Abstract:
The present invention relates to a method for detecting an antigen with use of an antibody and an enzyme. Specifically, the present invention provides a method for detecting an antigen without use of a labeled-antibody. the method comprises immersing particles in a first buffer solution which is predicted to contain the antigen; wherein an antibody and a multi-copper oxidase CueO are immobilized on each surface of the particles, and the antibody reacts specifically with the antigen. The method further comprises the following steps recovering the obtained particles; mixing the particles recovered, an oxidation-reduction indicator (reductant), and a second buffer solution so as to prepare a reaction solution; measuring an activity degree of the multi-copper oxidase CueO contained in the reaction solution; determining that the first buffer solution contains the antigen based on the above activity degree.

Description:
RELATED APPLICATIONS 
       [0001]    This is a continuation of International Application No. PCT/JP2011/007240, with an international filing date of Dec. 22, 2011, which claims priority of Japanese Patent Application No. 2011-125972, filed on Jun. 6, 2011, the contents of which are hereby incorporated by reference. 
     
    
     TECHNICAL FIELD 
       [0002]    The present invention relates to a method for detecting an antigen with use of an antibody and an enzyme. 
       BACKGROUND ART 
       [0003]    Japanese Patent Publication No. 4271086 (hereafter, Patent Literature 1; this literature corresponds to US Pre-Grant Patent Application Publication No. 2005/0282237) discloses an enzyme immunoassay method.  FIG. 2  shows a sandwich method, which is included in the enzyme immunoassay method. 
         [0004]    As shown in  FIG. 2 , a support  107  has an antibody  108  on its surface. The sample containing an antigen  109  is supplied to the surface of the support  107  to cause the antigen  109  to be bound specifically to the antibody  108 . Subsequently, the sample containing the unreacted antigen  109  was removed from the support by washing. 
         [0005]    Next, a labeled-antibody  111  comprising an enzyme  110  which detects the antigen  109  is supplied to the surface of the support  107  to form the complex composed of the antibody  108 , the antigen  109 , and the labeled-antibody  111 . Subsequently, the sample containing the unreacted labeled-antibody  111  was removed by washing. 
         [0006]    Finally, a substrate  112  of the enzyme  110  is supplied to the surface of the support  107 . The enzyme  110  reacts with the substrate  112  metabolically to form a product  113 . The luminescence degree or light absorption degree of the product  113  is measured so as to detect the antigen  109  indirectly. 
       CITATION LIST 
     Patent Literature 
       [0000]    
       
         [Patent Literature 1] Japanese Patent Publication No. 4271086. 
       
     
       SUMMARY OF INVENTION 
     Technical Problem 
       [0008]    However, the sandwich method requires not only the antigen  108  but also the labeled-antibody  111  comprising the enzyme  110 . It is necessary that the labeled-antibody  111  is supplied after the specific reaction generated between the antibody  108  and the antigen  109 . Furthermore, the unreacted labeled-antibody  111  is required to be removed. 
         [0009]    One of the purposes of the present invention is to provide a method for detecting an antigen without use of a labeled-antibody. 
       Solution to Problem 
       [0010]    The present invention is directed to a method for detecting an antigen, the method comprises steps of: 
         [0011]    (a) immersing particles in a first buffer solution which is predicted to contain the antigen; wherein 
         [0012]    an antibody and a multi-copper oxidase CueO are immobilized on each surface of the particles, and 
         [0013]    the antibody reacts specifically with the antigen; 
         [0014]    (b) recovering the particles obtained in the step (a); 
         [0015]    (c) mixing the particles recovered in the step (b), an oxidation-reduction indicator (reductant), and a second buffer solution so as to prepare a reaction solution; wherein 
         [0016]    the second buffer solution contains a substrate of the multi-copper oxidase CueO; and 
         [0017]    the second buffer solution has an ion strength falling within the range of not less than 0.3 mM and not more than 1.0 mM; 
         [0018]    (d) measuring an activity degree of the multi-copper oxidase CueO contained in the reaction solution obtained in the step (c) by an absorbance measurement technique; and 
         [0019]    (e) determining that the first buffer solution contains the antigen if the following formula is satisfied: 
         [0000]      the activity degree measured in the step( d )≧1.4×(blank value)
 
         [0020]    wherein the blank value represents the activity degree of the multi-copper oxidase CueO measured by an absorbance measurement technique in which the antigen is not used, however, the particles and the second buffer solution are used. 
       Advantageous Effects of Invention 
       [0021]    In an aspect, the present invention provides a method for detecting an antigen without use of a labeled-antibody. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0022]      FIG. 1  shows a reaction flowchart of the method for detecting an antigen according to the present invention. 
           [0023]      FIG. 2  shows a reaction flowchart of the conventional enzyme immunoassay method (Sandwich method). 
           [0024]      FIG. 3  shows an example of a particle support where a CueO and an antibody are immobilized. 
           [0025]      FIG. 4  shows the result of the absorbance measurement conducted in the example 1. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0026]    An illustrative embodiment of the present invention is described below with reference to the drawings. 
         [0027]      FIG. 1  shows a reaction flowchart of the method for detecting an antigen according to the present invention. 
         [0028]    (Step (a)) 
         [0029]    In the step (a), particles  101  are immersed in a first buffer solution which is predicted to contain an antigen  104 . 
         [0030]    As shown in  FIG. 3 , each particle  101  comprises a multi-copper oxidase  102  and an antibody  103  on its surface. Hereinafter, the multi-copper oxidase  102  is referred to as “CueO”. 
         [0031]    In light of increasing amounts of the immobilized antibody  103  and CueO  102 , it is preferable that each particle  101  is made of a particle having a diameter of 10-20 nanometers. 
         [0032]    As shown in  FIGS. 1 and 3 , the antibody  103  and CueO  102  are immobilized on the surface of the particles  101 . The antibody  103  reacts specifically with the antigen  104 . 
         [0033]    In light of easily immobilizing the antibody  103  and the CueO  102 , an example of the material of the particles  101  is noble metal. An example of the noble metal is gold, silver, or platinum. Gold is preferred. 
         [0034]    The antibody  103  may be a polyclonal antibody or a monoclonal antibody. A Fab fragment or a F(ab′)2 fragment of an antibody may be used as the antibody  103 , which is created through artificially removing its Fc or a portion thereof from the antibody. 
         [0035]    The CueO  102  is one enzyme which catalyzes an oxidation-reduction reaction. As shown in  FIG. 1 , the CueO  102  performs four-electron reduction of the substrate  105  composed of oxygen molecule and catalyzes the reaction in which water is generated as a product  106 . The active center of the CueO  102  is composed of four copper ions each having differential property called type 1, type 2, type 3a, and type 3b. 
         [0036]    It is preferred that the CueO derived from  Escherichia coli  is used. 
         [0037]    The method for immobilizing the antibody  103  and the multi-copper oxidase  102  on the particles  101  includes the following method (a): 
         [0038]    Method (a): a method using non-specific adsorption due to hydrophobic interaction generated between the antibody  103  and the multi-copper oxidase  102 . 
         [0039]    An example of the antigen  104  detected by the present invention is a virus, a  bacillus , a fungus, a protein, or an oligonucleotide. A virus, a  bacillus , or a fungus is preferred. 
         [0040]    An example of the first buffer solution is a Tris-buffered HCl solution, a Tris-buffered saline, or a phosphate buffer saline. Preferably, the first buffer solution has a near-neutral pH. 
         [0041]    The immersion causes the specific binding of the antibody  103  to the antigen  104 . Subsequently, the first buffer solution containing the unreacted antigen  104  was removed from the surface of the particles  101 . 
         [0042]    In the present invention, the specific binding of the antibody  103  to the antigen  104  increases the enzyme activity of the CueO  102 . 
         [0043]    (Step (b)) 
         [0044]    In the step (b), the particles  101  obtained in the step (a) are recovered. More particularly, the particles  101  can be recovered by centrifugation. A filter may be used to recover the particles  101 . 
         [0045]    (Step (c)) 
         [0046]    In the step (c), the particles  101  recovered in the step (b), an oxidation-reduction indicator (reductant), and a second buffer solution are mixed to prepare a reaction solution. The second buffer solution may contain the oxidation-reduction indicator (reductant) beforehand, prior to the addition of the particles  101  to the second buffer solution. Instead of this, the oxidation-reduction indicator (reductant) may be added to the second buffer solution, after the particles  101  are added to the second buffer solution. 
         [0047]    The second buffer solution contains a substrate  105  of the CueO  102 . An example of the substrate  105  is oxygen or proton. Oxygen and proton are contained in an ordinal and commercially-available buffer solution. 
         [0048]    An example of the oxidation-reduction indicator is 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic Acid (hereinafter, referred to as “ABTS”), p-Phenylenediamine, or 2,6-Dimethoxyphenol. ABTS is preferred, since ABST (reductant) is commercially-available and easy to be acquired. 
         [0049]    It is preferred that the second buffer solution has pH of not less than 4.5 and not more than 5.5. Sodium hydroxide, potassium hydroxide, ammonia, or trimethylammonium are used to adjust the pH of the second buffer solution. An example of the second buffer solution is an acetic acid buffer solution, a citrate buffer solution, a succinic acid buffer solution, a phthalic acid buffer solution, or 2-morpholinoethanesulfonic acid buffer solution, which is referred to as “MES”. An acetic acid buffer solution is preferred. 
         [0050]    The second buffer solution is required to have an ionic strength falling within a range of not less than 0.3 mM and not more than 1.0 mM. When the ionic strength is less than 0.3 mM, it is difficult to ensure the stability of the pH. When the ionic strength is more than 1.0 mM, as is clear from the examples described later, it is difficult to detect the antibody contained in the first buffer solution. 
         [0051]    (Step (d)) 
         [0052]    In the step (d), the activity degree of the CueO contained in the reaction solution obtained in the step (c) is measured by an absorbance measurement technique. 
         [0053]    It is preferred that a visible-ultraviolet spectrophotometer is used to measure the absorbance. 
         [0054]    A specific procedure for measuring the absorbance is described below. First, after the particles  101  are immersed in the second buffer solution in the step (c), the absorbance is measured continuously for a predetermined period, and a change amount of the absorbance per unit time is recorded. The oxidation-reduction indicator (reductant) contained in the reaction solution is oxidized by the CueO. Since the absorbance of the oxidation-reduction indicator in the oxidation state is different from the absorbance of the oxidation-reduction indicator in the reduction state, the change amount of the oxidation-reduction indicator is calculated from the absorbance. 
         [0055]    The activity degree of CueO is calculated from the change amount of the oxidation-reduction indicator oxidized per unit time and the amount of the CueO used in the measurement. 
         [0056]    (Step (e)) 
         [0057]    In the step (e), it is determined that the first buffer solution contains the antibody, if the following formula is satisfied. 
         [0000]      (the activity degree of the CueO measured in the step( d ))≧1.4×(blank value)
 
         [0058]    Here, the blank value is the activity degree of the CueO measured by the absorbance measurement technique where the antigen is not used, however, the particles  101  and the second buffer solution are used. 
         [0059]    The blank value may be calculated in parallel with the step (c). Alternatively, the blank value is calculated beforehand. 
         [0060]    The present invention is described in more detail by the following example 1. 
         [0061]    (Preparation of a CueO Solution) 
         [0062]    As described below, a CueO derived from  Escherichia coli  (K−12) was produced in the  Escherichia coli  as a recombinant CueO, and the CueO was purified. 
         [0063]    The gene of the CueO was amplified by a PCR method using  Escherichia coli  genomic DNA (LA genome DNA set for PCR, available from Takara Bio Inc.). 
         [0064]    In the PCR, a base sequence coding for a histidine tag was added to an oligo DNA primer, and the histidine tags were added to the carboxyl terminal side of the amino acid sequence of the CueO. 
         [0065]    The amplified DNA fragment was cloned (In-Fusion Dry-Down PCR Cloning Kits; available from Clontech laboratories, Inc.) to an expression vector (pRSFDuet-1, available from Merck KGaA). With use of the expression vector,  Escherichia coli  (BL21(DE3); available from Agilent Technologies, Inc.) was transformed. The transformed  Escherichia coli  were incubated for sixteen hours on a LB broth containing copper sulfate with a concentration of 1 mM. 
         [0066]    A periplasm fraction was extracted from the recovered fungus body, and CueO was purified from the extraction liquid with use of a histidine tag purification column (TALON CellThru Resin; available from Clontech laboratories, Inc.) and an anion-exchange column (HiTrap Q HP 5 mL; available from GE Healthcare). 
         [0067]    With use of a desalination column (HiTrap desalting; available from GE Healthcare), the solvent of the solution containing the purified CueO was substituted with 10 mM of tris-hydrochloric acid buffer solution (pH: 7.5). Subsequently, the CueO solution was condensed with use of the ultrafiltration unit (Amicon Ultra-4, MWCO: 30,000; available from Millipore Corp.). The condensed CueO solution has a concentration of 1 mg/mL. The concentration of the CueO solution was measured by the Bradford method using Bovine Serum Albumin (BSA) as a standard. The obtained CueO solution was stored at four degrees Celsius. 
         [0068]    (Preparation of the Antibody) 
         [0069]    An anti-Bovine Serum Albumin antibody (anti-BSA antibody [Rabbit], 1 mg/mL, available from Bethyl Laboratories, Inc.) was used as antibody  103 . The antibody was stored at a temperature of 4 degrees Celsius. 
         [0070]    (Immobilization of the CueO  102  and the Antibody  103  to the Particles  101 ) 
         [0071]    A dispersion liquid of gold particles each having a diameter of 20 nanometers (Gold Colloid 20 nml; BBI Holdings Plc) was used. The gold particles served as the particles  101 . The dispersion liquid (12 milliliters) and the antibody (0.12 milliliters) were mixed and left alone for 40 minutes at a temperature of 23 degrees Celsius. Subsequently, the CueO solution (0.12 milliliters) was added and left alone for 40 minutes at a temperature of 23 degrees Celsius. Furthermore, a 10% aqueous solution (0.12 milliliters) of polyoxyethylene(20)sorbitan monolaurate (available from Affymetrix, Inc) was added and left alone for 40 minutes at a temperature of 23 degrees Celsius. 
         [0072]    Next, the CueO and the antibody which were not immobilized to the particles  101  were removed as below. 
         [0073]    The dispersion liquid of the gold particles containing the CueO, the antibody, and polyoxyethylene(20)sorbitan monolaurate was poured to four centrifuge tubes (PC tube 3.0 mL; available from Beckman Coulter, Inc.). Each centrifuge tube had the dispersion liquid at a volume of 3 milliliters. 
         [0074]    Glycerol at a volume of 0.05 milliliters (&gt;99.5%; available from Life Technologies Corp.) was submerged in the bottom of each dispersion liquid. The glycerol served as a cushion. 
         [0075]    Each centrifuge tube was inserted into a rotor (TLA-100, available from Beckman Coulter, Inc) and fixed to a centrifuge system (Optima TL; available from Beckman Coulter, Inc). The four centrifuge tubes were centrifuged at 70,000 rpm at a temperature of 4 degrees Celsius for 20 minutes. 
         [0076]    A supernatant was removed from each centrifuge tube to leave condensed gold particle dispersion liquids (approximately 0.01 milliliter×4). 
         [0077]    A Tris-buffered HCl solution (pH:8.0, 0.1 mM, 3 milliliters) containing polyoxyethylene(20)sorbitan monolaurate was added to the condensed gold particle dispersion liquid (0.01 milliliter). Hereinafter, this procedure is referred to as “procedure A”. Hereinafter, the resultant buffer solution is referred to as “T-buffer solution”. 
         [0078]    The gold particle dispersion added the T-buffer solution were poured to four centrifuge tubes (PC tube 3.0 mL; available from Beckman Coulter, Inc.). Each centrifuge tube had the dispersion liquid at a volume of 3 milliliters. Glycerol at a volume of 0.05 milliliters (&gt;99.5%; available from Life Technologies Corp.) was submerged in the bottom of each dispersion liquid. The glycerol served as a cushion. Each centrifuge tube was inserted into a rotor (TLA-100, available from Beckman Coulter, Inc) and fixed to a centrifuge rotor (Optima TL; available from Beckman Coulter, Inc). The four centrifuge tubes were centrifuged at 70,000 rpm at a temperature of 4 degrees Celsius for 20 minutes. A supernatant was removed from each centrifuge tube to leave condensed gold particle dispersion liquids (approximately 0.01 milliliter×4). Hereinafter, this procedure is referred to as “procedure B”. 
         [0079]    The procedure A and the procedure B were repeated again. 
         [0080]    The gold particle dispersion liquid thus condensed was collected from the four centrifuge tubes, and the T-buffer solution was added so that the volume was adjusted to 1.2 milliliters. The concentration of the CueO contained in the gold particle dispersion liquid thus obtained was measured by a SDS-PAGE technique. The result was 0.02 milligram/milliliter. The gold particle dispersion liquid thus obtained was stored at a temperature of 4 degrees Celsius. 
         [0081]    (Preparation of an Acetic Acid Buffer Solution) 
         [0082]    While a value of a pH was measured with use of a pH meter, sodium hydroxide was added to acetic acid so as to prepare a 1M acetic acid buffer solution with a pH of 3. The acetic acid buffer solution is diluted by 5% so as to prepare an acetic acid buffer solution (pH:3) with a concentration of 50 mM. Similarly, an 1M acetic acid solution with a pH of 6 was prepared. Furthermore, an acetic acid buffer solution (pH:6) with a concentration of 50 mM was prepared. 
         [0083]    (Preparation of an ABTS Solution) 
         [0084]    2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic Acid Ammonium Salt) (549 milligram, available from Tokyo Chemical Industry Co., Ltd., hereinafter referred to as “ABTS”) was dissolved with ultrapure water to prepare an ABTS solution (100 mM) with a volume of 10 milliliter. ABTS served as an oxidation-reduction indicator. 
         [0085]    The liquids shown in the following Table 1 were mixed to prepare an ABTS acetic acid buffer solution (pH:3) with a acetic buffer solution concentration of 1.0 mM. Similarly, An ABTS acetic acid buffer solution (pH:6) with a acetic buffer solution concentration of 1.0 mM was prepared. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 liquid 
                 Amount 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Acetic buffer solution (pH: 3) with a 
                 1 
                 milliliter 
               
               
                   
                 concentration of 50 mM 
               
               
                   
                 ABTS solution 
                 0.05 
                 milliliters 
               
               
                   
                 Ultrapure water 
                 47.95 
                 milliliters 
               
               
                   
                   
               
             
          
         
       
     
         [0086]    The liquids shown in the following Table 2 were mixed to prepare an ABTS acetic acid buffer solution (pH:3) with a acetic buffer solution concentration of 0.5 mM. Similarly, An ABTS acetic acid buffer solution (pH:6) with a acetic buffer solution concentration of 0.5 mM was prepared. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 liquid 
                 Amount 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Acetic buffer solution (pH: 3) with a 
                 0.5 
                 milliliter 
               
               
                   
                 concentration of 50 mM 
               
               
                   
                 ABTS solution 
                 0.05 
                 milliliters 
               
               
                   
                 Ultrapure water 
                 48.45 
                 milliliters 
               
               
                   
                   
               
             
          
         
       
     
         [0087]    The liquids shown in the following Table 3 were mixed to prepare an ABTS acetic acid buffer solution (pH:3) with a acetic buffer solution concentration of 5 mM. Similarly, An ABTS acetic acid buffer solution (pH:6) with a acetic buffer solution concentration of 5 mM was prepared. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Liquid 
                 Amount 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Acetic buffer solution (pH: 3) with a 
                 5 
                 milliliter 
               
               
                   
                 concentration of 50 mM 
               
               
                   
                 ABTS solution 
                 0.05 
                 milliliters 
               
               
                   
                 Ultrapure water 
                 43.95 
                 milliliters 
               
               
                   
                   
               
             
          
         
       
     
         [0088]    The liquids shown in the following Table 4 were mixed to prepare an ABTS acetic acid buffer solution (pH:3) with a acetic buffer solution concentration of 10 mM. Similarly, An ABTS acetic acid buffer solution (pH:6) with a acetic buffer solution concentration of 10 mM was prepared. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                 Liquid 
                 Amount 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Acetic buffer solution (pH: 3) with a 
                 10 
                 milliliter 
               
               
                   
                 concentration of 50 mM 
               
               
                   
                 ABTS solution 
                 0.05 
                 milliliters 
               
               
                   
                 Ultrapure water 
                 38.95 
                 milliliters 
               
               
                   
                   
               
             
          
         
       
     
         [0089]    While a pH was measured with a pH meter, the ABTS acetic acid buffer solution (pH:3) with a acetic acid buffer solution concentration of 1.0 mM was added to the ABTS acetic acid buffer solution (pH:6) with a acetic acid buffer solution concentration of 1.0 mM so as to prepare an ABTS acetic acid buffer solution (pH:5.5) with a acetic acid buffer solution concentration of 1.0 mM. Similarly, an ABTS acetic acid buffer solution (pH:4.5) with a acetic acid buffer solution concentration of 1.0 mM was prepared. 
         [0090]    Similarly, ABTS acetic acid buffer solutions (pH:5.5) each having a concentration 0.5 mM, 5 mM, and 10 mM were prepared. Furthermore, ABTS acetic acid buffer solutions (pH:4.5) each having a concentration 0.5 mM, 5 mM, and 10 mM were prepared. 
         [0091]    (Measurement of the Activity of CueO by an Absorbance Measurement Technique) 
         [0092]    The activity of the CueO was measured by an absorbance measurement technique with use of the oxidant-reduction indicator. For the absorbance measurement, an ultraviolet-visible spectrophotometer (UV-1600PC, available from Shimadzu Corp.) was used. As a measurement vessel, a cell (Disposable cell semi-micro, available from Kartell spa) having a light path length of 1 centimeter was used. 
         [0093]    (Measurement of a Blank Value) 
         [0094]    First, a blank value was measured as below. In the measurement of the blank value, an antigen was not used. 
         [0095]    After the ABTS acetic acid buffer solution (1.0 mM; pH:4.5, 0.98 milliliter) was added to a cell, the cell was set in a visible-ultraviolet spectrophotometer. Next, the gold particle dispersion liquid (0.02 milliliter) was added to the cell. The absorbance of the ABTS acetic acid buffer solution was measured continuously (interval: 0.1 second) at a wavelength of 420 nanometers for sixty seconds.  FIG. 4  shows the measurement result. 
         [0096]    From the change amount of the absorbance measured for sixty seconds, the activity degree of the CueO was calculated on the basis of the following mathematical formula. About the unit of the activity degree of the CueO, 1 U means that the amount of the ABTS oxidized for sixty seconds is equal to 1 micromole. The result of the CueO activity measurement is shown in Table 5, which is described later. 
         [0000]      Activity of CueO(U/mg)= A/εlB    
         [0097]    where 
         [0098]    A represents an absorbance (nanometer), 
         [0099]    ε represents a molar absoption coefficient of the ABTS at a wavelength of 420 nanometers, which was 36,000 (M −1  cm −1 ) in the present example, 
         [0100]    l represents a light path length, which was 1 centimeter in the present example, and 
         [0101]    B represents the concentration of the CueO, which was 0.02 milligram/milliliter in the present example. 
         [0102]    In this manner, the blank value was measured. 
         [0103]    (Preparation of an Antigen-Antibody Mixture) 
         [0104]    After silica beads coated with BSA (diameter: 300 nanometers; 25 milligram/milliliter; available from micromod Partikeltechnologie GmbH) was added to the gold particle dispersion liquid (0.6 milliliter) as an antigen, the gold particle dispersion liquid was incubated at a temperature of 23 degrees Celsius and for thirty minutes. The gold particles each capturing the antigen was precipitated by centrifugation (2500 rpm, 23 degrees Celsius, 3 minutes), and the supernatant was removed. The T-buffer solution (1 milliliter) was added to the precipitate thus obtained, and the T-buffer solution was suspended. The gold particles were precipitated again by centrifugation (2500 rpm, 23 degrees Celsius, 3 minutes). The T-buffer solution (1 milliliter) was added to the obtained precipitate, and the T-buffer solution was suspended. The gold particles were precipitate again by centrifugation (2500 rpm, 23 degrees Celsius, 3 minutes). The T-buffer solution (0.6 milliliter) was added to the obtained precipitate, and the T-buffer solution was suspended. Thus, an antigen-antibody mixture was prepared. 
         [0105]    (Preparation of the Reaction Solution and Measurement of the Absorbance) 
         [0106]    After the ABTS acetic acid buffer solution (1.0 mM; pH:4.5, 0.98 milliliter) was added to a cell, the cell was set in a visible-ultraviolet spectrophotometer. Next, the gold particle dispersion liquid (0.02 milliliters) was added to the cell. The absorbance of the ABTS acetic acid buffer solution was measured continuously (interval: 0.1 second) at a wavelength of 420 nanometers for sixty seconds. From the measurement result, the activity of the CueO was calculated as a measurement value. The measurement value thus calculated was shown in the following Table 5, which is described later. 
         [0107]    An amplification ratio was defined in the following equation: 
         [0000]      (The amplification ratio)=(the measurement value)/(the blank value). 
         [0108]    Amplification ratios were calculated as described above using the ABTS acetic acid buffer solutions (pH:4.5, 5.0, and 5.5) each having an acetic acid buffer solution concentration of 1.0 mM and the ABTS acetic acid buffer solutions (pH:4.5, 5.0, and 5.5) each having an acetic acid buffer solution concentration of 10 mM. These results are shown in Table 1. 
         [0000]    
       
         
               
               
             
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 5 
               
             
             
               
                   
                   
               
               
                   
                 pH 
               
             
          
           
               
                   
                 4.5 
                 5.5 
               
               
                   
                   
               
             
          
           
               
                 Acetic acid 
                 0.5 
                 1 
                 5 
                 10 
                 0.5 
                 1 
                 5 
                 10 
               
               
                 buffer solution 
               
               
                 concentration (mM) 
               
               
                 Blank value[A] 
                 1.9 
                 2.0 
                 1.9 
                 1.9 
                 0.32 
                 0.34 
                 0.31 
                 0.29 
               
               
                 (U/mg) 
               
               
                 Measurement 
                 2.7 
                 2.8 
                 1.9 
                 1.9 
                 0.55 
                 0.57 
                 0.34 
                 0.32 
               
               
                 Value[B] (U/mg) 
               
               
                 Amplification 
                 1.4 
                 1.4 
                 1.0 
                 1.0 
                 1.7 
                 1.7 
                 1.1 
                 1.1 
               
               
                 ratio[B/A] 
               
               
                   
               
             
          
         
       
     
         [0109]    As is clear from Table 5, when the concentration of the acetic acid buffer solution (more exactly, the concentration of the acetic acid contained in the ABTS acetic acid buffer solution) is 1.0 mM or less, the amplification ratio is 1.4 or more. On the contrary, the concentration of the acetic acid buffer solution is 5 mM or more, the amplification ratio is 1.1 at most. 
         [0110]    Accordingly, a skilled person would understand from Table 5 that the antigen is detected if the amplification ratio is 1.4 or more. Furthermore, a skilled person would understand easily that the labeled-antibody  111  is not used in the present invention. 
         [0111]    The present invention is used for a biosensor. The present invention is very useful for quick immunodiagnosis with multiplex assay. 
       REFERENTIAL SIGNS LIST 
       [0000]    
       
         
           
               101 : particle 
               102 : multi-copper oxidase CueO 
               103 : antibody 
               104 : antigen 
               105 : substrate 
               106 : product 
               107 : support 
               108 : antibody 
               109 : antigen 
               110 : enzyme 
               111 : labeled-antibody 
               112 : substrate 
               113 : product