JP2006343214A - Immunoreaction measuring method, and reagent for immunoreaction measurement used therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunoreaction measuring method capable of reducing decline of a bonding force of an antibody to an antigen by a chelating function, and improving easily a measured value, and a reagent for immunoreaction measurement used therefor. <P>SOLUTION: This immunoreaction measuring method wherein pH in a reaction system when an antigen-antibody reaction is generated is set to be acid, which is a method for measuring an antigen or an antibody which is a material to be measured included in a sample, includes a process A for mixing together benzoic acid or a benzoic acid salt, an antibody or an antigen which is a specific bonding material to be bonded specifically to the material to be measured, and the sample; and a process B for detecting an antigen-antibody complex generated from the antigen-antibody reaction between the material to be measured and the specific bonding material in the reaction system constituted in the process A and including the sample, the specific bonding material, and the benzoic acid or the benzoic acid salt. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an immune reaction measurement method capable of measuring an antigen or antibody which is a substance to be measured contained in a sample, and an immune reaction measurement reagent used therefor.

  In the medical field, in order to diagnose various diseases and examine the course of disease states, it is widely used to examine the content of proteins characteristic of each disease present in human body fluids.

  For the measurement of the content of these proteins, mainly an immune reaction measurement method using a highly specific antigen-antibody reaction is widely used. At present, various methods are also used for an immune reaction measurement method. Has been developed.

  Among them, measurement methods for detecting an aggregate complex produced by a reaction between an antigen and an antibody, such as a specific gravity method, a turbidimetric method, and a slide aggregation method, are well known. Since these are performed in a state where the antigen and the antibody are uniformly dispersed in the solution, they are collectively referred to as a homogeneous immune reaction measurement method.

  In these reactions, the reaction system generates turbidity depending on the amount of antigen and antibody due to the formation of aggregated complexes. The nephelometry and turbidimetry are methods of optically measuring this. The nephelometry is based on the amount of light scattered in the reaction system, and the nephelometry is based on the amount of transmitted light reduced by scattering in the reaction system. This is a measurement method based on the original. In general, the same reaction system can be used as the measurement target of both methods, and the target that can be measured by either method can be measured by the remaining one. The slide agglutination method is a method for judging the turbidity caused by the formation of the agglomerated complex by visual observation on a slide glass or the like, and the reaction system can be the same as the specific gravity method or the turbidimetric method.

  In the conventional homogeneous immune reaction measurement method, attempts have been made to use various additives in order to promote antigen-antibody reaction and to measure trace components with high sensitivity. As a well-known example, water-soluble polymers such as polyethylene glycol, dextran, polyvinylpyrrolidone, and polyvinyl chloride are mixed in the reaction system to promote the formation of aggregated complexes by antigen-antibody reaction, and the reaction time and measurement. A method for improving the sensitivity is mentioned. Among these water-soluble polymers, polyethylene glycol is known to be highly effective even at a relatively low concentration, and a method of using polyethylene glycol having an average molecular weight of 6,000 at a concentration of 2 to 6% by weight is widely used. In particular, a concentration of 4% by weight is considered to be highly effective with less non-specific turbidity.

  The promoting effect of the water-soluble polymer on the antigen-antibody reaction generally has a large molecular weight and tends to increase as the concentration increases.

  In the conventional homogeneous immune reaction measurement method, the higher the signal intensity depending on the degree of the antigen-antibody reaction, that is, the concentration of the antigen, the better the S / N ratio can be maintained, and the stable measurement can be performed. it can. However, when trying to obtain the above effect by further promoting the antigen-antibody reaction, it is necessary to add a higher concentration or higher molecular weight water-soluble polymer in the conventional addition of the water-soluble polymer, Since the viscosity of the solution in which the water-soluble polymer is dissolved increases, there is a problem that handling in the analysis operation becomes difficult.

  In homogeneous immune reaction measurement methods, a phenomenon called a zone phenomenon is generally known. The zone phenomenon is a phenomenon in which an aggregated complex is less likely to occur when either one is present in excess of the equivalent range where an antigen and an antibody form the largest aggregated complex. Because of this phenomenon, it becomes difficult to distinguish between the measurement results when the antigen is at a high concentration and at a low concentration. Therefore, correct quantification and determination depending on the antigen concentration cannot be performed, and in order to avoid this, there is a problem that the measurement concentration range is limited.

Therefore, a method for enhancing the antigen-antibody reaction using a polycarboxylic acid such as dicarboxylic acid or tricarboxylic acid under acidic conditions is also disclosed (for example, see Patent Documents 1, 2, and 3).
International Publication No. 03/010541 Pamphlet International Publication No. 03/056333 Pamphlet International Publication No. 2004/053489 Pamphlet

  According to the method using a polyvalent carboxylic acid described in Patent Document 1, 2, or 3, the antigen-antibody reaction is enhanced, and thus the measurement value can be easily improved. Furthermore, the zone phenomenon occurring in the antigen excess region can be alleviated.

However, in the method using the above polyvalent carboxylic acid, the polyvalent carboxylic acid has a chelating action and efficiently deprives divalent and trivalent metal ions such as Ca ²⁺ and Fe ³⁺ present in the reaction system. There is a nature. For this reason, if the antigen retains a metal ion in its molecular structure, the metal ion is deprived. If the antigen is a substance that retains a metal ion in the molecular structure and causes a change in the molecular structure due to the desorption of the metal ion, there is a problem that the binding force of the antibody to the antigen may be reduced. It was.

  In view of the above-described conventional problems, the present invention provides an immunoreaction measurement method capable of reducing a decrease in binding force of an antibody to an antigen due to a chelating action and easily improving a measurement value, and an immunoreaction measurement reagent used therefor The purpose is to provide.

  In order to solve the above-mentioned conventional problems, the immunoreaction measurement method of the present invention is a method for measuring an antigen or antibody which is a substance to be measured contained in a sample, comprising benzoic acid or a salt of benzoic acid and , The step A of mixing the sample with an antibody or antigen that is a specific binding substance that specifically binds to the substance to be measured, and the sample, the specific binding substance and the step constituted by the step A In a reaction system containing benzoic acid or a salt of benzoic acid, the method includes a step B of detecting an antigen-antibody complex generated by an antigen-antibody reaction between the substance to be measured and the specific binding substance, and the antigen-antibody reaction occurs When the pH of the reaction system is set to acidic.

  In addition, the reagent for measuring immune reaction according to the present invention is a reagent for measuring an antigen or antibody that is a substance to be measured contained in a sample, and is used for benzoic acid or a salt of benzoic acid and a substance to be measured. The antibody or antigen, which is a specific binding substance that specifically binds, is prepared so that the pH when the antigen-antibody reaction between the substance to be measured and the specific binding substance occurs is acidic.

  According to the immune reaction measurement method of the present invention and the immune reaction measurement reagent used therefor, it is possible to reduce a decrease in the binding force of an antibody to an antigen due to a chelating action, and to easily improve a measurement value.

  The present invention relates to an immune reaction measurement method capable of reducing a decrease in binding ability of an antibody to an antigen due to a chelating action and easily improving a measurement value, and an immune reaction measurement reagent used therefor. In addition, the present invention relates to an immune reaction measurement method capable of alleviating a zone phenomenon occurring in an antigen excess region and an immune reaction measurement reagent used therefor.

  The present inventors added a benzoic acid or a salt of benzoic acid to the reaction system during the antigen-antibody reaction to keep the pH of the reaction system acidic, thereby reducing the decrease in the binding ability of the antibody to the antigen due to chelation. In addition, the present inventors have found that the measured value of the immune response due to the antigen-antibody binding can be improved, and that the zone phenomenon occurring in the antigen excess region can be alleviated.

  As shown in FIG. 1, an immune reaction measurement method according to an embodiment of the present invention is a method for measuring an antigen or antibody that is a substance to be measured contained in a sample, which comprises benzoic acid or a salt of benzoic acid and , The step A of mixing the sample with an antibody or antigen that is a specific binding substance that specifically binds to the substance to be measured, and the sample, the specific binding substance and the step constituted by the step A In a reaction system containing benzoic acid or a salt of benzoic acid, the method includes a step B of detecting an antigen-antibody complex generated by an antigen-antibody reaction between the substance to be measured and the specific binding substance, and the antigen-antibody reaction occurs When the pH of the reaction system is set to acidic. Here, the reaction system may contain both benzoic acid or a salt of benzoic acid.

  Moreover, it is preferable that buffer capacity is given by the benzoic acid or salt of benzoic acid contained in the reaction system, and the pH of the reaction system is set to be acidic. In this way, it is not necessary to add another buffer in order to set the pH of the reaction system to be acidic, the decrease in the binding power of the antibody to the antigen due to chelation is reduced, and the measurement of the above immune reaction The effect of improving the value can be efficiently exhibited. In order to provide buffering ability with benzoic acid or a benzoic acid salt contained in the reaction system, the concentration of the benzoic acid or the benzoic acid salt is preferably 0.01 M or more.

  In step A, a buffer may be further mixed.

  In addition, the reagent for measuring an immune reaction in one embodiment of the present invention is a reagent for measuring an antigen or antibody that is a substance to be measured contained in a sample, and includes benzoic acid or a salt of benzoic acid, and a target. It contains an antibody or antigen that is a specific binding substance that specifically binds to the measurement substance, and is prepared so that the pH when the antigen-antibody reaction between the measured substance and the specific binding substance occurs is acidic. . Here, both benzoic acid or a salt of benzoic acid may be contained. Further, it is preferable that the buffering ability is given by benzoic acid or a salt of benzoic acid, and the pH is adjusted to be acidic when the antigen-antibody reaction between the substance to be measured and the specific binding substance occurs. Further, it may contain a buffer.

  As the buffer used in the immunoreaction measurement method and the immunoreaction measurement reagent of the present invention, those known in the art can be used, for example, phosphorous composed of monosodium dihydrogen phosphate, disodium hydrogen phosphate and the like. Examples include acid-based buffers, sodium cacodylate, and 2- (N-morpholino) ethanesulfonic acid. In this case, the amount of the buffer to be included is the type of the buffer used, the amount of the sample (analyte) containing the object to be measured, and the antigen to be measured with respect to the reaction system or the antibody or antigen supply method for the antibody. Depending on the above, adjustments may be made so that the effects of the present invention can be obtained.

  In the immune reaction measurement method of the present invention, the pH of the reaction system is preferably set to 4.5. At the above pH, the effect of improving the measured value of the immune reaction by benzoic acid or a salt of benzoic acid is enhanced, and the effect of alleviating the zone phenomenon occurring in the antigen excess region is increased.

  The reagent for immune reaction of the present invention is preferably prepared so that the pH when the antigen-antibody reaction occurs is set to 4.5 for the above reasons.

  Examples of the benzoic acid or benzoic acid salt used in the immune reaction measurement method and immune reaction reagent of the present invention include benzoic acid, benzoic anhydride, ammonium benzoate, potassium benzoate, sodium benzoate, lithium benzoate and the like. Can be mentioned. These are all commercially available and can be used alone or in combination.

  In addition, the reaction system and the reagent for immune reaction of the method for measuring immune reaction of the present invention are added with other arbitrary components known in the art within a range in which the effect of the present invention can be obtained according to its use. Can be done. For example, when applied to a homogeneous immune reaction measurement method such as a specific gravity method, a turbidimetric method, a slide agglutination method, etc., polyethylene glycol is added to the reaction system and immune reaction reagent of the immune reaction measurement method of the present invention. obtain. The content is preferably 2 to 6% by weight with respect to the reaction system in the immune reaction measurement method of the present invention from the viewpoint of low non-specific aggregation and a high effect of improving measurement sensitivity. More preferably, the concentration is 4% by weight. Similarly, in the reagent for immune reaction of the present invention, the concentration at which an antigen-antibody reaction occurs is preferably 2 to 6% by weight, and more preferably 4% by weight.

  In order to reduce non-specific turbidity due to self-aggregation of antigen or antibody, the reaction system and immune reaction reagent of the immune reaction measurement method of the present invention include Tween 20, octyl glucoside, sodium lauryl sulfate (SDS), sucrose mono Surfactants such as laurate and CHAPS can be added. The content is preferably 0.3% or less, preferably 0.1% or less, based on the reaction system in the immune reaction measurement method of the present invention, from the viewpoint that there is little inhibition of the antigen-antibody reaction. Is more preferable. Similarly, in the reagent for immune reaction of the present invention, the content is preferably 0.3% or less, more preferably 0.1% or less, when the antigen-antibody reaction occurs.

  The measurement system to which the immune reaction measurement method and the immune reaction measurement reagent of the present invention are applied is not particularly limited, and in particular, a uniform method such as a ratio method, a turbidimetric method, a slide agglutination method having a zone phenomenon occurring in an antigen excess region. It is preferable because a higher effect can be expected with respect to the measurement system. In particular, when applied to the Hiei method and the turbidimetric method, in which measurement by an automatic measuring instrument is widespread, it is particularly preferable because it can reduce or simplify the process required to determine the zone phenomenon occurring in the antigen excess region.

  In the immune reaction measurement method of the present invention, the antigen-antibody complex is preferably an aggregated complex. In Step B, it is preferable to detect the aggregated complex by measuring an optical parameter caused by the aggregated complex, and it is more preferable that the optical parameter is scattered light intensity or transmitted light intensity. .

  In the immune reaction measurement method and immune reaction measurement reagent of the present invention, the substance to be measured is preferably an antigen having a structure that holds a metal ion therein. An antigen that retains a metal ion in its molecular structure may change its molecular structure when the metal ion is desorbed. Benzoic acid is a monovalent carboxylic acid, and its chelating action is weaker than that of polyvalent carboxylic acid, which can alleviate the elimination of metal ions from the antigen and alleviate the structural change of the antigen. It is possible to mitigate a decrease in the binding force of the antibody to the antigen. Thereby, even if the substance to be measured is an antigen having a structure that holds a metal ion therein, the reaction enhancement effect can be efficiently exhibited.

In particular, C-reactive protein (hereinafter abbreviated as CRP) is a protein that undergoes a structural change depending on the presence or absence of Ca ^{2+ in} the molecule, and has a high measurement frequency as an infectious disease marker. Preferably there is.

  The antigen or antibody that is a substance to be measured of the immune reaction measurement method and the reagent for measuring immune reaction of the present invention is not particularly limited, and may be any substance that can be generally measured using an antigen-antibody reaction, such as a protein, Examples include nucleic acids, lipids, bacteria, viruses and haptens. Of these, proteins are preferred because they are the main measurement targets in clinical tests using antigen-antibody reactions. Examples of proteins include hormones such as LH (luteinizing hormone), FSH (follicle stimulating hormone), hCG (chorionic gonadotropin), various immunoglobulin classes and subclasses, complement components, markers of various infectious diseases, CRP, Examples include albumin, rheumatoid factor, blood group antigen and the like. Among these, it is preferable that the substance to be measured is human albumin or CRP.

  The antibody used in the immunoreaction measurement method and immune reaction measurement reagent of the present invention is not particularly limited, and any antibody of IgG, IgM, IgE, IgA, or IgD may be used as long as it specifically binds to the antigen. May be. Of these, IgG antibodies are preferred because they have few non-specific reactions, are relatively commercially available, and are easily available. The species of animal from which the antibody is derived is also not particularly limited, but antibodies derived from rabbits, goats, and mice are relatively easy to obtain and are preferred because there are many examples of use.

  The immune reaction measurement method of the present invention can be typically performed as follows. Benzoic acid or a salt of benzoic acid is added to a buffer containing a buffering agent so that the pH of the reaction system is kept acidic, preferably, the pH is kept at 4.5. Benzoic acid or a salt of benzoic acid may also serve as a buffer. A reaction system was formed by mixing the buffer and the antigen or antibody that is the substance to be measured and the antibody or antigen that is a specific binding substance that specifically binds to the substance to be measured. Measure immune response.

  The method of adding benzoic acid or a salt of benzoic acid, the method of adding a buffer to keep the pH of the reaction system acidic, and the method of adjusting the pH of the reaction system are not limited to the above. In order to satisfy the above-mentioned requirements, benzoic acid or a salt of benzoic acid and a buffer may be present in advance in the antibody or antigen-containing solution against the antigen.

  The reagent for immune reaction of the present invention can be typically produced as follows.

  In the case where an antibody or antigen against an antigen or antibody to be measured and benzoic acid or a salt of benzoic acid are prepared separately, it is as follows. An antibody or antigen-containing solution against an antigen or antibody to be measured may have any composition as long as the effect of benzoic acid or a salt of benzoic acid is obtained. The solution containing benzoic acid or a salt of benzoic acid is buffered so that the pH is preferably maintained at 4.5 so that the buffering capacity necessary for keeping the pH during the antigen-antibody reaction acidic may be provided. It is prepared by preparing an agent and benzoic acid or a salt of benzoic acid and dissolving them in pure water. If the said requirements are satisfy | filled, a buffer, a benzoic acid, or a salt of benzoic acid may exist in a separate solution, respectively. Further, benzoic acid or a salt of benzoic acid may also serve as a buffer.

  In addition, benzoic acid or a salt of benzoic acid may be present in the antibody or antigen-containing solution against the antigen to be measured or the antibody, in which case the requirements shown above should be satisfied. A solution containing benzoic acid or a salt of benzoic acid thus prepared, and benzoic acid by substituting the low molecular component by dialysis or gel filtration of the antigen to be measured or a solution containing an antibody against the antibody or antigen. Alternatively, a salt of benzoic acid may be included.

  As described above, according to the immune reaction measurement method and immune reaction measurement reagent of the present invention, benzoic acid or a salt of benzoic acid is present in the reaction system of the immune reaction, and the pH of the reaction system is set to be acidic. Thus, it is possible to reduce a decrease in the binding force of the antibody to the antigen due to the chelating action, and to improve the measured value of the immune reaction due to the binding of the antigen antibody. This can also alleviate the zone phenomenon that occurs in the antigen excess region. In the conventional method of adding a water-soluble polymer, in order to improve the measurement value and maintain a good S / N ratio in the measurement of antigen-antibody reaction, a water solution having a higher concentration or higher molecular weight can be used. There is a problem that it is necessary to add a polymer, increase the viscosity of the solution, and it becomes difficult to handle the analytical operation. However, benzoic acid or a salt of benzoic acid used in the present invention is a low molecular weight substance. Therefore, the viscosity of the solution is not increased and the handling in the analysis operation is facilitated.

  In addition, by mitigating the zone phenomenon that occurs in the antigen excess region and reducing the drop in the measured value at a high concentration of the substance to be measured, it is possible to widen the region where the measured value is judged as positive, The concentration range can be expanded.

  In addition, when an antigen undergoes a structural change due to a chelating action, benzoic acid is a monovalent carboxylic acid, and the chelating action is weaker than that of a polyvalent carboxylic acid, and the elimination of metal ions from the antigen is alleviated. Therefore, the decrease in the binding force of the antibody to the antigen can be mitigated. Thereby, the reaction enhancement effect can be exhibited efficiently.

  EXAMPLES Hereinafter, although this invention is demonstrated using an Example, this invention is not limited only to these.

Example 1
Hereinafter, a method for constructing a reagent when human albumin is used as a substance to be measured will be described. In this example, a method for preparing a reagent comprising an antibody solution and a buffer containing benzoic acid or a salt of benzoic acid, which can be used for the measurement by the slide agglutination method, the turbidimetric method and the specific gravity method will be described.

  For the preparation of the buffer solution shown below, pure water filtered with Milli-Q SP TOC (manufactured by Millipore) was used. In addition, reagents such as salts and buffering agents not specifically described below are obtained from Wako Pure Chemical Industries, Ltd., polyethylene glycol 6,000 uses a first grade reagent, and others use a special grade reagent. did.

First, an antibody solution was prepared. Rabbit anti-human albumin polyclonal antibody was purified using protein A column chromatography from antiserum collected from rabbits immunized with human albumin (manufactured by Wako Pure Chemical Industries). The protein A-immobilized gel packed in the column was Amersham Pharmacia. The equilibration buffer used for purification was 1.5M glycine, 3.0M NaCl, pH 8.9, and the elution buffer was 0.1M citric acid, pH 4.0. I used something. Purification was performed as follows. Flow the equilibration buffer 5 times the gel volume packed in the column to equilibrate the column, then double the volume of antisera containing 10-20% of the total binding capacity of the antibody with the equilibration buffer. The antibody in the serum was bound to protein A by diluting and flowing through the column. Subsequently, the equilibration buffer was allowed to flow until no serum component that did not adsorb to protein A came out of the column, and the column was washed. Subsequently, an elution buffer was passed through the column to elute the antibody bound to protein A. The eluted antibody fraction was put in a dialysis tube having a molecular weight cut off of 10,000, and about 100 times volume of PBS buffer solution containing 0.04 wt% NaN ₃ (8 g / l NaCl, 0.2 g / l KCl, 1. The buffer component was replaced by dialyzing several times with 15 g / l Na ₂ HPO ₄ · 12H ₂ O, 0.2 g / l KH ₂ PO ₄ , pH 7.4). Subsequently, the antibody concentration was estimated by measuring the absorbance at 280 nm and adjusted with the same buffer as that used in dialysis to give an antibody concentration of 2.4 mg / ml, which was used as an antibody solution. The antibody concentration is not particularly limited to this. Although the prepared antibody solution can be stored at room temperature, it is preferably stored at a lower temperature and more preferably stored at 4 ° C. from the viewpoint of preventing antibody denaturation.

  A buffer containing benzoic acid or a salt of benzoic acid was prepared as follows. As the benzoic acid or benzoic acid salt, sodium benzoate was used, and a buffer solution of each substance was formed.

  The buffer solution using sodium benzoate was constructed as follows. At the final concentration, 0.05M sodium benzoate and 4% polyethylene glycol 6,000 were weighed and dissolved in about 90% pure water of the preparation target volume. An aqueous HCl solution was added thereto to adjust the pH to 4.5, and the target volume was adjusted with pure water. The prepared buffer was stored at room temperature.

  By combining at least one of the buffer solution containing benzoic acid or benzoic acid salt configured as described above with an antibody solution, an immune reaction reagent can be configured.

  In order to form a reaction system, the reagent used in this example comprises a reagent containing an antibody solution, a buffer containing benzoic acid or a salt of benzoic acid, and a sample (analyte) containing an antigen. Use by mixing. The mixing method may be any method. The mixing ratio can be determined according to the measurement range of the required antigen concentration. By measuring the immune reaction caused by the binding of the antigen and the antibody generated in the reaction system formed by mixing, the antigen concentration in the specimen can be known.

  Although not shown in the present embodiment, the antibody may be immobilized on a fine particle carrier such as latex, gold colloid, or magnetic fine particle, or the antibody, the dye, the fluorescent substance, the luminescent substance, etc. may be labeled. .

  The buffer component and pH of the antibody solution are not limited to the above composition and pH. For example, in the case of constituting a one-component reagent, benzoic acid or a salt of benzoic acid is included in the antibody solution, and the reaction system. In order to maintain the pH of the solution acidic, dialysis may be performed with an acidic buffer containing benzoic acid or a salt of benzoic acid.

  In this example, sodium benzoate was used to prepare a buffer solution containing benzoic acid or a salt of benzoic acid. However, other benzoic acid or a salt of benzoic acid may be used, for example, benzoic acid or benzoic acid. Any of acid anhydride, ammonium benzoate, potassium benzoate, lithium benzoate and the like may be used. These can also be used in combination. In this case, the pH adjustment is indicated by HCl or the like when the pH when dissolved in pure water is more alkaline than the pH intended for adjustment, and above when the pH is acidic. It may be performed using a hydroxide or the like, and may be performed by adjusting the mixing ratio of the benzoic acid or benzoic acid salt exemplified above.

  Further, in this example, the preparation method in the case where the main buffer capacity of a buffer solution containing benzoic acid or a salt of benzoic acid is imparted by benzoic acid or a salt of benzoic acid is shown, but the concentration to be added to the reagent is It is not particularly limited. Further, other buffering agents may be used to impart the main buffering ability to the reagent, or benzoic acid or a salt of benzoic acid may be coordinated with another buffering agent to impart the buffering ability.

(Example 2)
In this example, the effect of an acidic reaction system containing benzoic acid or a salt of benzoic acid on an antigen-antibody reaction is shown in comparison with a neutral reaction system generally used in an immune reaction measurement method. The comparison was performed by measuring human albumin by an immuno-ratio method. As the reagent for constituting an acidic reaction system containing benzoic acid or a salt of benzoic acid, the reagent constituted in Example 1 was used.

  As Comparative Example 1, 3- (N-morpholino) propanesulfonic acid (manufactured by Dojin, hereinafter abbreviated as MOPS) was used as a buffer solution for forming a neutral reaction system, and 0.05M MOPs was used. A composition having a composition of 4% by weight polyethylene glycol 6,000 and pH 7.4 was prepared.

Human albumin (manufactured by Wako Pure Chemical Industries, Ltd.) used as an antigen was added to a PBS buffer containing 0.04% by weight of NaN ₃ at concentrations of 0, 5, 10, 20, 30, 50, 100, and 300 mg / dl. It dissolved so that it might become. The antibody and sample antigen solution were stored at 4 ° C. until use, and each buffer was stored at room temperature.

A self-made measuring device was used for the measurement. The configuration of the self-made measuring device was as follows. The light source is a 15 mW semiconductor laser pointer with a wavelength of 680 nm modulated at 270 Hz (manufactured by Kiko Giken, model number MLXS-D-12-680-35), and the detector is a silicon photodiode for visible infrared precision photometry (manufactured by Hamamatsu Photonics). Model No. S2387-66R). The cell was formed in a cubic shape with a capacity of about 200 μl by laminating optical glass plates having a thickness of 0.1 cm. In each arrangement, the cell is arranged at 0.5 cm from the light source so that one surface thereof is perpendicular to the light source, and the detector is 5.5 cm away from the cell in a direction forming an angle of 90 ° with the light source. A light shielding cylinder was provided between the detector and the cell so that stray light was not incident on the detector. The current signal depending on the amount of light detected by the detector is amplified to a voltage signal of 100 times through an amplifier circuit using a current-voltage conversion circuit (10 ⁶ V / A) and an operational amplifier, and then a lock-in amplifier (NF circuit design block) Phase sensitive detection through a model No. 5610B), which can be incorporated into a computer by GPIB control.

  Measurement of each concentration of human albumin solution was as follows. The mixing ratio of the reaction system was 178 μl for the buffer, 9 μl for the human albumin solution, and 9 μl for the antibody solution.

  The buffer, human albumin solution, and antibody solution were added to the cell in the above volume and mixed with stirring to cause an antigen-antibody reaction. The measurement of scattered light started 10 seconds before adding the antibody solution and continued for 300 seconds at 0.5 second intervals. The measured value was obtained as a voltage value. The measured value after 300 seconds was taken as the measured value in each concentration of human albumin solution. The measurement was performed at room temperature.

  FIG. 2 is a graph showing the results of measuring the human albumin solution of each concentration for each buffer solution. From FIG. 2, it is clearly higher in the case where the measurement is performed using the buffer solution containing sodium benzoate which is the present example than in the case where the antigen-antibody reaction is measured using the buffer solution containing the mops which is Comparative Example 1. The measured value is shown. Moreover, when the buffer solution containing sodium benzoate was used, the decrease in the measured value due to the zone phenomenon occurring in the antigen excess region was suppressed as compared with the case of using the buffer solution containing the mops.

  From the above results, it was confirmed that the measured value of the antigen-antibody reaction could be improved by the immune reaction measurement method of the present invention. It was also confirmed that this could alleviate the zone phenomenon that occurs in the antigen excess region.

  It was also confirmed that the measured value of the antigen-antibody reaction can be improved by the immune reaction reagent of the present invention. It was also confirmed that this could alleviate the zone phenomenon that occurs in the antigen excess region.

(Example 3)
In this example, the effect of an acidic reaction system containing benzoic acid or a salt of benzoic acid on an antigen-antibody reaction is shown in comparison with an acidic reaction system using a polyvalent carboxylic acid or a salt of a polyvalent carboxylic acid. The comparison was performed by measuring human CRP by immunocomparison method. Since human CRP has a structure consisting of five subunits having the same structure, it is a substance having a plurality of binding sites for one type of antibody. For this reason, immunoturbidimetry and specific measurement can be performed using a monoclonal antibody.

For the measurement, anti-human CRP monoclonal antibody produced from a hybridoma cell having the accession number FERM BP-6621 of the Biotechnology Institute of Industrial Technology, Ministry of International Trade and Industry (currently the National Institute of Advanced Industrial Science and Technology (AIST)) Was used. This antibody is sensitive to structural changes caused by the presence or absence of Ca ²⁺ retention in human CRP molecules, and binds when Ca ²⁺ is present in the molecule and does not bind when Ca ²⁺ is not present in the molecule. Has properties. The antibody solution was prepared from mouse ascites by the same procedure as in Example 1.

  The buffer solution containing benzoic acid or a salt of benzoic acid was the one configured in Example 1.

  Moreover, as Comparative Example 2, phthalic acid was used as the polyvalent carboxylic acid, and a buffer solution containing this was prepared as follows. In the final concentration, potassium hydrogen phthalate was weighed to 0.05M and polyethylene glycol 6,000 to 4% by weight, and dissolved in about 90% pure water of the preparation target volume. An aqueous NaOH solution was added thereto to adjust the pH to 4.5, and the target volume was adjusted with pure water. The prepared buffer was stored at room temperature.

The CRP solution of each concentration used for the measurement was prepared by diluting purified human CRP (manufactured by Chemicon International) with a PBS buffer containing 0.04 wt% NaN ₃ . Human CRP solutions with concentrations of 0, 5, 10, 20, 30, 50, and 100 mg / dl were prepared. The antibody and sample antigen solution were stored at 4 ° C. until use, and each buffer was stored at room temperature.

  The measurement apparatus of Example 2 was used for the measurement. Measurement of the CRP solution at each concentration was performed as follows. The mixing ratio of the reaction system was 178 μl of buffer solution, 9 μl of human CRP solution, and 9 μl of antibody solution.

  A buffer, an antibody solution, and a human CRP solution were added to the cell in the above volumes and mixed with stirring to cause an antigen-antibody reaction. The measurement of scattered light started 10 seconds before adding the antibody solution and continued for 300 seconds at 0.5 second intervals. The measured value was obtained as a voltage value. The measured value after 300 seconds was taken as the measured value for each concentration of human CRP solution. The measurement was performed at room temperature. The results are shown in FIG.

FIG. 3 clearly shows a higher measured value in the case of measurement using the buffer solution containing benzoic acid in this example than in the case of using the buffer solution containing phthalic acid in Comparative Example 2. It was. This is because when a buffer solution containing phthalic acid is used, Ca ²⁺ in the human CRP molecule is released due to the chelating action of phthalic acid, resulting in a structural change in human CRP and binding of anti-human CRP monoclonal antibody to human CRP. On the other hand, when a buffer containing benzoic acid is used, benzoic acid has a lower chelating action than phthalic acid, and Ca ²⁺ in the human CRP molecule is released when a buffer solution containing benzoic acid is used. This is because the change can be suppressed and the decrease in the binding force of the antibody can be reduced.

  From this result, it can be confirmed that according to the immune reaction measurement method of the present invention and the reagent for measuring immune reaction used therefor, it is possible to reduce the decrease in the binding power of the antibody to the antigen due to the chelating action and to easily improve the measurement value. It was.

  The immune reaction measurement method and immune reaction measurement reagent according to the present invention are useful for measuring an antigen in a sample, particularly for measuring an antigen in a biological sample such as a human body fluid.

The flowchart showing each process of the immune reaction measuring method of one Embodiment of this invention. The graph which shows the result of having performed the human albumin measurement by the immuno-ratio method about the immune-reaction measuring method using the reagent for immune-reaction measuring in one Example of this invention, and a comparative example The graph which shows the result of having performed the human CRP measurement by the immuno-ratio method about the immune reaction measuring method using the reagent for immune reaction measurement in the other Example of this invention, and another comparative example

Claims (14)

A method for measuring an antigen or antibody, which is a substance to be measured, contained in a sample,
A step of mixing benzoic acid or a salt of benzoic acid, an antibody or antigen that is a specific binding substance that specifically binds to the substance to be measured, and the sample,
And an antigen produced by an antigen-antibody reaction between the substance to be measured and the specific binding substance in the reaction system comprising the sample, the specific binding substance, and the benzoic acid or benzoic acid salt, which is constituted by the step A. Comprising detecting the antibody complex B.
And an immune reaction measurement method in which the pH of the reaction system when the antigen-antibody reaction occurs is set to be acidic. The method of measuring an immune reaction according to claim 1, wherein a buffer is further mixed in the step A. The method for measuring an immune reaction according to claim 1 or 2, wherein the pH of the reaction system is set to 4.5. The immune reaction measuring method according to any one of claims 1 to 3, wherein the reaction system contains 2 to 6% by weight of polyethylene glycol. The method for measuring an immune reaction according to claim 1, wherein the antigen-antibody complex is an aggregated complex. The method for measuring an immune reaction according to claim 5, wherein in step B, the aggregated complex is detected by measuring an optical parameter resulting from the aggregated complex. The method for measuring an immune reaction according to any one of claims 1 to 6, wherein the substance to be measured is an antigen having a structure holding a metal ion therein. The immune reaction measuring method according to any one of claims 1 to 7, wherein the substance to be measured is a human C-reactive protein. A reagent for measuring an antigen or antibody that is a substance to be measured contained in a sample,
Benzoic acid or a salt of benzoic acid,
And an antibody or antigen that is a specific binding substance that specifically binds to the substance to be measured,
A reagent for measuring an immune reaction prepared so that the pH when an antigen-antibody reaction between the substance to be measured and the specific binding substance occurs is acidic. The reagent for measuring immune reaction according to claim 9, further comprising a buffer. The reagent for measuring an immune reaction according to claim 9 or 10, which is prepared so that a pH at which an antigen-antibody reaction occurs is set to 4.5. The reagent for measuring an immune reaction according to any one of claims 9 to 11, further comprising polyethylene glycol, wherein the concentration of the polyethylene glycol when an antigen-antibody reaction occurs is 2 to 6% by weight. The reagent for measuring an immune reaction according to any one of claims 9 to 12, wherein the substance to be measured is an antigen having a structure holding a metal ion therein. The reagent for measuring immune reaction according to any one of claims 9 to 13, wherein the substance to be measured is a human C-reactive protein.

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