JPH1048210A - Method for adjusting sensitivity in analysis of specific bond - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sensitivity adjusting technology for immunochromatograph. SOLUTION: Sensitivity is adjusted in the analysis of specific bond using immunochromatograph or the like. A sensitivity adjusting substance labeled with a specific bonding substance unspecifiable from the background and same as that employed in a specifiable labeling substance is caused to react together with the specifiable labeling substance. An analyzer where this method is applied is also provided. According to the method, the sensitivity can be adjusted surely without requiring any additional reaction time and an apparatus where this method is applied can be manufactured easily.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chromatographic analysis utilizing a binding reaction between a ligand and a receptor by specific affinity. More specifically, the present invention relates to a technique that enables control of measurement sensitivity particularly in specific binding analysis. A typical example of the specific binding analysis in the present invention is an immunochromatography method.
Components targeted for specific binding analysis have various characteristics. For example, a component that serves as an indicator of a disease often has clinical significance when detected at a certain concentration or higher. For such a substance, it is necessary to adjust the sensitivity of the reaction system so as not to be positive in a normal range.

Among the specific binding assays, a measurement method using an antigen-antibody reaction is an analysis technique utilizing a high specificity of an immune system, and strictly discriminates substances having similar structures and activities, and has high sensitivity. Can be realized. In particular, if a luminescence reaction or amplification of various signals is used as a label detection system, it is possible to expect even higher sensitivity than the RIA method using a conventional radioisotope as a label. In addition, in a reaction system such as an immunoturbidimetric method or a latex agglutination method, in addition to these measuring systems, a quantitative numerical value can be obtained with high accuracy by performing an optical measurement. In addition, the widespread use of monoclonal antibody preparation technology has enabled production of antibodies having various binding activities in large quantities with uniform quality, so that high specificity and reproducibility can be easily realized.

[0003] Against this background, techniques for measuring antigenic substances, which are indicators of various diseases, based on immunological reactions using antibodies have been widely put into practical use. It is known that measurement of an antigenic substance in a biological sample such as blood, feces, or urine helps to diagnose various diseases and health conditions. Pregnancy is suspected, for example, when detecting chorionic hormone in urine. Hemoglobin in feces is an index for screening for digestive ulcers, tumors and the like.

[0004] Incidentally, immunological analysis methods include RIA,
Apart from obtaining quantitative values by ELISA, immunoturbidimetry, or latex agglutination, qualitative reactions such as slide agglutination and immunochromatography are applied to realize simple tests by simple operations. ing. In particular, the immunochromatography method can perform analysis simply by dropping a liquid serving as a sample, and can easily judge the analysis result. It is useful for effective health care.

[0005]

2. Description of the Related Art The immunochromatography method is based on the following reaction principle. For example, when an antigen is to be detected by a sandwich method, a chromatographic medium having a region (an immobilized region) in which an antibody against the antigen is immobilized is prepared. The chromatographic medium is made of a material capable of transferring a liquid fluid, and generally holds the labeled antibody in a dry state upstream of the immobilization region.
When a liquid serving as a sample is dropped on the portion holding the labeled antibody, the liquid sample is transferred to the fixing region. At this stage, the labeled antibody is dissolved by the liquid sample, and if an antigen to be detected is present, a binding reaction occurs.
Eventually, when the liquid sample reaches the immobilization region, the labeled antibody is captured by the immobilization region via the antigen if present.
Conversely, when no antigen is present, the labeled antibody is removed downstream of the fixed region without being captured by the fixed region. When an antibody is to be detected, a labeled antigen is used instead of a labeled antibody. On the other hand, when applying the principle of the competition method, the labeled antigen is used for measuring the antigen

As described above, analysis methods based on qualitative reaction principles, such as immunochromatography, often require adjustment of the analysis sensitivity. In the case where a certain substance is not detected under normal conditions and appears only in a pathological case, the sensitivity adjustment is not an important issue and a high sensitivity is required because it is only necessary to reliably analyze the presence of the substance. However, such substances are rare; rather, the boundary between normal and abnormal is generally a matter of quantity. When performing qualitative analysis of such substances, the sensitivity must be adjusted to give negative results only in the normal concentration range and only in the abnormal concentration range.

[0007] The basic approach to sensitivity adjustment is to reduce the number of analytes participating in the analysis system. For example, in immunochromatography, a method of starting analysis after contacting a sample to be analyzed with an immobilized antibody is known [1] [2]. With this method, the sensitivity can be easily reduced. However, additional reaction time is required in order to contact the immobilized antibody for capture in advance.
If the additional reaction time is insufficient, there arises a problem that an expected decrease in sensitivity may not be realized. In order to secure an additional reaction time, a special structure for holding the liquid sample in the region where the capture antibody is immobilized for a certain period of time is required. The same effect can be expected when the capture antibody is used in a free state instead of being immobilized. However, nitrocellulose, which is generally used for a chromatographic medium, has a property of adsorbing an antibody. Therefore, in order to retain the antibody in a free state, it is necessary to perform blocking before retaining the antibody.

[0008] In addition, for example, an indirect binding mode is used for a label or a solid phase, and a design is made so that a competitive reaction occurs in this portion, and as a result, the same effect as diluting a sample is obtained. Attempts to do so are known [3]. in this way,
Although the dilution effect can be achieved without narrowing the measurement range, the reaction system becomes complicated because an indirect binding reaction is required for labeling and immobilization. Therefore, in principle, the sensitivity can be adjusted, but in reality, a sophisticated manufacturing technique is required.

Another background art is an immunochromatographic method in which an inert latex which is not directly involved in a reaction is reacted with a sample together with a latex on which an immune component is immobilized.
[4] is known. However, the inert latex particles in this known technique are intended to suppress non-specific reactions and do not have a sensitivity adjusting function. In addition, there is a known technique [5] [6] in which particles of a plurality of colors are used in combination to realize simultaneous measurement of a plurality of items. However, the particles with colors distinguishable from each other used in these reports merely function as different labels and do not have a function of adjusting the sensitivity.

[0010]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a new technique capable of easily realizing sensitivity adjustment in immunochromatography. More specifically, an object of the present invention is to provide a sensitivity adjustment technique that can exert a reliable sensitivity adjustment function without giving an additional reaction time and can easily manufacture a specific binding analyzer even on a commercial basis. It is an issue.

[0011]

The present invention relates to a method for adjusting sensitivity in a specific binding assay for analyzing either a ligand or a receptor having a specific affinity as a component to be analyzed. a) containing the same receptor or ligand used for the identifiable labeling substance with an indistinguishable label that is indistinguishable from the background and is insoluble and capable of fluid transport in a chromatographic medium. This is a sensitivity adjustment method using a sensitivity adjusting substance and reacting the sensitivity adjusting substance with a component to be analyzed in advance or reacting it with an identifiable labeling substance. a) a receptor for a ligand to be analyzed,
Alternatively, when a receptor is used as an analyte, a corresponding ligand is labeled with an insoluble label that can be identified from its background, and b) a liquid containing the analyte is used as the above-mentioned identifier. Fluidly transports the sample liquid with a chromatographic medium that can be fluidically transported together with the sample liquid; c) identifiable via the analyte component at a position on the fluid transport path where the identifiable labeling substance and the liquid are reached by the fluid transport. An immobilization region to which a receptor is immobilized in a form capable of binding a label; d) an identifiable labeling substance immobilized via the analyte in the immobilization region; e) no binding reaction in the immobilization region. The identifiable labeling substance is removed from the immobilization area by fluid movement; Bond, or an identification label substance not Azukara the coupling reaction in a fixed area for detecting the labeled component insoluble as an index into

The sensitivity adjusting method of the present invention is effective in specific binding analysis using a chromatographic medium such as immunochromatography. Specific binding analysis was performed by analyzing antigens and haptens and their antibodies, saccharide-lectin, avidin (or its derivative) and biotin (or its derivative), hemoglobin-haptoglobin, single-stranded nucleic acids having base sequences complementary to each other, enzymes, This is a method in which one component is analyzed using specific affinity between a ligand and a receptor such as a hormone and a receptor thereof.
Hereinafter, the application of the sensitivity adjustment method of the present invention will be described using a specific example of an immunochromatography method using a sandwich method as a reaction format.

In the immunochromatography method (in the case of the sandwich method), a labeled antibody and an immobilized antibody are reacted with an antigen to form a sandwich-like immune complex, and the formation of this complex is labeled on a chromatographic medium. Confirm by detecting. When the sensitivity adjusting method of the present invention is applied to immunochromatography, the same antibody as the labeled antibody labeled with an indistinguishable label that cannot be distinguished from the background is used as the sensitivity adjusting substance. Here, the background can be defined as a material of the chromatographic medium in a fixed area of the chromatographic medium. Therefore,
A label that cannot be distinguished from the background can indicate, for example, colorless latex particles for a white chromatographic medium. Since nitrocellulose itself, which is generally used as a chromatographic medium, is white, the most basic choice for making a macroscopic determination is a colorless or white marker. However, since nitrocellulose can be colored, choices other than white are also conceivable. For example, if the chromatographic medium is black, white latex can be used as an identifiable marker. Note that the condition that the marker cannot be distinguished from the background does not limit the color of the indistinguishable marker to the same color as the chromatographic medium or colorless. While indistinguishable signs are recognized from the background by strong contrast, indistinguishable insignificant signs can be virtually shaded with weak contrast.

Materials such as latex particles and metal colloids can be used for the identifiable marker and the indistinguishable marker which can be used for visual judgment. In particular, latex particles made of a polymer such as polystyrene are commercially available in various colors and have a wide selection range. On the other hand, since metal colloids each have a unique color tone, an arbitrary color tone cannot always be obtained. But gold, silver,
Copper, platinum, silver iodide, silver bromide, copper hydroxide, iron oxide,
It is known that a wide range of compounds such as aluminum hydroxide, chromium hydroxide, mercury sulfate, barium sulfate, titanium dioxide and the like can be used for labeling in immunochromatography. In addition, some metal colloids, such as gold, which change color from yellow-orange to blue-violet depending on the particle size, are also known. Therefore, although the color tone is limited, a considerably wide color tone can be selected from among the metal colloids. In the present invention, a hydrophobic dye, a pigment, a nonmetallic colloid such as selenium, tellurium, or sulfur, and a dye-encapsulated liposome can be used as a labeling substance. For hydrophobic dyes and pigments, many compounds are already known as labels [7]. In the case of a dye-encapsulated liposome, any color tone such as colorless or a fluorescent dye can be used.

When a sign is read mechanically, rather than visually, the selection of the indistinguishable sign is further expanded. For example, if a substance that shows a specific reflection intensity at a specific wavelength is used as an identifiable label, the non-identifiable label only needs to be a substance that shows only the same reflection intensity at the measurement wavelength as the background. It doesn't matter. The same applies to the case where a component that produces fluorescence or luminescence at a specific wavelength is used as the labeling substance. In this case, it suffices that the material has no optical activity at the wavelength used for measuring the label, and the fluorescence (emission) characteristics at other wavelengths are irrelevant. In immunochromatography, not only optically active components but also magnetic labels can be used.
When measuring a magnetically active label, the inactive label only needs to be magnetically inactive, and its optical properties are irrelevant.

The identifiable label or the indistinguishable label of the present invention, which is characterized by the above-described discriminability from the background, can fix at least one of a ligand and a receptor. The liquid must be able to move through the chromatographic medium by the sample liquid while the liquid is fixed. By allowing the fluid to move together with the sample, it is possible to promote a specific binding reaction with the component to be analyzed even during the movement.

Since these labeling substances are actually transferred in a chromatographic medium, it is preferable that the difference in the fluid transfer characteristic between the sensitivity adjusting substance and the labeling component does not increase. When the two fluids move as much as possible, the sensitivity controlling substance and the analyte component can sufficiently react with each other, resulting in a reliable sensitivity controlling action. In order to satisfy such a condition, the most basic method is to use the same material for the unidentifiable substance and the identifiable substance. For example, if a colored latex is used as an identifiable substance, latex particles having the same physical properties as possible except for the difference in color may be used. Of course, it is possible to empirically search for similar fluid transfer characteristics even for completely different materials. Alternatively, even if the movement characteristics are slightly different, it is needless to say that a much more reliable sensitivity control action can be expected as compared to a case where the sensitivity control antibody is completely fixed to the chromatographic medium, for example.

In the case of immunochromatography, an antigen corresponds to a ligand and an antibody corresponds to a receptor. When an antigen is to be analyzed, an antibody is used as a reagent component. In this case, the sensitivity adjusting substance of the present invention may be obtained by binding the same antibody as the antibody constituting the labeling component to the indistinguishable labeling substance. By using a common antibody,
A more reliable sensitivity adjustment action can be expected. For example, when using a monoclonal antibody bound to a labeling component,
It is desirable to use a monoclonal antibody that recognizes at least the same epitope for sensitivity adjustment. Alternatively, a polyclonal antibody may be used for sensitivity adjustment. In this case, a polyclonal antibody having a reaction characteristic capable of sufficiently competing with the epitope of the antibody used for labeling is selected.

On the other hand, it is also conceivable to mix the polyclonal antibody and the monoclonal antibody, or mix the polyclonal antibody with the monoclonal antibody to constitute the labeling component. At this time, an antibody common to at least one of the mixed antibodies may be used for sensitivity adjustment. If only one type does not provide sufficient sensitivity adjustment,
If the antibody used for other mixing and the common antibody are combined for sensitivity adjustment, a stronger sensitivity adjustment effect can be expected.

Further, the sensitivity-adjusting substance of the present invention must be brought into contact with the analyte in the order defined as the sample liquid. That is, the sensitivity adjusting substance in the present invention needs to be brought into contact with the analyte before the label component comes into contact with the analyte, or together with the label. Even if the sensitivity adjusting substance is acted on after the analyte and the label have come into contact with each other, most of the analyte has already reacted with the label and the required sensitivity adjusting action can no longer be expected. . In order to easily realize such a reaction sequence, the following means is effective.
The most basic method is a method in which a sensitivity adjusting substance is added to a sample liquid in advance, and this is analyzed by a normal binding analysis operation. In such a procedure, it is not necessary to prepare a new reagent kit. In the above-described immunochromatography method, it is possible to previously hold a sensitivity adjusting substance in a part of the chromatographic medium. The position where the sensitivity adjusting substance is held may be upstream of the position where the label component is held with respect to the immobilization region, or may be the same position as the label component. When the sensitivity adjusting substance is held upstream, it comes into contact with the analyte before the labeling component, and on the other hand, when it is held at the same position, it comes into contact with the analyte. In any case, a sufficient reaction time can be ensured before reaching the immobilization region by fluid movement, as compared with the prior art in which a capture antibody is immobilized for sensitivity adjustment.

By adopting such a reaction sequence, a part of the component to be analyzed binds to the sensitivity adjusting substance. Analyte components bound to the sensitivity modifier are captured in the immobilization region in the same manner as those bound to the label component. However, the substance provided as a label to the sensitivity adjusting substance is a substance that cannot be distinguished from the background, and as a result, it cannot be caught as a signal and the apparent sensitivity decreases.
In the present invention in which a sensitivity adjusting mechanism is realized by such a mechanism, the amount of the sensitivity adjusting substance used is empirically set so as to realize a desired sensitivity.
Specifically, a sample containing a component to be analyzed at a concentration to be made negative may be prepared, and the amount of the sensitivity adjusting substance used may be set so that the sample is determined to be negative. The amount of the sensitivity modifier used depends on the titer of the antibody used on the label side, the amount of the labeled component used, the cut-off value of the target analyte (ie, the positive / negative determination standard value), the expected sample liquid Set in consideration of volume, etc.

As the chromatographic medium, a material capable of smoothly moving these components in fluid while causing an immunological reaction between the labeled antibody and the antigen is used. Specifically, materials such as nitrocellulose, filter paper, and nonwoven fabric can be exemplified. If these materials are blocked with albumin, milk components, normal animal serum, and the like, if necessary, nonspecific adsorption can be prevented, and an effect of promoting the development of a sample solution can be expected. As the components other than these capturing agents, those used in known immunochromatography can be used as they are.

The immobilized antibody in the immobilized region does not necessarily need to be immobilized in advance, and may be indirectly captured while moving in a chromatographic medium using protein A or avidin-biotin system. It is possible. The technique of immobilizing an antibody directly or indirectly at an arbitrary position on a chromatographic medium to obtain an immobilized antibody is already known. Even when the method of indirect immobilization is used, it is preferable that the mixture of the labeled antibody and the antigen reacts with the antibody to be immobilized after coming into contact with the capture agent.

The sensitivity adjusting method of the present invention can be applied to various components which have been analyzed in a known specific binding assay such as immunochromatography. Such analytes can include hormones, tumor markers, microbial antigens, various blood components, and the like.
More specifically, human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), luteinizing hormone (LH), insulin, glucagon, thyroid stimulating hormone (TSH), and corticosteroids are analyzed as hormones. Also known as Above all, hC using urine as a sample
The analysis of G is an important index for the diagnosis of pregnancy, and is a main test item for which an analyzer that can perform a simple test for general households is commercially available. For various blood components, for example, a method for detecting hemoglobin based on an immunological reaction is applied to a method for detecting fecal occult blood. By detecting hemoglobin in feces by immunological techniques, it becomes possible to test fecal occult blood without dietary restriction, and it is now an important test item for colorectal cancer screening.

Although the above description has been made with reference to an example in which an antigen is an analysis target, an antibody can also be an analysis target based on the same principle. Detection of antibodies that recognize a particular antigen provides important information in diagnosing infectious diseases. In the detection of an antigen-specific antibody, a labeled antigen is used instead of a labeled antibody, and an immobilized antibody that recognizes an antibody class (or subclass) to be analyzed is used.

In addition to the above-described sandwich method in which two or more binding sites on a substance to be analyzed are used in a chromatographic medium to form a sandwich structure, the principle of competition of binding components for one binding site is described. Assay formats, such as competitive methods and inhibition reactions, are available. The sensitivity adjustment method of the present invention can be applied to these assay formats in addition to the sandwich method. That is, the present invention provides a sensitivity adjustment method in a competition method or an inhibition reaction method in addition to the sensitivity adjustment method in the sandwich method. In the competition method or the inhibition reaction method, the identifiable label or the non-identifiable label of the present invention is given to the same component as the component to be analyzed.
The binding of these labeled ligands to the receptor in the immobilized region is inhibited by the analyte (ligand) present in the sample, whereby an analytical system is established (the receptor is the analyte). At this time, the sensitivity adjusting substance in the present invention works in the direction of inhibiting the binding of the labeling component to the immobilized region together with the component to be analyzed, resulting in the effect of adjusting the sensitivity. Here, the same components as the components to be analyzed used as the labeling component or the sensitivity controlling substance do not necessarily have to be the same compound, as long as the binding activity of the immobilized region to the capture component is substantially equivalent. Specifically, if an antibody is used as the capture component of the fixed region, the fragment may be a simple fragment of the antigen, rather than a complete molecule, as long as it has an antigenic determinant recognized by the antibody.

The components necessary for the construction of the immunochromatographic method to which the sensitivity adjusting method of the present invention is applied can be combined in advance to form a kit for a specific binding analyzer. That is, the present invention relates to a specific binding analyzer for analyzing one of a ligand and a receptor having a specific affinity as a component to be analyzed, comprising the following components g) -j), Utilizing an insoluble and sensitivity-regulating substance capable of transporting fluid in a chromatographic medium, labeled with the same receptor or ligand as that used for the discriminating labeling substance with an indistinguishable label that cannot be discriminated, A specific binding analyzer provided at a position where the substance can be reacted with the analyte in advance or with an identifiable labeling substance; g) a receptor for a ligand which is an analyte;
Alternatively, when the receptor is used as the analyte, the corresponding ligand is labeled with an insoluble label that can be identified from its background. H) A fluid containing the analyte is transported together with the above-mentioned identifier. I) a chromatographic medium capable of fluid-transferring a liquid containing an analyte component together with the identifiable labeling substance; j) the identifiable labeling substance and the liquid on a fluid transfer path are fluid An immobilized area where the receptor is immobilized in a position that can be bound by the analyte via the analyte, to a location reached by transport.

The specific binding analyzer according to the present invention must be provided at a position where the sensitivity modifier can be reacted with the analyte in advance or with the identifiable labeling substance. If such a configuration is specifically shown, for example, the following configuration can be given. Generally, the chromatographic medium constituting the specific binding analyzer has a region holding at least a label component upstream of the fixed region. It is possible to adopt a configuration in which the sensitivity adjusting substance is retained upstream of the immobilized region with respect to the region retaining the labeled component, or retained in a chromatographic medium together with the labeled component. In a specific binding analyzer using a chromatographic medium, a region for receiving a sample liquid called a liquid receiving portion may be provided further upstream than a region holding a labeling component. In such a case, the sensitivity adjusting substance may be held at an arbitrary position between the liquid receiving part and the area holding the labeling component. In the specific binding analyzer according to the present invention, the entire chromatographic medium or a part thereof can be covered with a liquid-impermeable material for the purpose of preventing unnecessary evaporation of the sample liquid. Further, a liquid absorbing pad for absorbing an excess amount of the liquid sample applied to the chromatographic medium can be kept in contact with an arbitrary portion of the chromatographic medium. The structure of such a specific binding analyzer is already known [8].

[0029]

The sensitivity adjusting substance of the present invention realizes a sensitivity adjusting mechanism which does not require an additional reaction time. Although the present invention also proposes an embodiment in which the sensitivity adjusting substance contacts the analyte component prior to the labeling component, it is not necessary to provide an additional reaction time in this embodiment. The sensitivity adjusting substance moves in fluid while reacting with the analyte component, and eventually comes into contact with the labeling component, but thereafter always reacts competitively with the labeling component until it passes through the immobilization region. Based on such a principle, it is possible to reliably control the sensitivity without giving an additional reaction time. The sensitivity principle of the present invention is such that the sensitivity adjusting substance in the present invention does not come into contact with the analyte component prior to the labeling component, and a sufficient sensitivity adjusting effect can be expected even in a mode in which both come into contact with the analyte component. This is because it has been adopted. In the prior art, since the antibody for adjusting the sensitivity is fixed to the chromatographic medium and allowed to act, the effect of reliably adjusting the sensitivity as in the present invention cannot be expected.

[0030]

Since the sensitivity adjustment method of the present invention employs the above-described reaction principle, it is possible to easily realize a reliable sensitivity adjustment which is hardly expected by a known method. The present invention is particularly advantageous when producing a specific binding analyzer having a sensitivity adjusting function. The specific binding analyzer provided with the sensitivity adjusting function provided by the present invention is a special antibody for securing the reaction time because the antibody for sensitivity adjustment is not fixed and reacts while moving with the sample liquid. Demonstrates reliable sensitivity adjustment without the need for a structure.

Further, since the sensitivity-adjusting substance of the present invention is retained in the chromatographic medium in a form in which it is bound to some insoluble substance in advance, even when applied to a protein-adsorbing chromatographic medium such as nitrocellulose, for example. No special operation such as blocking is required, and the number of manufacturing steps is reduced. In particular, when an embodiment in which the present invention is applied to a chromatographic medium together with a labeling component is employed, an additional manufacturing step for the sensitivity adjusting function hardly occurs. These features are advantageous as compared to a method in which the antibody is simply used in a free state.

[0032]

【Example】

1. Preparation of anti-human hemoglobin A0 polyclonal antibody A human hemoglobin A0 solution purified from human hemoglobin lysate was mixed with an equal amount of Freund's complete adjuvant to form an emulsion. After immunizing 1 ml of this emulsion with New Zealand White Rabbit, booster immunization was performed three times every two weeks,
One week later, blood was collected from the ear vein and centrifuged to obtain antiserum. The gamma globulin fraction was salted out from the obtained antiserum by a 40% saturated ammonium sulfate salting out method, and the precipitate obtained by centrifugation was subjected to 0.02M containing 0.15M sodium chloride.
Dissolved in phosphate buffer pH 7.2 (hereinafter abbreviated as PBS)
After dialysis against the same buffer for 24 hours, the protein concentration was adjusted to 1% by absorbance at 280 nm to obtain an anti-human hemoglobin A0 polyclonal antibody.

2. Preparation of anti-human hemoglobin A0 monoclonal antibody BALB / c mice were immunized with the same emulsion as in 1.
Boosters are given three times every two weeks, and three days after the final immunization, the spleen is removed and the method of Keller and Milstein [9]
Was fused with P3-NS1 / 1-Ag4-1 myeloma according to the procedure described above. EL
Antibody-producing hybridomas were selected by the ISA method and cloned by the limiting dilution method. Among the several antibody-producing clones obtained as a result of the screening, those clones which did not rapidly aggregate when reacted with hemoglobin in a state sensitized to latex particles as a label were selected and used in the following experiments. The obtained clone was administered to the abdominal cavity of BALB / c mice to which pristane had been administered intraperitoneally in advance, and ascites was collected 3 weeks later. 4 from this ascites
The gamma globulin fraction was salted out by a 0% saturated ammonium sulfate salting-out method, and the precipitate obtained by centrifugation was subjected to a 0.02 M phosphate buffer pH 7.2 containing 0.15 M sodium chloride (hereinafter referred to as P
BS), dialyzed against the same buffer for 24 hours, and adjusted to a protein concentration of 1 by absorbance at 280 nm.
% To obtain an anti-human hemoglobin A0 monoclonal antibody.

3. Red polystyrene latex (average particle size 0.303 μm) in which the monoclonal antibody obtained by using the sensitivity modifier and the labeling component 2 is dispersed in an equal volume of PBS buffer to 10% w / v, or from the same material The mixture was mixed with a colorless latex and subjected to physical adsorption at 37 ° C. for 1 hour. The precipitate obtained by centrifugal washing with the same PBS buffer was dispersed in a PBS buffer containing 1% BSA, and left standing at room temperature for 1 hour to block. Wash again by centrifugation with PBS buffer, and finally disperse in the same buffer so that the latex concentration becomes 2% w / v. Colored latex-labeled monoclonal antibody (hereinafter abbreviated as labeled antibody) or colorless latex labeling sensitivity The substance was referred to as a control substance (hereinafter abbreviated as sensitivity control substance). The obtained labeled antibody was mixed with the sensitivity controlling substance at a ratio of 1: 0, 2: 1, and 1: 1, and a 5% (w / v) PBS solution of sucrose was applied in advance and dried to obtain a porous polyethylene. 3 on sheet (1mm thick, 1 × 1cm)
00 μl was dropped and dried at room temperature to prepare a labeled antibody region.

4. Preparation of an immunochromatography apparatus A nitrocellulose membrane filter (manufactured by Toyo Roshi) with a pore size of 5 μm was cut into 1 × 4.5 cm and 2 cm from the end.
Spot 3 μl of the anti-hemoglobin polyclonal antibody diluted to 0.6% at the position of
A was immersed in a PBS solution for 30 minutes. After the surface of the membrane was washed with water, it was air-dried at room temperature to obtain a developed area.
Further, a thick filter paper 17Cr (manufactured by Whatman) was cut into a size of 1 × 3 cm, and arranged downstream of the developing area to form a liquid absorbing area. Each of the regions was arranged with its ends overlapped by 5 mm with the developing region (nitrocellulose sheet) facing down in the order of labeled antibody region-developing region-liquid absorption region, and fixed on a polyethylene terephthalate support with double-sided tape.

5. Detection of hemoglobin As a sample, hemolyzed blood was diluted with a PBS solution containing 0.1% BSA to prepare a sample containing 25 to 1600 µg / ml of hemoglobin. Hemoglobin concentration was measured by the cyanmethemoglobin method [10]. 100 samples in sample supply area
μl was dropped, and the change in color in the fixed region of the developed region (the site where the polyclonal antibody was fixed) was visually observed. The results are shown in Table 1. In the table, "-" indicates that color development was not confirmed in the fixed area, "±" indicates that color development was slightly observed, and "+"
Indicates that a clear color development was recognized. As can be seen from Table 1, it is clear that the analysis sensitivity can be freely adjusted by the mixing ratio of the sensitivity adjusting substance.

[0037]

[Table 1]

Reference [1] JP-A-6-341989 [2] JP-A-8-101197 [3] JP-A-4-351962 [4] Published JP-A-4-504766 [5] JP-A-1-152366 [6] JP-A-8-108 -94618 [7] JP-A-56-160655 [8] Publication No. 1-503174 [9] Nature 256, p495, 1975 [10] Medical Physics Vol. 2, p1072, 1950

Claims (8)

[Claims] 1. A method for adjusting sensitivity in a specific binding assay for analyzing one of a ligand and a receptor having a specific affinity as a component to be analyzed, comprising the following conditions a) to f): The same receptor or ligand used for the identifiable labeling substance is labeled with an indistinguishable label that cannot be distinguished from the background. Sensitivity adjusting method in which a sensitivity adjusting substance is reacted in advance with an analyte or with an identifiable labeling substance a) a receptor for a ligand which is an analyte,
Alternatively, when a receptor is used as an analyte, a corresponding ligand is labeled with an insoluble label that can be identified from its background, and b) a liquid containing the analyte is used as the above-mentioned identifier. Fluidly transports the sample liquid with a chromatographic medium that can be fluidically transported together with the sample liquid; c) identifiable via the analyte component at a position on the fluid transport path where the identifiable labeling substance and the liquid are reached by the fluid transport. An immobilization region to which a receptor is immobilized in a form capable of binding a label; d) an identifiable labeling substance immobilized via the analyte in the immobilization region; e) no binding reaction in the immobilization region. The identifiable labeling substance is removed from the immobilization area by fluid movement; Bond, or an identification label substance not Azukara the coupling reaction in a fixed area for detecting the labeled component insoluble as an index into 2. A labeling substance which can be distinguished from the background is given a color different from the background color so as to be identifiable to the naked eye, while a sensitivity adjusting substance is given the same color as the background color or is colorless and visually unavoidable. 2. The sensitivity adjustment method according to claim 1, wherein the sensitivity cannot be identified by 3. The method according to claim 1, wherein white nitrocellulose or a derivative thereof is used as the chromatographic medium, and the distinguishable labeling component is selected from the group consisting of colored latex particles, colored metal colloids, and colored pigments. Item 2 sensitivity adjustment method 4. The method according to claim 3, wherein the identifiable label component and the non-identifiable label component are made of the same material. 5. The method according to claim 3, wherein the indistinguishable label component that cannot be distinguished from the background is selected from the group consisting of colorless or white latex particles, metal colloids, and pigments. 6. The combination of a ligand and a receptor having a specific affinity is selected from the group consisting of antigen-antibody, saccharide-lectin, nucleic acid-nucleic acid having a complementary sequence, and avidin-biotin. 1. Sensitivity adjustment method 7. A specific binding analyzer for analyzing one of a ligand and a receptor having a specific affinity as a component to be analyzed, which comprises the following components g) -j), Utilizing an insoluble and sensitivity-regulating substance capable of transporting fluid in a chromatographic medium, labeled with the same receptor or ligand as that used for the discriminating labeling substance with an indistinguishable label that cannot be discriminated, A specific binding analyzer provided at a position where the substance can be reacted with the analyte in advance or with an identifiable labeling substance; g) a receptor for a ligand which is an analyte;
Alternatively, when the receptor is used as the analyte, the corresponding ligand is labeled with an insoluble label that can be identified from its background. H) A fluid containing the analyte is transported together with the above-mentioned identifier. I) a chromatographic medium capable of fluid-transferring a liquid containing an analyte component together with the identifiable labeling substance; j) the identifiable labeling substance and the liquid on a fluid transfer path are fluid An immobilized area where the receptor is immobilized in a position that can be bound by the analyte via the analyte, to a location reached by transport. 8. A method for adjusting sensitivity in a specific binding assay for analyzing one of a ligand and a receptor having a specific affinity as a component to be analyzed, comprising the following conditions m) -r): The same ligand or receptor used for the identifiable labeling substance is labeled with an indistinguishable label that cannot be distinguished from the background. Reacting the sensitivity modifier with the immobilization region in advance,
Or a method for adjusting the sensitivity by reacting with the immobilized region together with an identifiable labeling substance. M) It is possible to identify the ligand or ligand analog as the analyte or the receptor or receptor analog when the receptor is the analyte, from the background. Using an identifiable labeling substance labeled with an insoluble labeling component, and n) transferring the sample liquid with a chromatographic medium capable of fluidly transferring the liquid containing the analyte with the identifiable labeling substance; At the position on the transfer path where the identifiable labeling substance and the liquid reach by the fluid transfer, a receptor corresponding to the analyte component, or a ligand when the receptor is the analyte, the ligand is the analytic component and the identifiable labeling substance. Has a fixed area that is fixed in such a way that it can be combined, p) identifiable When the labeling substance is immobilized together with the analyte in the immobilization region, or when the receptor is used as the analysis component, the labeling substance constitutes a competitive reaction system by reacting with the ligand. The unrecognizable labeling substance is removed from the immobilized area by the fluid transfer. R) The binding of the recognizable labeling substance to the immobilization area or the recognizable labeling substance not participating in the binding reaction in the immobilization area Is detected using insoluble labeled components as indices