JPWO2006004039A1 - Immunoassay apparatus and method - Google Patents

Abstract

An object is to provide a simple immunoassay device for quantification that is simple to use, can be measured in a single step, can be measured in a short time, and can handle various antigens. The present invention includes (1) a reaction solution addition site between a sample and a labeled first antibody that specifically binds to an antigen, or a sample addition site that contains the labeled first antibody and is movable when the antibody is dissolved in the sample. A first part, (2) a second part containing a second antibody that specifically binds to the first antibody in a form that cannot be transferred to the third part, and (3) a label of the first antibody is detected. The first portion of the labeled antibody comprises an F (ab ′) fragment and a labeled substance. Anti-idio in which the second antibody is an anti-idiotype antibody to the first antibody and cannot bind to the conjugate of the antigen and the first antibody, with a binding ratio of 1: 1 to 1: n (n is an integer) The objective is achieved by an immunoassay device, which is a type antibody. [Selection] Figure 3

Description

  The present invention relates to an immunoassay device used in the field of clinical tests and the like, and relates to an immunoassay device capable of quantitatively measuring an object to be measured in a sample simply, rapidly and with high sensitivity.

  As a method for measuring a substance contained in a biological sample such as blood and urine, a simple immunoassay device that is simple to use and can be measured in a short time has been developed. This apparatus has been widely used for emergency inspections, bedside inspections, etc., and its importance and usefulness have increased.

  As a simple immunoassay device, a measuring device based on the principle of immunochromatography, flow-through method, immunosensor method or the like is known. The method used in these devices is the principle that after adding a sample, the solution moves up and down or left and right while carrying out an antigen-antibody reaction, and finally a signal corresponding to the amount of the object to be measured is obtained. And its structure is relatively simple. As a signal detection method, a method is generally used in which colored particles typified by colloidal gold or colored latex are labeled on an antibody and the coloration of a detection line or a detection surface is read. In addition, although the measurement operation is complicated, coloration by an enzyme reaction or the like is also used.

  These simple immunoassay devices are basically used for qualitative purposes for determining the presence or absence of a measurement object. In recent years, however, attempts have been made to quantify using these devices in order to carry out more useful diagnoses. These attempts are based on the fact that the color tone of the detection line after measurement changes depending on the amount (concentration) of the measurement object. Specifically, as a method of reading the color tone of the detection line, a method of measuring with a reflectometer, a method of analyzing an image with a CCD image sensor (JP-A-8-334511, JP-A-2000-266751), a method based on electrical conductivity (JP 2001-337065) has been developed.

  Further, in the same immunoassay apparatus, prior art disclosing an apparatus relating to a method of capturing unreacted labeled antibody among all labeled antibodies and measuring unlabeled labeled antibody (that is, reacted labeled antibody). There is. For example, US Pat. No. 5,705,338, JP-A-4-16745, and JP-A-2003-262636. However, these measuring devices do not have sufficient quantitative properties. Furthermore, the use of anti-idiotype antibodies in the same prior art is disclosed in JP-A-7-151757.

JP-A-8-334511 JP2000-266751 JP 2001-337065 A US5705338 JP-A-4-16745 JP 2003-262636 A JP-A-7-151757

  The above-described quantification attempt is an application study to measure and quantify the detection result obtained from the qualitative device. Therefore, it is difficult to say that the color tone or intensity difference of coloring is delicate, the quantitative range is narrow, and sufficient quantitative properties are ensured. In addition, when an enzyme reaction is used as a detection method, it is effective for quantification and high sensitivity, but operations such as washing operation and addition of a test solution are required, and there are also problems such as background increase.

  The present invention has been made in view of the above-described problems, and an object of the present invention is to provide advantages of a simple immunoassay device, that is, simple use method, one-step operation, measurement in a short time, and the like. And providing a simple immunoassay device for quantification that can handle various antigens.

  As a result of intensive studies, the present inventor has conceived that simple immunoassay for quantification can be performed by configuring the apparatus as having three different parts in the apparatus, thereby completing the present invention. It was.

That is, the present invention is an immunoassay device capable of measuring the amount of antigen by specifically binding an antigen as a measurement object in a sample and a labeled antibody and measuring the label of the conjugate. The device has three parts (1) to (3) below, and the solution can move in order through each part. The labeled first antibody has an antibody portion F. (Ab ′) fragment and F (ab ′) fragment and label have a binding ratio of any of 1: 1 to 1: n (n is an integer), and the second antibody is an anti-idio against the first antibody. It is an immunoassay device that is a type antibody and is a kind of anti-idiotype antibody that cannot bind to a conjugate of an antigen and a first antibody.
(1) a site for adding a solution obtained by reacting a sample with a labeled first antibody capable of specifically binding to an antigen, or a labeled first antibody, The first antibody, which is a site for adding the sample, is contained in such a form that the antibody can be dissolved and moved in the sample. (2) A second antibody capable of specifically binding to the first antibody A second part (3) containing a reagent for detecting a label contained in the first antibody or a part thereof, which is a part contained in such a form that it cannot move to the third part, A third site which is a site for detecting the label of 1 antibody

  The present invention also reacts an antigen to be measured in a sample with a labeled antibody that specifically binds to the antigen, and captures the unreacted labeled antibody with an anti-idiotype antibody against the antibody. An immunoassay method for detecting a binding product between an antigen and a labeled antibody, wherein the antibody portion of the labeled antibody is an F (ab ′) fragment and the binding ratio of the F (ab ′) fragment to the labeled product is 1. 1 to 1: n (n is an integer), and the anti-idiotype antibody is an anti-idiotype antibody of a kind that cannot bind to a conjugate of an antigen and a labeled antibody. You can also

  The immunoassay device provided by the present invention enables quantitative measurement with a remarkably simple operation quickly and with high sensitivity. Therefore, it can be used for emergency examinations and bedside examinations, and is extremely useful in the medical field. Moreover, it can be easily applied to various antigens to be measured and is highly versatile.

  Embodiments of the present invention will be described below. An example of the device of the present invention is schematically shown in FIG. In FIG. 1, the device 4 includes a first part 1, a second part 2, and a third part 3. The sample solution provided to the first part is sequentially developed or sent, passes through the second part, and reaches the third part. Each part of the first part 1, the second part 2 and the third part 3 can be composed of various materials and forms, among which a glass fiber membrane, a porous membrane, a filter paper, a nylon membrane It is preferable to comprise a porous carrier such as Here, each part can be formed on one porous carrier, but an optimum carrier can be selected and used for each part. When changing the material for each part, it is necessary to arrange the parts so that they are in contact with each other so as not to obstruct the flow of the solution. Examples of the combination of carriers at each part include an embodiment in which the first part is a glass fiber membrane, the second part is a nitrocellulose film, and the third part is a filter paper. Moreover, it is preferable to arrange | position each site | part on the support body 5 which consists of plastic sheets etc., and an example of such an apparatus is shown in FIG. In FIG. 2, a first part 1, a second part 2, and a third part 3 are arranged on a support 5, and these constitute a device 4.

  Next, each part will be described. In the present invention, (1) the sample solution is added directly to the first site, and (2) the sample solution and the labeled first antibody (hereinafter labeled antibody) are reacted in a separate container in advance. It may be added to the first site later. In the former case, the first site contains a labeled antibody. On the other hand, in the latter case, the labeled antibody is not included in the first site. At this site, after the sample solution is added, the labeled antibody and the conjugate of the antigen and the labeled antibody move to the second site without being retained.

  In the second site, the anti-idiotype antibody is present in a state (1) directly immobilized on the carrier or device, or (2) immobilized on particles such as beads or latex. That is, the anti-idiotype antibody contained in the second site exists in a form that cannot move to the third site. A known method can be used for immobilizing the anti-idiotype antibody, and a physical adsorption method or a covalent bond method is appropriately selected. When an anti-idiotype antibody is immobilized on particles such as beads or latex, take measures such as making the pore size of the carrier at the second site or the third site smaller than the particle or narrowing the channel width. There is a need. At the second site, only the unreacted labeled antibody is captured, and the conjugate of the antigen and the labeled antibody moves to the third site without being captured.

  At the third site, the measurement of the label contained in the conjugate of the transferred antigen and the labeled antibody is performed. For the measurement of the label, a known detection method can be used according to the label, and necessary reagents can be included in the site. These reagents can be contained entirely in the third part, or a part of the reagents can be contained in the second part, the first part or another part. . The detection is performed using a general device such as a reflectometer, a spectrophotometer, or a fluorescence detector.

  The measurement object in the present invention refers to a test object in a general clinical test or the like, and specifically, various components contained in blood, urine, saliva, secretion, etc., and solids such as tissues and stool These are various components contained in the extract from In the present invention, since only one type of antibody is required for the measurement object, not only a substance having a large molecular weight capable of binding a plurality of monoclonal antibodies but also a small molecule (also referred to as a hapten) that can bind only one monoclonal antibody. Substances) can also be measured.

  In the present invention, the labeled first antibody binds various labels used in ordinary immunoassay methods to the F (ab ′) fragment of a monoclonal antibody against an antigen as a measurement target in a sample. It has been made. Furthermore, the optimal binding ratio between the F (ab ′) fragment of the antibody and the labeled substance is a state in which the antibody is bound at any ratio of 1: 1 to 1: n (n is an integer, preferably 1 to 5). is there. A known method can be used for F (ab ′) fragmentation of the monoclonal antibody and binding of the label to the F (ab ′) fragment.

  The use of such a labeled antibody in this apparatus is very advantageous for improving sensitivity and improving quantitativeness. As the monoclonal antibody, IgG is generally used. Since a labeled antibody using IgG as it is usually has a bivalent binding property, when it is reacted with an antigen as a measurement object, the binding ratio of the labeled antibody to the antigen is 1: 1 or 1: 2. Combined materials will be mixed. This is because not all antigens always bind to the binding site contained in one molecule of IgG antibody. When a bound substance having a mixed binding ratio passes through the second site, a 1: 1 bound substance is also captured in addition to an unreacted substance, which causes a decrease in sensitivity and quantitativeness. In contrast, in the present invention, since a monovalent binding F (ab ′) fragment is used, the binding ratio between the labeled antibody and the antigen is only 1: 1, except for unreacted ones. As a result, sensitivity and quantitativeness can be improved.

  As the labeling substance, those used for general immunoassay, such as enzymes, colored particles, fluorescent substances, luminescent substances, and ferrocene, can be used. Although it can be appropriately selected according to the required measurement sensitivity and the like, it is preferable to use an enzyme that is not easily affected by the matrix. As the enzyme, those which hardly exist in the living body or in the sample are preferable, and for example, peroxidase, glucose oxidase and the like are preferable. On the other hand, when an enzyme contained in a living body is used, an antibody against the enzyme may be included in the first site or the second site to remove the human-derived enzyme, or the enzyme It is also possible to take advantage of the specificity for the substrate or coenzyme. For example, when NAD is used as a coenzyme with glucose-6-phosphate dehydrogenase derived from Leuconostoc mesenteroides, this human-derived enzyme is NADP-dependent, so there is no problem even if it exists in the sample.

  The reagent for detecting the label is appropriately selected according to the label. When an enzyme is used for the label, usually one or more selected from NAD or NADP or a derivative thereof, an enzyme substrate or reagents for secondary generation of an enzyme substrate, a chromogenic substrate, diaphorase, and peroxidase It is a reagent. A specific example is described below.

  When peroxidase is used for the label, a chromogenic substrate, hydrogen peroxide, or reagents for secondary generation of hydrogen peroxide is required. In this case, the chromogenic substrate is 1,2-phenylenediamine (OPD), 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), 2,2′-azinobis-3-ethylbenzothiazoline-6- A sulfonic acid (ABTS) etc. can be used. Moreover, as a reagent for generating hydrogen peroxide secondary, for example, glucose and glucose oxidase can be used. When glucose oxidase is used for the label, a chromogenic substrate, glucose and peroxidase are required, and the same chromogenic substrate as that when the label is peroxidase can be used. In addition, when glucose-6-phosphate dehydrogenase is used as a label, glucose-6-phosphate and NAD or a derivative thereof are required. When performing detection by coloration, diaphorase and a chromogenic substrate are further required. It becomes. In this case, a reducing coloring agent such as nitro blue tetrazolium (NBT) can be used as the coloring substrate.

  Needless to say, auxiliary reagents such as reagent stabilizers and surfactants can also be added. If there are problems with the stability of these reagents when they are mixed, the reagents are dispersed in the first part, the second part, or another part. It is also possible to leave.

  The second antibody in the present invention is a monoclonal antibody that recognizes the unique structure of the antigen-binding site of the antibody portion of the labeled antibody, and is generally called an anti-idiotype antibody. Anti-idiotype antibodies are known to exist in a type that can inhibit binding to the target antibody by an antigen and a type that cannot be inhibited. The anti-idiotype antibody that can be used in the present invention refers to an antibody whose binding can be inhibited by an antigen, that is, a type that cannot bind to a conjugate of an antigen and a labeled antibody.

  This anti-idiotype antibody can be easily prepared according to a usual method. For example, when an anti-idiotype antibody of a mouse-derived monoclonal antibody is produced, if the monoclonal antibody that serves as the antigen is administered to the mouse as a KLH complex, it can be produced according to a conventional method for producing a monoclonal antibody. Therefore, the anti-idiotype antibody that can be used in the present invention can be easily and produced in a large amount in the same manner as a normal monoclonal antibody.

  In the present invention, the amount of the anti-idiotype antibody used for the second site needs to capture the unreacted labeled antibody almost completely, so that an excessive amount is required relative to the amount of the labeled antibody. The amount is preferably 10 times or more in terms of mole. Here, an antigen can be used in place of the anti-idiotype antibody, but in this case, from a practical viewpoint, it is limited to a stable antigen and an antigen that can be prepared in large quantities at a low cost. Since it is limited, no antigen is used in the device of the present invention. In addition, the amount of labeled antibody used in the present invention may be at least the upper limit of the antigen measurement range, and preferably about 2 to 100 times the upper limit of the antigen amount in terms of mole.

  The immunoassay apparatus of the present invention has been described above. FIG. 3 schematically shows the measurement principle of the present invention. The upper part (A) in the figure shows the entire device 4 in a perspective view. An arrow X in the figure indicates the direction in which the sample flows. Further, the lower part (B) in the figure shows the state of the reaction occurring in the apparatus over time. That is, at T = t0, the antigen 6 and the labeled antibody 7 are added to the first site 1. When an appropriate time has passed and T = t1, the sample passes through the second part 2 and the third part 3. At this time, the labeled antibody 7 that has not reacted with the antigen 6 is bound to the second antibody 8 and cannot move from the second site 2 to the third site 3. On the other hand, the labeled antibody 7 that has reacted with the antigen 6 passes through the second portion 2 and reaches the third portion 3. Thus, only the labeled antibody 7 that has reacted with the antigen 6 is detected at the third site 3.

  On the other hand, since the immunoassay method of the present invention can be easily carried out using the immunoassay device of the present invention, further explanation is omitted. Hereinafter, the present invention will be described in more detail with reference to examples of the present invention. However, the present invention is not limited to these examples.

Example 1 Production of anti-idiotype antibody against anti-human CRP mouse monoclonal antibody (1) Production of immunogen 0.1 M phosphorus containing 5 mg of commercially available anti-human CRP mouse monoclonal antibody (manufactured by Oriental Yeast) and 4 mg of KLH (manufactured by Calbiochem) 3 μL of glutaraldehyde (70%) was added to 2 mL of acid buffer (pH 6.7), and reacted at room temperature for 1 hour. After the reaction, it was dialyzed against PBS buffer to make an immunogen.

(2) Immunization A 0.5 mg / mL solution of the above immunogen and 1 mL of complete Freund's adjuvant were mixed and emulsified, and 200 μL was intraperitoneally administered to 8-week-old BALB / c mice. Thereafter, 200 μL of the above immunogen-incomplete Freund's adjuvant mixed emulsion was administered intraperitoneally three times every two weeks. Furthermore, two weeks after the third administration, 100 μL of the above-mentioned 0.5 mg / mL immunogen was intravenously injected.

(3) Cell fusion of spleen cells Three days later, the spleen was removed from the mouse, and the spleen cells were suspended in DMEM medium and washed. On the other hand, mouse myeloma cell line P3X63Ag8 (Dainippon Pharmaceutical Co., Ltd.) was cultured so as to be in a logarithmic growth phase according to cell fusion, and collected by centrifugation. Cell fusion was performed using the PEG method (PEG 4000).

(4) Production and screening of hybridoma Hybridomas were cultured in DMEM medium containing 10% FCS and HAT medium, and screened by the following method. The primary screening of the hybridoma culture supernatant is a purified F (ab ′) ₂ fragment obtained by digesting pepsin from an anti-human CRP mouse monoclonal antibody as a solid phase antigen, and a commercially available HRP-labeled goat anti-mouse IgG (Fc) as a secondary antibody. This was carried out by sandwich ELISA using an antibody (manufactured by ICN). Furthermore, as a secondary screening, CRP was reacted with the solid phase antigen in advance and the same operation was carried out to determine whether or not it was a type that caused binding inhibition by CRP. As a result of cloning and screening of positive wells, two clones of 13D and 14A were obtained as hybridomas that produce an antibody that specifically recognizes the anti-human CRP monoclonal antibody and inhibits binding by CRP.

(5) Preparation of antibody Among the hybridoma cell lines obtained above, 13D was expanded and the anti-idiotype antibody was collected. The culture supernatant was purified with a protein A column (manufactured by Amersham Japan).

Example 2 Production of HRP-labeled anti-human CRP antibody (1) Pepsin digestion 1 mL of 0.1 M acetate buffer (pH 4.0) was mixed with 5 mg of anti-human CRP mouse monoclonal antibody and 0.3 mg of pepsin (manufactured by Sigma), The reaction was carried out at 24 ° C. for 24 hours. After the reaction, it was purified by gel filtration to obtain 1.8 mg of F (ab ′) ₂ fragment. As the anti-human CRP mouse monoclonal antibody, a commercially available product similar to the above was used.

(2) Reduction The F (ab ′) ₂ fragment solution was replaced with a 0.1 M phosphate buffer (pH 6.5) containing 5 mM EDTA using an ultrafiltration membrane. A 0.1 M cysteamine solution was added to the antibody solution and reacted at 37 ° C. for 2 hours. After the reaction, it was purified by gel filtration to obtain 1.0 mg of F (ab ′) fragment.

(3) Maleidation of HRP 5 mg of peroxidase (HRP: manufactured by Roche) was dissolved in 750 μL of 0.1 M phosphate buffer (pH 7.0). 2 mg of N-hydroxysuccinimide ester (Zieben Chemical Tokyo) was dissolved in 120 μL of DMF, 75 μL was added to the HRP solution, and reacted at 30 ° C. for 1 hour. After the reaction, it was purified by gel filtration to obtain 2.6 mg of maleimidated HRP.

(4) Preparation of conjugate 1.0 mg of F (ab ′) fragment and 1.0 mg of maleimidated HRP were mixed and reacted for 24 hours in the refrigerator. After the reaction, it was purified by gel filtration and after concentration, a 1.2 mg / mL HRP-labeled anti-human CRP antibody solution was obtained.

Example 3 Production of CRP Measuring Device (Test Piece) (1) Preparation of Label Area Filter Paper (First Site) 10 mg of BSA and 10 mg of glucose were dissolved in 10 mL of PBS buffer. Next, the HRP-labeled anti-human CRP antibody prepared in Example 2 was diluted 5000 times with this solution. The solution was further impregnated with filter paper (manufactured by Whatman), dried, and then cut into 12 mm × 5 mm.

(2) Preparation of capture area membrane (second site) 5 μL of the anti-idiotype antibody solution 1 mg / mL solution prepared in Example 1 was added to a high flow membrane (Millipore) cut to 10 mm × 5 mm. After drying at room temperature, it was placed in a 3 mg / mL casein solution, allowed to stand for 30 minutes, then taken out and dried.

(3) Preparation of detection area filter paper (third part)
5 mg of TMBZ and 30 units of pyranose oxidase were dissolved in 1 mL of PBS buffer. This solution was impregnated with filter paper (3 hours manufactured by Whatman), dried, and cut into 7 mm × 5 mm.

(4) Assembling the test piece A double-sided adhesive tape was applied to a plastic plate (60 mm × 5 mm) as a support with a width of 25 mm from the upper end. Next, the upper end of the plastic plate was left about 5 mm, and the membrane (2) was attached and fixed. Next, the filter paper of (3) was stuck and fixed so as to overlap the upper end of the membrane of (2) by about 2 mm. Further, the filter paper of (1) was stuck and fixed so that the lower end of the membrane of (2) overlapped by about 2 mm.

Example 4 CRP Quantitative Measurement A solution containing 0, 3.25, 7.5, 15, 30, 60, 120 ng / mL CRP was used as a specimen. The sample was added to 30 μL of the label area filter paper at the sample addition position to start the reaction, and after 5 minutes, the reflectance at 690 nm of the detection area was measured with a color difference meter. A calibration curve showing the relationship between the obtained reflectance and the concentration of human CRP is shown in FIG. According to this, a good calibration curve was shown from a low concentration, and it was found that CRP can be quantified.

It is a figure which shows typically an example of the apparatus of this invention. It is a figure which shows typically another example of the apparatus of this invention. It is a figure which shows the measurement principle of the apparatus of this invention. 6 is a calibration curve showing the relationship between the reflectance obtained in Example 4 and the concentration of human CRP.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... 1st site | part, 2 ... 2nd site | part, 3 ... 3rd site | part, 4 ... Apparatus, 5 ... Support body, 6 ... Antigen, 7 ... Labeled antibody, 8 ... 2nd antibody

Claims (6)

An immunoassay device capable of specifically binding an antigen as a measurement target in a sample and a labeled antibody and measuring the amount of the antigen by measuring the label of the conjugate. It has the following three parts (1) to (3) inside, and the solution is capable of moving through each part in order. The labeled first antibody has an F (ab ′) fragment in the antibody part. And the binding ratio of the F (ab ′) fragment to the label is any one of 1: 1 to 1: n (n is an integer), and the second antibody is an anti-idiotypic antibody against the first antibody and An immunoassay device that is an anti-idiotype antibody of a kind that cannot bind to a conjugate of an antigen and a first antibody.
(1) a site for adding a solution obtained by reacting a sample with a labeled first antibody capable of specifically binding to an antigen, or a labeled first antibody, The first antibody, which is a site for adding the sample, is contained in such a form that the antibody can be dissolved and moved in the sample. (2) A second antibody capable of specifically binding to the first antibody A second part (3) containing a reagent for detecting a label contained in the first antibody or a part thereof, which is a part contained in such a form that it cannot move to the third part, A third site which is a site for detecting the label of 1 antibody The device according to claim 1, wherein the second antibody is immobilized on a carrier or particles at the second site. The device according to claim 1 or 2, wherein the first antibody is labeled with an enzyme. The reagent for detecting the label contained in the first antibody is NAD or NADP or a derivative thereof, an enzyme substrate or a reagent for secondary generation of an enzyme substrate, a chromogenic substrate, diaphorase, or peroxidase. The apparatus of claim 3, wherein the apparatus is one or more reagents selected from: The apparatus according to any one of claims 1 to 4, wherein the three portions are each made of any one of a glass fiber membrane, a porous membrane, a filter paper, and a nylon membrane. An antigen that is a measurement object in a sample is reacted with a labeled antibody that specifically binds to the antigen, and the unreacted labeled antibody is captured with an anti-idiotype antibody against the antibody, and the antigen that has not been captured is labeled. An immunoassay method for detecting a binding substance with an antibody, wherein the antibody portion of the labeled antibody is an F (ab ′) fragment, and the binding ratio of the F (ab ′) fragment to the labeling substance is 1: 1 to 1: n (n is an integer), and the anti-idiotype antibody is an anti-idiotype antibody of a kind that cannot bind to a conjugate of an antigen and a labeled antibody.

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