of the above reaction.

The present invention relates to a method for measuring an affinity substance, comprising the following steps.
(1) Incubating a reaction liquid in which carrier particles bound with a binding partner having binding activity with a measurement target affinity substance and a measurement target affinity substance are mixed,
(2) a step of applying a voltage pulse to the reaction solution in step (1),
(3) After step (2), aggregates of carrier particles formed by binding with the affinity substance to be measured, and carriers that did not bind to the affinity substance to be measured and did not form an aggregate Counting either or both of the particles, and
(4) After step (3), determining the level of the substance to be measured based on either or both of the aggregate formation level and the level of carrier particles that did not form the aggregate

  A feature of the present invention is to incubate a reaction liquid in which a carrier particle bound with a binding partner having binding activity with a measurement target affinity substance and a measurement target affinity substance is mixed before applying a voltage pulse. is there. The present inventors have clarified that the formation of aggregates after application of the voltage pulse is promoted by incubation of the reaction solution before application of the voltage pulse. That is, the reaction is promoted by incubation before the voltage pulse is applied.

  In the present invention, the incubation of the reaction solution is performed at a temperature of room temperature or higher, for example. The incubation temperature is desirably as high as possible as long as the activity of various reaction components contained in the reaction solution can be maintained. The time for incubation is not limited. That is, the incubation can be performed at a temperature that does not cause denaturation of the reaction components. The longer the incubation time, the stronger the promoting effect. Therefore, it is desirable to set in advance the conditions of temperature and time that can be expected to have the required level of promoting effect.

Specifically, for example, the temperature condition for incubation can be usually 37-90 ° C, preferably 40-90 ° C, or 45-80 ° C. For example, a protein antigen that is an affinity substance can be measured based on the present invention using an antibody that is a binding partner. It is known that proteins constituting antibodies and antigens are denatured at high temperatures. However, for example, the incubation condition of 56 ° C. for 30 minutes, which is a general condition for deactivating serum, does not denature many proteins. Furthermore, the present inventors have confirmed that protein denaturation can be neglected even at a temperature of about 90 ° C., if the treatment is performed for a short time under low protein concentration conditions such as immunoassay. For example, in the range of 45 to 80 ° C., it was confirmed that incubation for 5 to 180 seconds can obtain almost the same level of reaction promoting effect (FIG. 3). Accordingly, preferable incubation conditions in the present invention are preferably 45-80 ° C., more preferably 50-65 ° C., for 5 seconds or longer, for example, 5-30 seconds.
It goes without saying that a longer incubation time is included in the present invention. However, when shortening of the reaction time is desired, by adopting a short time of 5 seconds or more, the target reaction promoting effect can be expected without sacrificing the reaction time. For example, under high temperature conditions exceeding 80 ° C., protein denaturation can be avoided by setting the incubation time to 5-180 seconds.

  Alternatively, high temperature conditions are not a problem when the method of the present invention is applied to the reaction of a heat-resistant substance. For example, DNA is extremely stable even under high temperature conditions. Therefore, when the binding between DNAs is to be measured by aggregation of carrier particles, a higher temperature can be selected as the temperature for incubation.

  In the present invention, the mechanism by which the reaction is promoted by incubation can be explained as follows. Carrier particles aligned by application of a voltage pulse form an agglomerate by the binding partner immobilized thereon being cross-linked with a binding partner on another carrier particle via an affinity substance. A series of reactions were thought to occur when the carrier particles were aligned. However, according to the knowledge obtained by the present inventor, it was confirmed that the incubation before applying the voltage pulse contributes to the efficiency of the reaction. It was also clarified that an antigen was bound to the antibody on the carrier particles during this incubation. In other words, it was considered that the antibody was made to capture the antigen before the carrier particles were aligned by applying the voltage pulse, thereby improving the reaction efficiency.

  In the state where the carrier particles are aligned, it is considered that the opportunity of contact between the binding partner on the carrier particles and the affinity substance present in the reaction solution is limited. In the reaction solution to which the voltage pulse is applied, the alignment and dispersion of the carrier particles are repeated by intermittent application of the electric field. As a result, the chance of contacting the binding partner with the affinity substance is increased. On the other hand, under the condition where no voltage pulse is applied, it is considered that the degree of freedom of the carrier particles is much greater than when the voltage pulse is applied. As a result, the chance of contacting the affinity partner with the binding partner on the carrier particle is increased. Carrier particles having a binding partner that has captured the affinity substance are aligned with the other carrier particles in the electric field by application of a voltage pulse. At this time, since the binding partner has already captured the affinity substance, an aggregate is rapidly formed by the binding with the binding partner of another approaching carrier particle.

That is, in the method of detecting the aggregation of carrier particles after application of the voltage pulse and measuring the affinity substance using the aggregation as an index, the aggregates of the carrier particles are formed through the following primary reaction and secondary reaction. You can say.
Primary reaction: a reaction in which a binding partner on a carrier particle captures an affinity substance. At this time, it is not always necessary to form a crosslinked structure between the carrier particles via the affinity substance.
Secondary reaction: A reaction in which binding partners on a plurality of carrier particles bind to an affinity substance. As a result, a crosslinked structure (that is, an agglomerate) between the carrier particles via the affinity substance is formed.
The secondary reaction is promoted by the application of voltage pulses. However, specific conditions for promoting the primary reaction have not been clarified. Therefore, the present invention can be regarded as providing conditions for promoting the primary reaction. That is, it can be said that the incubation step before the application of the voltage pulse in the present invention is a step for promoting the primary reaction.

  In the present invention, the water-soluble polymer compound can be added to the reaction solution before the voltage pulse is applied. By detecting the binding between the affinity substance and the binding partner by the particle agglutination reaction in the presence of the water-soluble polymer compound, the agglutination reaction can be enhanced or stabilized. The concentration of the water-soluble polymer compound in the reaction solution can be appropriately selected from 0.05 to 5%, for example. More preferably, it is 0.1 to 3%, More preferably, it is 0.3 to 1%. Compounds with strong agglutination enhancement tend to increase the likelihood of non-specific agglutination at concentrations above 5%. In addition, at a low concentration of 0.05% or less, the effect may not be sufficiently expected.

As the water-soluble polymer compound, polyethylene glycol, dextran, carboxymethyl cellulose and the like can be used. The molecular weight of polyethylene glycol is preferably 6,000 to 2,000,000. One type of these water-soluble polymer compounds may be used, or two or more types may be combined. In order to add the water-soluble polymer compound to the reaction solution, a necessary amount can be added in advance to the reagent containing the particle carrier. Alternatively, the water-soluble polymer compound can be mixed as a reagent different from the particle carrier. For example, a water-soluble polymer compound can be added to a sample diluent. Further, it can be added to a plurality of reagents to be mixed and a diluent.
Preferably, a two-reagent system is used, which is contained in a first reagent such as a buffer and mixed with a second reagent containing a carrier and used for measurement. At this time, a non-specific absorbent that absorbs a non-specific substance such as a heterophile antibody or a substance for absorbing a rheumatoid factor can be added to the first reagent.

The improvement in reaction efficiency by incubation before application of a voltage pulse based on the present invention can also be applied to an aggregation inhibition reaction. That is, this invention provides the measuring method of an affinity substance including the following processes. The incubation conditions are the same as those described above. Also in the aggregation inhibition reaction system, a water-soluble polymer compound can be added to the reaction solution.
(1 ′) a step of incubating a reaction liquid containing at least a binding partner having binding activity with a measurement target affinity substance and a measurement target affinity substance before or after mixing with the agglutination reagent component. The carrier particles are aggregated by an agglutinating reagent, and the aggregation is inhibited by an affinity substance to be measured.
(2 ′) applying a voltage pulse to the reaction solution in step (1 ′) in the presence of the agglutinating reagent component;
(3 ′) After step (2 ′), any of the aggregates of the carrier particles formed by binding to the agglutinating reagent and the carrier particles whose aggregation is inhibited by binding to the affinity substance to be measured or Counting both, and
(4) After the step (3 ′), a step of determining the level of the substance to be measured based on either or both of the aggregate formation level and the level of the carrier particles that did not form the aggregate.

  As described above, incubation of the reaction solution before application of the voltage pulse is effective for promoting the agglutination reaction of the carrier particles. At this time, as described above, the higher the incubation temperature, the greater the effect. On the other hand, the temperature of the reaction solution to which the voltage pulse is applied rises due to Joule heat. When current flows through the conductor, the heat generated in the conductor is Joule heat. The present inventor has found that a high temperature condition in incubation acts on the agglutination reaction in an accelerated manner, whereas an increase in temperature when a voltage pulse is applied acts on the agglutination reaction in an inhibitory manner. Therefore, it is advantageous to keep the temperature of the reaction solution low during voltage application.

  That is, the present invention relates to a method for aggregating carrier particles, comprising a step of applying a voltage pulse to a reaction solution containing a carrier partner bound to a specific substance and having a binding activity with a specific substance and the specific substance. A method is provided in which the temperature of the reaction solution is 0 ° C. to 20 ° C. while a voltage is applied.

The method of the present invention can be used in a method for measuring an affinity substance using, for example, aggregation of carrier particles as an index. More specifically, the present invention provides a method for measuring an affinity substance comprising the following steps.
(1) A voltage pulse is applied under a condition of 0 ° C. to 20 ° C. to a reaction solution in which a carrier particle bound with a binding partner having a binding activity with a measurement target affinity substance and a measurement target affinity substance are mixed. Process,
(2) After step (1), aggregates of carrier particles formed by binding with the affinity substance to be measured, and carriers that did not bind to the affinity substance to be measured and did not form an aggregate Counting either or both of the particles, and
(3) After the step (2), determining the level of the substance to be measured based on either or both of the aggregate formation level and the level of the carrier particles that did not form the aggregate

Or this invention provides the measuring method of an affinity substance including the following processes.
(1 ′) A voltage is applied to a reaction liquid containing at least a binding partner having a binding activity with a measurement target affinity substance, a measurement target affinity substance, and an aggregating reagent component under a condition of 0 ° C. to 20 ° C. Applying a pulse;
(2 ′) After step (1 ′), any of the aggregates of carrier particles formed by binding to the agglutinating reagent and carrier particles whose aggregation is inhibited by binding to the affinity substance to be measured or Counting both, and
(3) After the step (2 ′), a step of determining the level of the substance to be measured based on either or both of the aggregate formation level and the level of the carrier particles that did not form the aggregate.

  In this invention, the temperature at the time of voltage pulse application is 0-20 degreeC normally, for example, 0-15 degreeC, Preferably it is 1-8 degreeC, or 2-4 degreeC. The temperature of the reaction solution rises by applying the voltage pulse. Therefore, in order to keep the temperature of the reaction solution low, it is advantageous to use a cooling means. As a suitable cooling means for locally creating a low temperature environment, for example, a Peltier element can be shown. The Peltier element is an electronic element composed of a semiconductor using the Peltier effect discovered by Peltier (Jean Charles A. Peltier). When a direct current is passed through N-type and P-type semiconductors, the temperature is absorbed by one of the semiconductors and heat is dissipated by the other (heat exchange phenomenon). The temperature on the side where the temperature is absorbed drops and is cooled. Commercially available Peltier elements usually have a cooling capacity of around −10 ° C. The cooling capacity of the Peltier element can be freely controlled by the current supplied to the semiconductor. Therefore, while the voltage pulse is applied, the temperature of the reaction solution can be maintained within a predetermined temperature range by monitoring the temperature of the reaction solution with a temperature sensor and operating the Peltier element as necessary.

  Alternatively, when the reaction liquid is sufficiently cooled when the voltage pulse is applied and the temperature of the reaction liquid is within a predetermined range even after the voltage pulse is applied, the cooling at the time of applying the voltage pulse is not necessarily required. For example, if the temperature of the reaction solution at the end of application of the voltage pulse is 20 ° C. or less, the necessary temperature condition can be satisfied without cooling during application. As long as the reaction solution is sufficiently cooled in advance, and if necessary, the temperature rise in the environment in which the reaction solution is placed can be suppressed, it is not essential to actively cool during the voltage pulse application.

  In the present invention, the reaction solution to which the voltage pulse is applied can be incubated in advance. The conditions for incubation are as described above. If the reaction is incubated at a high temperature of 37-90 ° C., cool it well before applying the voltage pulse. Usually, since the volume of the reaction solution is 1 mL or less, the reaction solution can be cooled in a very short time. The condition that the reaction solution incubated at high temperature is cooled and a voltage pulse is applied at 0-20 ° C. is a preferable condition in the present invention.

  The mechanism by which the increase in temperature at the time of voltage pulse application acts on the aggregation reaction in an inhibitory manner can be considered as follows. In the reaction solution to which the voltage pulse is applied, the alignment and dispersion of the carrier particles are repeated. In order to increase the chance of contact between the binding partner on the carrier particles and the affinity substance (or the aggregating reagent component) in the reaction solution, carrier particle dispersion is effective. At the same time, in order to crosslink a plurality of carrier particles by binding with an affinity substance (or an aggregating reagent component) to form an aggregate, the alignment of the carrier particles is effective. However, when the movement of the carrier particles in the reaction solution is intense, the carrier particles may not be sufficiently aligned. It can be said that the state in which the temperature of the reaction liquid is increased is a state where the Brownian motion of the carrier particles contained in the reaction liquid becomes intense and alignment of the carrier particles at the time of voltage application is difficult. As a result, alignment of carrier particles due to application of a voltage is inhibited, and agglutination reaction is inhibited. When the temperature of the reaction liquid at the time of applying the voltage pulse is controlled according to the present invention, the effect of aligning the carrier particles by applying the voltage can be sufficiently obtained, and the inhibition of the aggregation reaction due to the temperature rise can be suppressed.

  In order to suppress the movement of the carrier particles in the reaction solution to which the voltage pulse is applied, it is also effective to increase the viscosity of the reaction solution. The reaction solution for a general agglutination reaction is less than 0.75 mPas. With such a viscosity, the movement of the carrier particles is not suppressed, and the agglutination reaction may be inhibited. In contrast, the present inventor confirmed that the agglutination reaction can proceed efficiently at a viscosity of 0.8 mPas or more. That is, the present invention relates to a method for aggregating carrier particles, comprising a step of applying a voltage pulse to a reaction solution containing a carrier partner bound to a specific substance and having a binding activity with a specific substance and the specific substance. And providing a method characterized in that the viscosity of the reaction solution is 0.8 mPas or more while a voltage is applied.

More specifically, the present invention provides a method for measuring an affinity substance including the following steps, wherein the viscosity of the reaction solution is 0.8 mPas or more.
(1) a step of applying a voltage pulse to a reaction liquid in which carrier particles bound with a binding partner having binding activity with a measurement target affinity substance and a measurement target affinity substance are mixed;
(2) After step (1), aggregates of carrier particles formed by binding with the affinity substance to be measured, and carriers that did not bind to the affinity substance to be measured and did not form an aggregate Counting either or both of the particles, and
(3) After the step (2), determining the level of the substance to be measured based on either or both of the aggregate formation level and the level of the carrier particles that did not form the aggregate

Alternatively, the present invention provides a method for measuring an affinity substance comprising the following steps, wherein the reaction solution has a viscosity of 0.8 mPas or more.
(1 ') a step of applying a voltage pulse to a reaction liquid containing at least a binding partner having a binding activity with a measurement target affinity substance and a measurement target affinity substance and an aggregating reagent component;
(2 ′) After step (1 ′), any of the aggregates of carrier particles formed by binding to the agglutinating reagent and carrier particles whose aggregation is inhibited by binding to the affinity substance to be measured or Counting both, and
(3) After the step (2 ′), a step of determining the level of the substance to be measured based on either or both of the aggregate formation level and the level of the carrier particles that did not form the aggregate.

  In the present invention, the viscosity of the reaction solution is usually 0.8 mPasm or more, for example, 1-3 mPas, preferably 1-2 mPas. The viscosity of the reaction solution can be adjusted by adding a compound capable of adjusting the viscosity. As the compound capable of adjusting the viscosity, any compound that does not interfere with the binding between the affinity substance and the binding partner can be used. For example, the viscosity of the reaction solution can be increased by adding bovine serum albumin, casein, glycerin, sucrose, choline chloride or the like. The addition amount of these compounds can be suitably selected from 0.05 to 5%, for example. More preferably, it is 0.1 to 3%, More preferably, it is 0.3 to 1%. Even if the composition of the reaction solution is the same, the viscosity generally increases as the temperature of the reaction solution decreases. Therefore, application of a voltage pulse under a low temperature condition is also effective in increasing the viscosity of the reaction solution.

  A person skilled in the art can set an appropriate addition amount by adding these compounds to the reaction solution and measuring the viscosity under a temperature condition in which a voltage pulse is applied. Methods for determining the viscosity of a liquid are known. In general, a rotary viscometer or an ultrasonic viscometer is used.

  In the present invention, the affinity substance and the binding partner having binding activity with the affinity substance include any combination of substances capable of constituting a binding reaction. That is, when a substance binds to one substance, one is an affinity substance and the other is a binding partner. The affinity substance and binding partner in the present invention may be a natural substance or an artificially synthesized compound. In addition, the affinity substance and the binding partner can be purified substances, and impurities can coexist. Furthermore, the affinity substance and the binding partner may be present on the surface of the cell or virus.

In the present invention, as an example of the binding reaction between the affinity substance and the binding partner, for example, the following reaction can be shown. Any substance constituting these reactions can be used as an affinity substance or a binding partner in the present invention.
Reaction of antigen or hapten with antibody (immune reaction)
Hybridization between nucleic acids having complementary base sequences Reaction between lectin and its receptor Reaction between lectin and sugar chain Reaction between ligand and receptor
Reaction of DNA and transcriptional regulators

Among the above binding reactions, as a preferable binding reaction in the present invention, for example, an immune reaction can be shown. The following substances can be shown as antigens constituting the immune reaction. These antigens may be present not only on the antigen molecule itself but also on fragments thereof or on the cell surface. Note that these substances are examples of antigenic substances, and it goes without saying that the present invention can be applied to other antigenic substances. For example, any antigenic substance that can be measured based on an immunological agglutination reaction using latex, blood cells or the like as a carrier can be used as the affinity substance in the present invention.
Tumor marker:
AFP, CEA, CA19-9, PSA and other coagulation filament markers:
Infectious disease markers such as protein C, protein S, antithrombin (AT) III, FDP, FDP-D-dimer:
Hormones such as CRP, ASO, HBs antigens:
Tissue components such as thyroid stimulating hormone (TSH), prolactin, and insulin:
Myoglobin, myosin, hemoglobin, etc. Other:
Nucleic acids such as DNA

  An antigenic substance and an antibody that recognizes the antigenic substance can be used either as an affinity substance and the other as a binding partner. In the present invention, an affinity substance is referred to as an affinity substance when the substance is a measurement target. On the other hand, the binding partner refers to a substance that can be used as a probe for measuring an affinity substance and has a binding activity for the affinity substance. Therefore, when measuring an antigen, an antibody can be used as a binding partner. Conversely, when measuring an antibody, an antigen recognized by the antibody can be used as a binding partner. For example, any antibody capable of measurement based on immunological agglutination using latex, blood cells or the like as a carrier can be used as the affinity substance in the present invention. Antibodies against HBs (hepatitis B virus surface antigen), HBc (hepatitis B virus core antigen), HCV (hepatitis C), HIV (AIDS virus), TP (syphilis), etc. are measured by immunological aggregation reaction ing.

  Several reaction principles are known for measuring the reaction between an affinity substance and a binding partner using the aggregation of carrier particles as an indicator. Any of these reaction principles can be applied to the present invention. Hereinafter, the measurement principle using the reaction between the affinity substance and the binding partner using the aggregation of the carrier particles as an index will be exemplified.

Direct agglutination reaction:
Aggregation of the carrier particles due to the reaction between the substance to be measured and the binding partner on the carrier particles is detected. For example, this principle includes the case where an antigen molecule is measured by an antibody that is a binding partner. Or conversely, this principle also includes the case where an antibody as an affinity substance is measured using aggregation of carrier particles bound with an antigen as an index. In a direct agglutination reaction, the level of agglutination and the amount of an affinity substance that is a measurement target substance are usually directly proportional.

Aggregation inhibition reaction:
A low molecular weight antigen called a hapten is difficult to form a cross-linked structure through an antigen necessary for aggregation of carrier particles. Therefore, hapten cannot be detected by the principle of direct agglutination reaction. Therefore, an agglutination reaction is used by binding a polyhapten in which a plurality of haptens or fragments containing epitopes thereof are bound to a carrier and an antibody on the carrier particle. Polyhaptens can crosslink a plurality of antibody molecules, thus aggregating carrier particles. However, the presence of the hapten prevents the reaction between the polyhapten and the antibody and prevents the carrier particles from aggregating. The level of aggregation inhibition is directly proportional to the presence of the hapten. In other words, the amount of the substance to be measured and the level of the aggregation reaction are inversely proportional.

Examples of the antigen to be measured classified as a hapten include the following components.
hormone:
Estrogen and estradiol drugs:
Theophylline

  In the present invention, in order to measure a hapten based on the principle of an aggregation inhibition reaction, a component capable of aggregating carrier particles bound with an antibody against the hapten is necessary. In the present invention, a component capable of aggregating carrier particles bound with an antibody against a hapten is referred to as an agglutinating reagent. An agglutination reagent is defined as a reagent that has specific affinity for an antibody and has the effect of crosslinking carrier particles by binding to the antibody. The polyhapten described above can be used as an agglutinating reagent in the measurement of hapten.

In the present invention, the binding partner is used by binding to carrier particles. Examples of the carrier particles of the present invention include latex particles, kaolin, gold colloid, red blood cells, gelatin, and liposomes. As the latex particles, those generally used in the aggregation reaction can be used. Polystyrene latex, polyvinyl toluene latex, and polymethacrylate latex particles are known. Preferred particle carriers are polystyrene latex particles. Latex particles in which a functional group is introduced on the surface of the latex particle by copolymerization of a monomer having a functional group can also be used. For example, -COOH, -OH, -NH _2, latex particles having a functional group -SO ₃ and the like are known. A binding partner can be chemically bonded to latex particles having a functional group.

  For example, in the case of latex particles, the average particle size of the carrier particles is preferably 0.3 to 20 μm. When the average particle size is 0.3 μm or less, or 20 μm or more, it is difficult to form a pearl chain. For example, in the case of latex particles, the average particle size of the carrier particles is more preferably 2 to 10 μm. Also, smaller carrier particles can be used by using elliptical particles with a large dielectric polarization.

  Thus, compared with the carrier particles in the known latex agglomeration turbidimetric method having a particle size of 0.05 to 0.6 μm, particles having a large size of 1 μm or more can be used in the method of the present invention. This is because the use of the step of applying the voltage pulse promotes the agglutination reaction, and as a result, a sufficient reaction proceeds in a short time even for large-sized particles. Large carrier particles lead to the following advantages. First, the aperture size for measuring particles can be increased. As a result, the aperture is less likely to be clogged. Further, the increase in the carrier particles facilitates the discrimination from the measurement interfering substance contained in the body fluid. As a result, measurement accuracy is improved.

  In the present invention, when other particles are used as carrier particles instead of latex particles, particles having the same size as latex particles can be used. For example, when particles such as kaolin, colloidal gold, gelatin, or liposome are used as the carrier particles, the average particle size of the carrier particles is preferably 0.3 to 20 μm.

The binding partner and the particle carrier can be bound by a method corresponding to each material. A person skilled in the art can appropriately select a method for combining the two. For example, proteins such as antigens, antibodies, or fragments thereof can be physically adsorbed on latex particles. In latex particles having a functional group on the surface, a substituent capable of covalent bonding with the functional group can be chemically bonded. For example, protein —NH ₂ can be bound to latex having —COOH.

  The carrier particles bound with the binding partner can be blocked as necessary. Specifically, non-specific protein binding to the carrier particle surface can be prevented by treating the carrier particle surface with an inert protein. As the inactive protein, bovine serum albumin, skim milk powder or the like can be used. Furthermore, in order to improve the dispersibility of the carrier particles, a surfactant or saccharide can be added to the dispersion medium. In addition, an antibacterial agent can be added to the particle carrier to prevent the growth of microorganisms.

  The present invention includes the step of aligning the affinity substance and the carrier particles in an electric field. A method for aligning carrier particles in an electric field and performing an agglutination reaction is known (Japanese Patent Laid-Open No. 7-83928). That is, the carrier particles can be aligned along the electric field by applying a voltage pulse to the reaction liquid containing the affinity substance and the carrier particles.

  At this time, when the principle of the aggregation inhibition reaction is used, the affinity substance and the carrier particles are aligned in the presence of the aggregation reagent. The agglutinating reagent can be brought into contact after the contact between the carrier particles and the affinity substance to be measured. Alternatively, the three components can be contacted simultaneously by adding carrier particles after previously mixing the affinity substance to be measured and the agglutinating reagent.

  An AC component or a DC component can be used for the voltage pulse. Both can be arbitrarily combined. An AC voltage is preferred because the reaction solution is susceptible to electrolysis. As the AC voltage, a square wave, a rectangular wave, a sine wave, or the like can be used. The power supply frequency of the AC voltage can be arbitrarily set depending on the ionic strength of the reaction solution (reagent). The AC voltage is applied so as to obtain an electrolytic strength of 5 to 50 V / mm when indicated by a peak value. When the electrolytic strength is less than 5 V / mm, the support is less likely to be pearl chained, and therefore the aggregation reaction is not sufficiently promoted. When the electrolytic strength exceeds 50 V / mm, the reaction solution is likely to be electrolyzed, making it difficult to measure the aggregation reaction. More preferably, it is applied so as to obtain an electric field strength of 10 to 20 V / mm. The AC frequency is preferably 10 KHz to 10 MHz. More preferably, the frequency is 50 KHz to 1 MHz.

  As the concentration of the carrier particles in the reaction system is higher, the pearl chain is more easily formed, so that the agglutination reaction is promoted. Further, the higher the concentration of the carrier particles, the larger the aggregation rate of the carrier particles when redispersed when there is no biologically specific reactive substance. For example, in the case of latex particles, the concentration of carrier particles in the reaction system is preferably 0.01 to 2% by weight, more preferably 0.05 to 1% by weight.

  In the present invention, a salt that promotes the aggregation reaction can be added to the reaction solution. For example, the aggregation reaction can be promoted by adding a salt at a relatively high concentration of 10 mM or more. However, it is not preferable that the salt concentration exists in the reaction system at a concentration of 600 mM or more because electrolysis of the reaction solution easily occurs. A more preferable salt concentration is 10 to 300 mM, and a most preferable salt concentration is 25 to 150 mM. When there is a possibility that the biological sample itself contains a salt that promotes the agglutination reaction, the salt concentration of the reagent may be adjusted so that the final salt concentration in the reaction solution falls within the above range. When a direct current component is used as a voltage pulse, electrolysis occurs even in a reaction solution having a salt concentration of about 6 mM. Therefore, it is difficult to measure a biological specific agglutination reaction in the presence of salt.

The salt in the present invention may be selected from those that promote biological specific aggregation reaction. Examples include, but are not limited to, sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, and ammonium chloride. A salt showing a value of 100 cm ² / (Ω · mol) or more in an aqueous solution having a molar electric conductivity of 10 mM and 25 ° C. is preferable as the salt used in the present invention. More specifically, for example, sodium chloride, potassium chloride, ammonium chloride and the like can be shown as preferable salts.

  In the present invention, the specimen containing an affinity substance is not limited. That is, any sample containing an affinity substance to be measured can be used as a specimen. For example, blood samples, samples collected from a local area such as the pharynx, saliva, sputum, urine, or stool are typical biological samples. In addition, any biological material collected from a living body can be used in the present invention as a specimen for measuring a biological substance. Furthermore, cultures that can be obtained by culturing these biological samples can also be used as specimens of the present invention. The biomaterial can be used as a specimen as it is or after being processed as necessary. For example, a biomaterial can be made into a specimen through processes such as fractionation, dilution, dissolution, and extraction.

  In the present invention, the specimen may be a stock solution, or it is automatically diluted and used for measurement. The dilution factor can be arbitrarily set. When there are a plurality of types of reagents necessary for the reaction, it is possible to add two or more reagents sequentially. It is desirable that the specimen and the reagent are mixed in advance before applying the voltage. Both can be physically mixed using a stir bar. Or both can be mixed by an electrical method. As an electrical method, a method of physically moving the position of the carrier particle by intermittently applying voltage pulses in different directions can be exemplified.

  Each process which comprises the measuring method of this invention is demonstrated concretely below. The reaction liquid in which necessary components are mixed together with the sample moves to a tank in which electrodes are arranged, and is applied with a voltage pulse. In the case of incubating the reaction solution in advance before the application of the voltage pulse, the reaction solution is incubated after the transfer to the tank equipped with the electrode and before or at any stage. When an electric field is applied, the carrier particles cause dielectric polarization and are attracted electrostatically to form a linear shape. This phenomenon is called pearl chain formation. Thereafter, when the electric field is stopped, the carrier particles arranged in a straight line are instantaneously redispersed. On the other hand, when the binding partners are bound to each other via an affinity substance during the pearl chain formation, the carrier particles remain in an aggregated state without being dispersed even after the electric field is stopped. The presence of the affinity substance can be detected or measured by measuring the aggregate formed in this way and / or the carrier particles not aggregated.

  The measurement method of the present invention includes any one of an aggregate of carrier particles formed by binding to an affinity substance to be measured and a carrier particle that has not bound to an affinity substance to be measured and has not formed an aggregate. Or both are counted as indicators. In the present invention, the particles can be measured after the electric field is stopped. Alternatively, particles placed in an electric field can be measured without stopping the electric field. For example, particles placed in an electric field can be counted by taking them out of the electric field. Further, a step of dispersing the particles can be performed before counting the particles. By the dispersion step before counting, particles aggregated due to non-specific factors can be dispersed. As a result, improvement in measurement accuracy can be expected. The particles are dispersed by stirring or dilution of the reaction solution.

A known method can be used to count the aggregated single particles. For example, a method for determining the level of aggregation based on two-dimensional information is known. That is, a microscopic image of the reaction solution is scanned, and either agglomerates and non-aggregated particles or both occupied per unit area are counted.
Alternatively, in the present invention, the carrier particles can be counted using three-dimensional information as an index. In the present invention, counting using three-dimensional information as an index means measuring three-dimensional information of particles and / or aggregates and counting the particles and / or aggregates based on the results. Counting carrier particles based on three-dimensional information is preferable as a counting method in the present invention.

  The method for measuring the three-dimensional information is not limited. Further, the counting in the present invention means obtaining the number of particles and / or aggregates. The number of particles and / or agglomerates can be measured solely by number. Alternatively, aggregated particles can be measured separately from non-aggregated particles. Furthermore, about the aggregated particle | grains, the number of the aggregates can also be measured for every number of aggregated particles. Several methods for counting particles using three-dimensional information as an index are known.

  The particle counting method that can be used in the present invention is advantageously a measuring method based on a physical principle. In the present invention, the physical measurement method refers to a measurement method capable of evaluating physical information unique to particles or agglomerates. The physical information unique to the particles or agglomerates can be rephrased as true measurement results. On the other hand, the method of analyzing the two-dimensional information acquired from the image information detects an overlap of particles that are not actually aggregated as an aggregate. Such a detection result cannot be said to be physical information unique to the particle.

  Use of a flow system is advantageous for physically measuring particles or agglomerates. The flow system is a system that can analyze physical information of particles passing through a fine flow cell. By using the flow system, physical measurement can be easily performed. That is, the physical measurement in the present invention includes a step of measuring and counting three-dimensional information of either or both of particles and agglomerates by a flow system. As a method for physically counting particles using three-dimensional information as an index, for example, the Coulter principle or the laser diffraction scattering method can be shown.

The Coulter principle (USPA2656508, 1953) is an analysis method in which electrodes are placed on both sides of an aperture (pore) and the volume of the particle is detected based on a change in electrical resistance due to the particle passing through the aperture. When a minute current is passed between both electrodes through the electrolytic solution, when the particles suspended in the electrolytic solution are sucked and pass through the aperture, the electrolytic solution corresponding to the particle volume is replaced by the particles. As a result, a change occurs in the electrical resistance between both electrodes, and by measuring this change, the particle count and size (volume) can be measured. There is a capacitance method as a method for detecting the volume, but the electric resistance method is mostly used.
The aperture size can be appropriately adjusted according to the particle to be analyzed. When detecting the aggregation of carrier particles used for general immunological particle aggregation reaction, the aperture size is usually 30 to 1000 μm, preferably 50 to 200 μm.

  The aperture size is advantageously several to several hundred times, preferably 5 to 50 times the average particle size of the carrier particles. In this case, a signal proportional to the volume can be detected, and accurate and sensitive measurement can be realized. The sensitivity is better when the aperture size magnification is smaller than the particle diameter. However, when the aperture size is too small, the particles are easily clogged, and when the aperture size is too large, the particle detection sensitivity is lowered, which is not preferable.

By counting the aggregated particles in this way, the ratio of the aggregated particles can be known. The ratio of aggregated particles refers to the ratio of aggregated particles to all counted particles. Further, the ratio of the aggregated particles is referred to as an aggregation rate. Furthermore, a standard curve can be obtained by obtaining the aggregation rate for a standard sample whose concentration is known in advance and plotting the relationship between the two on a graph. On the other hand, if the aggregation rate of the specimen is collated, the concentration of the measurement target affinity substance contained in the specimen can be clarified.
Alternatively, the standard curve can be expressed as a regression equation. If the regression equation is obtained, the concentration of the affinity substance to be measured can also be calculated by substituting the aggregation rate into the regression equation.

On the other hand, the laser diffraction scattering method is to measure the particle count and the average diameter by detecting fluctuations generated when a particle is irradiated with a laser. In any case, in order to increase the measurement accuracy, it is preferable to use a method of diluting reactive particles, applying ultrasonic waves, and / or a sheath follow method in order to suppress erroneous measurement of particles.

In addition, the following method can be shown as a method for measuring the particle volume.
Centrifugal sedimentation method: A method of measuring the particle size distribution by the Stokes equation showing the relationship between the sedimentation rate of particles in a liquid and the particle size. (In the light transmission centrifugal sedimentation method, Stokes' law is applied, and if particles with the same specific gravity are used, the larger particles are settled faster than the smaller particles. Analyzed as change, particle size distribution can be obtained.)

Capillary method: When the Reynolds number of the viscous fluid flowing through the capillary is small, a Poiseuille fluid is generated there. This flow is faster in the center of the capillary and slower in the tube wall, so that large particles flow in an average fast flux and small particles in an average slow flux. In other words, when particles flow through a capillary of a certain length, they are separated and detected by size due to the difference in moving speed.

Three-dimensional image analysis: A plurality of pieces of image information taken from different directions can be analyzed to obtain three-dimensional information of particles. Alternatively, three-dimensional information of particles can be obtained by scanning image information on the xy plane in the z-axis direction.

  In the measurement method of the present invention, aggregated (or not) carrier particles are counted. Depending on the counting result, the affinity substance to be measured is measured qualitatively or quantitatively. In qualitative measurement, the presence of aggregated particles means the presence of an affinity substance to be measured. Alternatively, in the case of an aggregation inhibition reaction, the presence of the measurement target is proved when the inhibition of aggregation is detected.

  In quantitative measurement, the level of aggregation can be correlated with the amount of affinity substance to be measured. More specifically, the measurement method of the present invention is performed on a sample in which the amount of the affinity substance is known in advance, and the relationship between the detection result of the aggregated particles and the amount of the affinity substance is clarified. Next, the same measurement is performed on the sample, and the amount of the affinity substance can be clarified from the detection result of the aggregated particles based on the deposition. Even in the case of an agglutination inhibition reaction, a quantitative measurement is possible in the same manner.

  As a counting method of particles and / or agglomerates, in a means for counting a certain number of particles, an arithmetic expression according to the purpose such as the number of particles aggregated to two or more / total number of particles or single particle / total number of particles is used. You can choose. The total number of particles may be the number of all particles measured within a certain measurement time. If the total amount of the reaction solution is to be analyzed, It can also be the total number. If the total volume of the reaction solution is clear, the total number of particles contained in the reaction solution can be approximately calculated by counting a part thereof.

Alternatively, the affinity substance can be detected or measured based on the number of particles and / or aggregates detected per certain time by an electric resistance method, a laser diffraction scattering method, or the like. That is, since the single particles are aggregated by the aggregation reaction to form aggregates, the number of particles counted per time is reduced. Alternatively, the time required to count a predetermined number of particles and / or agglomerates can be used as an index. When such a counting method is applied to the present invention, the relationship between the number of particles and / or aggregates and the amount of affinity substance can be expressed as a regression equation.
In the particles sensitized with the antibody, the proportion of aggregates composed of two or more particles increases depending on the concentration of the antigen. The aggregation rate represented by the number of particles aggregated into two or more / the total number of particles converges to 1.00 (100%).

  Regardless of the Coulter principle or the laser diffraction scattering method, the method of measuring the three-dimensional information of particles is expected to provide high-accuracy analysis with a simple equipment configuration compared to the method of analyzing two-dimensional image data. it can. As already described, in the analysis of two-dimensional image data, the volume of the reaction solution is limited. On the other hand, since the method of measuring three-dimensional information can apply a flow analysis method, the volume of the reaction solution is not limited. Further, the physical shape of the reaction space is not limited. For these reasons, the device configuration is simple. In addition, the ability to freely set the amount of reaction solution contributes to the improvement of reproducibility and detection sensitivity.

  Alternatively, the present invention can also be applied to an aggregation inhibition reaction system. The principle of the immunological particle agglutination method based on the agglutination inhibition reaction using an agglutination reagent has already been described. Through the above steps, the present invention can be applied to immunological particle agglutination reactions. The application of the voltage pulse, the formation level of the agglomerates, or the analysis of the level of the carrier particles that have not formed the agglomerates can be performed by the method specifically described above.

  In the case of carrying out the present invention based on the principle of the aggregation prevention reaction, it is desirable to select conditions under which more aggregates in which two or more particles aggregate are generated. Alternatively, a method of evaluating the aggregation level using the number of single particles / total number of particles as an index is preferable. When based on the principle of the aggregation inhibition reaction, the use of such an arithmetic expression can be expected to have higher sensitivity than the analysis based on the arithmetic expression of the number of particles aggregated to two or more / the total number of particles.

  In addition, the present invention provides an apparatus for carrying out the above measurement method. That is, the present invention relates to an apparatus for aggregating carrier particles, comprising carrier particles bound with a binding partner having binding activity with a specific substance, and means for applying a voltage pulse to a reaction solution containing the specific substance. And an apparatus characterized by having means for heating the temperature of the reaction solution to 37 ° C to 90 ° C.

Alternatively, the present invention relates to the binding between the carrier particle having a binding activity with the affinity substance to be measured and containing the following elements, and the affinity substance to be measured. A measuring apparatus for measuring aggregation by an agglutinating reagent as an index is provided. A configuration example of the measuring apparatus of the present invention is shown in FIG. 1 or FIG.
a: space for holding the reaction solution,
b: means for incubating the temperature of the reaction solution at 37 ° C. to 90 ° C.
c: means for applying a voltage pulse to the reaction solution,
d: means for setting the temperature of the reaction solution to 0 ° C. to 20 ° C. when a pulse voltage is applied, and
e: Means for counting either or both of carrier particles and agglomerates of carrier particles contained in the reaction solution

  In the present invention, a: a space for holding the reaction liquid can be any space for holding the reaction liquid. In order to react a small amount of sample, it is advantageous to have a small volume of space. For example, a space of about 1 μL to 10 mL, preferably about 10 to 500 μL can be used. This space can be equipped with a sample or reagent supply means or a carrier particle measurement means described later, if necessary. The reaction solution accommodated in the space is composed of carrier particles bound with a binding partner having binding activity with the measurement target affinity substance, and a sample containing the affinity substance to be measured. Alternatively, an agglutination reagent component is further added in the aggregation inhibition reaction system.

  In the present invention, a: a space for holding the reaction solution is equipped with a means for b: incubation of the reaction solution at 37 ° C. to 90 ° C. In order to maintain the reaction solution at a predetermined temperature, for example, a temperature sensor and a heating means can be used. A heater or a Peltier element can be used as the heating means.

  Next, the means for applying a voltage pulse to the c: reaction solution in the present invention will be described. The voltage pulse is applied through an electrode in contact with the reaction solution. Electrodes for aligning carrier particles with an electric field are also used, for example, in the prior art documents mentioned above. These known electrodes can be used in the present invention. The apparatus of the present invention can be equipped with a power source for supplying a voltage to the electrodes.

The electrode for applying a voltage pulse in the apparatus of the present invention is composed of at least one set (two) of electrodes. More than two electrodes can also be provided to provide a plurality of voltage pulses in different directions. For example, three electrodes A, B, and C can be arranged, and voltage pulses in three directions between A-B, B-C, and A-C can be applied. In addition, two sets (four) of electrodes can be arranged to apply a direct voltage pulse.
Furthermore, a mechanism for driving the electrodes can be provided to apply voltage pulses in different directions. For example, voltage pulses can be applied from a plurality of different directions by rotating the electrode in the reaction solution.

  The apparatus of the present invention comprises d: means for setting the temperature of the reaction solution to 0 ° C. to 20 ° C. when a pulse voltage is applied. A temperature sensor and a Peltier device can be used as a preferable means for maintaining the reaction solution at 0 ° C. to 20 ° C.

  Furthermore, the apparatus of the present invention includes a means for counting e: one or both of the carrier particles and the aggregates of the carrier particles contained in the reaction solution. The counting means can be installed in the space. Alternatively, the reaction liquid held in the space can be counted after being taken out of the space and introduced into the counting means.

  As a means for counting carrier particles aggregated or not aggregated using the three-dimensional information as an index, a measuring means using the Coulter principle or the laser diffraction scattering method can be used. For the Coulter principle, for example, the reaction solution in the space is introduced into an aperture provided with electrodes for the Coulter principle, and necessary analysis is performed. Similarly, when using the laser diffraction scattering method, the reaction solution may be introduced into the optical cell for analysis and analyzed.

  In the present invention, carrier particles that have been pearl chained in an electric field can be counted after being redispersed as necessary. The device of the present invention can be equipped with a mechanism for redispersion of carrier particles. The carrier particles can be redispersed by dilution or sonication.

The elements (a) to (e) constituting the device of the present invention can be arranged in one continuous flow path. Or each element is comprised as a discontinuous space, and the measuring method of this invention can also be implemented by moving a reaction liquid between each element.
The apparatus of the present invention can be combined with an additional mechanism for carrying out the above measurement method. Additional mechanisms that can be combined with the apparatus of the present invention are illustrated below.
Sample sorting mechanism Sample dilution mechanism Measurement result recording mechanism Measurement result display mechanism Measurement result printing mechanism Hereinafter, the present invention will be described in more detail based on examples.

Example 1 Promotion of antigen-antibody reaction (1) Preparation of latex reagent sensitized with AFP antibody
0.1 mg of anti-alpha fetoprotein (AFP) antibody (manufactured by Dako) was dissolved in 1 mL of glycine buffer (containing 50 mM glycine, 50 mM sodium chloride, 0.09% sodium azide, hereinafter abbreviated as GBS), and 2.0 μm latex ( Sekisui Chemical Co., Ltd., 1% solid content suspension) was added and stirred at 37 ° C. for 2 hours. The sensitized latex was then centrifuged to remove the supernatant. The precipitate was suspended in 1 mL of 0.5% bovine serum albumin glycine buffer (0.5% BSA-GBS) to prepare an anti-AFP antibody-sensitized latex reagent.

(2) Measuring apparatus The affinity substance (AFP) was measured by antigen-antibody reaction using the apparatus shown in FIG.
The apparatus shown in FIG. 1 dispenses and mixes a specimen and reagent 1 (buffer solution), and further dispenses and mixes reagent 2 (latex reagent) to produce a reaction solution. A temperature control mechanism 2 is provided. However, in the case of a one-reagent system, dispensing of reagent 1 (buffer solution) can be omitted. Next, the reaction solution is moved to the reaction vessel 3 (pulse application vessel), and a voltage pulse is applied to the reaction solution via the electrode 4 for several seconds to several tens of seconds. Carrier particles placed in an electric field are pearl chained. The reaction liquid to which the voltage pulse is applied is diluted in the dilution tank 5, and the aggregation state of the carrier is measured using the particle size distribution meter 6. FIG. 1B is a diagram showing a cross section of the pulse application tank. The distance between the electrodes is 0.8 mm, the electrode thickness is 0.03 mm, and the electrode length is 20 mm.

(3) Measuring method
Using 0.5% BSA-GBS, the AFP antigen solution was diluted to prepare specimen solutions having concentrations of 0, 0.0075, and 0.015 ng / mL. 3 μL of these specimens and 3 μL of the aforementioned anti-AFP antibody-sensitized latex reagent were placed in a test tube, mixed and incubated at 45 ° C. to 80 ° C. for 20 seconds, and immediately injected into a reaction cell with electrodes. Using the apparatus of (2), a voltage pulse was applied for 30 seconds with an electrolytic voltage of AC voltage (rectangular wave) ± 12 V / mm having a frequency of 200 KHz. Immediately after application for 30 seconds, the electric field is turned off, the reaction solution is diluted with physiological saline, and the particle size distribution of latex particles is measured using a Coulter Multisizer, and the agglutination ratio (AR;%) of the latex is measured. Was determined by the following equation.
AR = (number of particles aggregated into two or more) / (total number of particles) × 100 (%)

The results are shown in FIG. As shown in FIG. 2, in the method of the present invention, a high aggregation rate was confirmed as compared with the conventional method.
Conditions of the present invention: Incubate reaction solution before applying voltage pulse at high temperature (45 ° C, 62 ° C, 80 ° C) for 20 seconds Conventional method condition: Reaction solution before applying voltage pulse is room temperature (25 ° C ) And no high-temperature treatment As a result, it can be seen that the present invention can measure with higher sensitivity than the conventional method. Further, in a general latex agglutination method without applying a pulse voltage, the incubation 37 ° C. 20 min, the reaction of the low concentration range of 0.015 n g / mL was observed.

Comparative Example 1
Each specimen of Example 1 and 3 μL of anti-AFP-sensitized latex reagent were placed in a test tube, incubated at 25 ° C. for 20 seconds, injected into a reaction cell with electrodes, and a high frequency voltage was applied in the same manner as in Example 1. The procedures other than the incubation at 25 ° C. were the same as those in Example 1. The results are shown in FIG.

Comparative Example 2
Each specimen of Example 1 and 3 μL of anti-AFP-sensitized latex reagent were placed in a test tube and incubated at 37 ° C. for 20 minutes. 0.5 μL of this reaction solution was diluted in 20 mL of physiological saline, and the particle size distribution of latex particles was measured using a Coulter Multisizer as in Example 1, and the aggregation rate was similarly calculated. The results are shown in FIG. FIG. 2 shows that the present invention can be measured with higher sensitivity than the conventional method (without temperature treatment before applying the pulse voltage).

Example 2 Promotion of antigen-antibody reaction (1) Preparation of AFP antibody-sensitized latex reagent In the same manner as in Example 1, an anti-AFP antibody-sensitized latex reagent was prepared.
(2) Measuring apparatus The affinity substance (antigen-antibody reaction) was measured using the apparatus of FIG.
(3) Measuring method
Using 0.5% BSA-GBS, the AFP antigen solution was diluted to prepare specimen solutions having concentrations of 0, 0.0075, and 0.015 ng / mL. Take 3 μL of these specimens and 3 μL of the anti-AFP antibody-sensitized latex reagent described above, mix and incubate at 62 ° C. for 0 to 180 seconds, immediately inject a reaction cell with electrodes, and use the above-mentioned device to frequency 200 kHz An alternating voltage (rectangular wave) of ± 12 V / mm was applied for 30 seconds at an electrolytic strength to form a pearl chain. Immediately after the application for 30 seconds, the electric field was turned off, the reaction solution was diluted in physiological saline, and the particle size distribution of latex particles was measured using a Coulter Multisizer, and the agglutination ratio (AR;%) of latex was measured. Was determined by the following equation.

AR = (number of particles aggregated into two or more) / (total number of particles) × 100 (%)

(4) Results The results are shown in FIG. As shown in FIG. 3, in the conditions of the present invention, the aggregation rate was higher than that of the conventional method.
Conditions of the present invention: Incubate reaction solution before applying voltage pulse at high temperature (62 ° C.) for 5 to 180 seconds (5 sec, 20 sec, 180 sec) Conditions of conventional method: No high temperature treatment before applying voltage pulse ( 0sec)
That is, according to the present invention, it can be seen that measurement can be performed with higher sensitivity than the conventional method.

Example 3 Promotion of antigen-antibody reaction (1) Preparation of PSA antibody-sensitized latex reagent (reagent 2)
0.1 mg of anti-PSA antibody (Dako) is dissolved in 1 mL of glycine buffer (containing 50 mM glycine, 50 mM sodium chloride, 0.09% sodium azide, hereinafter abbreviated as GBS), and 2.0 μm latex (manufactured by Sekisui Chemical Co., Ltd.) (1% solid content suspension) was added and stirred at 37 ° C. for 2 hours, and then the sensitized latex was centrifuged to remove the supernatant. The precipitate was suspended in 1 mL of 0.5% bovine serum albumin glycine buffer (0.5% BSA-GBS) to prepare an anti-PSA antibody-sensitized latex reagent.

(2) Preparation of Tris hydrochloric acid buffer (reagent 1) containing PEG 20000
0.5% bovine serum albumin 50 mM Tris HCl buffer solution (50 mM Tris, 50 mM sodium chloride, 0.09% sodium azide contained, pH 8.4) contains 0.1 to 1.0% polyethylene glycol (molecular weight 20000, hereinafter abbreviated as PEG 20000) A reaction promoting reagent was prepared. As a control method, a PEG 20000 reagent with 0 concentration was prepared.

(3) Measuring apparatus The affinity substance (antigen-antibody reaction) was measured using the apparatus of FIG. (Temperature control mechanism 2 was set to room temperature.)

(4) Measuring method
Using 0.5% BSA-GBS, the PSA antigen solution was diluted to prepare a sample solution having a concentration of 0 and 9.5 ng / mL. After mixing 1 μL of these specimens and 3 μL of 0.5% BSA-Tris HCl buffer containing 0-1.0% PEG 20000, take it into the 3 μL test tube of the anti-PSA antibody-sensitized latex reagent described above, and immediately inject the reaction cell with electrodes. It was. Using the above-described apparatus, a voltage pulse was applied for 30 seconds with an electrolytic strength of AC voltage (rectangular wave) ± 12 V / mm having a frequency of 200 KHz. Immediately after application for 30 seconds, the electric field is turned off, the reaction solution is diluted in physiological saline, the particle size distribution of latex particles is measured using a Coulter Multisizer, and the latex agglutination ratio (AR;%) is determined. It was determined by the following formula.
AR = (number of particles aggregated into two or more) / (total number of particles) × 100 (%)
(5) Results The results are shown in FIG. FIG. 4 shows that the present invention can be measured with higher sensitivity than the control method.

Example 4 Promotion of aggregation reaction (1) Preparation of anti-AFP antibody-sensitized latex reagent An anti-AFP antibody-sensitized latex reagent was prepared in the same manner as in Example 1.
(2) Measuring apparatus AFP was measured by antigen-antibody reaction using the apparatus shown in FIG.
5A dispenses and mixes the sample and reagent 1 (buffer solution), and further dispenses and mixes reagent 2 (latex reagent) to produce a reaction solution. A tank and a temperature control mechanism are provided. In practice, however, dispensing of the buffer solution can be omitted in the case of one reagent system. Next, the reaction solution is moved to the reaction tank 2 (pulse application tank), and a voltage pulse is applied through the electrode 3 for several seconds to several tens of seconds. At this time, the reaction vessel is cooled to 4 ° C. by the temperature control unit. The reaction liquid to which the voltage pulse is applied is diluted in the dilution tank 5, and the aggregation state of the carrier is measured using the particle size distribution meter 6. FIG. 5B is a diagram showing a cross section of the pulse application tank. The distance between the electrodes is 0.8 mm, the electrode thickness is 0.03 mm, and the electrode length is 20 mm.

(3) Measuring method
Using 0.5% BSA-GBS, the AFP antigen solution was diluted to prepare a sample solution having a concentration of 0 and 0.0075 ng / mL. 3 μL of these specimens and the above-described anti-AFP antibody-sensitized latex reagent 3 μL test tube were mixed, immediately mixed, and injected into a reaction cell with electrodes. Using the above-mentioned apparatus, a voltage pulse was applied to the reaction solution for 30 seconds with an electrolytic strength of AC voltage (rectangular wave) ± 12 V / mm having a frequency of 200 KHz. At this time, the temperature of the reaction cell was maintained at 4 ° C. Immediately after application for 30 seconds, the electric field is turned off, the reaction solution is diluted in physiological saline, the particle size distribution of latex particles is measured using a Coulter Multisizer, and the latex agglutination ratio (AR;%) is determined. It was determined by the following formula.
AR = (number of particles aggregated into two or more) / (total number of particles) × 100 (%)
The above operation was repeated 5 times in the same manner, and the average value of the measurement results and the average value ± 2.6SD were obtained and shown in FIG.

Comparative Example 3
The procedure of Example 4 was followed except that each sample of Example 4 and the anti-AFP-sensitized latex reagent were used and the reaction cell temperature was set to 22 ° C. The results are shown in FIG.

Comparative Example 4
Each specimen of Example 4 and 3 μL of the anti-AFP-sensitized latex reagent were placed in a test tube and incubated at 37 ° C. for 20 minutes. 0.5 μL of this reaction solution was diluted in 20 mL of 20 mL of physiological saline, and the particle size distribution of latex particles was measured using a Coulter Multisizer as in Example 4, and the aggregation rate was calculated in the same manner. Further, the measurement was repeated 5 times in the same manner as in Example 4, and the results are shown in FIG.

If the measured value (background signal) when the antigen concentration is 0 can be discriminated from the value obtained when measuring a certain concentration of antigen, the antigen at this concentration can be measured. The minimum measurable antigen concentration is the detection limit. For example, if there is no overlap between the following values A and B, an antigen of 0.0075 ng / mL or more can be detected.
Measured value A: When the antigen amount was 0 ng / mL and 0.0075 ng / mL were repeatedly measured, “average value of aggregation rate obtained by measuring 0 ng / mL + 2.6 SD
Measured value B: average value of aggregation rate measured at 0.0075 ng / mL−2.6SD
Comparing the detection limit under each condition from FIGS. 6, 7, and 8, it can be said that the detection limit of the method of the present invention (FIG. 6) is 0.0075 ng / mL. On the other hand, this concentration cannot be detected by the conventional method (FIGS. 7 and 9). From this, it can be seen that the present invention in which the reaction solution during application of the voltage pulse is kept at a low temperature can be measured with higher sensitivity in a very short time compared to the conventional method.

Example 5 Promotion of Aggregation Reaction (1) Preparation of Anti-PSA Antibody Sensitized Latex Reagent (Reagent 2) In the same manner as in Example 3, an anti-PSA antibody sensitized latex reagent was prepared.
(2) Preparation of Tris hydrochloric acid buffer (reagent 1)
Tris hydrochloride buffer (50 mM Tris, 50 mM sodium chloride, 0.09% sodium azide, 0.25% PEG 20000 contained, pH 8.4) and bovine serum albumin (hereinafter abbreviated as BSA) 0.5%, 2.5%, 5%, 7.5 Reagent 1 containing 10% and 10% was prepared, respectively.
(3) Measuring apparatus The affinity substance (antigen-antibody reaction) was measured using the apparatus of FIG. The temperature control mechanism 2 was set to room temperature.

(4) Measuring method
Using 0.5% BSA-GBS, the PSA antigen solution was diluted to prepare specimen solutions having concentrations of 0, 9.5, and 32 ng / mL. Mix 1 μL of these samples with 3 μL of Tris buffer containing 0.5%, 2.5%, 5%, 7.5%, or 10% BSA, and then add 3 μL of the anti-PSA antibody-sensitized latex reagent described above. Immediately thereafter, a reaction cell with electrodes was injected. Using the above-described apparatus, a voltage pulse was applied for 30 seconds with an electrolytic strength of AC voltage (rectangular wave) ± 12 V / mm having a frequency of 200 KHz. Immediately after application for 30 seconds, the electric field is turned off, the reaction solution is diluted in physiological saline, the particle size distribution of latex particles is measured using a Coulter Multisizer, and the latex agglutination ratio (AR;%) is determined. It was determined by the following formula.

AR = (number of particles aggregated into two or more) / (total number of particles) × 100 (%)
The BSA concentrations in the final reaction solution are 0.5%, 1.4%, 2.4%, 3.5%, and 4.6%. Here, a BSA concentration of 0.5% in the final reaction solution was compared as a control method.
(5) Temperature measurement of reaction liquid The temperature change of the reaction liquid when a pearl chain was formed was measured by the measurement method described above.
(6) Viscosity measurement of the final reaction solution The viscosity of the reaction solution having the above-mentioned final reaction solution (BSA concentration 0.5% to 4.6%), BSA concentration 0.3% and 6.8% at 4 to 52 ° C was measured using a vibration viscometer (Visco Mate).

Table 1
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Before application of BSA concentration in final reaction solution
0.5% 24 ° C 37 ° C
1.4% 24 ℃ 38 ℃
2.4% 25 ℃ 37 ℃
3.5% 25 ℃ 38 ℃
4.6% 24 ℃ 37 ℃
-------------------------------------------

(7) Results The results are shown in FIGS. 9 and 10 and Table 1.
As shown in FIG. 9, the aggregation rate of each concentration increased as the BSA concentration in the final reaction solution was increased from 0.5% to 3%. Since the aggregation rate tended to increase even at PSA 0 ng / mL (blank), the blank-corrected aggregation rate was compared. As a result, it was confirmed that the aggregation rate was remarkably increased by increasing the BSA concentration in the final reaction solution from 0.5% to 3%. That is, it can be seen that the present invention can further increase the sensitivity.

  FIG. 10 shows the relationship between the BSA concentration and the viscosity in the final reaction solution, and the relationship between the temperature and the viscosity in the reaction solution. First, from FIG. 9 and FIG. 10, under conditions where temperature control is not performed during pearl chain formation, the aggregation rate increases by increasing the BSA concentration in the final reaction solution from 0.5% to 3%. At this time, the viscosity in the final reaction liquid increases from 0.75 to 0.9 mPas. That is, it can be seen that the sensitivity can be increased by adjusting the viscosity in the final reaction solution from 0.75 to 0.9 mPas.

  Table 1 shows the results of measuring the temperature change of the reaction liquid when an alternating voltage was applied to form a pearl chain. The temperature of the reaction solution before application was room temperature (about 25 ° C.), but increased to about 37 ° C. after application. On the other hand, in the conventional method, the BSA concentration in the final reaction solution was about 0.5%, but the viscosity of the reaction solution was less than 0.8 mPas (0.6 to 0.75 mPas) due to the temperature rise by application of a voltage pulse. I understand that there was. From the above, it can be seen that the viscosity of the reaction solution can be measured with higher sensitivity by applying an AC voltage under the condition of 0.8 to 0.9 mPas.

  Furthermore, it can be seen from Table 1 that the viscosity of the reaction solution according to the present invention for maintaining the temperature of the reaction solution during application of the voltage pulse shown in Example 4 at a low temperature was 1.4 mPas. From these, it can be seen that the viscosity in the reaction solution can be measured with higher sensitivity by applying an AC voltage under the condition of 0.8 to 3 mPas. From the above results, it was confirmed that the preferred viscosity for improving the sensitivity was 1 to 3 mPas, more preferably 1 to 2 mPas.

  The measuring method or measuring apparatus of the present invention is useful for measuring any substance having affinity. Specifically, information useful for diagnosis of various diseases can be obtained by analyzing a sample collected from a living body. More specifically, hormones, tumor markers, enzymes, drugs, infectious pathogens, or antibodies against them are routinely measured in medical institutions. Any of these components to be measured is included in the affinity substance in the present invention. Alternatively, microorganisms or drugs contained in biological samples, foods, or samples collected from the environment can be measured or detected by the present invention.

(A) is a figure which shows the structure of the apparatus based on this invention. Moreover, (B) shows the cross section of the pulse application tank which comprises the apparatus based on this invention. Reference numerals in the drawings indicate elements described in the description of the reference numerals. The measurement result (relationship with pre-processing temperature) obtained when the measuring method of this invention was implemented with the measuring apparatus which has the structure of FIG. 1 is shown. In the figure, the vertical axis indicates the aggregation rate P / T (%), and the horizontal axis indicates the AFP concentration (ng / mL). The measurement result (relation with the pre-processing time under high temperature) obtained when the measuring method of this invention was implemented with the measuring apparatus which has the structure of FIG. 1 is shown. The measurement result (relationship with a reaction accelerator) obtained when the measuring method of this invention was implemented with the measuring apparatus which has the structure of FIG. 1 is shown. Each plot in the figure shows the following results. □: antigen amount 0 ng / mL (blank value) ○: antigen amount 9.5 ng / mL ●: blank correction (9.5-0 ng / mL) (A) is a figure which shows the structure of the apparatus based on this invention. Moreover, (B) shows the cross section of the pulse application tank which comprises the apparatus based on this invention. Reference numerals in the drawings indicate elements described in the description of the reference numerals. The measurement result obtained when the measuring method of this invention was implemented with the measuring apparatus which has the structure of FIG. 5 is shown. In the figure, the vertical axis indicates the aggregation rate P / T (%), and the horizontal axis indicates the AFP concentration (ng / mL). The result of the control method of FIG. 6 is shown. In the figure, the vertical axis indicates the aggregation rate P / T (%), and the horizontal axis indicates the AFP concentration (ng / mL). The result of the control method of FIG. 6 is shown. In the figure, the vertical axis indicates the aggregation rate P / T (%), and the horizontal axis indicates the AFP concentration (ng / mL). FIG. 9 is a graph showing the effect of bovine serum albumin (BSA) added to the reaction solution on the aggregation rate of carrier particles in the measurement of prostate specific antigen (PSA) according to the present invention. In the figure, the vertical axis represents the aggregation rate (%), and the horizontal axis represents the final concentration of BSA in the reaction solution. Each column shows the results of PSA 0 ng / mL, 9.5 ng / mL, and 32 ng / mL in order from the left. In the graph, the aggregation rate at PSA 0 ng / mL at each BSA concentration, the difference in aggregation rate at 9.5 ng / mL (-●-), and the difference in aggregation rate at 32 ng / mL (-○-) are combined. Indicated. 3 is a graph showing the influence of the concentration of bovine serum albumin (BSA) in the reaction solution on the viscosity of the reaction solution. In the figure, the vertical axis represents viscosity (mPas) and the horizontal axis represents temperature (° C.).

Explanation of symbols

FIG.
1: Dispensing + stirring means 2: Temperature control mechanism
3: Reaction tank 4: Electrode (pulse application means)
5: Dilution means 6: Particle size distribution measurement means

FIG.
1: Dispensing + stirring means 2: Reaction tank
3: Electrode (pulse application means) 4: Temperature control mechanism
5: Dilution means 6: Particle size distribution measurement means

Claims (22)

A method for measuring an affinity substance, comprising the following steps.
(1) a step of incubating a reaction liquid in which a carrier particle bound with a binding partner having a binding activity with a measurement target affinity substance and a measurement target affinity substance is mixed at 37 to 90 ° C .;
(2) a step of applying a voltage pulse to the reaction solution in step (1),
(3) After step (2), aggregates of carrier particles formed by binding with the affinity substance to be measured, and carriers that did not bind to the affinity substance to be measured and did not form an aggregate Counting either or both of the particles, and
(4) After step (3), determining the level of the substance to be measured based on either or both of the aggregate formation level and the level of carrier particles that did not form the aggregate The method according to claim 1, wherein step (1) is a step of incubating the reaction solution at 45 to 80 ° C. The method according to claim 2, wherein step (1) is a step of incubating the reaction solution at 50 to 65 ° C. The method according to claim 1, wherein the reaction solution contains a water-soluble polymer. The method according to claim 1, wherein the viscosity of the reaction solution in step (2) is adjusted to 0.8 to 3 mPas. The method for measuring an affinity substance according to claim 1, wherein step (2) is performed at 0 to 20 ° C. The method for measuring an affinity substance according to claim 6, wherein the step (2) is performed at 0 to 10 ° C. The method according to claim 1, wherein either or both of the aggregate and the carrier particles that did not form the aggregate are counted using the three-dimensional information as an index. The method according to claim 1, wherein the binding between the affinity substance and the binding partner is binding by an antigen-antibody reaction. The method according to claim 9, wherein the affinity substance is an antigen, and the binding partner is an antibody or a fragment containing an antigen-binding region thereof. The method according to claim 9, wherein the affinity substance is an antibody or a fragment containing an antigen-binding region thereof, and the binding partner is an antigen or a fragment containing an antigenic determinant thereof. The method of claim 1, wherein the voltage pulse is an alternating voltage pulse. A method for measuring an affinity substance, comprising the following steps.
(1 ′) 37-90 ° C. before or after mixing a reaction liquid containing carrier particles bound with at least a binding partner having a binding activity with a measurement target affinity substance and a measurement target affinity substance with an agglutinating reagent component In which the carrier particles are aggregated by an agglutinating reagent and the aggregation is inhibited by an affinity substance to be measured.
(2 ′) applying a voltage pulse to the reaction solution in step (1 ′) in the presence of the agglutinating reagent component;
(3 ′) After step (2 ′), any of the aggregates of the carrier particles formed by binding to the agglutinating reagent and the carrier particles whose aggregation is inhibited by binding to the affinity substance to be measured or Counting both, and
(4) After the step (3 ′), a step of determining the level of the substance to be measured based on either or both of the aggregate formation level and the level of the carrier particles that did not form the aggregate. The method according to claim 13, wherein in the step (1 '), after the reaction solution is incubated, an agglutinating reagent is mixed before the step (2'). 14. The method of claim 13, comprising the further step of incubating the agglutination reagent after mixing and before step (2 '). The method according to claim 13, wherein in the step (1 '), the reaction solution is incubated in the presence of an agglutinating reagent, and then the step (2') is performed. Incubating a reaction solution containing a binding partner having binding activity with a specific substance and the specific substance at 37 to 90 ° C., and then aggregating the carrier particles, including a step of applying a voltage pulse Way for. The method according to claim 17 , wherein the temperature of the reaction solution is 0 ° C. to 20 ° C. while applying a voltage. The method according to claim 17 , wherein the binding between the binding partner and the specific substance is an antigen-antibody reaction. The method of claim 17 , wherein the voltage pulse is an alternating voltage pulse. The method according to claim 17 , wherein a water-soluble polymer is added to the reaction solution. The method according to claim 17 , wherein the viscosity of the reaction solution is adjusted to 0.8 to 3 mPas.

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