JPH09281104A - Method for preventing pseudonegative reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent a pseudonegative reaction caused in the case of excessive existence of analyzing object components by combining under the existence of a trap agent capable of trapping analyzing object components in isolation state and not capable of trapping complex produced when an analyzing object component combines with a corresponding component, or bringing it into contact with the trap agent after the combining reaction. SOLUTION: A complex produced when analyzing object component combines with a corresponding combining component, causes combining reaction under the existence of a trap agent not capable of trapping. For the combining analysis, a trap agent is reacted after the contact between the first receptor and analyzing object rigand and a receptor combining with the second receptor at a plurality of different sites is utilized. The first receptor exists in marked state and the second receptor exists in solid-phased state. For the trap agents having molecular sieve effect, dextran is used for example. For the mark, latex, pigment, metal colloid, enzyme as well are used.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a binding assay for a ligand or a receptor, which utilizes a binding reaction due to a specific affinity between the ligand and the receptor. A typical example of the binding analysis utilizing the affinity between the ligand and the receptor is an immunological analysis method utilizing the antigen-antibody reaction.

Among the binding assays utilizing the ligand-receptor reaction, the assay method utilizing the antigen-antibody reaction is an analytical technique utilizing the high specificity of the immune system, and is strictly applicable to substances having similar structures and activities. It is possible to identify and to realize high sensitivity. Further, if the luminescence reaction and various signal amplifications are used as a detection system for the label, it is expected that the sensitivity will surpass that of the conventional RIA method using a radioisotope as a label. In addition, the widespread use of monoclonal antibody production technology has made it possible to easily produce a large amount of antibodies having various binding activities with uniform quality, thus achieving high specificity and reproducibility.

[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 the feces also serves as an indicator of digestive ulcers and tumors.

The present invention relates to a technique for preventing the false negative reaction observed when the component to be measured is present in excess in its binding partner in the ligand-receptor binding assay. More specifically, the present invention relates to a method of preventing a false negative reaction in an antigen excess region of an antigen measuring method utilizing an antigen-antibody reaction, and an application of this method of preventing a false negative reaction to an immunological measuring method.

[0005]

[Problems of the prior art] An antigen measuring method utilizing an antigen-antibody reaction can be expected to have linearity when the antigen to be measured is in a certain concentration range. That is, the measured value of the label increases (or decreases) in a fixed relationship as the antigen concentration increases. There are two types of antigen measuring methods, such as the sandwich method and direct agglutination method, in which the measured value increases with the antigen concentration, and the competition method and agglutination inhibition reaction, which show an inverse relationship with the antigen concentration.

However, in the sandwich method or the direct agglutination method, if the antigen to be measured is present in excess, the expected linearity may not be maintained and the measured value may be similar to that at low concentration. That is, although the antigen to be measured is present in a large amount, the result is that the apparent amount is small. Such a phenomenon is known as a false negative reaction due to excess antigen.

In the case of the sandwich method, since the antigen is present in excess, another antigen molecule binds to both the labeled antibody and the solid-phase antibody, and is referred to as “solid-phase antibody-antigen” and “antigen-labeled antibody”. Two immune complexes are formed, and the sandwich structure "solid phase antibody-antigen-labeled antibody" that should be originally formed is not formed, leading to a false negative. Similarly, in the immunochromatography method, the labeled antibody is originally captured by the solid phase antibody through the antigen, but when a large amount of the antigen is present, the free antigen that could not be completely reacted with the labeled antibody is against the solid phase antibody. As a result, the labeled antibody is prevented from binding to the solid phase antibody through the antigen. The competition with the solid-phase antibody referred to here includes the case where the antigen reacted with the labeled antibody and the free antigen compete with each other, and a part of the excess antigen exists in the solid-phase antibody region before the labeled antibody in time. It is considered that the phenomenon in which the binding site is reached and the binding site is occupied.

In the latex agglutination reaction, the particles originally form a crosslinked structure through the formation of an immune complex, but when the antigen is present in excess, the particles are covered with the antigen and the crosslinked structure is formed. It cannot be formed. The false-negative reaction is called prozone phenomenon or hook action in the sandwich method or latex agglutination reaction, and various countermeasures have been tried.

One of the countermeasures is a method of alleviating the antigen excess state by adding an antibody that is auxiliary to the original reaction. For example, in the case of the latex agglutination reaction, the same antibody as the antibody to the measurement target antigen sensitized to the particles is added to the reaction system in a free state. If the antigen is present in excess, the excess antigen also reacts with the free antibody, reducing the effect on particle aggregation [1]. This principle is also applied to immunochromatography. In other words, by reacting a free antibody against the antigen to be measured with the labeled antibody together with the antigen, a false negative reaction countermeasure is taken [2].
However, the free antibody added to the reaction system is a factor that lowers the sensitivity when the antigen concentration is low, so it is difficult to expect the expansion of the measurement range. If the measurement is carried out with a reaction solution to which no free antibody is added, the measurement range can be expected to be expanded, but two types of reaction systems must be prepared, which is not realistic. Applying this idea, a method [3] has also been proposed in which a sample solution containing an antigen to be measured is treated with an immobilized antibody capable of capturing the antigen in advance and then the desired immunological reaction is performed [3]. This method is effective when the antigen is present in excess, but it also acts to reduce the sensitivity, so expansion of the measurement range is not expected.

The biggest problem of the false negative reaction in the antigen excess region is that the apparent measured value becomes low. Conversely, even if the upper limit of the measurement range is exceeded, if the range is confirmed to be over, the wrong judgment will not be made. Based on this way of thinking, apart from the method of suppressing the influence of excess antigen, whether free antigen or antibody is further added to the reaction solution after measurement and the change in the measured value is observed to determine whether it is in the antigen excess region. The technology to judge is known [4] [5]. If it can be determined that the antigen is in excess, it is possible to prevent an erroneous determination result by diluting the sample and re-measuring. However, in order to realize such a method, it is unavoidable to use a reaction principle that allows the reaction solution after the measurement to carry out the reaction again. In other words, it can be applied in the reaction principle of a homogeneous system such as latex agglutination reaction or immunoturbidimetric method, but in principle it is difficult to apply in a heterogeneous reaction system such as the solid phase sandwich method. In addition, the determination of the antigen excess region is often performed by applying the measured values obtained at various timings to a special calculation formula, and therefore a programmable dedicated measuring device is generally required.

[0011]

The first object of the present invention is to prevent false negative reactions in ligand-receptor binding assays without sacrificing assay sensitivity.
A second object is to provide a method for preventing false negative reactions due to an excessive amount of analytes by a versatile method that does not require a special measurement device and can be applied to various measurement principles. Further, the third object of the present invention is to provide a new immunological measurement technique to which this false negative reaction preventing method is applied.

[0012]

Means for Solving the Problems The present invention relates to a binding assay for a ligand or a receptor utilizing a binding reaction due to a specific affinity between a ligand and a receptor, and a binding component corresponding to a binding component corresponding to an analyte component. It is a technique to prevent false negative reactions that occur when it exists in excess, and it is possible to capture the analyte in the free state, but the complex formed by binding the analyte with the corresponding binding component is This is a method of preventing a false negative reaction in which the binding reaction is performed in the presence of a capture agent that cannot be captured, or the binding reaction is followed by contact with the capture agent.

The present invention is characterized in that, by using such a special capturing agent, for example, a false negative reaction in an antigen excess region of an immunological analysis method is prevented. As the scavenger, those having a so-called molecular sieving effect capable of fractionating various components based on the difference in molecular weight are useful. As such a capture agent, a gel using a material such as dextran, cross-linked agarose, cellulose, polyacrylamide, or hydrophilic vinyl polymer can be used. These gels are widely used as gel filtration chromatography for separating and purifying compounds based on the difference in molecular weight. The capture agent of the present invention does not necessarily need to strongly adsorb the component to be analyzed in the free state. For example, the gel exemplified as a specific scavenger causes a difference in elution rate at a specific molecular weight, and does not adsorb a substance. However, the difference in the elution rate can be regarded as temporarily capturing a specific fraction, which is a preferable capturing agent of the present invention. In addition to gel, activated carbon or the like can be used as this type of scavenger.

When a gel is used as a capture agent, the gel can capture the analyte component in a free state, but cannot capture the complex formed by the analyte component binding to the corresponding binding component. . For example, an antibody (receptor) that specifically recognizes an antigen (ligand)
When the analysis is to be performed by, the capture agent selected is one that is used to fractionate the molecular weight of the antigen. Since adult hemoglobin (HbA) has a molecular weight of about 65,000, Sephadex G75, which has a molecular weight cut-off of 3 × 10 ³ to 8 × 10 ⁴ for globular proteins, also has a molecular weight of 1 × 1 for globular proteins.
Sephacryl S-200HR having a molecular weight cut-off of 0 ³ to 1 × 10 ^5.
(All manufactured by Pharmacia) can be used as a scavenger. Other glycoprotein hormones LH, FSH, T
Since SH and hCG have a molecular weight of 20,000 to 30,000, Sephadex G50F (1.5 g x 10 globular protein), which has the same molecular weight as hemoglobin or a small molecular weight cutoff
^{3 to} 3 × 10 ⁴ , also made by Pharmacia) can be combined as a scavenger. In gel filtration chromatography, these components are trapped in the gel during passage and are eluted later than the components with higher molecular weight.
The antibody that is the receptor is IgG and has a molecular weight of 1.6 × 10
^Since it has a molecular weight of 5, it has a molecular weight that is an order of magnitude higher than that of the hormone to be analyzed, and both are separated by contact with the gel.

In addition, in the binding assay, the antibody that is the receptor is often labeled with a macromolecule such as an enzyme or latex, so that the component to be analyzed in the free state and that bound to the receptor are different from each other. There is a large difference in molecular weight, and both can be fractionated more clearly. For example, horseradish peroxidase (hereinafter abbreviated as HRP), which is often used as a labeling enzyme, has a molecular weight of about 44,000, and Aspergillus β-galactosidase has a molecular weight of about 105,000. Even if the same β-galactosidase is derived from E. coli, its molecular weight reaches 540000. When IgG is labeled with such enzymes, it is often used as F (ab ′) 2 obtained by digesting and removing the Fc region.
Fc is excluded from the viewpoint of molecular weight because it becomes a conjugate with an enzyme.
As a result, it has a form of compensating for a portion, and as a result, it has a molecular weight greatly different from that of the component to be analyzed.

As the label, insoluble substances such as latex, pigment or metal colloid are used in addition to the enzyme. If these labels are used, it can be said that they are huge molecules with a size that can be traced with the naked eye, so they have a molecular weight that is far from that of the free analyte, and the fractionation by the capture agent should be performed promptly. You can

On the other hand, depending on the substance, a combination in which a large difference in molecular weight cannot be expected between the complex of the component to be analyzed and its binding partner is expected. Even in such cases, introducing a label with a large molecular weight,
Alternatively, by combining with a carrier such as a high molecular weight polysaccharide or a polymer having a larger molecular weight, it becomes possible to fractionate both due to the difference in molecular weight.

In addition to the glycoprotein hormone, a combination of a ligand (or receptor) to be analyzed and a capture agent can be exemplified. -Ligand-Example of a capture agent suitable for human serum albumin (MW69000) Sephadex G-75 3 x 10 ³ -8 x 10 ⁴ Superdex 75pg 3 x 10 ³ -7 x 10 ⁴ Sephacryl S-200HR 5 x 10 ³ -2 .5 × 10 ⁵ -receptor-human IgG (MW156000-161000) and human IgA (MW16000)
Examples of capture agents suitable for 0) Sephadex G-150 5 × 10 ³ -3 × 10 ⁵ Sephacryl S-300HR 1 × 10 ⁴ −1.5 × 10 ⁶ Examples of capture agents suitable for human IgM (MW 1000000) Sephadex G-100 4 × 10 ³ −1.5 × 10 ⁵ Sephacryl S-200HR 5 × 10 ³ −2.5 × 10 ⁵

The method for preventing false negative reaction of the present invention is effective in ligand-receptor binding analysis by various reaction formats such as sandwich method, immunochromatography method, immunoturbidimetric method, and latex agglutination method. The present invention is particularly useful in the so-called sandwich method, in which the binding assay utilizes receptors that bind to at least two different sites of the target ligand. In the sandwich method, multiple receptors bind to a ligand. The capture agent in the present invention exerts a greater effect by acting during the reaction step with the plurality of receptors. Among them, in the case of the immunochromatography method, the present invention can be applied without adding any additional operation, and thus can be said to be the most preferable embodiment. The application of the false negative reaction prevention method of the present invention will be described below by taking an immunochromatographic method as a specific reaction format.

In the immunochromatography method, a labeled antibody and an immobilized antibody are reacted with an antigen to form a sandwich-shaped immune complex, and the formation of this complex is confirmed by detecting the label on a chromatographic medium. To do. When the false negative reaction prevention method of the present invention is applied to an immunochromatographic method, a capturing agent is allowed to act after contact between a labeled antibody and an antigen to be measured, and then a reaction with a solid-phased antibody is carried out to cause additional effects such as washing. It is preferable because no step is required.
Specifically, a mixed solution of a labeled antibody and a sample solution containing an antigen to be measured is moved in a fluid-movable chromatographic medium to reach a solid-phased antibody site. The mixed solution is arranged so as to pass through the capture agent before reaching. The scavenger does not necessarily have to be firmly fixed. That is, even if the mixture of the labeled antibody and the antigen is contacted and then moved to the region of the immobilized antibody as it is, if the moving speed is slower than that of the labeled antibody, the analysis result is not affected. The antigen-antibody reaction can be performed even in the coexistence of the capture agent. In short, after the labeled antibody and the antigen to be analyzed are in contact with the labeled antibody, the antigen to be analyzed, which is present in a free state and does not completely react with the labeled antibody, is captured as part of the labeled antibody before reaching the immobilized antibody. It is sufficient that the reacted antigen can react preferentially with the immobilized antibody.

In order to realize such a constitution, the gel described above may be dispersed and filled in the chromatographic medium, or the chromatographic medium itself may be partially constituted by gel. is there. Since it is difficult to maintain the shape as a chromatographic medium by gel alone, wrap the gel with a liquid impermeable material, or coat the gel on a liquid impermeable support and dry it. Can be placed. Further, it is also possible to dispose a suspension in which a gel is dispersed in a porous carrier composed of polyethylene or polyurethane and to dry the suspension if necessary. When the capture agent is composed of a material different from the chromatographic medium, the capture agent is arranged so as to be in fluid contact with the chromatographic medium that constitutes another region described later. Specifically, the two are overlapped in a plane, the two are partially overlapped,
Alternatively, it is possible to adopt a form of contacting in a cross section.
In addition to this, the gel can be placed in a tubular structure, and the upper and lower open ends thereof can be closed with a chromatographic medium to form the structure of the present invention. In this embodiment, the reaction is started by supplying the sample to the lower chromatographic medium.
A sample that reaches a gel having a tubular structure causes the gel to swell. The volume-increased gel eventually reaches the upper chromatographic medium, and thus the upper and lower chromatographic media are fluidly connected with the gel being the trapping agent sandwiched therebetween. By the way, the commercially available gel can be selected not only by the material and the molecular weight cut off but also by the particle size. Since the particle size has a great influence on the elution rate, it is advisable to select an appropriate particle size in consideration of the characteristics of the solution as the analytical sample and the required development rate. Specifically, diluted serum, urine,
Alternatively, for an aqueous sample such as a dispersion solution of feces, a particle size of 20 to 200 μm can be exemplified.

The chromatographic medium is made of a material capable of smoothly fluidizing these components while causing an immunological reaction between the labeled antibody and the antigen. Specifically, materials such as nitrocellulose, filter paper, and nonwoven fabric can be exemplified. These materials can prevent non-specific adsorption by blocking with sugar, albumin, milk components, serum of normal animals, etc. if necessary, and are expected to promote the development of sample solution. it can. As the components other than these capturing agents, those used in known immunochromatography can be used as they are. The immobilized antibody does not necessarily have to be immobilized in advance,
It is also possible to indirectly capture while moving in the chromatographic medium using protein A, avidin-biotin system or the like. 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.

Up to this point, the case where an antigen is an analysis target has been described as an example, but 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 principle, the reverse combination, that is, a combination in which the antibody that recognizes the antibody is labeled and the antigen is immobilized, is considered, but it seems to be disadvantageous in terms of sensitivity and economy. This is because a large amount of antibody is contained in the blood sample in addition to the antibody to be analyzed, and an amount of antibody that reacts with all of these is required.

The components necessary for the constitution of the immunochromatographic method to which the false negative reaction preventing method of the present invention is applied can be combined in advance to form a kitted immunochromatographic apparatus. The configuration of the chromatographic apparatus according to the present invention will be specifically described. That is, the immunochromatographic apparatus according to the present invention has the following composition 1-3, and an immunochromatographic apparatus that comes into contact with a solid-phased antibody located downstream due to fluid migration of an immune complex of a labeled antibody and an antigen to be measured. In the device, a mixture of a labeled antibody and an antigen to be measured captures an antigen that is a substance to be measured, but does not substantially capture an antigen in a state of forming an immune complex with the labeled antibody in the coexistence of a capture agent. Or an immunochromatographic device which is brought into contact with the immobilized antibody after being brought into contact with the capture agent. Configuration 1: Labeled antibody that recognizes the antigen to be measured Configuration 2: Chromatographic medium that can move the immune complex of the labeled antibody and the antigen to be measured in fluid Configuration 3: Immobilized antibody immobilized on the chromatographic medium

The immunochromatographic apparatus according to the present invention supplies a liquid serving as a sample to a liquid receiving part, reacts an antigen to be measured existing in the sample liquid with a labeled antibody, and moves a chromatographic medium in a fluid to capture it. When it reaches the area of the immobilized antibody after passing through the area where the agent is placed, the immune complex with the labeled antibody (measurement target antigen-labeled antibody) binds to the immobilized antibody via the measurement target antigen and the reaction is terminated. .
If the antigen to be measured does not exist, the labeled antibody will not be captured by the immobilized antibody. This chromatographic device
The entire chromatographic medium, or a portion thereof, can be covered with a liquid impermeable material to prevent unnecessary evaporation of the 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. As such an immunochromatographic device, for example, Publication No. 1-503174 is known.

In this other assay format, it is possible to apply the method of preventing false negative reactions of the present invention by providing a step of passing a capture agent after the binding reaction. For example, in the ELISA sandwich method using an enzyme-labeled antibody and an immobilized antibody, a mixed solution containing a sample and an enzyme-labeled antibody is treated with a capture agent and then reacted with the immobilized antibody. For the treatment with the capturing agent, a method of passing through a gel packed column is convenient.

In the method for measuring an antigen to which the method for preventing a false negative reaction of the present invention is applied, various publicly known antigenic substances can be measured. For example, specifically hemoglobin,
Haptoglobin, albumin, globulin, α-1 antitrypsin, α-2 macroglobulin, transferrin, lactoferrin, chorionic hormone, luteinizing hormone, and estrogen can be listed. The sample containing these is not particularly limited. Ie blood,
It can show a wide range of samples such as serum, plasma, urine, feces, saliva, and samples obtained directly from living organisms, cultures of microorganisms such as bacteria and animal cells, environmental samples derived from foods, waters and soils, etc. it can.

Further, the principle of the present invention can be applied to a binding assay utilizing a ligand-receptor reaction other than the antigen-antibody reaction. For example, in a hybridization assay in which a nucleic acid such as DNA is subjected to binding analysis by hybridization with a probe having a complementary sequence, or in a binding analysis using a combination of hormone and its receptor, or avidin and biotin and derivatives thereof, analysis is performed. When the target component is present in excess, the present invention can be expected to be effective.

[0029]

The capturing agent of the present invention prevents false negative reaction by capturing an excessive amount of the component to be analyzed so that it cannot participate in the binding reaction. The action of the capture agent in the present invention does not work after the component to be analyzed binds to the binding partner to form a complex. Therefore, after the binding reaction is performed once or by allowing the capture agent to act while the binding reaction is performed, it is possible to suppress only the influence of the excessive analyte component without affecting the sensitivity of the analysis system. Based on such a principle, the method of preventing a false negative reaction of the present invention can be expected to exert its maximum effect by allowing a capture agent to act between binding reaction steps in an assay system including a plurality of binding reaction steps.

By the way, a technique for separating the products of the binding reaction based on the difference in molecular weight is already known. For example, gels were used to separate nucleic acids that formed double strands by hybridization from single-stranded nucleic acids [6] [
7]. Also, in immunological analysis methods, attempts to use gels to separate immune reaction products are known.
[8]. However, the gels used in these analytical techniques are used only for the final separation of reaction products. Therefore, it does not suggest the solution of the problem of the present invention, which is to suppress the influence of the components to be analyzed present in excess. Furthermore, by combining a high-molecular-weight substrate that cannot enter the gel, a binding assay that does not require B / F separation has been proposed [9]. This conventional technique also merely performs fractionation with the binding reaction product, and does not suggest solving the problem of the present invention. There is also a report [10] in which the gel is simply used as a carrier for thin-phase chromatography, but it does not show any application to binding analysis using the ligand-receptor reaction.

[0031]

INDUSTRIAL APPLICABILITY The present invention prevents the false negative reaction in the antigen excess region of the antigen measuring method utilizing the antigen-antibody reaction without sacrificing the analytical sensitivity. Further, the present invention prevents a false negative reaction in the antigen excess region by a versatile method that does not require a special measurement device and can be applied to various measurement principles. The present invention cannot quantify an analyte that is present in excess. However, when a false-negative reaction occurs, there is a risk of making a completely wrong decision because it gives a result as if it were not present, even though the analyte is present at a high concentration. By preventing the false negative reaction in the excess range in the present invention, it can be reliably known that the analyte exists at a concentration exceeding at least the upper limit of the quantification range. Subsequently, the present invention will be described in more detail based on examples.

[0032]

【Example】

1. Preparation of anti-human hemoglobin A0 polyclonal antibody J. Biol. Chem. 235, p2327 from human hemoglobin hemolysate
The human hemoglobin A0 solution purified according to the method described in (1978) 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 3 times every 2 weeks, and 1 week after the final immunization, 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 washed with 0.15 M sodium chloride-containing 0.0
It was dissolved in a 2 M phosphate buffer (pH 7.2) (hereinafter abbreviated as PBS), dialyzed against the same buffer for 24 hours, and the protein concentration was adjusted to 1% by the absorbance at 280 nm to give 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.
Three booster immunizations were performed every two weeks, and three days after the final immunization, the spleen was removed and subjected to P3-NS1 / 1-Ag4 according to the method of Keller and Milstein described in Nature 256, p495 (1975).
-1 fused with myeloma. Antibody-producing hybridomas were selected by the ELISA 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. The gamma globulin fraction was salted out from this ascites by the 40% saturated ammonium sulfate salting-out method, and the precipitate obtained by centrifugation was mixed with 0.02M containing 0.15M sodium chloride.
Dissolved in phosphate buffer pH 7.2 (hereinafter abbreviated as PBS)
After dialysis against the same buffer solution for 24 hours, the protein concentration was adjusted to 1% by the absorbance at 280 nm to obtain an anti-human hemoglobin A0 monoclonal antibody.

3. Colored latex label of anti-human hemoglobin A0 monoclonal antibody Red polystyrene latex (average particle size 0.303 μm) in which the monoclonal antibody obtained in 2 was dispersed in an equal volume of PBS buffer to 10% w / v It was mixed with and physically adsorbed 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. Again P
Centrifuge and wash with BS buffer, and finally disperse with the same buffer to a latex concentration of 2% w / v, and then color latex labeled monoclonal antibody (hereinafter abbreviated as labeled antibody)
And 100 μl of the obtained labeled antibody was previously added to 5% (w / v)
The sucrose PBS solution was applied slowly to the dried porous polyethylene sheet (thickness 1 mm, 8 mm × 1.5 cm) and dried at room temperature to form a labeled antibody region.

4. Preparation of Immunochromatography Device Sephadex G75 (Pharmacia) was used as a capture agent according to the present invention to prepare a capture region for an immunochromagraph. Prepare a 1 x 5 cm, 2 mm thick polystyrene plate with a 5 mm diameter hole at a position 1 cm from the end in the longitudinal direction.
Below this, the end of the labeled antibody region prepared in 3 was placed at a position to close the bottom of the hole, and 10 mg of Sephadex G75 was put in the hole. In this state, the other end of the labeled antibody region is about 5 mm protruding from the end of the polystyrene plate.
And Sephadex G75 put in the hole functions as a trapping area. Nitrocellulose membrane with a pore size of 5 μm
A filter (made by Toyo Roshi Kaisha, Ltd.) was cut into 1 x 4.5 cm and anti-hemoglobin diluted to 0.6% at a position 2 cm from the edge.
3 μl of the polyclonal antibody was spotted, air-dried at room temperature, and immersed in a PBS solution of 2% BSA 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. It is arranged so that the end of the development area is located above the hole provided in the polystyrene plate, and the thick filter paper 17
A cut piece of Cr (manufactured by Whatman) having a size of 1 × 3 cm was placed downstream of the spot where the monoclonal antibody was spotted in the development area to form a liquid absorption area. In this state, the whole device was wrapped in parafilm and fixed.

5. Detection of Hemoglobin (Preventive Effect on False Negative Reaction in Excess of Antigen) 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 is based on Medical Phys
It was measured by the cyan methemoglobin method described in ics Vol. 2, p1072 (1950). 150 μl of the sample was slowly added dropwise to the labeled antibody region (the part protruding from the polystyrene plate) of the immunochromatographic apparatus prepared in 4, and the color change at the antibody-immobilized site in the development region was observed. By this operation, Sephadex G75 absorbs the water supplied from the labeled antibody region, swells rapidly, and eventually contacts the developing region.
After that, the sample is supplied to the development zone via Sephadex G75, but at this time, the elution rate between hemoglobin that has reacted with the labeled antibody and hemoglobin that exists in the free state without reacting in Sephadex G75 There is a difference. In other words, free hemoglobin is captured by Sephadex G75. As a target, the one in which the labeled antibody region and the development region were directly connected without providing the capture region was used. The results are shown in Table 1. The symbols in the table indicate the strength of coloring by the red latex in the development area, in order of strong coloring +
+, +, ±,-.

[0037]

[Table 1]

As can be seen from Table 1, when the concentration exceeds 400 μg / ml, the coloring strength corresponding to the concentration cannot be obtained when there is no scavenger. On the other hand, in the present invention using a scavenger, strong coloring was observed up to a much higher concentration, and it was confirmed that the false negative reaction prevention effect was high.

6. Detection of hemoglobin (influence on sensitivity in low-concentration sample) Using the immunochromatographic device of the present invention which is the same as in 4, sensitivity to a method of adding a free antibody that has been previously proposed as a measure against false negative reaction in an antigen excess region Were compared. The object used had the same structure as the immunochromatographic apparatus for the object used in 5 (that is, without the Sephadex G75). However, in this example, as a measure against false negatives, 2 μl of the polyclonal antibody (diluted to 0.5%) obtained in 1 was dropped on the labeled antibody region and dried to form the labeled antibody region. Analysis was carried out in the same manner as 5 for a hemoglobin solution of 0-160 ng / ml. The results are shown in Table 2. As is clear from Table 2, the immunochromatographic device according to the present invention maintains sufficient sensitivity. From these results, it was confirmed that the present invention can effectively prevent false negative reactions without sacrificing sensitivity.

[0040]

[Table 2]

List of cited documents [1] JP-A-6-324043 [2] JP-A-4-351962 [3] JP-A-6-341989 [4] JP-A-60-196669 [5] JP-A-2-87063 [6 ] JP-A 63-245698 [7] JP-A 1-299462 [8] JP-A 61-110059 [9] JP-A 2-176465 [10] J. Chromatog. 28, pp2-11, 1967

Claims (12)

[Claims] 1. A binding assay for a ligand or a receptor utilizing a binding reaction based on a specific affinity between the ligand and the receptor, which occurs when the analyte component is present in excess relative to the corresponding binding component. A technique that prevents false-negative reactions that can capture analytes in the free state, but cannot capture the complex formed by binding of the analytes with the corresponding binding component in the presence of a capture agent. Method for preventing false negative reaction in which binding reaction is performed or after the binding reaction is contacted with the capture agent 2. The binding assay utilizes a receptor that binds to at least two different sites of the analyte ligand, wherein the capture agent is allowed to act after contact between the first receptor and the analyte ligand, and then the second ligand. A method for preventing a false negative reaction according to claim 1, wherein the method reacts with a receptor. 3. The first receptor is present in a solution in a labeled form or in a form capable of being labeled, and the second receptor is present in a solid-phased state or in a solid-stateable state. Method for preventing false negative reaction according to claim 2 4. The binding assay utilizes a ligand recognized by a receptor to be analyzed and an anti-receptor recognizing the receptor to be analyzed, and a capture agent is allowed to act after contacting either of the ligand and the anti-receptor. The method for preventing false negative reactions according to claim 1, wherein the reaction with the other is then carried out. 5. A capture agent is a substance to be analyzed in a free state,
The method for preventing a false negative reaction according to claim 1, which has a molecular sieving effect capable of fractionating the complex with the binding partner based on the molecular weight. 6. The false product according to claim 5, wherein the scavenger having a molecular sieving effect is a gel composed of a material selected from the group consisting of dextran, crosslinked agarose, cellulose, polyacrylamide, and hydrophilic vinyl polymer. How to prevent negative reactions 7. The method for preventing a false negative reaction according to claim 1, wherein the ligand is an antigen, the receptor is an antibody, and the binding reaction with a specific affinity is an antigen-antibody reaction. 8. A binding assay is an immunochromatographic method for analyzing an antigen that traps an antigen reacted with a labeled antibody with a solid-phased antibody, wherein a capture agent is allowed to act after the reaction between the labeled antibody and the antigen, and then the labeled The method for preventing a false negative reaction according to claim 7, wherein an antibody-antigen complex is contacted with the immobilized antibody. 9. The false-negative reaction according to claim 7, wherein the mixture containing the labeled antibody and the antigen is brought into contact with the immobilized antibody in the coexistence of a capture agent, or reacts with the immobilized antibody after contact with the capture agent. How to prevent 10. A labeled antibody that recognizes an antigen to be measured, a chromatographic medium capable of fluid transfer of an immune complex of the labeled antibody and the antigen to be measured, and a solid-phased antibody immobilized on the chromatographic medium. An immunochromatographic device configured to contact with a solid-phased antibody located downstream by fluid transfer of an immune complex between a labeled antibody and an antigen to be measured, wherein a mixture of the labeled antibody and the antigen to be measured is measured. Antigen that is the target substance is captured, but antigen that is in the state of forming an immune complex with the labeled antibody is not substantially captured. In the presence of a capture agent, or after contact with the capture agent, the immobilized antibody is immobilized on the solid-phased antibody. Immunochromatographic device in which the capturing agent is arranged at a contact position 11. The antigen to be measured is hemoglobin, haptoglobin, albumin, globulin, α-1 antitrypsin, α-2 macroglobulin, transferrin,
The immunochromatographic device according to claim 10, which is an antigen selected from the group consisting of lactoferrin, chorionic hormone, luteinizing hormone, and estrogen. 12. In a method for measuring an antigen by an immunochromatography method including the following steps, an antigen as a substance to be measured is captured, but an antigen in a state of forming an immune complex with a labeled antibody is not substantially captured. Using a capture agent, a mixture of a labeled antibody and an antigen to be measured in the coexistence of the capture agent,
Alternatively, a method for measuring an antigen that is brought into contact with a solid-phased antibody after being brought into contact with the capture agent 1) Step of reacting a labeled antibody with an antigen to be measured Step 3) When the immune complex is generated in Step 1, the step of capturing with an immobilized antibody capable of binding this immune complex 4) The label captured by the immobilized antibody Of observing the presence of the antigen to be measured