MXPA97001443A - Assay and equipment method for using the ens - Google Patents

Abstract

In a test method which uses a chromatography strip, which has a binding region in a position where a retention substance 2 capable of binding to an analyte specifically, is immobilized on its chromatography carrier and in which a tracer formed of a retention substance 1, capable of binding retention substance 2 in competition with the analyte specifically and a marker, is placed in an area upstream of the binding region in advance or is added at the time of sample addition , in such a way that its development can be effected, the gelatin is added in an area upwards of the binding region at the time of the addition of the sample or placed in advance on the

Description

ß ASSAY METHOD AND EQUIPMENT FOR USING THE TEST DESCRIPTION This invention relates to a test method and equipment for using the assay, more particularly to a test 5 test method in which a chromatography strip and equipment are used to use the test to be used in the test method. A method in which a chromatography strip is used, is known as a quick and easy test method.

As described in Japanese Patent Application Kokai No. 60-19226, Japanese Patent Application Kokai No. 1-63865 and Japanese Patent Application Kokai No. 3-176659, the chromatography strip has a chromatography carrier in which, for example, a tracer consisting of a A substance capable of specifically binding to an analyte, called retention substance 1 in the present invention and a label is arranged in such a way that its chromatographic development can be carried out or in which a tracer is added at the time of the addition of a sample and one The substance capable of specifically binding to an analyte, called the retention substance 2 in the present invention, is immobilized on its downstream region. When a sample solution is added to a region where the tracer is disposed on the chromatography carrier or a The tracer and a sample solution are added to the carrier of ß the chromatography at the same time, an analyte binds to the tracer and the tracer to which the analyte is bound and the tracer to which the analyte is not bound, both are revealed in the longitudinal direction, namely in the direction 5 downwards. When the tracer reaches a region where the retention substance 2 is immobilized, only the analyte (to which the tracer is attached) joins the retention substance 2 and the tracer is accumulated therein, although the tracer to which the analyte it is not united, it flows 10 out in the downward direction. In this way, the presence of the analyte and its amount in the aqueous sample solution can be found by the development of color or the like in the region where the retention substance 2 is immobilized. In addition, in relation to the present invention, the Japanese Patent Application Kokai No. 3-176659 describes covering approximately the entire portion or a part of a region, called the marker region in the present invention, with gelatin. Such a type of test method in which a chromatography strip is used, can perform the test within a short period of time and its handling is easy, but still requires improvements such as further increase in detection sensitivity of the analytes In consecuense, The problems to be solved by the present invention are to provide a test method in which a chromatography strip is used and the detection sensitivity and the like, are improved and to provide equipment for use in testing, which it is used in the test method in which detection sensitivity and the like are improved. The inventors of the present invention have found that when gelatin is added to a specified position of a chromatography carrier at the time of 10 addition of the sample or arranged in advance on a specified position of the chromatography carrier, various points of the test methods and equipment for using the prior art assay can be improved, for example, the detection sensitivity 15 considerably increased, the detection time can be shortened and the pro-zone phenomenon in the interleaved method is reduced. Accordingly, an aspect of the present invention is a test method which uses a strip of 20 chromatography, in which its chromatography carrier has at least one binding region in which a retention substance 2, capable of binding to an analyte, is immobilized directly or indirectly by means of a retention substance 3, capable of join both of the analyte and the substance 2 or in which a retention substance 4 capable of specifically binding to a retention substance 1, which binds specifically to the analyte or to the retention substance 2 in competition with the analyte, is immobilized in a downstream area of the retention substance. the region where the retention substance 2 is immobilized and a tracer consisting of the retention substance 1 and a marker is disposed on an upward area of the binding region in advance or added at the time of the addition of a sample, in such a way that the tracer can be revealed chromatographically; which comprises adding gelatin to an area upstream of the binding region at the time of addition of a sample or arranging the gelatin in advance in an area upstream of the binding region, but excluding a region where the tracer is arranged in advance (which will be mentioned as the marker region from now on). Another aspect of the present invention is a kit for using the assay, which comprises at least the chromatography strip mentioned above and gelatin or a chromatography strip with the gelatin disposed, in which the gelatin is available in advance in an area upstream of the binding region of the chromatography carrier mentioned in the above, but excluding the marker region.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an illustration describing the chromatography strip. Figure 2 is a graph showing the results of an assay carried out by the assay method of the present invention (Example 2). Figure 3 is a graph showing the results of an assay carried out by the assay method of the present invention (Example 3). The analyte whose presence or amount in a sample is detected by the present invention, is not particularly limited with the proviso that any compound which binds specifically to the analyte is present. Their examples include a compound in which biotin is Introduced (to which avidin binds specifically), a compound which contains avidin, complementary DNA, a compound or hapten which has antigenicity, a compound which binds specifically to antibodies of a certain antigen, an antibody, a antibody fragment which 20 binds specifically to its antigen, lectin which binds specifically to a sugar ligand and a sugar ligand which binds specifically to the lectin, as well as a ligand which binds specifically to a receptor and a receptor, which binds specifically to a ligand. As 25 something routine, the detection sensitivity becomes * high when these binding reactions have high reactivity or affinity. Their illustrative examples include microorganisms; virus; antigens such as specific antigens of pathogenic microorganisms, viruses and the like 5 and prostate cancer tumor marker antigens and the like; antibodies such as those which are induced by microbial, viral and similar infections, autoantibodies and similar antibodies and antibodies specific for acute phase and protein antigens. 10 similar; fecal hemoglobin or hemoglobin specific antigen; cells such as a cell which carries a specific antigen, a cell which has a specific sugar ligand and a cell which has a specific receptor; hormones such as thyroid hormone, hormone 15 parathyroid, hormone that stimulates gonads and the like and physiologically active proteins or peptides such as cytokine, monoclin and the like; proteins or glycoproteins such as enzymes, proteoglycan, gamma-seminoprotein, amyloid precursors and the like; chemical substances 20 low molecular weight or drugs in blood; factors that control signal transfer such as agonists, antagonists and the like; factors that control blood clotting / thrombolysis; factors that control the expression of the gene; cell adhesion molecules; 25 lectins such as immunomodulatory lectin and the like; and »RNA, DNA and similar nucleic acids. Particularly, faeces, urine and the like are advantageous as non-invasive samples. The retention substance 1 is formed of a Compound which binds specifically to an analyte by retaining it or which performs a specific binding (retention of) to a retention substance 2, which will be described later, in competition with an analyte. Is * say, when the analyte is an antigen, the substance of Retention 1 is an antibody or fragment of the antibody, which binds specifically to the antigen (hereinafter, the term "antibody" includes fragments of antibody which specifically bind to the antigen) or an antigen or a compound which binds to an antibody Specifically (hereinafter, the term "antigen" includes compounds which specifically bind to the antibody), and is an antigen or antibody when the analyte is an antibody. Also, when the analyte is a ligand (or a compound that has a ligand), the substance 1 of Retention is a receptor (or a compound that has a receptor) or a ligand, and is a ligand or a receptor when the former is a receptor; in some cases, retention substance 1 performs a specific binding to a retention substance 3 by means of an analyte, resulting in its Link to the retention substance 2 by means of the retention substance 3. The linkage of an analyte with the retention substance 1 can be either reversible or irreversible, but the sensitivity of the detection decreases when the affinity is too low. 5 A marker is added to the retention substance 1 to form a tracer. In addition to a marker for the retention substance 1, it can be carried out by the absorption or inclusion of the marker with the retention substance 1 or by its linkage via the hydrogen bond or linkage. 10 covalent. In brief, a marker can be added, such that retention substance 1 is not easily released from it and can be recognized by an analyte or retention substance 2. When retention substance 1 is bound to a The analyte, the retention substance 2, which will be described later or the retention substance 4, which will also be described later, the marker generates a signal, namely a color, which indicates its presence. Preferably, the marker can be formed of microparticles which 20 are insoluble in water and have little water repellency. Examples of desirable markers include gold colloid and similar metal colloids and selenium colloid and similar non-metal colloids; organic colloids; coloring particles; colored polystyrene and organic polymers 25 similar color; and similar oil microparticles and liposomes, in which a color agent is encapsulated. When colloids and the like do not reveal color, a coloring agent is added. Examples of the coloring agent include those which reveal a color in the visible region, as well as fluorescent materials and substances which reveal color in the ultraviolet region. The substances which reveal color, include fluorescence by enzyme and the like, arranged in a binding region or the like are also included 10 in the coloring agent of the present invention. The tracer, either by itself or in bound form to an analyte, is revealed and displaced in the chromatography carrier when a sample solution is added to the chromatography strip. The plotter is used as a 15 solution (or a suspension), but can be preserved as a powder by lyophilization or a similar means. There are two methods for arranging the tracer in such a way that its chromatographic development can be effected in the chromatography carrier with an aqueous solvent. At The first method, a section of the chromatography carrier, which has been impregnated with an aqueous tracer solution and then dried is disposed in advance in a base material or the chromatography carrier in an upward position of a binding region, the which will be described later, 25 forming an appropriate distance from the link region.

* In this case, the position where the plotter is arranged, is called the marker region. In the second method, the tracer is added to the chromatography carrier at the time of adding the sample. In this case, therefore, the chromatography strip does not have a marker region. The retention substance 2 binds to the tracer, which has an analyte by means of the analyte, or to the tracer which has the retention substance 1 by means of the retention substance 1. In this latter case in which its The binding is effected by means of the retention substance 1, it is bound to the retention substance 2 in competition with the analyte. In any case, the retention substance 2 binds the analyte specifically. In another way, you can join an analyte indirectly by its specific link to the 15 retention substance 3, which binds specifically to the analyte. An illustrative example of retention substance 2, is an antibody (as defined above, the term "antibody" as used herein also includes an antibody fragment, which binds to the antibody. 20 antigen specifically) when the analyte is an antigen. When the retention substance 2 is an antibody and the retention substance 1 is also an antibody, it is advantageous that they recognize different epitopes of the antigen to be detected. In the same way, it is an antigen (like 25 defined above, the term "antigen" as used herein, also includes a compound which binds to an antibody specifically) when the analyte is an antibody, the former is a ligand when the latter is a receiver and the first is a receiver when the latter is a 5 ligand. In addition, when the retention substance 2 binds to the retention substance 3 specifically, avidin, antibiotin antibody, lectin and the like can be exemplified as the retention substance 2. The immobilization of the retention substance 2 10 on the chromatography carrier can be effected either by covalent bond between an appropriate functional group of the chromatography carrier and a functional group of the retention substance 2 or by absorption of the retention substance 2 strongly to the chromatography carrier.

Alternatively, the retention substance 2 may be bound or absorbed to microparticles, which are then dispersed and immobilized in the chromatography carrier. In this regard, the term disposition of the means of immobilization of a substance, in such a way that 20 is substantially not dispersed or developed by aqueous solvents. The area where the retention substance 2 is immobilized is called the binding region. When the retention substance 1, the retention substance 2, the retention substance 3 or the substance of 25 retention 4 is an antibody, in most cases a monoclonal antibody is used, but even a serum antibody can be used if it is necessary to carry out appropriate means such as absorption treatment, selection of antigen or animal species and Similar. The retention substance 3 is a conjugate body, which comprises a portion that binds to an analyte specifically and another portion that binds to the substance of ß retention 2 specifically. As described in detail in relation to the retention substance 1, the portion that is It binds an analyte specifically, it is an antigen when the analyte is an antibody, an antibody when the latter is an antigen, a receptor when the latter is a ligand or a ligand when the latter is a receptor. The portion that binds to the retention substance 2 is specifically, for For example, biotin, when the retention substance 2 is avidin, antibiotin antibody or avidin when the latter is biotin or a sugar ligand when the latter is lectin. The retention substance 3 is disposed in advance in any region optionally selected in the 20 area upwards of the binding region of the chromatography carrier, such that it can be developed and displaced in the chromatography carrier when an aqueous sample solution is added to the chromatography strip, or is added to the chromatography carrier in the chromatography carrier. moment of the 25 addition of the sample. The layout method is as described in detail in relation to the tracer layout. The retention substance 4 binds to the retention substance 1 specifically. The chromatography carrier is not particularly limited, with the proviso that it can reveal solutes with an aqueous solvent. For example, it may consist of a nonwoven fabric made of cellulose, nitrocellulose, F cellulose acetate, polyacrylate, glass, agarose, 10 polyurethane or the like or a combination thereof with an inorganic powder. The chromatography strip is obtained, for example by laminating the chromatography carrier onto an appropriate base material when necessary. As is universally known, gelatin is obtained from vertebrates such as fish, mammals and the like. The melting temperature of the gelatin gel is preferably 15 ° C or more, more preferably 25 ° C or more. According to the invention, the The melting temperature of the gelatin gel is measured by the following method. The gelatin is dissolved in a 10 mM phosphate buffer (pH 7.4) containing 0.9% sodium chloride at a final concentration of 10 g / dL. A 1 ml portion of the The gelatin solution prepared in this manner is placed in a test tube 100 mm in length and 12.5 mm in diameter, and the test tube is sealed with a stopper. The test tube is completely soaked in a 0 ° C water bath to effect the gelation of the gelatin. Then, the temperature of the water bath is slowly increased at a rate of 2 minutes by 1 ° C to measure the first drop temperature of the gelatin gel, when the test tube is turned over. Gelatin is used when a sample solution is added or placed in advance on the carrier 10 chromatography. When used at the time of sample addition, it can be dissolved in the sample solution or added separately from the sample solution as an aqueous gelatin solution. As the aqueous solvent to be used in the preparation of the gelatin solution As it is aqueous, it is generally advantageous to use the same aqueous solvent used in the preparation of the sample solution. When gelatin is used at the time of sample addition, the gelatin concentration in the gelatin solution is preferably within the range of 20 0.1 to 1.5% by weight. When the gelatin is placed beforehand, it is also advantageous to place the gelatin beforehand in a quantity of% by weight mentioned in the foregoing by solution of the sample. When the gelatin is added at the time of When adding a sample solution, it is added to a ß position upwards of the binding region. When the gelatin is arranged in advance, it is arranged in a position above the binding region, but excluding the marker region. As the illustrative addition or disposition position 5, the most suitable position is optionally selected in advance and more excellent results are obtained in some cases, when placed in region 7. When the chromatography strip disposed with gelatin in which the gelatin is arranged on the carrier d The chromatography in advance is placed in a position selected in advance within the upstream area of the binding region but excluding the marker region so that it can be developed with an aqueous solvent. And to say, a section of the chromatography carrier, which Once it has been impregnated with an aqueous solution of gelatin and then dried, it is placed in a base material or the chromatography carrier. Also, when the retention substance 4, which binds to the retention substance 1 is immobilized Specifically in the downward area of the chromatography carrier where the retention substance 2 is attached, to prepare the binding region, it is advantageous to add gelatin in advance to, or over an area between the region where the retention substance 2 the region where the retention substance is immobilized is immobilized. When the gelatin is used by other methods than those of the present invention, for example, when the gelatin is placed in the marker region in advance, it causes prolonged detection time. , decreased detection sensitivity and similar problems in many cases. On the other hand, when gelatin is used, for example in ß at the time of sample addition, such problems do not 10 occur even if it is added to the marker region. The reason for this is that when the gelatin is placed on the marker region in advance, the gelatin exerts certain influences on the marker region, although it retains the chromatography strip. The aqueous solvent of the sample solution may be water, but a buffer is generally used. The aqueous solvent may have a pH value within the range of preference of 5 to 9. The sample solution is added to the region Marker or an area upstream of the region, when the marker region is present or in an area upstream of the binding region, when the marker region is not present. The term "addition" as used herein, means a case in which the solution of the sample is 25 places on the chromatography strip or a case in which most of the tip upwards of the chromatography strip is soaked in the solution of the sample. When the tracer and / or the retention substance 3 are not placed in advance in the chromatography carrier, these can be added at the time of the addition of the sample. With regard to their method of addition, they are usually added together with the sample. The equipment for using the assay of the present invention includes two boxes. In the first box, at least one first chromatography strip that does not have gelatin placed in advance and the gelatin is used in combination. In this case, an aqueous solvent and the like can be used additionally in combination. Also, the gelatin can be dissolved or not dissolved in the aqueous solvent. The second box is formed of a chromatography strip with placed gelatin. Also in this case, an aqueous solvent and other similar constituents can be used in combination. In any of the foregoing cases, the tracer and / or retention substance 3, when not disposed on the chromatography carrier in advance, can be used in the form of powder prepared by lyophilization or a similar medium or as an aqueous solution. (or a suspension).

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is further described with reference to the drawings. Figure 1 represents a chromatography strip in which the marker region is placed. The chromatography strip 1 is formed from the chromatography carrier 2 and, as the occasion demands, a base material. The chromatography carrier 2 has the marker region 3, which is arranged in such a way that the tracer can be developed with an aqueous solvent. The upstream region 5 of the marker region 3 is arranged as the occasion demands and becomes the addition position of the sample solutions in many cases when placed. This region can perform the filtration of the suspended materials in the sample solution and can also contain auxiliary substances, effective to carry out the test uniformly. Examples of such auxiliary substances include those which can perform the binding reaction of an analyte with the more specific or quantitative retention substance 1, as well as color reagents of insoluble labels and the like. When a sample solution is added to an area upstream of the marker region 3 or to the marker region 3, an analyte contained in the sample, if it can be bound to the retention substance 1, is marked by the tracer. Next, each of the tracer to which the analyte was bound, the remaining tracer to which the analyte did not bind and the remaining analyte is displaced by the chromatographic development 5 towards the right-hand direction in Figure 1, namely the direction towards down and reaches the link region 4 placed in an area downstream of the marker region 3. The marker region 3 and the link region 4 are not directly linked together, but are arranged 10 keeping a. some space which is optionally selected. The region 7, which is used to make this space, can have gelatin placed on it or the marker substance 3 which is disposed on it in such a way that it can perform the chromatographic development. In In many cases, a colored agent of the marker can be additionally placed on it. Link region 4 includes two cases. That is, in the first case, the tracer which binds with an analyte is retained in this region and reveals color or tracer 20 competes with the analyte and is retained in this region to reveal color. In the second case, the retention substance 4 is immobilized on a downward area of a region, where the retention substance is immobilized (this region corresponds to the binding region in the first case 25 mentioned in the above). In this case, the arrangement is optionally made as the occasion demands, but when it is placed and used for the detection of color development, it is used as link region 4. In the second case, the plotter which is not attached and retained by retention substance 2 due to its competition with an analyte is retained by retention substance 4 in this binding region and reveals color. When the retention substance 3 is placed, the β retention substance 3 is bound to an analyte, which is 10 bound to the tracer and the tracer / analyte / retention substance 3 complex formed, is therefore bound to the retention substance 2 by means of the retention substance 3 and is stopped in the binding region 4. Alternatively, the retention substance 3 binds to the retention substance 2 and then binds to an analyte specifically in the binding region 4. The region 6 downstream of the binding region 4, is positioned as the occasion demands, for make flow 20 out the tracer to which an analyte was not bound by means of the retention substance 1, when the retention substance 1 is able to bind with the analyte, or to flow out soluble components to part of the analyte contained in the analyte. Sample solution. In some cases, a The substance which reveals color, when the aqueous solvent of ß an aqueous sample solution is flowed in, such as a pH indicator or the like, is placed in region 6 downwards, thereby making possible the judgment of the reliability of detection. When the chromatography strip does not have the marker region 3, the assay is carried out in the same manner as described above, except that the tracer is added at the time of sample addition. ß Example 1 10 Preparation of the tracer formed from the human anti-hemoglobin antibody labeled with selenium colloid: The selenium colloid is prepared by stirring 91 mM L-sodium ascorbate and 32 mM selenium oxide at approximately 4 ° C for 15 minutes and then at approximately 42 ° C during 15 approximately 70 hours. The selenium colloid thus obtained is diluted with bis-tris lOmM buffer (pH 7.0) to such a concentration that its absorbance at 550 nm becomes 15. The monoclonal antibody of mouse anti-human hemoglobin 20 (0.02%) is added to the diluted preparation and the mixture is stirred at room temperature for 1 hour. Next, the anti-human hemoglobin antibody thus obtained, labeled with selenium colloid, it is washed with 10 mM Tris-HCl buffer (pH 7.2) and used as a tracer. Preparation of the marker region: A tracer suspension is obtained by adding the tracer to a buffer solution of 10 mM Tris-HCl (pH 7.2) containing 1% casein and adjusting the absorbance of the mixture to 1.0 at a wavelength 550 nm. A fiberglass membrane (Lypore 9524, manufactured by LYDALL U.S.A.) is soaked in the suspension prepared in this way to completely impregnate it with the suspension and then the fiberglass membrane dries 10 to be used as a marker region. Preparation of the binding region: The mouse anti-human hemoglobin monoclonal antibody having a binding site, which is different from that of the anti-human hemoglobin antibody mentioned in 15 above, was mixed with 30 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, at a final concentration of 2 mg / ml. Separately from this, a laminate (PET100 PE LR007A, manufactured by Lintech) having a thickness of 100 μm and a rectangular size of 0.4 x 6.0 cm, adheres to 20 a nitrocellulose membrane (manufactured by Schleicher &Schuell, USA) used as a chromatography carrier having a rectangular size of 0.4 x 4.5 cm and a pore size of 5 μm, such that its upper ends are coupled when its longer sides are placed in shape 25 longitudinal. The mixed antibody solution is splashed in a line in a region of approximately 1 cm from the bottom of the nitrocellulose film to which the laminate adhered and then the spots are completely dried to immobilize the anti-human hemoglobin antibody. . Preparation of the chromatography strip: A pad is cut into square size of ß 0.4 x 0.4 cm that has the anti-human hemoglobin antibody labeled with the selenium colloid (marker region) 10 adheres to the laminate on the underside of the nitrocellulose in which the anti-human hemoglobin antibody has been immobilized, so that it slightly contacts the nitrocellulose. As a prefilter, the non-woven hydrophobic fabric (Sontala 8801, manufactured by Du 15 Pont, U.S.A.) is cut in a size of 0.4 x 1.3 cm and adheres to the laminate on the underside of the pad, in such a way that it makes contact with the nitrocellulose slightly, so that a chromatography strip is obtained. 20 Assay: 0.1 M Tris-HCl buffer (pH 7.6) containing 0.1% bovine serum albumin (manufactured by Seikagaku Kogyo, Tokyo), 0.9% sodium chloride and 0.1% sodium azide, supplemented in addition with human hemoglobin (manufactured 25 by Sigma, U.S.A.), is used as the sample. This is added ß pig skin gelatin (manufactured by Sigma, U.S.A) to a final concentration of 0.7%. A 25 μl portion of the sample solution is applied to the pre-filter of the chromatography strip. After 7 minutes of the addition of the sample, the result is judged by observing the "reddish color" caused by the selenium colloid with the naked eye. The minimum concentration of hemoglobin by which the "color ß reddish "can be observed with the naked eye was used as the sensitivity.10 The results of the trial are shown in Table 1. As is evident from Table 1, the sensitivity increased by a factor of approximately 80 times when using gelatin (a) compared to a case in which gelatin (b) was not used Table 1 Example 2 The procedure of Example 1 was repeated, except that the trial is started 1 minute after the addition of the sample until 12 minutes later, at 1 minute intervals.

* The results are shown in Figure 2. As is evident from Figure 2, the test time was shortened to approximately 1/3 when the gelatin was used (line a) compared to a case in which the 5 gelatin (line b). Example 3 The procedure of Example 1 was repeated, except that the concentration of human hemoglobin in the sample solution was changed to the values shown in Figure 3. The results are shown in Figure 3. As is evident from the Figure 3, the upper limit of the measurable human hemoglobin concentration when using gelatin (line a) is approximately 10 times greater than the measurable upper limit when gelatin was not used (line b). In another 15 words, it is clear that the pro-zone phenomenon is reduced when gelatin is used. Example 4 The procedure of Example 1 was repeated, except that human hemoglobin was detected using gelatin preparations shown in Table 2. In this case, the concentration of human hemoglobin is set at 10 ng / ml and the amount of gelatin which is to be added to the sample solution is changed to 1.0%. The results are shown in Table 2. The melting temperature of the gelatin was measured by the method described above. > Table 2 INDUSTRIAL APPLICABILITY By the use of the test method or the test equipment of the present invention, the detection sensitivity of the analytes can be increased. Also, the time required for its detection can be shortened. In addition, the so-called pro-zone phenomenon in the intercalated method is reduced.

Claims (10)

1. > CLAIMS 1. A test method which uses a chromatography strip, in which its chromatography carrier has at least one binding region in which a retention substance capable of binding to an analyte is immobilized directly or indirectly by means of a retention substance capable of binding both of the analyte and the retention substance, or in which a retention substance capable of specifically binding to a retention substance, 10 binds specifically to the analyte or retention substance, in competition with the analyte is immobilized in an area downstream of the region, where the retention substance is immobilized, and a tracer formed from the retention substance and the marker is arranged in a 15 upstream of the binding region in advance or added at the time of addition of a sample, such that the tracer can be revealed chromatographically; The method is characterized in that it comprises adding gelatin to an area upstream of the 20 region at the time of adding a sample or placing gelatin in an upward area of the binding region, but excluding a region where the tracer is placed in advance.
2. 2 . The test method according to claim 1, characterized in that the gelatin gel melts at 15 ° C or more.
3. 3. The test method according to claim 1, characterized in that the gelatin gel melts at 25 ° C or more.
4. 4. The test method according to claim 1, 2 or 3, characterized in that the sample is feces or urine.
5. 5. The test method according to claim 1, characterized in that when a region where the retention substance capable of specifically binding to the retention substance, is immobilized in a downward area of a region where the substance of The retention is immobilized, used as the binding region, the gelatin is added or placed in advance to or in an area between the region where the retention substance is immobilized and the region where the retention substance is immobilized.
6. 6. The equipment for use in an assay characterized in that it comprises at least the chromatography strip according to claim 1 and a gelatin or a chromatography strip with placed gelatin, in which the gelatin is placed in advance in a area 25 upwards of the link region according to claim 1, but excluding a region where the tracer is placed in advance.
7. 7. The equipment for using the test according to claim 7, characterized in that the gel of 5 gelatin melts at 25 ° C or more.
8. 8. The equipment to be used in the test according to claim 7, characterized in that the gelatin gel melts at 15 ° C or more.
9. 9. The equipment to be used in the test according to claim 6, 7 or 8 characterized in that the sample is feces or urine.
10. 10. The equipment to be used in the test according to claim 1, characterized in that when a region where the retention substance capable of 15 specifically binds to the retention substance, is immobilized in a downward area of a region where the retention substance is immobilized, is used as the binding region, the gelatin is added or placed in advance to or over an area between the region where the substance of The retention is immobilized and the region where the retention substance is immobilized.