JP4288672B2 - Method for measuring affinity substances by particle aggregation and dilution - 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 / Matsuzawa et al., Chemistry and Industry, Vol. 36, No. 4) No., 1982). 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. In the report of Cambiaso et al., A reagent having an antibody bound to latex having a particle size of 0.8 μm was reacted with an antigen at 37 ° C. for 20 minutes. 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. Then, if the carrier particles are redispersed after the electric field is stopped, agglomerates of the carrier particles that are reaction products can be detected.

Japanese Patent Laid-Open No. 7-83928 Cambiaso et al., J. Immunol. Methods 18, 33, 1977 Matsuzawa et al., Chemistry and Industry, Vol.36, No.4, 1982

  As described above, in the method of measuring the affinity substance by counting the aggregation of the carrier particles, the discrimination result between the aggregate and the carrier particles that have not aggregated has a great influence on the measurement result. However, even when the carrier particles do not form aggregates, they may be counted as aggregates if they are in a positional relationship where the particles appear to overlap. The present inventors have confirmed that the problem of overlap between particles can be solved by counting aggregated particles based on the three-dimensional information of the particles. However, even in the analysis based on the tertiary information, a plurality of particles may be detected simultaneously when the particle concentration is high. That is, it was confirmed that a plurality of carrier particles that are not aggregated may be counted as aggregates.

  If the particle concentration is set low in advance, it may be possible to avoid overlapping of particles in identification of aggregated particles. However, pearl chain formation is difficult under conditions where the particle concentration is low. A certain level of particle concentration is required for the particle aggregation reaction by pearl chain formation. In other words, it is ideal if the particle concentration can be increased in the pearl chain formation step and then the particle concentration can be decreased in the aggregated particle detection step. In order to lower the particle concentration, for example, a reaction solution containing carrier particles may be diluted.

  However, in reality, the aggregate formed by immunological binding collapses due to dilution of the reaction solution. As a result, the number of aggregated particles is counted lower than actual. That is, a negative error occurs due to dilution. An object of the present invention is to provide a method for measuring an affinity substance that is not easily affected by dilution in detecting an agglomerate formed by pearl chain formation, and an apparatus therefor.

In order to solve the above-mentioned problems, the present inventors have repeatedly studied a process for detecting an aggregate of carrier particles. Then, in the step of diluting the reaction liquid after pearl chain formation, it was considered that the disadvantages associated with dilution could be eliminated by utilizing means capable of strengthening the bonds that form aggregates. Furthermore, an effective means for preventing the disadvantage caused by dilution was clarified, the effect was confirmed, and the present invention was completed. That is, the present invention relates to the following measuring method and measuring apparatus.
[1] In the method for measuring an affinity substance comprising the following steps (1)-(3) or (1 ′)-(3 ′), the affinity substance is introduced before step (3) or (3 ′). And a step of diluting the reaction solution by means for enhancing the binding between the agglutinating reagent and the binding partner.
(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 step (2), determining the level of the substance to be measured based on either or both of the level of aggregate formation and the level of carrier particles that did not form aggregates, or
(1 ′) a step of applying a voltage pulse to a reaction liquid in which a carrier particle bound with a binding partner having a binding activity to a measurement target affinity substance and a measurement target affinity substance are mixed with an agglutinating reagent component. The carrier particles are aggregated by an agglutinating reagent and the aggregation is inhibited by an affinity substance to be measured;
(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 ′), 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 The method according to [1], wherein the step of diluting the reaction solution is a step of mixing the reaction solution and the dilution solution under the condition of applying the voltage pulse.
[3] The method according to [2], wherein the voltage pulse is an AC voltage.
[4] The method according to [3], wherein the AC voltage has a frequency of 2 KHz to 20 MHz.
[5] The method according to [2], wherein the step of diluting the reaction solution includes a step of further diluting the carrier particles after the electric field is stopped after mixing the reaction solution and the dilution solution under the application of the voltage pulse. .
[6] The step of diluting the reaction solution may be performed by adding a binding enhancer that enhances the binding between the affinity substance to be measured and the binding partner or between the agglutination reagent and the binding partner to the reaction solution and then mixing with the reaction solution, Alternatively, the method according to [2], which is a step of diluting the reaction solution with a diluent containing the binding enhancer.
[7] In the step of diluting the reaction solution, the reaction solution is mixed with the dilution solution after adding a binding enhancer that enhances the binding between the affinity substance to be measured and the binding partner or between the agglutination reagent and the binding partner to the reaction solution. Or the method according to [1], which is a step of diluting the reaction solution with a diluent containing the binding enhancer.
[8] The method according to [7], wherein the binding between the affinity substance to be measured and the binding partner, or the binding between the aggregation reagent and the binding partner is an immunological binding.
[9] The method according to [8], wherein the antigen is a protein antigen and the binding enhancer is one or both compounds selected from glutaraldehyde and carbodiimide.
[10] The method according to [7], wherein the step of diluting the reaction solution is a step of mixing the reaction solution and the diluting solution under a voltage pulse application condition.
[11] The method according to [1], wherein the voltage pulse in the step (1) or (1 ′) is an AC voltage pulse.
[12] The method according to [1], wherein the voltage pulse is applied a plurality of times in the step (1) or (1 ′).
[13] The method according to [12], wherein the step (1) or (1 ′) includes a step of applying the next voltage pulse after dispersing the carrier particles after applying the voltage pulse.
[14] The method according to [12], wherein the plurality of voltage pulses are voltage pulses in different directions.
[15] The method according to [1], wherein the carrier particles have an average particle size of 1 μm or more.
[16] The method according to [15], wherein the average particle size of the carrier particles is 1 μm to 20 μm.
[17] The method according to [1], wherein in the step (2) or (2 ′), either or both of the aggregate and the carrier particles that have not formed the aggregate are counted using the three-dimensional information as an index. .
[18] The method according to [17], wherein in step (2) or (2 ′), the three-dimensional information of the agglomerates or carrier particles is physically measured.
[19] A method for physically measuring three-dimensional information is any one selected from the group consisting of an electrical resistance method, a laser diffraction scattering method, and a three-dimensional image analysis method. The method described.
[20] A carrier particle bound with a binding partner having a binding activity with an affinity substance to be measured, including the following elements, and an affinity substance to be measured: an affinity substance or an agglutinating reagent of the carrier particle A measuring device for measuring the aggregation by the index.
a: To hold a carrier particle bound with a binding partner having binding activity with an affinity substance to be measured, and a reaction liquid containing a sample containing the affinity substance to be measured, or additionally a reaction liquid containing an agglutinating reagent Space,
b: means for applying a voltage pulse to the reaction solution,
c: means for diluting the reaction solution, and
d: a means for counting either or both of carrier particles and agglomerates of carrier particles contained in the reaction liquid [21] means for diluting the reaction liquid is The apparatus according to [20], which is a means for mixing a diluent.
[22] The means for diluting the reaction solution includes a means for adding a binding enhancer that enhances the binding between the affinity substance to be measured and the binding partner, or the agglutinating reagent and the binding partner to the reaction solution. [20] The apparatus according to [20].

In the reaction solution to which the voltage pulse is applied, an aggregate is formed by the binding reaction between the binding partner held on the carrier particles and the affinity substance (or the aggregation reagent). In the present invention, in detecting the aggregate, the aggregate is detected after diluting the reaction solution by means for enhancing the binding reaction. As means for strengthening the binding reaction, dilution of the reaction solution under a voltage pulse application condition or a binding strengthening agent can be used. By employing means to enhance the binding reaction, various obstacles associated with the dilution of the agglomerates can be avoided.
That is, the diluted carrier particles are unlikely to be detected by overlapping each other. As a result, it is possible to prevent an error in erroneously detecting particle overlap as an aggregate. On the other hand, the structure of the agglomerates is maintained by strengthening the bonds. Therefore, it is possible to avoid the problem that the agglomerates collapse and become undetectable with dilution. Thus, according to the present invention, improvement in reproducibility and sensitivity can be realized.
According to the present invention, for example, an increase in sensitivity or an improvement in reproducibility of a measurement method using an immunological binding reaction using the aggregation of carrier particles as an index 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)-(3) or (1 ′)-(3 ′), before the step (3) or (3 ′): It is a method comprising a step of diluting the reaction solution by means for enhancing the binding between the substance and the binding partner or between the agglutination reagent and the binding partner.
(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 step (2), determining the level of the substance to be measured based on either or both of the level of aggregate formation and the level of carrier particles that did not form aggregates, or
(1 ′) a step of applying a voltage pulse to a reaction liquid in which a carrier particle bound with a binding partner having a binding activity to a measurement target affinity substance and a measurement target affinity substance are mixed with an agglutinating reagent component. The carrier particles are aggregated by an agglutinating reagent and the aggregation is inhibited by an affinity substance to be measured;
(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 step (2 ′), determining the level of the substance to be measured based on either or both of the level of aggregate formation and the level of carrier particles that did not form aggregates

  The dilution step of the reaction solution in the present invention may be performed by any means capable of enhancing the binding between the affinity substance and the binding partner or the aggregation reagent and the binding partner before the step (3) or (3 ′). Can be implemented. For example, a method of diluting the reaction solution under the condition of applying a voltage pulse is a preferable dilution method in the present invention. More specifically, dilution is performed by adding the reaction solution to the diluted solution to which the voltage pulse is applied. The diluent is placed between the electrodes and a voltage pulse is applied. In other words, a diluent is placed between the opposing electrodes.

  In the dilution step of the present invention, the size of the electrodes and the interval between the electrodes are not particularly limited as long as the reaction solution is diluted under a condition where a voltage pulse is applied to the reaction solution. That is, the initial contact between the reaction liquid after the pearl chain formation and the diluent is performed in an electric field sandwiching the counter electrode. For example, in the dilution method shown in FIG. 6 (B), the size of the electrode is (width) 2 to 12 mm x (length) 10 to 50 mm x (thickness) 0.01 to 0.04 mm, and the distance between the electrodes is 5 to 20 mm. Almost the same effect has been confirmed.

  The voltage pulse is preferably an alternating voltage. The application condition of the voltage pulse can be any condition that does not induce electrolysis of the reaction solution and the diluted solution. The voltage of the voltage pulse is, for example, 0.1 V to 1.2 V, more preferably 0.3 to 0.9 V. The frequency of the voltage pulse is, for example, 2 KHz to 20 MHz, and more preferably 10 KHz to 500 KHz. An arbitrary waveform can be used for the voltage pulse. Specifically, a square wave, a sine wave, a triangular wave, etc. can be shown. A more preferred voltage pulse is a square wave. In the present invention, it is desirable that the voltage pulse is applied at least in a time zone including the moment when the reaction solution and the diluent come into contact. Even if the application time is very short, it is possible to expect a strengthening action of the bond in dilution. More specifically, it is 0.5-30 seconds, usually 1-10 seconds, for example 1-5 seconds.

  A solution containing a salt can be used as the diluent. Specifically, physiological saline, glycine buffer, phosphate buffer, or the like to which a salt of 50 mM to 600 mM is added can be used. Examples of the salt include sodium chloride, potassium chloride, calcium chloride and the like. The diluent may contain a preservative such as sodium azide, a surfactant such as Triton X-100, glycerin, sucrose, and the like.

  In the present invention, the binding reaction between the affinity substance (or aggregating reagent) and the binding partner constituting the aggregate is enhanced. The dipole moment effect due to the electric field caused by the voltage pulse is thought to strengthen the coupling between the two. In any case, it was confirmed that the dilution of the agglomerates was suppressed by dilution under the voltage pulse application condition, and dilution of the reaction solution could be realized while maintaining the agglomerates.

  Alternatively, as a diluting means capable of enhancing the binding between the affinity substance and the binding partner or the aggregating reagent and the binding partner, a binding enhancer capable of enhancing the binding between the two can be used. In the present invention, the bond strengthening agent refers to a component that can reinforce the bond between the two by addition to the reaction solution. For example, protein-protein bonds are enhanced by the addition of compounds such as glutaraldehyde or carbodiimide. Therefore, the immunological binding between the protein antigen and the antibody can be enhanced by glutaraldehyde or carbodiimide. These compounds are preferred as the bond enhancer in the present invention.

  The binding enhancer acts on the functional group of the affinity substance and the binding partner to chemically bond them together. Alternatively, in the aggregation inhibition reaction system, the binding between the aggregation reagent and the binding partner is enhanced. As a result, the aggregate formed by the combination of the two acquires a physically high degree of stability. For example, if the affinity substance or the agglutinating reagent and the binding partner are proteins, there are amino groups and carboxyl groups in the molecule. These functional groups are crosslinked with chemical substances such as glutaraldehyde and carbodiimide.

  The concentration of the bond strengthening agent in the reaction solution can be appropriately set according to the type of the bond strengthening agent. More specifically, for example, in the case of glutaraldehyde, the final concentration in the reaction solution is usually 0.1 to 25%, preferably 0.2 to 18%. The binding enhancer may be added to the reaction solution prior to dilution of the reaction solution containing the conjugate of the affinity substance and the binding partner. The reaction solution after the addition of the binding enhancer can be diluted after incubation at 37 ° C. for several seconds to 20 seconds, preferably 2 to 10 seconds, or 2 to 5 seconds. When glutaraldehyde or carbodiimide is used as the binding strengthening agent, it can be diluted immediately after being added to the reaction solution.

  The addition of a bond strengthening agent is effective as a dilution step in the present invention. In the present invention, a bond strengthening agent can be further combined with the dilution step under the voltage pulse application condition described above. That is, after adding the binding enhancer to the reaction solution, the reaction solution can be diluted under the voltage pulse application conditions according to the conditions described above. Alternatively, the reaction solution can be diluted under the voltage pulse application condition according to the conditions described above by using a diluent to which a binding enhancer is added. By combining the two, the strengthening action of the bond is strengthened.

In the present invention, the dilution of the reaction liquid refers to reducing the concentration of carrier particles in the reaction liquid by mixing the reaction liquid and the diluted liquid. The concentration of carrier particles in the reaction solution is determined by the amount of sample and the amount of carrier particles supplied as a reagent. Furthermore, in the present invention, the concentration of the carrier particles in the reaction solution is usually set in a range in which the aggregation of the carrier particles can be promoted by pearl chain formation. Specifically, the concentration of the carrier particles in the reaction solution is usually 0.01 to 5% by weight, more preferably 0.1 to 2% by weight. On the other hand, it can be diluted, for example, 100 times or more, usually 1000 times or more, specifically 1000 to 100000 times, preferably 2000 to 40000 times. The concentration of the carrier particles after dilution is 0.1 × 10 ^{−5 to} 0.005% by weight, preferably 0.00001 to 0.001% by weight.

  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 agglomerated particles and the amount of an affinity substance that is a measurement target substance are usually directly proportional. That is, when the aggregate formation level is high, the affinity substance level (ie, concentration) is high. Conversely, when the level of carrier particles that did not form agglomerates is high, the level (ie, concentration) of the affinity substance is low.

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. That is, when the aggregate formation level is high, the affinity substance level (ie, concentration) is low. Conversely, when the level of carrier particles that did not form agglomerates is high, the level (ie concentration) of the affinity substance is high.

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.

  Whether it is a direct agglutination reaction or an agglutination inhibition reaction, a standard curve containing a certain amount of affinity substance in advance is measured in the same reaction system, and the level of agglomerates or carrier particles that have not agglomerated is measured to obtain a standard curve. Alternatively, a regression equation can be created. The level of the affinity substance in the sample can be determined by applying either the aggregate formation level obtained by measuring the sample or the level of the carrier particles not aggregated to the standard curve or regression equation. .

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.

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 a step of applying a voltage pulse to a reaction solution containing carrier particles in which an affinity substance and a binding partner are bound. A method of applying a voltage pulse to the reaction solution to cause an agglutination reaction is known (Japanese Patent Laid-Open No. 7-83928). Application of the voltage pulse aligns the carrier particles along the electric field and promotes the binding reaction between the affinity substance and the binding partner on 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. Then, an aggregate is formed by the reaction between the agglutinating reagent and the binding partner, and the affinity substance inhibits the binding between the two.

  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.

In the present invention, the number of times that the voltage pulse is applied to the reaction solution in the step (1) or (1 ′) is not limited. That is, the voltage pulse can be intermittently applied one or more times, for example, 1 to 20 times, usually 1 to 10 times, or 1 to 5 times. By intermittent application, the dispersion and alignment of the carrier particles is repeated. As a result, the chance of contact between the binding partner on the carrier particle and the affinity substance or the agglutinating reagent is increased. That is, a reaction promoting effect can be expected by applying intermittent voltage pulses. In the present invention, when the voltage pulse is applied a plurality of times, the voltage pulse can be applied to the reaction solution from different directions. Specifically, by arranging a plurality of sets of electrodes in the reaction liquid and changing the electrodes to be energized, voltage pulses in different directions can be given to the reaction liquid. Alternatively, the direction of the voltage pulse can be changed by moving the electrode in the reaction solution. A similar effect can be obtained by fixing the electrode and moving the space containing the reaction solution.
Furthermore, the carrier particles can be dispersed during the application of a plurality of voltage pulses. By interposing the dispersion step, an effect of further increasing the opportunity of contact between the binding partner and the affinity substance or the agglutinating reagent can be expected. The carrier particles can be dispersed during application of the voltage pulse by stirring or shaking the reaction solution or applying vibration to the reaction solution.

  As the concentration of the carrier particles in the reaction system is higher, the pearl chain is more easily formed, and the agglutination reaction is promoted. Also, the higher the carrier particle concentration, the greater the aggregation rate of the carrier particles when redispersed when no affinity substance is present. For example, in the case of latex particles, the concentration of the carrier particles in the reaction system is preferably 0.01 to 5% by weight, more preferably 0.1 to 2% 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.

  The present invention also provides a pearl chain by applying a voltage pulse to the reaction liquid in which a carrier particle bound to a binding partner having binding activity with a measurement target affinity substance and a measurement target affinity substance are mixed. Therefore, the agglomerates are formed by a specific reaction, and even if the voltage pulse is stopped, these re-dispersions do not occur. However, when a relatively strong physical external force is applied, the agglomerates may be collapsed. In order to stabilize the aggregates formed by this specific reaction, the functional groups of the proteins attached to the carrier particles that form the particles are further reinforced with chemical bonds. It is characterized by doing. The operation according to the present invention is preferably carried out before the carrier particles are diluted.

  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.

Further, the present invention relates to the binding between the carrier particle having a binding activity with the affinity substance to be measured, which contains 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.
a: To hold a carrier particle bound with a binding partner having binding activity with an affinity substance to be measured, and a reaction liquid containing a sample containing the affinity substance to be measured, or additionally a reaction liquid containing an agglutinating reagent Space,
b: means for applying a voltage pulse to the reaction solution,
c: means for diluting the reaction solution, and
d: Means for counting either or both of carrier particles and aggregates 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.

  Next, b: means for applying a voltage pulse to the 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. Furthermore, the apparatus of the present invention can comprise means for stirring the reaction liquid, means for shaking the reaction liquid, or means for applying vibration to the reaction liquid. All of these means are useful as means for dispersing carrier particles between a plurality of voltage pulses.

  The apparatus of the present invention has c: means for diluting the reaction solution. In the present invention, the means for diluting the reaction solution may hereinafter be referred to as dilution means. For example, the diluting means is configured by a mechanism for holding the diluting liquid, collecting at least a part of the reaction liquid, and mixing it with the diluting liquid. The diluting means is composed of a space that can accommodate a diluting solution that provides a predetermined dilution factor. The dilution factor in the present invention is, for example, 100 times or more, usually 1000 times or more, specifically 1000 to 100,000 times, preferably 2000 to 40000 times.

The diluting means of the present invention preferably comprises a mechanism that can reinforce the bonds forming the agglomerates. Specifically, a diluting means capable of mixing the reaction liquid and the diluting liquid under the voltage pulse application condition is a preferable diluting means in the present invention.
For example, an electrode for applying a voltage pulse to the diluent can be disposed in the space holding the diluent. Alternatively, the dilution means in the present invention can include a mechanism for adding a binding enhancer to the reaction solution. The structure for strengthening the bonds forming these agglomerates can be equipped alone or in combination.

  Furthermore, the apparatus of the present invention includes means for counting d: either or both of the carrier particles and the aggregates of the carrier particles contained in the reaction solution. The counting means includes, for example, a mechanism for analyzing carrier particles contained in the diluted reaction solution. Alternatively, the particles can be counted after taking out the diluted reaction solution from the space holding the diluent and introducing it into the counting means. Carrier particles or aggregates can be analyzed based on two-dimensional image information or three-dimensional physical information.

  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. A carrier particle and / or agglomerate analysis mechanism using three-dimensional information as an index is preferable as a counting means in the present invention.

  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 (d) constituting the apparatus 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]
(1) Preparation of anti-AFP antibody-sensitized latex reagent
0.1 mg of anti-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 2.06 μm latex (manufactured by Polyscience) 1 mL of a 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-AFP antibody-sensitized latex reagent.
(2) Measuring apparatus Biological specific agglutination reaction (antigen-antibody reaction) was measured using the apparatus shown in FIG. Dispense and mix specimen and reagent 1 (buffer: 2 reagent system. Not required for 1 reagent system / latex reagent only), then dispense reagent 2 (latex reagent) and mix The dispensing / stirring tank temperature control mechanism 1 is provided, and then the reaction liquid is moved to the reaction tank (pulse application tank) 3 and a voltage pulse is applied through the electrode 4 for several seconds to several tens of seconds to make a pearl chain. Make it. The reaction solution is pearl chained and then diluted (dilution tank 5), and the aggregation state of the carrier is measured using a particle size distribution meter 6. (Temperature control mechanisms 1 and 2 were set to OFF.)
FIG. 1B is a diagram showing an outline of the dilution tank 5. The diluted solution is dispensed into the dilution container 103, and the reaction solution is dispensed between the counter electrodes 101.
(3) Measuring method
AFP control serum L (16 ng / mL), M (125 ng / mL), H (1000 ng / mL) and free serum (0 ng / mL) were measured as sample liquids. Take 3 μL of the sample and 3 μL of the anti-AFP antibody-sensitized latex reagent described above in a test tube, mix them, immediately inject them into the reaction cell with electrodes, and use the above-mentioned apparatus to make an alternating voltage (rectangular wave) ± 12 V / frequency with a frequency of 200 KHz. A pearl chain was formed by applying an electrolytic strength of mm for 30 seconds. Immediately after the application for 30 seconds, the electric field was turned off and the reaction solution was diluted in physiological saline. For dilution, use the apparatus shown in Fig. 1 (B), add the reaction solution while applying AC voltage (rectangular wave) ± 0.7V at a frequency of 200KHz to the diluted solution via the electrode, and then use Coulter Multisizer. Was used to measure the particle size distribution of the latex particles, and the agglomeration rate (AR) of the latex was determined by the following equation.
AR = (number of particles aggregated into two or more) / (total number of particles) × 100 (%)
(4) The results are shown in FIG.

(Comparative Example 1)
The measurement was carried out in substantially the same manner as in Example 1 using each specimen of Example 1 and the anti-AFP-sensitized latex reagent. The same procedure as in Example 1 was performed except that the electric field was not applied in the step of diluting the reaction solution after forming the pearl chain. The results are shown in FIG.
As shown in FIG. 2, the measurement result (aggregation rate) of AFP 1000 ng / mL is 40.9% in the conventional method, which is a control, compared to 53.4% in this method. That is, in the method of the present invention, compared to the conventional method, the collapse of the aggregates that disintegrate when the reaction solution is diluted, that is, the aggregates of the specifically aggregated carrier particles, can be reduced by about 20%. The measurement result (aggregation rate) of AFP 0 ng / mL is 10.6% in the conventional method as a control compared to 7.4% in the present method. The aggregation rate when a sample of 0 ng / mL is measured can be considered as a background associated with nonspecific aggregation. That is, according to the method of the present invention, nonspecific aggregation is suppressed. From the above results, the measurement method of the present invention has a large slope of the aggregation rate and good linearity. Therefore, according to the present invention, it is possible to measure in a wide concentration range with high sensitivity.

(Example 2)
(1) Preparation of anti-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 shown in FIG.
(3) Measuring method
AFP control serum L (16 ng / mL) and free serum (0 ng / mL) were measured as sample solutions. Take 3 μL of the sample and 3 μL of the anti-AFP antibody-sensitized latex reagent described above in a test tube, mix them, immediately inject them into the reaction cell with electrodes, and use the above-mentioned apparatus to make an alternating voltage (rectangular wave) ± 12 V / frequency with a frequency of 200 KHz. A pearl chain was formed by applying an electrolytic strength of mm for 30 seconds. Immediately after the application for 30 seconds, the electric field was turned off, and the reaction solution was diluted with 20 mL of physiological saline. For dilution, use the apparatus shown in Fig. 1 (B), add the reaction solution while applying AC voltage (rectangular wave) ± 0.7V at a frequency of 200KHz to the diluted solution via the electrode, and then use Coulter Multisizer. Was used to measure the particle size distribution of the latex particles, and the agglomeration rate (AR) of the latex was determined by the following equation.
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 and average value ± 2SD of the measurement results were obtained.
(1) The results are shown in FIG.

(Comparative Example 2)
The measurement was carried out in substantially the same manner as in Example 2 using each sample of Example 2 and the anti-AFP-sensitized latex reagent. The same procedure as in Example 2 was performed except that the electric field was not applied in the step of diluting the reaction solution after forming the pearl chain. The results are shown in FIG.

(Comparative Example 3)
The measurement was carried out in substantially the same manner as in Example 2 using each sample of Example 2 and the anti-AFP-sensitized latex reagent. The same procedure as in Example 1 was performed, except that a reaction solution diluted with an electric field previously applied under the same conditions as in Example 2 was used as the diluent. The results are shown in FIG.
As shown in FIG. 3, the measurement result (aggregation rate) of AFP 15.6 ng / mL is 32.6% in the conventional method which is a control compared to 35.7% in this method. That is, in the method of the present invention, compared with the conventional method, the collapse of the aggregates that disintegrate when the reaction solution is diluted, that is, the aggregates of the specifically aggregated carrier particles can be reduced by about 10%. Moreover, the measurement result (aggregation rate) of AFP 0 ng / mL is 11.1% in the conventional method compared to 5.6% in the present method. The aggregation rate when a sample of 0 ng / mL is measured can be considered as a background associated with nonspecific aggregation. That is, according to the method of the present invention, nonspecific aggregation is suppressed. Furthermore, the CV value when AFP 15.6 ng / mL was measured five times was 4.28% in the conventional method compared to 1.16% in this method, and the reproducibility of the measurement was significantly improved. Therefore, highly sensitive and highly accurate measurement is possible by the present invention.

[Example 3] Promotion of antigen-antibody reaction (1) Preparation of anti-PSA antibody-sensitized latex reagent (Reagent 2)
0.1 mg of anti-PSA antibody (manufactured by 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 2.06 μm latex (manufactured by Polyscience) (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)
Acceleration of reaction containing 0.25% polyethylene glycol (molecular weight 20000, hereinafter abbreviated as PEG 20000) in 50 mM Tris HCl buffer solution (50 mM Tris, 50 mM sodium chloride, 0.09% sodium azide, pH 8.4) in 0.5% bovine serum albumin Reagents were prepared.
(3) Measuring apparatus The affinity substance (antigen-antibody reaction) was measured using the apparatus shown in FIG.
(4) Measuring method
PSA control serum L (9.5 ng / mL), M (32 ng / mL), and free serum (0 ng / mL) were measured as sample solutions. Take 1 μL of the sample, 3 μL of the Tris hydrochloric acid buffer solution and anti-PSA antibody-sensitized latex reagent described above in a test tube, mix them, immediately inject them into the reaction cell with electrodes, and use the above-mentioned apparatus to create an alternating voltage (rectangular) with a frequency of 200 KHz. Wave) A pearl chain was formed by applying an electrolytic strength of ± 12 V / mm for 30 seconds. Immediately after the application for 30 seconds, the electric field was turned off and the reaction solution was diluted with 20 mL of GBS. The dilution factor at this time is about 3300 times. For dilution, use the apparatus shown in Fig. 1 (B), add the reaction solution while applying AC voltage (rectangular wave) ± 0.7V at a frequency of 200KHz to the diluted solution via the electrode, and then use Coulter Multisizer. Was used to measure the particle size distribution of the latex particles, and the agglomeration rate (AR) of the latex was determined by the following equation.
AR = (number of particles aggregated into two or more) / (total number of particles) × 100 (%)
(5) The results are shown in FIG.

(Comparative Example 4)
Measurement was carried out in substantially the same manner as in Example 2, using each specimen of Example 3, Tris buffer and anti-PSA-sensitized latex reagent. The same procedure as in Example 1 was performed except that the electric field was not applied in the step of diluting the reaction solution after forming the pearl chain. The results are shown in FIG.

(Comparative Example 5)
Measurement was carried out in substantially the same manner as in Example 2, using each specimen of Example 3, Tris buffer and anti-PSA-sensitized latex reagent. The same procedure as in Example 2 was performed, except that a reaction solution diluted with an electric field previously applied under the same conditions as in Example 3 was used as the diluent. The results are shown in FIG.
As shown in FIG. 4, the measurement result (aggregation rate) of PSA 32 ng / mL is 30.3% in the conventional method, which is a control, compared to 35.7% in this method. That is, in the method of the present invention, compared to the conventional method, the collapse of the aggregates that disintegrate when the reaction solution is diluted, that is, the aggregates of the specifically aggregated carrier particles, can be reduced by about 20%. Moreover, the measurement result (aggregation rate) of PSA 0 ng / mL is 2.45% in the conventional method compared to 1.73% in the present method. The aggregation rate when a sample of 0 ng / mL is measured can be considered as a background associated with nonspecific aggregation. That is, according to the method of the present invention, nonspecific aggregation is suppressed. From the above results, the measurement method of the present invention has a large slope of the aggregation rate and good linearity. Therefore, according to the present invention, it is possible to measure in a wide concentration range with high sensitivity.

(Example 4)
(1) Preparation of anti-AFP antibody-sensitized latex reagent In the same manner as in Example 1, an anti-AFP antibody-sensitized latex reagent was prepared. However, using 2.0 μm latex (manufactured by Sekisui Chemical Co., Ltd.), 3 μm (manufactured by Polyscience) and 4.5 μm (manufactured by Polyscience) as a 1.0% solids suspension, three types of anti-AFP antibody sensitivities A prepared latex reagent was prepared.
(2) Measuring apparatus The affinity substance (antigen-antibody reaction) was measured using the apparatus shown in FIG.
(3) Measurement method Measurement was performed in the same manner as in Example 1 using the three types of latex reagents described above.
(4) The results are shown in FIG. 5, FIG. 6 and FIG.

(Comparative Example 6)
The measurement was carried out in substantially the same manner as in Example 4 using each specimen of Example 4 and the anti-AFP-sensitized latex reagent. The same procedure as in Example 4 was performed, except that the electric field was not applied in the step of diluting the reaction solution after pearl chain formation. The results are shown in FIG. 5, FIG. 6, and FIG.
As shown in FIG. 5, the measurement result (aggregation rate) of AFP 15.6 ng / mL is 24.1% in the conventional method, which is a control, compared to 35.8% in this method. % Could be reduced. In FIG. 5, the measurement result (aggregation rate) of AFP 0 ng / mL is 8.0% in the conventional method compared to 5.3% in the present method. In FIG. 6, the measurement result (aggregation rate) of AFP 15.6 ng / mL is 23.6% in the conventional method, which is 29.5% of the present method, and the collapse of the specifically aggregated aggregate is reduced by 20%. I was able to. In FIG. 6, the measurement result (aggregation rate) of AFP 0 ng / mL is 17.8% in the conventional method compared to 13.6% in the present method. Further, in FIG. 7, the measurement result (aggregation rate) of AFP 15.6 ng / mL is 3.0% in the conventional method, which is a control compared to 11.5% in this method, and the collapse of the specifically aggregated aggregate is reduced by 70%. We were able to. In FIG. 7, the measurement result (aggregation rate) of AFP 0 ng / mL is 2.5% in the conventional method compared to 4.1% in the present method. That is, the present invention can reduce the collapse of the aggregates of the carrier particles that are specifically aggregated as compared with the conventional method. Furthermore, nonspecific aggregation that occurred during dilution could also be suppressed. Therefore, highly sensitive measurement is possible by the present invention.

Example 5
(1) Preparation of anti-AFP antibody-sensitized latex reagent
Use 0.1 mg of 2.2 mg anti-AFP antibody (Dako) and 0.5 mL of 1.716 μm latex (2.5% solids suspension from Polyscience) and carbodiimide kit (Polyscience) and follow the kit manual. An anti-AFP antibody-sensitized latex reagent was prepared by chemically bonding the anti-AFP antibody and latex particles.
(2) Measuring apparatus The affinity substance (antigen-antibody reaction) was measured using the apparatus shown in FIG.
(3) Measuring method
AFP control serum H (1000 ng / mL) was measured as a sample solution. Take 3 μL of specimen and 3 μL of anti-AFP antibody-sensitized latex reagent in the test tube, mix, immediately inject into electrode-equipped reaction cell, and use the above-mentioned device, AC voltage with a frequency of 200 KHz (rectangular wave) ± 12 V / mm A pearl chain was formed by applying an electrolytic strength of 30 seconds for 30 seconds. Immediately after the application for 30 seconds, the electric field was turned off, and 16 μL of glutaraldehyde 0.25% to 25% solution (hereinafter abbreviated as GA solution) was added to the reaction solution, followed by incubation at 37 ° C. for 60 seconds, and then diluted in 20 mL of physiological saline. With respect to this reaction diluted solution, the particle size distribution of latex particles was measured using a Coulter Multisizer, and the latex agglomeration rate (AR) was determined by the following equation.
AR = (number of particles aggregated into two or more) / (total number of particles) × 100 (%)
Next, ultrasonic waves were applied to the remainder of the reaction diluent for 30 seconds and 60 seconds, and the particle size distribution was measured in the same manner (disintegrating severe test).
(4) The results are shown in FIG.

(Comparative Example 7)
Measurement was carried out in substantially the same manner as in Example 5 using each sample of Example 5 and the anti-AFP-sensitized latex reagent. The same procedure as in Example 5 was performed except that the sample was diluted in physiological saline containing no GA. The results are shown in FIG. Comparing the agglomeration rate immediately after the dilution, the one diluted after the GA treatment had an agglomeration rate of 21%, whereas the untreated GA showed a decay of 5%, 16%. Furthermore, in the disintegration severe test, it can be seen that the disintegration was remarkably improved by the 25% GA treatment. From the above, it can be seen that the present invention can reduce the collapse of the aggregates of the carrier particles specifically aggregated compared to the conventional method and can measure with high sensitivity.

Example 6
(1) Preparation of anti-AFP antibody-sensitized latex reagent In the same manner as in Example 5, an anti-AFP antibody-sensitized latex reagent was prepared.
(2) Measuring apparatus The affinity substance (antigen-antibody reaction) was measured using the apparatus shown in FIG.
(3) Measuring method
AFP control serum H (1000 ng / mL) was measured as a sample solution. Take 3 μL of specimen and 3 μL of anti-AFP antibody-sensitized latex reagent in the test tube, mix, immediately inject into electrode-equipped reaction cell, and use the above-mentioned device, AC voltage with a frequency of 200 KHz (rectangular wave) ± 12 V / mm A pearl chain was formed by applying an electrolytic strength of 30 seconds for 30 seconds. Immediately after the application for 30 seconds, the electric field was turned off, and 16 μL of a 25% glutaraldehyde solution (hereinafter abbreviated as GA solution) was added to the reaction solution, incubated at 37 ° C. for 0-60 seconds, and then diluted in physiological saline. With respect to this reaction diluted solution, the particle size distribution of latex particles was measured using a Coulter Multisizer, and the latex agglomeration rate (AR) was determined by the following equation.
AR = (number of particles aggregated into two or more) / (total number of particles) × 100 (%)
Next, ultrasonic waves were applied to the remainder of the reaction diluent for 30 seconds and 60 seconds, and the particle size distribution was measured in the same manner (disintegrating severe test).
(4) The results are shown in FIG.

(Comparative Example 9)
Measurement was carried out in substantially the same manner as in Example 6, using each sample of Example 6 and the anti-AFP-sensitized latex reagent. The same procedure as in Example 6 was performed, except that the sample was diluted with physiological saline not containing GA. The results are shown in FIG. Comparing the aggregation rate immediately after dilution, the one diluted after 25% GA treatment had an aggregation rate of 21% regardless of the treatment time (15 seconds, 30 seconds, 60 seconds). The aggregation rate was 18% at 0 seconds (diluted immediately after mixing), and 16% without GA. Furthermore, in the disintegration severe test, it can be seen that the disintegration was improved almost equally by 25% GA treatment (treatment time 15 to 60 seconds). From the above, it can be seen that the present invention can reduce the collapse of the aggregates of the carrier particles specifically aggregated compared to the conventional method and can measure with high sensitivity.

Example 7
(1) Preparation of anti-AFP antibody-sensitized latex reagent In the same manner as in Example 1, an anti-AFP antibody-sensitized latex reagent was prepared. However, using 2.0μm latex (manufactured by Sekisui Chemical Co., Ltd.), 2.8μm (manufactured by Polyscience) and 1.7μm (manufactured by Polyscience) as a 1.0% solids suspension, three types of anti-AFP antibodies A sensitized latex reagent was prepared.
(2) Measuring apparatus The affinity substance (antigen-antibody reaction) was measured using the apparatus shown in FIG.
(3) Measurement method Measurement was carried out in the same manner as in Example 6 using the three types of latex reagents described above.
(4) The results are shown in FIG. 10, FIG. 11 and FIG.

(Comparative Example 10)
Measurement was carried out in substantially the same manner as in Example 7, using each specimen of Example 7 and the anti-AFP-sensitized latex reagent. The same procedure as in Example 6 was performed, except that the sample was diluted with physiological saline not containing GA. The results are shown in FIG. 10, FIG. 11 and FIG. As described above, from FIG. 10, FIG. 11 and FIG. 12, the present invention can reduce the collapse of the aggregates of the specifically aggregated carrier particles compared with the conventional method (diluent containing no binding enhancer), and can measure with high sensitivity. You can see that

[Example 8] Simultaneous reproducibility test (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 5.
(2) Measuring apparatus The affinity substance (antigen-antibody reaction) was measured using the apparatus shown in FIG.
(3) Measuring method
AFP control sera L and M were measured as sample solutions. Take 3 μL of specimen and 3 μL of anti-AFP antibody-sensitized latex reagent in the test tube, mix, immediately inject into electrode-equipped reaction cell, and use the above-mentioned device, AC voltage with a frequency of 200 KHz (rectangular wave) ± 12 V / mm A pearl chain was formed by applying an electrolytic strength of 30 seconds for 30 seconds. Immediately after the application for 30 seconds, the electric field was turned off, and the reaction solution was added with 16 μL of glutaraldehyde 25% solution (hereinafter abbreviated as GA solution), incubated at 37 ° C. for 0 to 60 seconds, and diluted with physiological saline. With respect to this reaction diluted solution, the particle size distribution of latex particles was measured using a Coulter Multisizer, and the latex agglomeration rate (AR) was determined by the following equation.
AR = (number of particles aggregated into two or more) / (total number of particles) × 100 (%)
The above operation was repeated 10 times to measure and perform a reproducibility test.
(4) The results are shown in Table 1.

(Comparative Example 11)
Measurement was carried out in substantially the same manner as in Example 8, using each specimen of Example 8 and the anti-AFP-sensitized latex reagent. The same procedure as in Example 8 was performed except that the sample was diluted with physiological saline containing no GA. The results are shown in Table 1. In the present invention, the CV value of simultaneous reproducibility was around 2%, but the CV value of the control method has a large variation of 7 to 11%. From the above, it can be seen that the present invention can be measured with better reproducibility than the control method.

  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.

FIG. 1A is a diagram showing an outline of an apparatus for measuring the aggregation state of a carrier. FIG. 1 (B) is a diagram showing an outline of the dilution tank 5 of FIG. 1 (A). FIG. 2 is a graph showing the carrier agglutination rate when the reaction is carried out using AFP control serum as a sample solution and anti-AFP antibody-sensitized latex reagent as a carrier. The aggregation rate when diluted by application is indicated by black diamonds, and the aggregation rate when diluted without application is indicated by white squares. The vertical axis indicates the aggregation rate, and the horizontal axis indicates the concentration of AFP. FIG. 3 is a graph showing the aggregation rate of the carrier when the reaction was carried out using AFP control serum as the sample solution and the anti-AFP antibody-sensitized latex reagent as the carrier. The aggregation rate when diluted by application is indicated by black circles, the aggregation rate when diluted without application is indicated by white squares, and the aggregation rate when diluted with a previously applied dilution is indicated by black triangles. The vertical axis indicates the aggregation rate, and the horizontal axis indicates the concentration of AFP. FIG. 4 is a graph showing the agglutination rate of the carrier when the reaction was carried out using the PSA control serum as the sample liquid, the anti-PSA antibody-sensitized latex reagent as the carrier, and the reaction promoting reagent added. The aggregation rate when diluted by application is indicated by black diamonds, the aggregation rate when diluted without application is indicated by black squares, and the aggregation rate when diluted by a previously applied diluent is indicated by white triangles. The vertical axis represents the aggregation rate, and the horizontal axis represents the PSA concentration. FIG. 5 is a graph showing the aggregation rate of the carrier when reacted with an AFP control serum using a 2.0 μm anti-AFP antibody-sensitized latex reagent as a carrier. The aggregation rate when diluted by application is indicated by black diamonds, and the aggregation rate when diluted without application is indicated by white squares. The vertical axis indicates the aggregation rate, and the horizontal axis indicates the concentration of AFP. FIG. 6 is a graph showing the aggregation rate of a carrier when reacted with an AFP control serum using a 3 μm anti-AFP antibody-sensitized latex reagent as a carrier. The aggregation rate when diluted by application is indicated by black diamonds, and the aggregation rate when diluted without application is indicated by white squares. The vertical axis indicates the aggregation rate, and the horizontal axis indicates the concentration of AFP. FIG. 7 is a graph showing the carrier aggregation rate when a 4.5 μm anti-AFP antibody-sensitized latex reagent is used as a carrier and reacted with AFP control serum. The aggregation rate when diluted by application is indicated by black diamonds, and the aggregation rate when diluted without application is indicated by white squares. The vertical axis indicates the aggregation rate, and the horizontal axis indicates the concentration of AFP. FIG. 8 shows the case where AFP control serum and anti-AFP antibody-sensitized latex reagent were reacted, followed by sonication with addition of 0.25%, 2.5%, or 25% glutaraldehyde, and without addition of glutaraldehyde. 2 is a graph showing the agglomeration rate of the carrier when sonication is performed. The vertical axis represents the aggregation rate, and the horizontal axis represents the ultrasonic treatment time. FIG. 9 shows the case where AFP control serum and anti-AFP antibody-sensitized latex reagent were reacted, glutaraldehyde was added and incubated for 0 seconds, 15 seconds, 30 seconds or 60 seconds, and then sonicated, and It is a graph which shows the aggregation rate of the support | carrier when ultrasonication is performed without adding glutaraldehyde. The vertical axis represents the aggregation rate, and the horizontal axis represents the ultrasonic treatment time. FIG. 10 shows the aggregation rate of the carrier when reacted with AFP control serum using a 2.0 μm anti-AFP antibody-sensitized latex reagent as a carrier and then incubated with glutaraldehyde and when glutaraldehyde was not added. It is a graph. The vertical axis represents the aggregation rate, and the horizontal axis represents the ultrasonic treatment time. FIG. 11 shows the agglutination rate of the carrier when reacted with AFP control serum using 2.8 μm anti-AFP antibody-sensitized latex reagent as a carrier and then incubated with glutaraldehyde and without glutaraldehyde added. It is a graph. The vertical axis represents the aggregation rate, and the horizontal axis represents the ultrasonic treatment time. FIG. 12 shows the agglutination rate of the carrier when reacted with AFP control serum using 1.7 μm anti-AFP antibody-sensitized latex reagent as a carrier and then incubated with glutaraldehyde and when glutaraldehyde was not added. It is a graph. The vertical axis represents the aggregation rate, and the horizontal axis represents the ultrasonic treatment time.

Claims (22)

In the method for measuring an affinity substance comprising the following steps (1)-(3) or (1 ′)-(3 ′), the affinity substance and the binding partner are used before step (3) or (3 ′). Or a method comprising diluting the reaction solution by means for enhancing the binding between the agglutinating reagent and the binding partner.
(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 step (2), determining the level of the substance to be measured based on either or both of the level of aggregate formation and the level of carrier particles that did not form aggregates, or
(1 ′) a step of applying a voltage pulse to a reaction liquid in which a carrier particle bound with a binding partner having a binding activity to a measurement target affinity substance and a measurement target affinity substance are mixed with an agglutinating reagent component. The carrier particles are aggregated by an agglutinating reagent and the aggregation is inhibited by an affinity substance to be measured;
(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 step (2 ′), determining the level of the substance to be measured based on either or both of the level of aggregate formation and the level of carrier particles that did not form aggregates The method according to claim 1, wherein the step of diluting the reaction solution is a step of mixing the reaction solution and the diluting solution under a voltage pulse application condition. The method according to claim 2, wherein the voltage pulse is an alternating voltage. The method according to claim 3, wherein the alternating voltage has a frequency of 2 KHz to 20 MHz. The method according to claim 2, wherein the step of diluting the reaction solution includes the step of further diluting the carrier particles after the electric field is stopped after mixing the reaction solution and the dilute solution under the condition of applying the voltage pulse. The step of diluting the reaction solution is performed by adding a binding enhancer that enhances the binding between the affinity substance to be measured and the binding partner, or between the agglutination reagent and the binding partner to the reaction solution and then mixing with the reaction solution, or The method according to claim 2, which is a step of diluting the reaction solution with a diluent containing a reinforcing agent. The step of diluting the reaction solution may be performed by adding a binding enhancer that enhances the binding between the affinity substance to be measured and the binding partner, or the aggregation reagent and the binding partner, and then mixing the reaction solution with the dilution solution, Alternatively, the method according to claim 1, which is a step of diluting the reaction solution with a diluent containing the binding enhancer. The method according to claim 7, wherein the binding between the affinity substance to be measured and the binding partner, or the binding between the agglutination reagent and the binding partner is an immunological binding. The method according to claim 8, wherein the antigen is a protein antigen, and the binding enhancer is one or both compounds selected from glutaraldehyde and carbodiimide. The method according to claim 7, wherein the step of diluting the reaction solution is a step of mixing the reaction solution and the diluting solution under the application condition of the voltage pulse. The method according to claim 1, wherein the voltage pulse in the step (1) or (1 ') is an AC voltage pulse. The method according to claim 1, wherein the voltage pulse is applied a plurality of times in the step (1) or (1 '). 13. The method according to claim 12, comprising applying the next voltage pulse after dispersing the carrier particles after applying the voltage pulse in step (1) or (1 '). The method according to claim 12, wherein the multiple voltage pulses are voltage pulses in different directions. The method according to claim 1, wherein the average particle diameter of the carrier particles is 1 μm or more. The method according to claim 15, wherein the average particle diameter of the carrier particles is 1 μm to 20 μm. The method according to claim 1, wherein in the step (2) or (2 '), 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 17, wherein in step (2) or (2 '), the three-dimensional information of the agglomerates or carrier particles is physically measured. The method according to claim 18, wherein the method for physically measuring the three-dimensional information is any method selected from the group consisting of an electrical resistance method, a laser diffraction scattering method, and a three-dimensional image analysis method. . The carrier particles bound to the binding partner having the binding activity to the affinity substance to be measured, including the following elements, and the affinity substance to be measured are agglutinated by the affinity substance or the aggregation reagent of the carrier particles. Measuring device for measuring as an index.
a: To hold a carrier particle bound with a binding partner having binding activity with an affinity substance to be measured, and a reaction liquid containing a sample containing the affinity substance to be measured, or additionally a reaction liquid containing an agglutinating reagent Space,
b: means for applying a voltage pulse to the reaction solution,
c: means for diluting the reaction solution, and
d: Means for counting either or both of carrier particles and aggregates of carrier particles contained in the reaction solution 21. The apparatus according to claim 20, wherein the means for diluting the reaction liquid is a means for mixing the reaction liquid and the dilution liquid under the condition of applying the voltage pulse. 21. The means for diluting the reaction solution includes means for adding to the reaction solution a binding enhancer that enhances the binding between the affinity substance to be measured and the binding partner, or between the agglutination reagent and the binding partner. The device described in 1.

Images