JP2007218593A - Immuno-chromatography measuring method for bio-pilin detection, and its device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and device for measuring bio-pilin usable for easy and rapid diagnosis in a clinical case without requiring a special instrument and expert technology. <P>SOLUTION: In the immuno-chromatography measuring method and device for bio-pilin detection, a film carrier 3 having a capture part 41 formed by previously fixing an anti-bilirubin antibody to a predetermined position and an indicator body indicating a conjugate of the bio-pilin and high molecular compound with an indicator material are prepared. Mixed liquid of the indicator body and a tested material is chromato-developed by the film carrier toward the capture part. The indicator body and the bio-pilin in the tested material are captured by the capture part competitively. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to an immunochromatographic measurement method and apparatus for biopyrin detection by a competitive method using a membrane carrier having a capture site to which an anti-bilirubin antibody is immobilized.

Increasing active oxygen in the human body has been shown to cause all diseases, including cancer, adult diseases, and skin problems. A state where active oxygen increases in the human body and the body is damaged is called oxidative stress. If the degree of oxidative stress in a living body can be grasped, the occurrence of a disease caused by oxidative stress can be predicted, treatment can be taken in advance for prevention, and it can be an index of health status.

Biopyrine is a general term for bilirubin oxidation products. When oxidative stress accumulates in the living body and active oxygen increases, bilirubin is oxidized, and as a result, biopyrine is detected in urine and the like. Thus, since the amount of biopyrine reflects the amount of active oxygen in the living body, the degree of oxidative stress in the living body can be grasped based on the amount of biopyrin.

Conventionally, biopyrin measurement methods such as HPLC using an ODS column and ELISA using a monoclonal antibody are known (Patent Documents 1 and 2, Non-Patent Documents 1 and 2). However, since these methods require special equipment, the operation is complicated and the measurement takes time.

Japanese Patent No. 3230062 Japanese Patent Laid-Open No. 8-151365 Takashi Suzuki et al., `` Evaluation of biopyrrin concentration in urine by stress marker '' Medicine and pharmacy; 42 (6): 981-987 Yamaguchi T et al., `` Psychological stress increase bilirubin metabolites in human urine. '' Biochemical and Biophysical Reseach Communications 293 (2002) 517-520

An object of the present invention is to provide a biopyrin measurement method and apparatus that can be used for simple and rapid diagnosis in a clinical scene without requiring special equipment and skilled techniques.

The present inventors have found that it is difficult to stably immobilize biopyrin, which is a low molecular substance and is very susceptible to oxidation, on a membrane carrier, but it is difficult to apply it to a high molecular substance such as bovine serum albumin (BSA). By binding, it was found that this conjugate can be stably solid-phased on a labeling substance such as colloidal gold, and that this label can be used for competitive immunochromatographic measurement with biopyrine, thereby completing the present invention. It came to.

That is, according to one aspect of the present invention, a membrane carrier having a capture site formed by preliminarily fixing an anti-bilirubin antibody at a predetermined position, and a label obtained by labeling a conjugate of biopyrin and a polymer compound with a labeling substance A mixture of the labeled body and the test sample is chromatographed on the membrane carrier toward the capture site, thereby competitively binding the labeled body and the biopyrin in the test sample. There is provided an immunochromatographic assay for detecting biopyrin characterized by comprising capturing at a capture site.

According to still another aspect of the present invention, an anti-bilirubin antibody, a labeled body obtained by labeling a conjugate of biopyrin and a polymer compound with an appropriate standard substance, and a membrane carrier are provided, and the anti-bilirubin antibody is preliminarily provided. It is fixed to the membrane carrier to form a capture site, and the labeling body is prepared so that it can be chromatographed on the membrane carrier from a position separated from the capture site toward the capture site. An immunochromatography measuring apparatus for detecting biopyrine is provided.

It is convenient to prepare the labeled body on a membrane carrier because the measurement can be performed simply by injecting the test sample into the membrane carrier. It is convenient to prepare the marker by impregnating an impregnating member disposed on the membrane carrier.

The anti-bilirubin antibody used in the present invention may be an antibody against bilirubin, and may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody from the viewpoint of reaction specificity. A polyclonal antibody can be obtained, for example, from an antiserum obtained by immunizing an animal according to a conventional method using purified bilirubin as an antigen together with an appropriate adjuvant. Monoclonal antibodies are obtained by immunizing animals such as mice using purified bilirubin as an antigen together with an appropriate adjuvant, and then fusing the spleen cells and myeloma cells of the immunized animals. It can be obtained by selecting the HAT-containing medium and growing it, and then selecting the grown strain using the purified bilirubin, for example, by enzyme-labeled immunization. At that time, as described in JP-A-1-175945, an antigen used for immunizing a mouse may be one obtained by covalently binding separated bilirubin and bovine serum albumin by the acid anhydride method. Good. In this case, instead of bovine serum albumin, polymer compounds such as mussel hemocyanin, ovalbumin, and rabbit serum albumin may be used. As the monoclonal antibody, those described in JP-A-1-175945 can be used.

Since bilirubin is a low molecular weight compound, the epitope of the bilirubin molecule is limited, and it is usually considered that it is also conserved in biopyrin, which is a bilirubin oxidation product. Therefore, the antibody obtained using bilirubin as an antigen usually recognizes both bilirubin molecules and biopyrine, that is, various bilirubin oxidation products. It is said that 10 or more types of biopyrine exist, and not all of the structures have been determined. Examples of bilirubin oxidation products contained in biopyrine include, for example, 1,14, described in JP-A-8-151365. 15,17-tetrahydro-2,7,13-trimethyl-1,14-dioxo-3-vinyl-16H-tripyline-8,12-dipropionic acid (1,14,15,17-tetrahydro-2,7, 13-trimethyl-1,14-dioxo-3-vinyl-16H-tripyrrin-8,12-dipropionic acid), and 1,14,15,17-tetrahydro-3,7,13-trimethyl-1,14-dioxo -2-vinyl-16H-tripyline-8,12-dipropionic acid (1,14 , 15, 17-tetrahydro-3,7,13-trimethyl-1,14-dioxo-2-vinyl-16H-tripyrrin-8,12-dipropionic acid). Therefore, the anti-bilirubin antibody of the present invention may be an antibody obtained by immunizing an animal using these bilirubin oxidation products as an antigen, that is, an anti-bilirubin oxidation product antibody or an anti-biopyrin antibody.

The labeled body used in the present invention can be obtained by binding biopyrin to a polymer substance and labeling this conjugate with a labeling substance. The biopyrin used here may be one that competes with biopyrin in the test sample, may be bilirubin itself, or may be any one of the bilirubin oxidation products, and a mixture thereof. There may be. As the high-molecular substance constituting the bound product, it is preferable to use a biopolymer such as a protein, particularly a protein having many hydrophilic groups on the surface. Examples of such proteins include serum albumins such as human serum albumin and bovine serum albumin and albumins such as egg albumin, keyhole rimpet hemocyanin, bovine gamma globulin, and the like. As a method for binding biopyrine and a polymer substance, an acid anhydride method described in JP-A-1-175945 can be used.

The labeling substance used for labeling is not particularly limited, and examples thereof include a color labeling substance, an enzyme labeling substance, a radiation labeling substance, and the like. Is preferred. Examples of the color labeling substance include colloidal metal and colored latex. Typical examples of colloidal metals include platinum colloids and gold colloids. The size of the colloidal metal particles is usually about 3 to 100 nm in diameter. Representative examples of the colored latex include synthetic latex such as polystyrene latex colored with respective pigments such as red and blue. Natural latex such as natural rubber latex can also be used as the latex. The size of the colored latex can be selected from a diameter of several nanometers to several hundred nanometers. Commercially available products can be used as they are for these color labeling substances, but they can be further processed in some cases or can be produced by methods known per se. Moreover, a fluorescent label etc. can also be used as a color label.

Labeling of the bound substance is performed according to a conventional method corresponding to each colored labeling substance. For example, when the color labeling substance is gold colloidal particles, usually, the combined substance and the colloidal gold solution are physically mixed by mixing the mixture and the gold colloid solution at room temperature to room temperature for several minutes, at most 10 minutes. It is possible to combine them.

According to the present invention, an anti-bilirubin antibody is immobilized in advance on a predetermined position of a membrane carrier to form a capture site, a labeled body of a conjugate of biopyrin and a polymer substance is prepared, and a test sample and the labeled body are prepared. The biopyrin in the test sample and the label are competitively captured at the capture site by immunochromatographic development of the mixed solution on the membrane carrier. Measurements can be performed easily and quickly in clinical situations without the need for equipment and skill.

A measuring apparatus that can be used for carrying out the immunochromatographic measuring method of the present invention can be easily carried out according to the configuration of a known immunochromatographic test strip. Specific examples thereof will be described below with reference to the drawings.

As a specific example of the immunochromatographic test strip, for example, a test strip 10 shown in FIG. In FIG. 1, numeral 1 is an adhesive sheet, 2 is an impregnation member, 3 is a membrane carrier, 4 is a capture site, 5 is an absorption member, and 6 is a sample addition member.

In the example shown in the figure, the membrane carrier 3 is composed of a strip-shaped nitrocellulose membrane filter having a width of 5 mm and a length of 36 mm, and is attached to the middle of the adhesive sheet 1 having the same width. On the membrane carrier 3, its chromatographic starting point side, that is, the left side in FIG. 1 (hereinafter referred to as “upstream side”. The opposite side, that is, the chromatographic development direction side in FIG. The anti-bilirubin antibody is immobilized at a position of 6.0 mm downstream from the end of the terminal, and a first capture site 41 for competitively capturing the label and biopyrin in the test sample is formed. Furthermore, a so-called control line 42 is provided at a position 14.5 mm downstream from the upstream end of the membrane carrier 3. This control line 42 is for confirming that the reaction has been performed irrespective of the presence or absence of biopyrin as an analysis target substance, and is a substance that immunologically specifically binds to any labeled substance ( It can be formed by immobilizing (except for the substance to be analyzed) on the membrane carrier 3. For example, when a goat antibody labeled as an arbitrary substance coexisting with the test sample is used, the control line 42 can be formed by applying and fixing an antibody against the goat antibody. In this case, examples of the labeling substance used for labeling include the same ones as described with respect to the above-mentioned labeling body.

In the apparatus of FIG. 1, the membrane carrier 3 uses a nitrocellulose membrane filter, but the substance to be analyzed contained in the test sample can be chromatographed and a substance such as an antibody forming the capture site 4 is fixed. As long as it is possible, it may be anything, and other cellulose membranes, nylon membranes, glass fiber membranes and the like can also be used.
In the apparatus of FIG. 1, as described above, an antibody obtained using bilirubin as an antigen can be used as the anti-biopyrin antibody.

The labeled body is mixed with a test sample and a developing solvent in an appropriate container separate from the immunochromatographic test strip 10 to prepare a mixed solution, and this mixed solution is used for adding a sample to the illustrated immunochromatographic test strip 10. The membrane carrier 3 may be chromatographed by injection into the member 6. However, when a mixed solution prepared by mixing the test sample and the developing solvent is injected into the sample addition member 6, the immunochromatography is performed so that the label is mixed with the mixed solution and chromatographed on the membrane carrier 3. It is preferable to arrange it integrally with the method test strip 10. For this purpose, in the apparatus of FIG. 1, the marker is impregnated in the impregnating member 2 connected to the upstream end of the membrane carrier 3 to constitute a part of the immunochromatographic test strip 10.

In the illustrated example, a 5 mm × 15 mm strip-shaped glass fiber nonwoven fabric is used as the impregnating member 2, but is not limited thereto, and examples thereof include cellulose cloth (filter paper, nitrocellulose membrane, etc.), polyethylene, Porous plastic cloths such as polypropylene can also be used.
Further, the impregnated member 2 is impregnated with the labeled body in a pre-labeled state with a substance that reacts with an antigen and an antibody immobilized on the control line 42 so that the control line 42 can confirm that the reaction has been completed. Has been.
The impregnated member 2 can be produced by impregnating a member such as the glass fiber nonwoven fabric with a suspension containing both the marker and the substance captured by the control line, and drying it.

As shown in FIG. 1, the immunochromatographic test strip 10 has a membrane carrier 3 stuck in the middle of the pressure-sensitive adhesive sheet 1, and on the upstream end of the membrane carrier 3 on the downstream side of the impregnating member 2. The ends can be overlapped and connected, and the upstream portion of the impregnating member 2 can be adhered to the pressure-sensitive adhesive sheet 1.
Furthermore, in the apparatus of FIG. 1, while mounting the downstream part of the sample addition member 6 on the upper surface of the impregnation member 2, the upstream part of the sample addition member 6 is adhered to the adhesive sheet 1, In addition, the upstream portion of the absorbing member 5 is placed on the upper surface of the downstream portion of the membrane carrier 3, and the downstream portion of the absorbing member 5 is attached to the adhesive sheet 1 so that the immunochromatographic test strip 10 is attached. It is composed.

As the sample addition member 6, for example, a porous synthetic resin sheet or film such as porous polyethylene and porous polypropylene, and a paper or woven or non-woven fabric made of cellulose such as filter paper and cotton cloth are used. be able to.
The absorbent member 5 may be made of any material that can absorb and hold liquid quickly, and examples thereof include cotton cloth, filter paper, and porous plastic nonwoven fabric made of polyethylene, polypropylene, etc., and filter paper is particularly suitable. is there.

The immunochromatographic test strip 10 of FIG. 1 can be used as it is as a dipstick type measuring device, or alternatively, it can be provided as a dipstick type measuring device lined or sandwiched with a suitable plastic sheet. . Preferably, the immunochromatographic test strip 10 of FIG. 1 is a measuring device housed in a suitable plastic case having a test sample injection part and a judgment part opened above the sample addition member 6 and the capture site 4 respectively. Provided. As described above, in the present invention, the labeled body is mixed with a test sample and a developing solvent in a suitable container separate from the immunochromatographic test strip 10, and then the mixed liquid is subjected to an immunochromatographic method. Since the membrane carrier 3 may be chromatographed by being injected into the sample addition member 6 of the test strip 10, when the immunochromatographic test strip 10 is accommodated in the case, the marker is impregnated in the impregnation member 2. It may be housed inside the case, or it may be housed in a suitable container separate from the case and housed outside the case.

Thus, a test sample composed of a biological sample of a subject is mixed with an appropriate developing solvent as necessary to obtain a mixed solution that can be chromatographed, and the mixed solution is then subjected to the immunochromatographic test strip shown in FIG. When injected onto the sample addition member 6, the mixed solution passes through the sample addition member 6 and mixes with the marker in the impregnation member 2.
At this time, if biopyrine is not present in the mixed solution, the labeled substance is chromatographed in the membrane carrier 3 to reach the capture site 4 and the anti-bilirubin antibody and antigen immobilized on the first capture site 41. Since it is captured by the antibody reaction, the first capture site 41 exhibits a strong signal. On the other hand, if bipyrin is present in the mixed solution, biopyrin and the labeled body are competitively captured at the first capture site 41, and thus the first capture site 41 exhibits a weak signal. At the same time, it can be confirmed that the chromatographic development has been performed normally by the second capture site 42 presenting a signal. Thus, by detecting the signal level of the first capture site and the signal level of the second capture site, the presence or absence of biopyrin in the biological sample can be identified.
If the colored labeling substance such as a colloidal gold is used as the labeling substance, the first and second capturing parts 41a and 41b are colored by the accumulation of the colored labeling substance. It can be judged immediately.

The test sample is not particularly limited as long as it is a biological sample from which biopilin is excreted, and examples thereof include blood (whole blood, serum, or plasma) in addition to urine. Urine is preferable because it can be collected without giving a stimulus. The test sample may be diluted with an appropriate diluent such as a developing solvent and injected into the membrane carrier. When whole blood is used as a test sample, and particularly when a colored labeling substance such as gold colloid is used as the labeling substance, it is preferable to arrange a blood cell capturing membrane member on the sample addition member. The blood cell trapping membrane member is preferably laminated between the impregnation member and the sample addition member. This prevents red blood cells from being developed on the membrane carrier, so that it is easy to confirm the accumulation of the colored label at the capture site of the membrane carrier. As the blood cell capturing membrane member, a carboxymethylcellulose membrane is used. Specifically, ion exchange filter paper CM (trade name) sold by Advantech Toyo Co., Ltd. or ion exchange cellulose sold by Whatman Japan Co., Ltd. Paper or the like can be used.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (Preparation of anti-bilirubin antibody)
According to the method described in JP-A-1-75945, the separated bilirubin and bovine serum albumin were covalently bound by the acid anhydride method to obtain bovine serum albumin-bound bilirubin.
Mice were immunized using this bovine serum albumin-bound bilirubin as an antigen, and monoclonal antibodies were prepared by a conventional method.
As a result, it was confirmed that the obtained monoclonal antibody 24G7 recognizes the bilirubin site, that is, the epitope shown in the frame in the following formula. This epitope is also present in several bilirubin oxidation products.

Example 2 ( Preparation of bovine serum albumin-bound bilirubin (BR-BSA))
The binding of bilirubin to bovine serum albumin (hereinafter referred to as “BSA”) was performed by slightly modifying the mixed anhydride method. That is, equimolar numbers of isobutyl chloroformate and tributylamine were added to bilirubin obtained in the same manner as in Example 1, and stirred at 20 ° C. or lower for 30 minutes. BSA was added to adjust the pH to 8.5, and the mixture was reacted at 4 ° C. with stirring for 4 hours. The reaction solution was dialyzed against water and then freeze-dried to obtain BR-BSA powder.
The obtained BR-BSA had about 39 molecules of bilirubin bound to 1 BSA molecule.

Example 3 (Preparation of colloidal gold-labeled BSA-bound bilirubin)
(1) Preparation of gold colloid solution To 99 ml of ultrapure water boiled by heating, 1 ml of 1% (v / w) chloroauric acid aqueous solution was added, and then 1% (v / w) ) 1.5 ml of an aqueous sodium citrate solution was added and heated to boil for 5 minutes, then allowed to cool at room temperature. Subsequently, 200 mM potassium carbonate aqueous solution was added to this solution to adjust to pH 9.0, and ultrapure water was added thereto to make a total volume of 100 ml to obtain a gold colloid solution.

(2) Preparation of colloidal gold-labeled BSA-bound bilirubin 3.5 ml of colloidal gold and 140 μl of BSA-bound bilirubin obtained in Example 2 at 0.25 mg / ml were mixed and allowed to stand at room temperature for 2 minutes to bind this BSA. After binding bilirubin to the gold colloidal particle surface, add 10% BSA aqueous solution so that the final concentration in the gold colloidal solution is 1%, and block the rest of the gold colloidal particles with this BSA, Colloid-labeled BSA-conjugated bilirubin antigen was prepared. This solution was centrifuged (7000 rpm, 25 ° C., 25 minutes) to precipitate colloidal gold-labeled BSA-bound bilirubin antigen, and the supernatant was removed to obtain colloidal gold-labeled BSA-bound bilirubin antigen. This gold colloid-labeled BSA-bound bilirubin antigen is suspended in 50 mM Tris-HCl buffer (pH 7.4) containing 10% saccharose, 1% BSA, and 0.5% Triton-X100. Got.

Example 4 (Preparation of gold colloid-labeled goat IgG antibody)
(1) Preparation of colloidal gold solution The gold colloid solution was prepared in the same manner as in Example 3.
(2) Preparation of gold colloid-labeled goat IgG antibody
After mixing 14 μl of 0.25 mg / ml goat IgG antibody and 3.5 ml of colloidal gold solution and letting it stand for 2 minutes at room temperature to bind all of this antibody to the gold colloid particle surface, the final concentration in the colloidal gold solution is 1 10% bovine serum albumin (BSA) aqueous solution is added so that the remaining surface of the gold colloidal particles is blocked with this BSA, and a gold colloid-labeled goat IgG antibody (hereinafter referred to as “gold colloid-labeled antibody”) is added. A solution was prepared. This solution was centrifuged (7000 rpm, 25 ° C., 25 minutes) to precipitate the colloidal gold labeled antibody, and the supernatant was removed to obtain a colloidal gold labeled antibody. The colloidal gold labeled antibody was suspended in a 50 mM Tris-HCl buffer (pH 7.4) containing 10% sucrose, 1% BSA, 0.5% Triton-X100 to obtain a colloidal gold labeled antibody solution.

Example 5 (Preparation of immunochromatographic test strip for biopyrin detection)
(1) Preparation of gold colloid label impregnated member
Gold colloid-labeled BSA-bound bilirubin obtained in Example 3 (solid-phase amount 10 μg / ml) 30 μl and a colloidal gold-labeled goat IgG antibody obtained in Example 4 were applied to a 5 mm × 20 cm strip-shaped glass fiber nonwoven fabric. (Immobilized amount 1 μg / ml) 15 μl was impregnated, dried at room temperature and cut into a size of 5 mm × 15 mm to obtain a colloidal gold impregnated member.

(2) Formation of test line 0.5 μl of the solution containing 10 mg / ml of anti-bilirubin monoclonal antibody 24G7 obtained in Example 1 was added to the end of the chromatographic development start side in membrane support 3 for chromatographic development shown in FIG. It was applied on a line at a position of 6.0 mm from the part and dried at room temperature to form a first capture site 41 for capturing biopyrin or gold colloid-labeled BSA-bound bilirubin.

(3) Formation of control line 0.5 μl of the solution containing the anti-goat IgG polyclonal antibody was placed on the line at a position 14.5 mm from the end of the chromatographic development start side in the membrane carrier 3 for chromatographic development of (2) above. It was applied and dried at room temperature to form a second capture site 42 that captures the gold colloid-labeled goat IgG antibody.

(4) Production of immunochromatographic test strip As shown in Fig. 1, on the adhesive sheet 1, the membrane carrier 3 obtained in (3) above, and the impregnated member obtained in (1) above 2, an absorption member 5 and a sample addition member 6 were arranged to produce an immunochromatographic test strip.

Example 6 (Measurement using a standard reagent)
The BSA-bound bilirubin obtained in Example 2 is diluted with a specimen diluent and prepared to a predetermined concentration (0 u / l, 0.8 u / l, 3.2 u / l, 12.8 u / l) did. Then, 100 μL of this test sample was dropped on the sample addition member 6 of the immunochromatographic test strip obtained in Example 5 with a micropipette, and 15 minutes later, the amount captured at the first capture site 41 was observed with the naked eye. The results are shown in Table 1.

From Table 1, it was shown that as the antigen concentration in the sample increased, the coloration on the test line became weaker, and the amount of biopyrin could be determined by the method of the present invention.

Example 7 (Measurement using urine of a healthy person)
Three types of urine collected from three healthy subjects were used to examine changes in measured values between urine samples.
That is, the BSA-bound bilirubin obtained in Example 2 was diluted with each urine specimen and prepared to a predetermined concentration (0 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, 80 μg / ml) to be tested. Liquid. As a control, the same BSA-conjugated bilirubin was diluted with a buffer solution (Buffer) and prepared to the prescribed concentrations (0 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, 80 μg / ml). It was. Then, 100 μL of this test sample was dropped on the sample addition member 6 of the immunochromatographic test strip obtained in Example 5 with a micropipette, and 15 minutes later, the amount captured at the first capture site 41 was observed with the naked eye. The results are shown in Table 2.

Table 2 shows that although there is a slight difference in reactivity depending on the urine sample, the reactivity is linked with the control, and there is no non-specific reaction due to the urine sample. Thereby, it was shown that there is no difference between samples when measuring biopyrin by the method of the present invention.

Example 8 (Measurement 1 Using Patient Specimen)
Urine was collected from patients in their 70s who underwent thoracoscopic surgery for 7 days, and ELISA and immunochromatography were compared. The ELISA method was performed using a commercially available kit (trade name: Biopyrrin EIA Kit, manufactured by Shino Test Co., Ltd.). The immunochromatography method was performed in the same manner as in Example 7 except that urine collected from a patient was directly dropped onto the sample addition member 6 of the immunochromatography test strip as a test sample solution. For the sample, the amount of creatinine in urine was measured with an automatic analyzer (Hitachi H7150) and corrected. The results are shown in Table 3 and FIG.

From Table 3 and FIG. 2, it was shown that the biopyrin measurement values of the ELISA method and the immunochromatography method correlated well. In addition, compared with ELISA, immunochromatography showed a tendency to show significant fluctuations in the amount of biopyrine.

Example 9 (Measurement 2 Using Patient Specimen)
Urine was collected from patients in their 50s who underwent thoracotomy for 10 days, and ELISA and immunochromatography were compared. The ELISA method was performed using a commercially available kit (trade name: Biopyrrin EIA Kit, manufactured by Shino Test Co., Ltd.). The immunochromatography method was performed in the same manner as in Example 7 except that urine collected from a patient was directly dropped onto the sample addition member 6 of the immunochromatography test strip as a test sample solution. For the sample, the amount of creatinine in urine was measured with an automatic analyzer (Hitachi H7150) and corrected. The results are shown in Table 4 and FIG.

From Table 4 and FIG. 3, it was shown that the biopyrin measurement values of the ELISA method and the immunochromatography method correlated well. In addition, compared with ELISA, immunochromatography showed a tendency to show significant fluctuations in the amount of biopyrine.

Since the present invention can measure biopyrin easily and quickly in a clinical scene without requiring special equipment and skilled techniques, it is useful for diagnosis of a patient's condition in a medical field and checking of a person's health condition.

a is a top view which shows the specific example of the immunochromatography measuring apparatus of this invention, b is a longitudinal cross-sectional view of the apparatus shown by a. 10 is a graph showing the results of Example 8. 10 is a graph showing the results of Example 9.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Adhesive sheet 2 Impregnation member 3 Membrane carrier 4 Capture part 5 Absorption member 6 Sample addition member 10 Immunochromatography test strip

Claims (10)

A membrane carrier having a capture site formed by preliminarily fixing an anti-bilirubin antibody at a predetermined position, and a labeled body in which a conjugate of biopyrin and a polymer compound is labeled with a labeling substance are prepared. And by allowing the mixture and the biopyrin in the test sample to be competitively captured at the capture site by allowing the mixture to be chromatographed on the membrane carrier toward the capture site. An immunochromatographic assay for detecting biopyrine. The method according to claim 1, wherein the anti-bilirubin antibody is a monoclonal antibody. The mixed solution further includes a substance labeled with a labeling substance, and the membrane carrier further includes another capture site formed by immobilizing a substance that immunologically binds to the substance. The measurement method according to claim 1. The measurement method according to claim 1, wherein the labeling substance is a metal colloid or latex. The measurement method according to claim 1, wherein the membrane carrier is a nitrocellulose membrane. It comprises at least an anti-bilirubin antibody, a labeled product obtained by labeling a conjugate of biopyrine and a polymer compound with an appropriate standard substance, and a membrane carrier. The anti-bilirubin antibody is previously immobilized on the membrane carrier to form a capture site. An immunochromatography measuring apparatus for biopyrin detection, wherein the labeled body is prepared so that it can be chromatographed on the membrane carrier from a position separated from the capture site toward the capture site. The measurement apparatus according to claim 6, wherein the anti-bilirubin antibody is a monoclonal antibody. Further, a substance labeled with a labeling substance is provided, and the labeled substance is prepared so that it can be chromatographed on the membrane carrier from a position separated from the capture site toward the capture site, The measuring apparatus according to claim 6, wherein the carrier further comprises another capture site formed by immobilizing a substance that immunologically binds to the substance. The measuring apparatus according to claim 6, wherein the labeling substance is a metal colloid or latex. The measuring apparatus according to claim 6, wherein the membrane carrier is a nitrocellulose membrane.

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