JPH04258766A - Simple immunity diagnostic method processing many objects - Google Patents

Abstract

PURPOSE:To analyze many objects easily and quickly in the analysis of a biological fluid and its product by the immunity diagnostic method. CONSTITUTION:An analysis object and a metallic colloidal grain marker or an oxygen marker are reacted in a reaction container fitted with a porous film solidified with a material specifically reacted with the analysis object on the bottom face, they are sucked and filtered to remove an unreacted material, a reaction product is adsorbed on the porous film, and the presence of the analysis object is judged by its coloring. The device and operation are simple, and the necessary analysis time can be shortened to several minutes through ten and several minutes.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method suitable for simple and rapid processing of multiple samples of specific proteins using immunodiagnostic methods.

[0002] Samples (analytes) to which the present invention is applied include all physiological fluids and their products, such as serum, plasma, whole blood, urine, and feces.

0003

BACKGROUND ART AND PROBLEMS TO BE SOLVED BY THE INVENTION Immunodiagnostic methods are very useful in diagnosing diseases, and various methods have been established and are in clinical use.

Currently, immunodiagnostic methods use enzymes, isotopes, fluorescent substances, etc. as labels to quantitatively detect specific proteins [enzyme immunoassay (EIA method),
Radioimmunoassay (RIA method), fluorescence immunoassay (
A method of qualitatively determining the presence or absence of specific proteins using the agglutination reaction of red blood cells or latex (FIA method)]
agglomeration method).

[0005] The former method has the advantage of being able to quantitatively test specific proteins in a sample, as well as being able to test a large number of samples at the same time. On the other hand, the latter method is easier to operate and does not require special equipment than the former method, but it is not suitable for processing a large number of samples at the same time. (Medical Tribune 1987
(June 4, 2016 issue) Therefore, the former method is suitable for hospitals and testing institutions that process large numbers of samples every day, such as during mass medical examinations, and the EIA method is most suitable for processing large numbers of samples. .

However, although the EIA method has the above-mentioned advantages, it has the drawbacks that it is complicated to operate and requires a long time for testing.In particular, shortening the time required for testing requires processing a large number of samples. This is one of the important issues for hospitals and testing institutions that carry out testing.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for shortening the time required for inspection without sacrificing any of the conventional advantages of the EIA method described above.

That is, in the present invention, in a reaction vessel (hereinafter referred to as a well) equipped with a porous membrane capable of binding to proteins on the bottom surface, a substance that reacts specifically with the analyte is preliminarily attached to the porous membrane. A reagent group consisting of a substance that specifically reacts with the analyte and the analyte labeled with a metal colloid particle or an enzyme (hereinafter referred to as a metal colloid particle labeled substance or an enzyme labeled substance) is placed in the well. Add dropwise to react, then remove unreacted substances by suction filtration, and in some cases, add a coloring agent to color the reaction product on the porous membrane, which can be qualitatively judged with the naked eye. Furthermore, they have found that the time required for testing can be reduced quantitatively by comparing with standard samples of known concentrations, leading to the completion of the present invention.

A well with a porous membrane attached to the bottom that can be used in the present invention is a multi-screen filter plate (
(manufactured by Nippon Millipore Limited). This plate usually consists of 96 wells, but the present invention is not particularly limited to this number.

Materials for the porous membrane include activated nitrocellulose, polyvinylidene fluoride (PVDF),
Nylon 66 or the like is preferred.

There are two types of substances that specifically bind to the analyte used in the present invention: substances that react with porous membranes and substances that are labeled with enzymes or metal colloid particles.

Substances that react with porous membranes include various monoclonal antibodies, various polyclonal antibodies, and various antigens.

There are various ways to immobilize the substance that specifically binds to the analyte on the porous membrane, but one example is to immobilize the substance that specifically binds to the analyte in phosphate buffered saline (PBS).
1-1.0 mg protein/ml, preferably 0.3-1.0 mg protein/ml
Add an appropriate amount of a solution of anti-human hemoglobin antibody prepared at 0.6 mg protein/ml to the well using a pipette, leave it at a constant temperature overnight, and then remove the solution.
Immobilize anti-human hemoglobin antibody.

Next, in order to prevent non-specific reactions caused by impurities in the specimen sample or reaction reagents, the porous membrane immobilized with anti-human hemoglobin antibody was mixed with about 3% skim milk (D
An appropriate amount of a PBS solution (manufactured by IFCO) is added dropwise to the well, and the mixture is left standing at 30 to 40°C for several hours. Further, the solution is removed and dried at room temperature to about 50°C or less. Note that drying is preferably carried out in a desiccator.

[0015] In this way, a porous membrane immobilized with an anti-human hemoglobin antibody that can be used in the present invention is obtained. Enzyme immunoassays and metal colloids can be used as means for determining the presence or absence of an analyte in a sample.

In the enzyme immunoassay, an analyte and an enzyme label are reacted on a porous membrane on which a substance that specifically reacts with the analyte is immobilized, and then a coloring agent is added to the porous membrane. The measurement principle is to color the reaction product bound to the analyte and detect the analyte.

On the other hand, in the metal colloid method, the analyte and the metal colloid particle label are reacted on a porous membrane in which a substance that specifically reacts with the analyte is immobilized,
The measurement principle is to detect the analyte by coloring the porous membrane with metal colloid particles.

Next, there are various types of metal colloid particles that can be used to detect specific proteins, and gold colloid particles are particularly suitable. When colloidal gold particles are brought into contact with proteins under certain conditions, they bind by electrostatic adsorption to form colloidal gold particle labels. Colloidal gold particles are obtained by reducing an aqueous solution of chloroauric acid. For example, a method using sodium citrate as a reducing agent [G. Frens, Nature (Phys
．． Sci. ) 241, 20-22, 1973] and the method using citric acid or tannic acid (J. W. Slo
t, H. T. Genze, Eur. J. C
ell. Biol. 38, 87-93, 1985) are known. Also, regarding the method of binding colloidal gold particles to proteins, see B. L. As reported by Wang et al. (Histochemistry
83, 109-115, 1985) On the other hand, there are various enzymes that can be used in the present invention, but peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, etc. are particularly suitable, and these enzymes are not commercially available. It can be easily obtained because it is available.

Typical examples of the analyte in the present invention include human hemoglobin in fecal occult blood and urinary occult blood in the human body. In addition, when diagnosing infectious diseases in laboratory animals (mice, rats, guinea pigs, rabbits, etc.), antibodies for pathogens are usually searched for, but the target substances for this antibody search are Sendai virus antibodies, mouse hepatitis virus antibodies, etc. , Mycoplasma pulmonis antibodies, etc.

[0020] Furthermore, as substances that specifically react with the analyte used in the labeling of the present invention, anti-human hemoglobin monoclonal antibodies and anti-human hemoglobin polyclonal antibodies are useful for human hemoglobin; For Sendai virus antibodies, mouse hepatitis virus antibodies, Mycoplasma pulmonis antibodies, etc., protein A, anti-mouse IgG, anti-rabbit IgG, etc. can be used for animals other than rats, and anti-rat IgG is used for rats. be done.

[0021] Since commercially available anti-human hemoglobin polyclonal antibodies contain impurities, it is preferable to use them after purification, but commercially available reagents are sufficient for use with other reagents.

[0022] Important conditions for the method of labeling these antibodies with enzymes are: 1) the activity of the enzyme and antibody does not decrease; 2) The bond is stable and can withstand long-term storage. 3) The operation should be as simple as possible and the labeled substance should be easily obtained. Enzyme labeling methods that have been developed to date that meet these conditions can be broadly classified into two types. One is a method of chemically linking an enzyme and an antibody, such as the glutaraldehyde method (S. Avrame et al.
as, Immunochemistry, 6,43
, 1969) and the periodate oxidation method (P.K. Naka
ne, A. Kawai, J. Histoche
m. Cytochem, 22, 1084, 1974)
falls under this category. The other method is to bind the enzyme and the first antibody using an antibody or the like as an intermediary, and this method uses the avidin-biotin method (S.M. Hsu, L. Rain).
e, H. Furger, Am. J. Clin. P
athol. , 75, 734-738, 1981). Note that in the present invention, measurement can be performed using any labeling method.

[0023] As one aspect of producing the labeled product used in the present invention, for example, a method for obtaining a peroxidase-labeled antibody is known. Specifically, all the amino groups of peroxidase are blocked with 1-fluoro-2,4-dinitrobenzene (FDNB), and then the adjacent hydroxyl group of the sugar moiety of the dinitrophenylated peroxidase is cleaved with periodic acid to remove the aldehyde group. Cause new birth. Excess periodic acid is decomposed by adding ethylene glycol to stop the reaction.

Next, 0.01M sodium carbonate buffer (p
After dialysis against H9.5), a Schiff base is formed by reacting the newly formed aldehyde group with the amino group of the antibody. The Schiff base reaction product is then reduced with sodium borohydride in order to chemically stabilize it. After reduction, excess sodium borohydride is removed by dialysis to obtain the desired enzyme-labeled antibody. Furthermore, it is preferable to use the labeled antibody purified by gel filtration.

In the case of peroxidase, hydrogen peroxide, 3,3'-5,5'-tetramethylbenzidine and 3,3'-diaminobenzidine are used as chromogenic substrates, and for alkaline phosphatase, In some cases, bromo-chloroindole phosphate nitroprusside tetrazolium chromogens are used.

Furthermore, the reaction conditions for the coloring agent may be the same as those for ordinary enzyme reactions, and although there are no particular restrictions on the temperature, it is most preferable to carry out the reaction at room temperature.

[0027] The test results of the present invention can qualitatively determine the coloring of the porous membrane with the naked eye. If quantification is required, it can be easily quantified by reacting a sample of the analyte with a known concentration with the sample under the same conditions and comparing the colors of the sample and the sample.

[0028] Furthermore, it can be confirmed whether the test is being performed normally by providing one or more controls. That is, this control is a porous membrane immobilized with a substance that always reacts with a metal colloid particle label or an enzyme label, such as goat anti-rabbit IgG, and the reaction with the label causes porosity. The film is always colored.

Next, an overview of the inspection method will be given. As for the testing method, conventional methods such as enzyme immunoassay and metal colloid method can be applied, but for example, enzyme immunoassay uses a microplate with a porous membrane attached to the bottom (Japan Millipore Ltd.). immobilize anti-human hemoglobin antibody on Next, the specimen (suspension in the case of solids)
Add 10 to 250 μl, preferably 50 to 200 μl, and react for 1 to 10 minutes, preferably 2 to 5 minutes. All operations can be carried out at a reaction temperature of 4 to 40°C, preferably 10 to 30°C.

After the reaction, the liquid in the wells is removed by suction filtration. Next, as a washing operation, phosphate buffered saline (PB
S) bovine serum albumin or a surfactant, e.g.
Tween 20 (manufactured by Wako Pure Chemical Industries), Triton X
(manufactured by Wako Pure Chemical Industries, Ltd.), preferably Twee
After dropping 50 to 250 μl of PBS containing n20 into the wells, the wells are filtered by suction. Note that the washing operation is not necessarily necessary if a specimen blank is provided.

Next, add 10 to 250 μl of a diluted solution of purified peroxidase-labeled anti-human hemoglobin antibody, preferably 5
Add 0-200 μl and incubate for 1-10 minutes, preferably 2-5
Let it react for a minute.

After the reaction, washing and suction filtration are performed in the same manner as above. Furthermore, 10 to 250 μl, preferably 50 to 200 μl, of a coloring agent used for peroxidase is added, and the mixture is allowed to react for 1 to 10 minutes, preferably 2 to 5 minutes. The color former may be a reagent commonly used for peroxidase, but 3,3'-diaminobenzidine is particularly suitable. Finally, the color former solution is removed by suction filtration, and the degree of coloration is evaluated.

On the other hand, in the gold colloid method, an anti-human hemoglobin antibody is similarly immobilized on a microplate with a porous membrane attached to the bottom. Next, add 10 to 250 μl of the specimen containing human hemoglobin, preferably 50 to 200 μl.
1 and reacted for 1 to 10 minutes, preferably 2 to 5 minutes. All operations can be carried out at a reaction temperature of 4 to 40°C, preferably 10 to 30°C.

After the reaction, suction filtration is performed to remove the liquid in the well. Next, as a washing operation, a solution prepared by adding bovine serum albumin, Tween 20, Triton X, etc. to PBS, preferably PBS 5 containing Tween 20, is used.
After adding 0-250 μl to the wells, filter by suction. Note that the washing operation is not necessarily necessary if a specimen blank is provided.

Next, a diluted solution of anti-human hemoglobin antibody-sensitized colloidal gold particles (colloidal gold labeled material) obtained by adding an anti-human hemoglobin antibody to the colloidal gold particle suspension was diluted to 10%.
~250μl, preferably 50~200μl, 1~
Allow to react for 10 minutes, preferably 2-5 minutes.

After the reaction, the solution is washed and suction-filtered in the same manner as above, and judged based on the degree of coloration of the porous membrane.

[0037]

[Examples] The present invention will be explained in detail below using examples.

Example 1 Measurement of human hemoglobin (enzyme immunoassay) <Purification of anti-human hemoglobin polyclonal antibody> A hemoglobin-Sepharose column (5 ml) in which human hemoglobin was bound to activated Sepharose 4B (manufactured by Pharmacia) was diluted with 5 mM hydroxide. Equilibration was performed with acid buffer (pH 8.0). Next, goat anti-human hemoglobin antibody (manufactured by Medical and Biological Research Institute)
1 ml was mixed with 1 ml of the same boric acid buffer solution and adsorbed onto the column. Thereafter, after washing with the same boric acid buffer, elution was performed with 0.5M glycine buffer (pH 3.0). 28
The active fraction was collected according to the absorbance value at 0 nm, and 60% saturated ammonium sulfate was added. The resulting suspension was centrifuged at 3,000 rpm for 30 minutes, and the precipitate was collected and dialyzed against PBS at 4°C overnight to obtain 6 mg of purified anti-human hemoglobin polyclonal antibody.

<Preparation of antibody-immobilized porous membrane> Anti-human hemoglobin antibody was phosphate-buffered in each well of a multiscreen filter plate (96-well filter plate HATF nitrocellulose, 0.45 μm, manufactured by Nippon Millipore Ltd.). 0 in physiological saline (PBS).
20 μl of a solution prepared to have a concentration of 5 mg protein/ml was added dropwise. As a control, goat anti-rabbit IgG prepared with PBS at a concentration of 0.5 mg protein/ml was dropped into other wells.

[0040] Thereafter, the mixture was allowed to react overnight at room temperature, and then 100 μl of skim milk (manufactured by DIFCO) prepared with PBS to a concentration of 3% was added dropwise, soaked for 3 hours at 37°C for masking, and dried. This was used as an antibody-immobilized porous membrane.

<Preparation of peroxidase-labeled anti-human hemoglobin antibody> To 5 mg of horseradish peroxidase (manufactured by Toyobo Co., Ltd., grade IC), 1 ml of 0.3 M sodium bicarbonate buffer (pH 8.1) was added and dissolved. Thereafter, 1% 1-fluoro-2,4-dinitrobenzene (
0.1 ml of ethanol solution (manufactured by Merck & Co., Ltd., FDNB) was added and reacted for 1 hour at room temperature. D with blocked amino group
1 ml of 0.06M sodium periodate was added to NP-peroxidase, and the mixture was reacted at room temperature for 30 minutes. For excess periodic acid, add 1 ml of 0.16M ethylene glycol,
The mixture was stirred at room temperature for 1 hour to decompose. Thereafter, dialysis was performed overnight at 4° C. using 0.01M sodium carbonate buffer (pH 9.5) to obtain a DNP/aldehyde-peroxidase reaction product. On the other hand, purified anti-human hemoglobin antibody was prepared in advance at 5 mg/ml in 0.01M sodium carbonate buffer (pH 9.5), and dialyzed against the same buffer at 4°C overnight. The DNP/aldehyde-peroxidase reaction product and purified anti-human hemoglobin antibody were mixed and reacted at room temperature for 3 hours. After the reaction, 5 mg of sodium borohydride was added, and the mixture was left at 4°C overnight. An additional 0.85%
Dialysis was performed overnight at 4° C. using 0.01 M sodium phosphate buffer (pH 7.1) containing sodium chloride. Although the obtained peroxidase-labeled anti-human hemoglobin antibody can be used as it is, the following treatment was performed to remove unreacted substances. That is, 0.01M sodium phosphate buffer containing 0.85% sodium chloride in advance (
Purification was performed by gel filtration using a Sephacryl S-200 column (2.6 cm x 100 cm, manufactured by Pharmacia) equilibrated with pH 7.1), and fractions with absorption at two wavelengths of 280 nm and 403 nm were collected and labeled with purified peroxidase. Anti-human hemoglobin antibodies were obtained.

<Coloring agent> 0.05M Tris-HCl
(pH 7.2) 10 mg of 3,3'-diaminobenzidine and 10 μl of 30% hydrogen peroxide were added to prepare a coloring agent.

Detection method for human hemoglobin: Add 0.2 ml of the analyte to each well of a microplate equipped with a porous membrane immobilized with anti-human hemoglobin antibody and goat anti-rabbit IgG as a control, and incubate for 2 minutes at room temperature. Made it react.

Next, the plate was placed on a multiscreen vacuum manifold (manufactured by Nippon Millipore Ltd.), and suction filtration was performed from the porous membrane side to remove the liquid in the wells.

0.1% Tween20/P in each well
Washing was performed by adding 0.2 ml of BS and performing suction filtration in the same manner.

Next, 0.2 ml of a diluted solution of purified peroxidase-labeled anti-human hemoglobin antibody was added to each well.
The reaction was carried out for 2 minutes at room temperature. After washing, color former (3,
0.2 ml of 3'-diaminobenzidine/hydrogen peroxide)
In addition, after coloring was allowed to develop for 2 minutes at room temperature, the coloring agent was removed by suction filtration to complete the reaction, and the degree of coloring of the porous membrane was evaluated.

As a result, for the sample containing human hemoglobin, yellowish brown coloration was observed in both wells in which anti-human hemoglobin antibody and goat anti-rabbit IgG were immobilized, whereas for the sample containing no human hemoglobin No coloration was observed, and coloration was observed only in the wells in which goat anti-rabbit IgG was immobilized.

Example 2 Measurement of human hemoglobin (gold colloid method) <Preparation of antibody-immobilized porous membrane> Multiscreen filter plate (96-well filter plate HATF nitrocellulose 0.45 μm, manufactured by Nippon Millipore Limited) To each well, 20 μl of a solution of anti-human hemoglobin monoclonal antibody (manufactured by MEDIX BIOTECH) prepared in phosphate buffered saline (PBS) at a concentration of 0.5 mg protein/ml was added dropwise. As a control, rabbit anti-mouse IgG prepared with PBS to a concentration of 0.5 mg protein/ml was added dropwise to other wells. After that, the reaction was allowed to proceed overnight at room temperature, and then 100 μl of a PBS solution containing 3% skim milk (manufactured by DIFCO) was added dropwise and masked by soaking at 37°C for 3 hours. The dried product was then immobilized with antibodies. It was made into a porous membrane.

<Preparation of colloidal gold particles> Distilled water 395m
1% chloroauric acid (manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution (5 ml) was mixed with the solution and heated to 60° C. (solution A).

Next, 20 ml of 1% sodium citrate, 1
% tannic acid (manufactured by Sigma) 2.5 ml, distilled water 7
7.5 ml were mixed and heated to 60°C (solution B). While stirring solution A, quickly mix solution B and stir until the color of the solution changes from pale yellow to purple or red.
The desired colloidal gold particles were obtained by heating until boiling.

<Preparation of colloidal gold particles sensitized with anti-human hemoglobin antibody> 100 ml of colloidal gold particle suspension was adjusted to pH 9.0 with 0.1M potassium carbonate while stirring. Next, anti-human hemoglobin monoclonal antibody (MEDI
After adding 460 μg (manufactured by X BIOTECH) of the
Stored at 8°C.

Assay method using colloidal gold particles 200 μl of the analyte was added to each well of a microplate equipped with a porous membrane immobilized with anti-human hemoglobin and rabbit anti-mouse IgG as a control, and incubated at room temperature for 2 minutes. Made it react.

Next, the plate was placed on a multiscreen vacuum manifold (manufactured by Nippon Millipore Ltd.), and suction filtration was performed from the porous membrane side to remove the liquid in the wells.

0.1% Tween20/P in each well
Washing was performed by adding 200 μl of BS and performing suction filtration in the same manner.

After adding 200 μl of the anti-human hemoglobin antibody-sensitized gold colloid solution and allowing the reaction to proceed for 2 minutes, the excess anti-human hemoglobin antibody-sensitized gold colloid solution was suction-filtered to complete the reaction and remove the porous membrane. Judgment was made based on the degree of coloration.

As a result, for the sample containing human hemoglobin, reddish-purple coloring was observed in both wells in which anti-human hemoglobin monoclonal antibody and rabbit anti-mouse IgG were immobilized, whereas No coloration was observed in the sample without the IgG, and coloration was observed only in the wells in which rabbit anti-mouse IgG was immobilized.

Example 3. Operation time for measuring human hemoglobin The operation time for the colloidal gold particle method and enzyme immunoassay of Examples 1 and 2 was measured. As a control, an oxygen immunoassay using a microplate (Checkmate Hemo, manufactured by Wakamoto Pharmaceutical Co., Ltd.) was used.

[0058] Human hemoglobin (manufactured by Sigma, twice crystallized product) was added at 1 μg/m in a PBS solution containing 0.1% BSA.
diluted to a concentration of
The same operation was performed on all 96 wells of Nippon Millipore Ltd.). The results are shown in Table 1.

Table 1 Operation time for measuring human hemoglobin Measurement method
Operation time (minutes)
────────────────────────
──────── Example 1 Gold colloid particle method 8
Example 2 Enzyme immunoassay
12 Comparative example Checkmate Hemo 180 ──────
──────────────────────────
-The results showed that while CheckMate Hemo, which was used as a control, required 180 minutes per 96 samples, the gold colloid particle method required 8 minutes, and the enzyme immunoassay method shortened the time to 12 minutes. That is, the operation time was shortened to 1/20 in the gold colloid particle method and to 1/15 in the enzyme immunoassay.

Example 4. Detection sensitivity of human hemoglobin Human hemoglobin (manufactured by Sigma, twice crystallized) was 0.
0.001-1000 in PBS solution containing 1% BSA
Each solution with a concentration of μg/ml was prepared and used as a sample solution. Similar operations were performed according to the methods of Examples 1 and 2. As a control, an enzyme immunoassay using a microplate (Checkmate Hemo, manufactured by Wakamoto Pharmaceutical Co., Ltd.) was used.

As a result, in the gold colloid particle method, 0.1μ
g/ml, and 0.01 μg/ml for enzyme immunoassay.
It was sensitive enough to measure up to Control checkmate
The measurable sensitivity of hemoglobin was 0.01 μg/ml, which was comparable with enzyme immunoassay.

Table 2. Measurement sensitivity for human hemoglobin──
──────────────────────────
────────
Hb concentration (μg/ml
) ──────
────────────────────
1000 10
0 10 1.0 0.1 0.0
1 0.001 0──────────────
──────────────────────Gold colloid particle method + + +
+ + − − −Enzyme immunoassay + + +
＋ ＋ ＋ −
-Checkmate Hemo + + +
＋ ＋ ＋ −
−────────────────────────
────────────Example 5. Detection sensitivity with human hemoglobin-added stool Human hemoglobin (converted amount from whole blood) in fecal occult blood-negative stool per 1 g of stool, 10.2
0, 60, 100, and 1000 μg were added and mixed well. This was collected on a stool collection stick, suspended in PBS, and 200 ml/well was collected in the same manner as in Examples 1 and 2.
A μl drop was added and the operation was performed.

As a result, the detection sensitivity for human hemoglobin-added stool was 1 per 1 g of stool using the colloidal gold particle method.
00 μg/g, and up to 20 μg/g could be detected by enzyme immunoassay.

[0064] This was comparable to the oxygen immunoassay since the control Checkmate Hemo had a sensitivity of 20 μg/g.

Table 2. Detection sensitivity in human hemoglobin-added feces──────────────────────
────────────
Hb addition concentration (μg/g stool)
────────
──────────────────
1000 10
0 60 20 10 0
──────────────────────────
────────Gold colloid method
＋ ＋ − − −
-Enzyme immunoassay +
＋ ＋ ＋ − −Checkmate Hemo ＋ ＋
＋ ＋ − −──────────
────────────────────────Example 6. Reaction with fecal samples Eight fecal samples were each collected on a stool collection stick, suspended in PBS, and operated in the same manner as in Examples 1 and 2. As a control, enzyme immunoassay using a microplate (
Checkmate Hemo (manufactured by Wakamoto Pharmaceutical Co., Ltd.) was used.

As a result, as shown in Table 3, 6 out of 8 fecal samples were positive. This is the same result as the control Checkmate Hemo, indicating that the analytical method of the present invention is useful in clinical testing.

Table 3. Reactions in fecal samples──────────────────────────
─────────
Sample N
o. Measurement method ──────────────────
────────────
1 2 3 4
5 6 7 8─────
──────────────────────────
────Gold colloid method +
＋ ＋ − ＋ ＋
+ -Enzyme immunoassay +
＋ ＋ − ＋ ＋
＋ −Checkmate Hemo ＋ ＋
＋ − ＋ ＋ ＋
−────────────────────
──────────────

[0068]

According to the present invention, it is possible to provide an analysis method useful for diagnosing diseases of the human body by utilizing a specific binding reaction. In particular, the method of the present invention allows a large number of specimens to be processed in an extremely short time.

Claims (1)

[Claims] Claim 1: A reaction vessel equipped with a porous membrane having the ability to bind proteins on the bottom surface, in which a substance that specifically reacts with an analyte is immobilized on the porous membrane, and the analyte is placed in the reaction vessel. , and a reagent group consisting of a substance that specifically reacts with the analyte labeled with metal colloid particles or an enzyme to cause a reaction, and then, after removing unreacted substances by suction filtration, in some cases, a coloring agent is further added. In addition, an immunodiagnostic method is characterized in that the porous membrane is colored to determine the presence or absence of an analyte.