HK1172681B - Fluoroimmunoassay method - Google Patents

Description

Fluorescence immunoassay method

Technical Field

The present invention relates to a novel antigen concentration measurement method which does not require a solid-phase formation step and a washing step, and a kit for carrying out the antigen concentration measurement method.

Background

Among the methods for measuring the concentration of an antigen or an antibody, the most widely used measurement method in clinical diagnosis, basic research, environmental investigation, and the like is an immunoassay called a sandwich ELISA method (or a sandwich RIA method) in which 2 kinds of monoclonal antibodies recognizing different epitopes of the same antigen or a monoclonal antibody and a polyclonal antibody are used. The details of the sandwich method are as follows. In the first step, a monoclonal/polyclonal antibody called a primary antibody is immobilized on a measurement plate, a sample containing an antigen is injected into the plate, and the antibody is allowed to bind to the antigen for a certain period of time. Next, as a second stage, impurities bound to the antibody and antigens non-specifically bound to the plate are washed away using a washing solution. In the third stage, a labeled secondary antibody solution to which a reporter molecule such as an enzyme, a fluorescent dye, or a radioisotope is previously bound is injected and reacted for a certain period of time, so that the labeled secondary antibody is further bound to the antigen filled with the primary antibody. After the reaction, the amount of the antigen in the sample is measured by removing the excess labeled antibody with a washing solution and measuring the amount of the reporter molecule bound to the measurement plate with enzyme activity, fluorescence, radioisotope, or the like.

As described above, in the usual sandwich ELISA method, 2 kinds of antibodies having different epitopes need to be used, but in the case of using, for example, a low-molecular compound or the like as an antigen, it is difficult to produce a plurality of kinds of antibodies recognizing different epitopes. Therefore, Shanghai et al established a method of immunoassay using high-precision low-molecular compounds called open sandwich method using heavy chain variable regions (VH) and light chain variable regions (VL) of 1 antibody (patent documents 1 and 2, and non-patent documents 1 and 2). The method comprises the following antigen concentration determination methods: a method for measuring the amount of a reporter molecule of a labeled polypeptide bound to an immobilized polypeptide, which comprises preparing a VH region polypeptide and a VL region polypeptide of an antibody that specifically recognizes an antigen, labeling one of the polypeptides with a reporter molecule to prepare a labeled polypeptide, immobilizing the other polypeptide on a solid phase to prepare an immobilized polypeptide, bringing a sample containing the antigen and the labeled polypeptide into contact with the immobilized polypeptide, and measuring the amount of the reporter molecule of the labeled polypeptide bound to the immobilized polypeptide. In addition, as a measurement method for measuring a low molecular compound, there are a liquid chromatography and the like in addition to an immunoassay method, but this method requires a highly accurate measurement instrument, requires a large amount of a specimen, takes a long time for measurement, and has low versatility.

As an immunoassay method for measuring the concentration of an antigen using an antibody labeled with a fluorescent dye, the following methods are known: immunoassay methods in which an antibody and an antigen are labeled with different fluorescent dyes, respectively, and a change in the efficiency of Fluorescence Resonance Energy Transfer (FRET) occurring between the fluorescent dyes is used as an index (non-patent documents 3 and 4); an immunoassay method using, as an index, a change in quenching efficiency that utilizes a phenomenon in which fluorescence of an antibody quenched by mixing a quenching substance with a fluorescently labeled antibody in advance is enhanced by introduction of a target detection substance; a fluorescence immunoassay method for measuring a decrease in fluorescence intensity caused by aggregation of a labeled antibody and an analyte using an antibody labeled with a fluorescent dye (patent document 3).

Documents of the prior art

Patent document

Patent document 1: japanese laid-open patent publication No. 10-78436

Patent document 2: japanese patent No. 3784111

Patent document 3: japanese laid-open patent publication No. 10-282098

Non-patent document

Non-patent document 1: shandong Tian macro, Chinese medicine, 35468, 27: 71-80(2007)

Non-patent document 2: lim SL, et al, Anal Chem, 79(16) 6193-200(2007)

Non-patent document 3: iijima I.and Hohsaka T, Chembiolchem, 17;10 (6): 999-1006(2009)

Non-patent document 4: kajihara D, et al, Nat methods, 3(11):923(2006)

Disclosure of Invention

Problems to be solved by the invention

As described above, the immunoassay methods known so far require a step of immobilizing an antibody or an antigen and a washing step of removing the adsorption of a nonspecific labeling compound. These steps are complicated and time-consuming to operate, and result in variation, and therefore, development of a liquid phase immunoassay method which does not require a solid phase immobilization step and a washing step is required. An object of the present invention is to provide an immunoassay method which does not require a solid-phase formation step and a washing step, can rapidly and easily perform quantitative measurement of a target substance in a liquid phase, and can visualize an antigen.

Means for solving the problems

First, the present inventors tried to establish an antibody/antigen binding activity evaluation system using antibodies VH and VL labeled with different fluorescent dyes, using Fluorescence Resonance Energy Transfer (FRET) efficiency as an index. In contrast to the FRET measurement, which uses a light chain region (CR110-VL) of an anti-BGP antibody labeled with a fluorescent dye CR110 and a heavy chain region (TAMRA-VH) of an anti-BGP antibody labeled with a fluorescent dye TAMRA, in the absence of an antigen, FRET from CR110 to TAMRA does not occur, whereas in the presence of an antigen, VH and VL form a complex of the three via the antigen, thereby predicting that FRET from CR110 to TAMRA may occur, CR110-VL and TAMRA-VH or unlabeled VH are incubated with BGP antigen peptides at different concentrations, and the change in fluorescence intensity of CR110 is analyzed by Fluorescence Intensity Distribution Analysis (FIDA). As a result, when CR110-VL and TAMRA-VH were reacted, the fluorescence intensity of CR110 decreased depending on the concentration of the BGP antigen peptide, and it was confirmed that CR110-VL and TAMRA-VH were combined with the antigen peptide to form a complex, and thus the predicted FRET from CR110 to TAMRA occurred.

On the other hand, it was surprising that in the case of reacting CR110-VL with unlabeled VH, the fluorescence intensity of CR110 increased depending on the concentration of BGP antigen peptide. In order to confirm whether the same phenomenon can be observed even in the case of using a labeled VH, the present inventors incubated TAMRA-VH and CR110-VL or unlabeled VL with BGP antigen peptides at different concentrations, and analyzed the change in fluorescence intensity of TAMRA. As a result, it was found that when TAMRA-VH was reacted with unlabeled VL, the fluorescence intensity of TAMRA-VH was also increased depending on the concentration of BGP antigen peptide. The above results are entirely unexpected, establishing the following hypothesis: it is possible that VL and VH act as quenchers for fluorescent dyes (CR110, TAMRA) and that this quenching is released only when VH and VL form a complex with an antigen peptide, thereby increasing the fluorescence intensity.

Based on the above hypothesis, the present inventors attempted to establish a novel measurement method (hereinafter, also referred to as "homogeneous fluorescent immunoassay method") utilizing a quenching phenomenon by reacting TAMRA-VH and BGP peptide at different concentrations in the presence or absence of unlabeled VL and measuring fluorescence intensity. As a result, it was found that the fluorescence intensity of TAMRA-VH in the presence of VL increases depending on the concentration of BGP peptide, and a highly sensitive homogeneous fluorescence immunoassay method could be constructed based on the analysis result of the ratio of the fluorescence intensity of TAMRA-VH in the presence/absence of VL (+ VL/-VL). Further, the present inventors confirmed that the sensitivity of the "homogeneous fluorescence immunoassay method" described above was increased by using ATTO655 as a fluorescent dye and labeling the fluorescent dye on VH via a spacer.

Furthermore, the present inventors have conducted experiments to confirm the quenching effect using a mutant VH in which 4 tryptophanes (hereinafter, sometimes also referred to as Trp or W) present in the VH are each mutated to phenylalanine (hereinafter, sometimes also referred to as Phe or F), and have found that tryptophanes at positions 36, 47 and 106 (corresponding to position 103 in the Kabat numbering system) in the amino acid sequence of the VH function as a quencher for a fluorescent dye. Since these tryptophans are highly conserved in the mouse antibody VH, it is understood that by applying the "homogeneous fluorescence immunoassay method" of the present invention, measurement of an antigen can be performed using various antibodies.

The present invention has been completed based on the above findings.

Effects of the invention

According to the present invention, an immunoassay method capable of quickly and easily performing quantitative measurement of a target substance in a liquid phase and a kit for performing antigen measurement using the same can be provided. The measurement method of the present invention is a method for detecting/measuring the binding between an antigen and an antibody VL or VH using the fluorescence intensity of a fluorescent dye labeled on the antibody VL or VH as an index, and is based on the following novel findings: since the fluorescent dye is in a quenched state when the antibodies VL and VH are not bound to each other, and the quenching of the fluorescent dye is released when the antibodies VL and VH are bound to each other by the antigen, the method does not require a solid phase formation step and a washing step which are essential in the conventional immunoassay method, and thus, a highly accurate measurement result with a small variation can be obtained in a short time.

Namely, the present invention relates to: (1) a kit for measuring and detecting an antigen concentration, comprising an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide, wherein either one of the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide is labeled with a fluorescent dye, characterized in that the antigen concentration can be measured or the antigen can be visualized by using as an index the fact that there is a positive correlation between the antigen concentration in a liquid phase and the fluorescence intensity of the fluorescent dye; (2) the kit for measuring and detecting an antigen concentration according to the above (1), wherein the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide are bound to form a single chain antibody.

Furthermore, the invention relates to: (3) the kit for measuring and detecting the concentration of an antigen as described in the above (1) or (2), wherein the fluorescent dye is a rhodamine-based fluorescent dye orOxazine fluorescent pigments; (4) the kit for measuring and detecting an antigen concentration according to the above (3), wherein the fluorescent dye is CR110, TAMRA or ATTO 655; (5) the kit for measuring and detecting the antigen concentration according to any one of the above (1) to (4), wherein the antibody heavy chain variable region polypeptide comprises a polypeptide having an amino acid sequence represented by SEQ ID No. 1, and the antibody light chain variable region polypeptide comprises a polypeptide having an amino acid sequence represented by SEQ ID No. 2; (6) the kit for measuring and detecting the antigen concentration according to any one of the above (1) to (4), wherein the antibody heavy chain variable region polypeptide comprises a polypeptide having an amino acid sequence represented by SEQ ID No. 6, and the antibody light chain variable region polypeptide comprises a polypeptide having an amino acid sequence represented by SEQ ID No. 7.

Further, the present invention relates to: (7) the method for measuring and detecting the concentration of the antigen is characterized by sequentially comprising the following steps (a) - (c): (a) contacting an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide labeled with a fluorescent dye, or an antibody heavy chain variable region polypeptide and an antibody light chain variable region polypeptide labeled with a fluorescent dye (a1) in a liquid phase with an antigen in a test substance, or (a2) with an antigen in a test non-human animal subject to which an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide labeled with a fluorescent dye, or an antibody heavy chain variable region polypeptide and an antibody light chain variable region polypeptide labeled with a fluorescent dye are administered, or (a3) with an antigen in a subject in vitro; (b) measuring the fluorescence intensity of the fluorescent dye in the case of (a1) and detecting the fluorescence of the fluorescent dye in the cases of (a2) and (a 3); (c) the amount of the antigen contained in the test substance was calculated in the case of (a1) above, and the antigen contained in the subject was visualized in the cases of (a2) and (a3) above, using as an index the fact that the concentration of the antigen in the liquid phase had a positive correlation with the fluorescence intensity of the fluorescent dye.

In addition, the present invention relates to: (8) the method for measuring and detecting the antigen concentration according to (7) above, wherein the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide are bound to form a single-chain antibody; (9) the method for measuring and detecting the concentration of an antigen as described in (7) or (8), wherein the fluorescent dye is a rhodamine-based fluorescent dye orOxazine fluorescent pigments; (10) the method for measuring and detecting an antigen concentration according to (9) above, wherein the fluorescent dye is CR110, TAMRA or ATTO 655; (11) the method for measuring and detecting the antigen concentration according to any one of (7) to (10), wherein the antibody heavy chain variable region polypeptide comprises a polypeptide having an amino acid sequence represented by SEQ ID No. 1, and the antibody light chain variable region polypeptide comprises a polypeptide having an amino acid sequence represented by SEQ ID No. 2; (12) the antigen according to any one of (7) to (10) aboveA method for measuring concentration of an antibody, characterized in that the antibody heavy chain variable region polypeptide comprises a polypeptide having an amino acid sequence represented by SEQ ID No. 6, and the antibody light chain variable region polypeptide comprises a polypeptide having an amino acid sequence represented by SEQ ID No. 7.

Drawings

FIG. 1 is a diagram schematically showing a CR 110-labeled anti-BGP antibody light chain variable region polypeptide (CR110-VL), a TAMRA-labeled anti-BGP antibody heavy chain variable region polypeptide (TAMRA-VH) and a complex thereof (CR110-VL/TAMRA-VH) of the present invention.

FIG. 2 is a graph showing the results of reacting CR110-VL with TAMRA-VH in the presence of BGP peptide at different concentrations and measuring the fluorescence spectrum using excitation light of 490 nm.

FIG. 3 shows the results of reacting CR110-VL with TAMRA-VH in the presence of different concentrations of BGP peptide, and measuring the change in fluorescence intensity at 525nm (F525) and 575nm (F575).

FIG. 4 is a graph showing the results of reacting CR110-VL with TAMRA-VH in the presence of BGP peptide at different concentrations, measuring the fluorescence intensities at 525nm and 575nm, and analyzing the change in the ratio of the fluorescence intensities (F575/F525).

FIG. 5 shows the results of reaction of the CR 110-labeled anti-BGP antibody light chain variable region polypeptide of the present invention and TAMRA-labeled anti-BGP antibody heavy chain variable region polypeptide in the presence of BGP peptides of different concentrations, and analysis by Fluorescence Intensity Distribution Analysis (FIDA) using a 488nm laser and a 510-560 nm fluorescence filter. In the figure, CR110-VL represents a CR 110-labeled anti-BGP antibody light chain variable region polypeptide, TAMRA-VH represents a TAMRA-labeled anti-BGP antibody heavy chain variable region polypeptide, and w.t. -VH represents an unlabeled anti-BGP antibody heavy chain variable region polypeptide.

FIG. 6 is a graph showing the results of reaction of the CR 110-labeled anti-BGP antibody light chain variable region polypeptide of the present invention and the TAMRA-labeled anti-BGP antibody heavy chain variable region polypeptide in the presence of BGP peptides at different concentrations, and analysis by Fluorescence Intensity Distribution Analysis (FIDA) using a laser at 543nm and a fluorescence filter at 560 to 620 nm. In the figure, CR110-VL represents a CR 110-labeled anti-BGP antibody light chain variable region polypeptide, TAMRA-VH represents a TAMRA-labeled anti-BGP antibody heavy chain variable region polypeptide, and w.t. -VL represents an unlabeled anti-BGP antibody light chain variable region polypeptide.

FIG. 7 shows the results of reacting the TAMRA-labeled anti-BGP antibody light chain variable region polypeptide of the present invention with BGP peptides at different concentrations in the presence or absence of an unlabeled anti-BGP antibody light chain variable region polypeptide, and measuring the fluorescence intensity using a 543nm He-Ne laser. In the figure, + VL indicates the result of the reaction in the presence of the unlabeled anti-BGP antibody light chain variable region polypeptide, and-VL indicates the result of the reaction in the absence of the unlabeled anti-BGP antibody light chain variable region polypeptide and measurement of the fluorescence intensity.

FIG. 8 is a graph showing the results of reacting the TAMRA-labeled anti-BGP antibody heavy chain variable region polypeptide of the present invention with different concentrations of BGP peptide in the presence or absence of an unlabeled anti-BGP antibody light chain variable region polypeptide and analyzing the ratio of fluorescence intensities (+ VL/-VL).

FIG. 9 is a diagram showing tryptophan residues present in the anti-BGP antibody heavy chain variable region polypeptide of the present invention. The figure shows the positions of tryptophan residues (W33, W36, W47, W106) in a three-dimensional structure prediction model of a complex of an anti-BGP antibody heavy chain variable region polypeptide and an anti-BGP antibody light chain variable region polypeptide (VH/VL complex) and an individual anti-BGP antibody heavy chain variable region polypeptide (VH). The positions of these tryptophan residues correspond to the amino acids at positions 33, 36, 47 and 103 of VH, respectively, in the numbering system of Kabat database.

Fig. 10 is a graph showing the results of reacting a wild-type anti-BGP antibody heavy chain variable region polypeptide (WT) or a mutant anti-BGP antibody heavy chain variable region polypeptide (W33F, W36F, W47F, W106F) with an anti-BGP antibody light chain variable region polypeptide in the presence of BGP peptides of different concentrations and measuring the fluorescence intensity using a 543nm He-Ne laser.

FIG. 11 is a graph showing the results of reacting a wild-type anti-BGP antibody heavy chain variable region polypeptide (WT) or a mutant anti-BGP antibody heavy chain variable region polypeptide (W33F, W36F, W47F, W106F) with an anti-BGP antibody light chain variable region polypeptide in the presence of BGP peptides at different concentrations, and analyzing the change in diffusion time (dispersion time) by Fluorescence Correlation Spectroscopy (FCS).

FIG. 12 is a diagram showing a model for predicting the three-dimensional structure of a complex of a fluorescently labeled anti-BGP antibody heavy chain variable region polypeptide (Fluorescent labeled BGP-VH) having a spacer attached thereto and an anti-BGP antibody light chain variable region polypeptide (BGP-VL) (Fluorescent labeled BGP-VH/VL).

FIG. 13 shows the results of reacting an ATTO 655-labeled anti-BGP antibody heavy chain variable region polypeptide with or without a spacer (GGGSGGGS; SEQ ID NO: 4) and an anti-BGP antibody light chain variable region polypeptide in the presence of BGP peptides at different concentrations and measuring the fluorescence intensity.

FIG. 14 is a graph showing the ratio of fluorescence intensities measured by reacting an ATTO 655-labeled anti-BGP antibody heavy chain variable region polypeptide with or without a spacer (GGGSGGGS; SEQ ID NO: 4) and an anti-BGP antibody light chain variable region polypeptide in the presence of different concentrations of a BGP peptide.

Fig. 15 is a diagram showing a model for predicting the three-dimensional structure of a fluorescently labeled single-chain antibody in which the fluorescently labeled antibody heavy chain variable region polypeptide of the present invention is bound to the antibody light chain variable region polypeptide.

Fig. 16 is a diagram schematically showing the one-dimensional structure of a fluorescently labeled single-chain antibody in which a fluorescently labeled antibody heavy chain variable region polypeptide of the present invention and an antibody light chain variable region polypeptide are bound to each other.

FIG. 17 is a graph showing the results of reacting an ATTO 655-labeled anti-BGP single-chain antibody with or without a spacer and BGP peptides at different concentrations and measuring the fluorescence intensity.

FIG. 18 is a graph showing the ratio of fluorescence intensities measured by reacting an ATTO 655-labeled anti-BGP single-chain antibody with or without a spacer and BGP peptides at different concentrations.

FIG. 19 shows the results of reaction of the ATTO 655-labeled anti-BGP single-chain antibody of the present invention with BGP peptide at different concentrations and detection of fluorescence using a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi software engineering Co., Ltd.). In the figure, FL92 represents a spacer comprising the amino acid sequence represented by SEQ ID NO. 3, and 2TAG represents a spacer comprising MX (X is a fluorescent-labeled amino acid).

FIG. 20 is a graph showing the results of reacting a fluorescently labeled anti-BGP single-chain antibody with or without a spacer with BGP peptides at different concentrations and quantifying fluorescence using a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi software engineering Co., Ltd.). In the figure, FL92 represents a spacer comprising the amino acid sequence represented by SEQ ID NO. 3, and 2TAG represents a spacer comprising MX (X is a fluorescent-labeled amino acid).

FIG. 21 is a graph showing the results of reacting a fluorescently labeled anti-BGP single-chain antibody with or without a spacer with BGP peptide at different concentrations and detecting fluorescence using a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi software engineering Co., Ltd.). In the figure, FL92 represents a spacer comprising the amino acid sequence represented by SEQ ID NO. 3, and 2TAG represents a spacer comprising MX (X is a fluorescent-labeled amino acid).

FIG. 22 is a graph showing the ratio of fluorescence intensities obtained by reacting a fluorescently labeled anti-BGP single-chain antibody containing a FL92 spacer (SEQ ID NO: 3) with BGP peptides at different concentrations and measuring the resultant using a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi software engineering Co., Ltd.) and MF20/FluoroPoint-Light (manufactured by Olympus Co., Ltd.).

FIG. 23 is a graph showing the results of reacting a TAMRA-labeled anti-BGP single-chain antibody with or without a spacer with BGP peptides at different concentrations and detecting fluorescence. G3S (1) represents the sequence of the spacer (linker) containing GGGS, G3S (2) represents the sequence of the spacer (linker) containing GGGSGGGS (sequence No. 4), and G3S (3) represents the sequence of the spacer (linker) containing GGGSGGGS (sequence No. 10).

FIG. 24 shows the results of the reaction of a TAMRA-labeled anti-BGP single-chain antibody protein with different concentrations of BGP in the presence of PBST buffer and human plasma at a final concentration of 50%, and the measurement of fluorescence intensity.

FIG. 25 is a graph showing the results of reacting an ATTO 655-labeled anti-bisphenol A (BPA) antibody heavy chain variable region polypeptide of the present invention with BPA at various concentrations in the presence or absence of an unlabeled anti-BPA antibody light chain variable region polypeptide and measuring the fluorescence intensity.

FIG. 26 is a graph showing the ratio of fluorescence intensities measured by reacting an ATTO 655-labeled anti-BPA antibody heavy chain variable region polypeptide with various concentrations of BPA in the presence or absence of an unlabeled anti-BPA antibody light chain variable region polypeptide.

FIG. 27 is a graph showing the results of reacting TAMRA-labeled anti-BPA single-chain antibodies with BPA peptides at different concentrations, with or without a spacer, and detecting fluorescence. G3S (2) shows the sequence of the spacer (linker) containing GGGSGGGS (seq id No. 4), G3S (3) shows the sequence of the spacer (linker) containing GGGSGGGSGGGS (seq id No. 10), and G3S (5) shows the sequence of the spacer (linker) containing GGGSGGGSGGGSGGGSGGGS (seq id No. 11).

Fig. 28 is a graph showing the results of reacting the TAMRA-labeled anti-HEL single-chain antibody with different concentrations of HEL protein and detecting fluorescence.

FIG. 29 is a graph showing the results of reacting a TAMRA-labeled anti-estradiol single chain antibody with estradiol at different concentrations and detecting fluorescence.

FIG. 30 is a graph showing the results of reacting a TAMRA-labeled anti-SA single-chain antibody with BSA or HSA at different concentrations and detecting fluorescence.

FIG. 31 is a drawing showing that tryptophan at position 36, 47 or 103 in the numbering system of the Kabat database is conserved in the amino acid sequence of the mouse antibody heavy chain variable region. This figure shows a) a model for predicting the three-dimensional structure of the anti-BGP mouse antibody heavy chain variable region, and the possibility that W36, W47, and W106 among tryptophan residues contained in the anti-BGP mouse antibody heavy chain variable region are particularly related to quenching.

FIG. 32 is a graph showing that W33 is highly conserved in a wide variety of mouse antibody heavy chain variable regions.

FIG. 33 is a graph showing that W36 is highly conserved in a wide variety of mouse antibody heavy chain variable regions.

FIG. 34 is a graph showing that W47 is highly conserved in a wide variety of mouse antibody heavy chain variable regions.

FIG. 35 is a graph showing that W106 is highly conserved in a wide variety of mouse antibody heavy chain variable regions. In the amino acid sequence of the VH, Trp106 corresponds to the position at position 103 in the numbering system of the Kabat database.

Detailed Description

The kit for measuring and detecting the concentration of an antigen of the present invention is not particularly limited as long as it is a kit as described below: the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide are provided, and either one of the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide is labeled with a fluorescent dye, and the measurement of the antigen concentration or the visualization of the antigen can be performed using, as an index, the fact that there is a positive correlation between the antigen concentration in the liquid phase and the fluorescence intensity of the fluorescent dye. That is, the kit is not particularly limited as long as it is a kit represented by the following (1) to (4): (1) a kit for measuring an antigen concentration, comprising an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide labeled with a fluorescent dye, wherein the kit is capable of measuring the antigen concentration using as an indicator a positive correlation between the antigen concentration in a liquid phase and the fluorescence intensity of the fluorescent dye; (2) a kit for detecting an antigen, comprising an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide labeled with a fluorescent dye, wherein the kit is capable of visualizing the antigen using as an indicator a positive correlation between the amount of the antigen in a subject and the fluorescence intensity of the fluorescent dye; (3) a kit for measuring an antigen concentration, comprising an antibody heavy chain variable region polypeptide and an antibody light chain variable region polypeptide labeled with a fluorescent dye, wherein the kit is capable of measuring the antigen concentration using as an indicator a positive correlation between the antigen concentration in a liquid phase and the fluorescence intensity of the fluorescent dye; (4) a kit for detecting an antigen, comprising an antibody heavy chain variable region polypeptide and an antibody light chain variable region polypeptide labeled with a fluorescent dye, wherein the kit is capable of visualizing the antigen using as an indicator that there is a positive correlation between the amount of the antigen in a subject and the fluorescence intensity of the fluorescent dye. The antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide may be prepared as two separate polypeptide fragments or as a single-chain antibody fused by a linker or the like, as long as they can form a complex with the same antigen molecule. The antigen is not particularly limited as long as it is an antigen specifically recognized by the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide, and examples thereof include: proteins, peptides, carbohydrates, lipids, glycolipids, low molecular compounds, and the like.

The method for measuring and detecting the concentration of an antigen of the present invention is not particularly limited as long as it is a method for measuring and detecting the concentration of an antigen characterized by comprising the following steps (a) to (c) in this order: (a) contacting an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide labeled with a fluorescent dye, or an antibody heavy chain variable region polypeptide and an antibody light chain variable region polypeptide labeled with a fluorescent dye (a1) in a liquid phase with an antigen in a test substance, or (a2) with an antigen in a test non-human animal subject to which an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide labeled with a fluorescent dye, or an antibody heavy chain variable region polypeptide and an antibody light chain variable region polypeptide labeled with a fluorescent dye are administered, or (a3) with an antigen in a subject in vitro; (b) measuring the fluorescence intensity of the fluorescent dye in the case of (a1) and detecting the fluorescence of the fluorescent dye in the cases of (a2) and (a 3); (c) the amount of the antigen contained in the test substance was calculated in the case of (a1) above, and the antigen contained in the subject was visualized in the cases of (a2) and (a3) above, using as an index the fact that the concentration of the antigen in the liquid phase had a positive correlation with the fluorescence intensity of the fluorescent dye. That is, there is no particular limitation as long as it is a method described below: a method for measuring an antigen concentration (hereinafter, sometimes referred to as "measurement method [ I ]"), comprising the steps of: a step (a1-1) of bringing an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide labeled with a fluorescent dye into contact with an antigen in a test substance in a liquid phase, a step (b) of measuring the fluorescence intensity of the fluorescent dye, and a step (c) of calculating the amount of the antigen contained in the test substance by using as an index the concentration of the antigen in the liquid phase and the fluorescence intensity of the fluorescent dye in a positive correlation; a method for measuring an antigen concentration (hereinafter, sometimes referred to as "measurement method [ II ]"), comprising the steps of: a step (a1-2) of bringing an antibody heavy chain variable region polypeptide and an antibody light chain variable region polypeptide labeled with a fluorescent dye into contact with an antigen in a test substance in a liquid phase, a step (b) of measuring the fluorescence intensity of the fluorescent dye, and a step (c) of calculating the amount of the antigen contained in the test substance by using as an index the concentration of the antigen in the liquid phase and the fluorescence intensity of the fluorescent dye in a positive correlation; an antigen detection method (hereinafter, sometimes referred to as "non-human animal detection method [ I ]"), comprising the steps of: a step (a2-1) of contacting an antigen in a test non-human animal subject to which an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide labeled with a fluorescent dye have been administered, (b) a step of detecting fluorescence of the fluorescent dye, and (c) a step of visualizing the antigen contained in the test non-human animal subject using as an index a positive correlation between the amount of the antigen in the test non-human animal subject and the fluorescence intensity of the fluorescent dye; an antigen detection method (hereinafter, sometimes referred to as "non-human animal detection method [ II ]"), comprising the steps of: a step (a2-2) of contacting an antigen in a test non-human animal subject to which an antibody heavy chain variable region polypeptide and an antibody light chain variable region polypeptide labeled with a fluorescent dye have been administered, a step (b) of detecting the fluorescence of the fluorescent dye, and a step (c) of visualizing the antigen contained in the subject using as an index a positive correlation between the amount of the antigen in the test non-human animal subject and the fluorescence intensity of the fluorescent dye; an antigen detection method (hereinafter, sometimes referred to as "in vitro detection method [ I ]"), comprising the steps of: a step (a3-1) of contacting an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide labeled with a fluorescent dye with an antigen in a subject in vitro, a step (b) of detecting fluorescence of the fluorescent dye, and a step (c) of visualizing the antigen contained in the subject using as an index the presence of a positive correlation between the amount of the antigen in the subject and the fluorescence intensity of the fluorescent dye; an antigen detection method (hereinafter, sometimes referred to as "in vitro detection method [ II ]"), comprising the steps of: the method for detecting the antigen in the subject comprises the steps of (a3-2) contacting the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide labeled with a fluorescent dye with the antigen in the subject in vitro, (b) detecting the fluorescence of the fluorescent dye, and (c) visualizing the antigen contained in the subject using as an index the presence of a positive correlation between the amount of the antigen in the subject and the fluorescence intensity of the fluorescent dye. The antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide may be prepared as two separate polypeptide fragments or as a single-chain antibody fused by a linker or the like, as long as they can form a complex with the same antigen molecule. The antigen is not particularly limited as long as it is an antigen specifically recognized by the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide, and examples thereof include: proteins, peptides, carbohydrates, lipids, glycolipids, low molecular compounds, and the like.

The antibody heavy chain variable region polypeptide is not particularly limited as long as it contains an amino acid sequence specific for the antibody heavy chain variable region encoded by exons of the V, D and J regions of the antibody heavy chain gene, and any amino acid sequence may be further added to the N-terminal and/or C-terminal side of the amino acid sequence specific for the antibody heavy chain variable region. The amino acid sequence specific to the antibody heavy chain variable region is preferably an amino acid sequence in which the amino acid at position 36, 47 or 103 in the numbering system of Kabat database is tryptophan, and specifically, an amino acid sequence represented by sequence number 1 or an amino acid sequence represented by sequence number 6 can be preferably exemplified.

The antibody light chain variable region polypeptide is not particularly limited as long as it contains an amino acid sequence specific to the antibody light chain variable region encoded by exons of the V region and the J region of the antibody light chain gene, and any amino acid sequence may be further added to the N-terminal and/or C-terminal side of the amino acid sequence specific to the antibody light chain variable region. Further, the amino acid sequence specific to the antibody light chain variable region is preferably an amino acid sequence in which the amino acid at position 35 in the numbering system of Kabat database is tryptophan, and specifically, an amino acid sequence represented by sequence number 2 and an amino acid sequence represented by sequence number 7 can be preferably exemplified.

The antibody light chain variable region polypeptide, the antibody heavy chain variable region polypeptide, and the single-chain antibody polypeptide containing both the antibody light chain variable region and the antibody light chain variable region can be produced by a known chemical synthesis method, a genetic recombination technique, a method of decomposing an antibody molecule with a protease, or the like, and among them, it is preferably produced by a genetic recombination technique which is relatively easy to handle and can be produced in large quantities. In the case of producing the above-mentioned polypeptide by a gene recombination technique, a DNA containing a nucleotide sequence encoding an antibody light chain variable region or an amino acid sequence specific to the antibody light chain variable region may be introduced into an appropriate vector to prepare an expression vector, and the desired polypeptide may be expressed by an expression system or a cell-free translation system using bacteria, yeast, insects, animal and plant cells, etc. as a host. When the polypeptide is expressed in a cell-free translation system, the polypeptide may be expressed in a reaction solution obtained by adding nucleoside triphosphate and various amino acids to a cell-free extract of, for example, Escherichia coli, wheat germ, rabbit reticulocyte, or the like.

The fluorescent dye is not particularly limited as long as it is a fluorescent dye that is quenched (quenched) in a state of being labeled on an antibody heavy chain variable region polypeptide or an antibody light chain variable region polypeptide, and examples thereof include those having rhodamine, coumarin, Cy, EvoBlue, Vahl,examples of the fluorescent dye or derivative of the fluorescent dye include a basic skeleton such as oxazine, carbopol (Carbopyronin), naphthalene, biphenyl, anthracene, phenanthrene, pyrene, and carbazole, and specifically include: CR 110: carboxyrhodamine 110, RhodamineGreen (trade name); TAMRA: carboxytetramethylrhodamine, TMR; ATTO655 (trade name); BODIPY FL (trade name): 4, 4-difluoro-5, 7-dimethyl-4-boron-3 a,4 a-diaza-sym indacene-3-propionic acid; BODIPY 493/503 (trade name): 4, 4-difluoro-1, 3,5, 7-tetramethyl-4-boron-3 a,4 a-diaza-sym indacene-8-propanoic acid; bodipy 6G (trade name): 4, 4-difluoro-5- (4-phenyl-1, 3-butadienyl) -4-boron-3 a,4 a-diaza-sym indacene-3-propionic acid; BODIPY 558/568 (trade name): 4, 4-difluoro-5- (2-thienyl) -4-boron-3 a,4 a-diaza-sym indacene-3-propionic acid; BODIPY 564/570 (trade name): 4, 4-difluoro-5-styryl-4-boron-3 a,4 a-diaza-sym indacene-3-propionic acid; BODIPY576/589 (trade name): 4, 4-difluoro-5- (2-pyrrolyl) -4-boron-3 a,4 a-diaza-sym indacene-3-propionic acid; BODIPY 581/591 (trade name): 4, 4-difluoro-5- (4-phenyl-1, 3-butadienyl) -4-boron-3 a,4 a-diaza-sym indacene-3-propionic acid; cy3 (trade name); cy3B (trade name); cy3.5 (trade name); cy5 (trade name); cy5.5 (trade name); EvoBlue10 (trade name); EvoBlue30 (trade name); MR 121; ATTO 390 (trade name); ATTO 425 (trade name); ATTO 465 (trade name); ATTO 488 (trade name);ATTO 495 (trade name); ATTO520 (trade name); ATTO 532 (trade name); ATTO Rho6G (trade name); ATTO 550 (trade name); ATTO 565 (trade name); ATTO Rho3B (trade name); ATTO Rho11 (trade name); ATTO Rho12 (trade name); ATTO Thio12 (trade name); ATTO 610 (trade name); ATTO 611X (trade name); ATTO 620 (trade name); ATTO Rho14 (trade name); ATTO633 (trade name); ATTO 647 (trade name); ATTO 647N (trade name); ATTO655 (trade name); ATTO Oxa12 (trade name); ATTO 700 (trade name); ATTO 725 (trade name); ATTO 740 (trade name); alexa Fluor 350 (trade name); alexa Fluor 405 (trade name); alexa Fluor 430 (trade name); alexa Fluor 488 (trade name); alexa Fluor 532 (trade name); alexa Fluor 546 (trade name); alexa Fluor 555 (trade name); alexa Fluor568 (trade name); alexa Fluor 594 (trade name); alexa Fluor 633 (trade name); AlexaFluor 647 (trade name); alexa Fluor 680 (trade name); alexa Fluor 700 (trade name); alexa Fluor 750 (trade name); alexa Fluor 790 (trade name); rhodamine Red-X (trade name); texas Red-X (trade name); 5(6) -TAMRA-X (trade name); 5TAMRA (trade name); SFX (trade name), among them, CR110 and TAMRA, which are rhodamine-based fluorescent dyes, and the likeATTO655 of oxazine fluorescent dye.

The method for labeling the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide with a fluorescent dye is not particularly limited, and the following methods can be used: a method of directly labeling by using functional groups at both ends or side chains of a polypeptide or indirectly labeling by using a crosslinking agent; a method of site-specifically labeling a polypeptide while synthesizing the polypeptide using an in vitro transcription-translation system, and the like. As a method for labeling using an in vitro transcription-translation system, there are known: amber suppression method (Ellman J et al (1991) Methods Enzymol.202:301-36), C-terminal labeling method (Japanese patent laid-open No. 2000-139468), N-terminal labeling method (U.S. Pat. No. 5643722, Olejnik et al (2005) Methods 36:252-260), etc., wherein DNA or mRNA in which the codon encoding the amino acid of the labeled target site is replaced with an amber codon as one of the stop codons is prepared, and a protein is synthesized from the DNA or mRNA by an in vitro transcription-translation system. In this case, a protein in which a labeled amino acid is introduced at a position substituted with an amber codon can be synthesized by adding a suppressor tRNA to which a labeled unnatural amino acid is bound to a protein synthesis reaction solution. In addition, in the C-terminal labeling method, a protein into which a label is specifically introduced at the C-terminal can be synthesized by performing translation from DNA or mRNA into the protein in an in vitro transcription-translation system in which a labeled puromycin is added at an optimum concentration.

The kit for measuring and detecting the concentration of an antigen of the present invention may include reagents and instruments generally used in such an immunoassay kit, such as a buffer solution, a tube or plate for measurement, and an antigen that can be used as a standard substance. The kit for measuring and detecting the concentration of an antigen of the present invention can be suitably used for the method for measuring and detecting the concentration of an antigen of the present invention.

In the step (a1-1) of the aforementioned measurement method [ I ] of the present invention, a complex of three of an antibody light chain variable region polypeptide, an antibody heavy chain variable region polypeptide labeled with a fluorescent dye, and an antigen specifically recognized by an antibody is formed in a solution by adding the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide labeled with a fluorescent dye to a buffer solution, a physiological saline solution, or the like, followed by addition of a test substance and incubation. In the step (a1-2) of the above measurement method [ II ], the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide labeled with a fluorescent dye are added to a buffer solution, a physiological saline solution or the like, and then a test substance is added thereto and incubated to form a complex of the three components, i.e., the antibody heavy chain variable region polypeptide, the antibody light chain variable region polypeptide labeled with a fluorescent dye, and the antigen specifically recognized by the antibody in the solution. Examples of the test substance include: there are some cases where the target antigen to be measured may be contained in body fluids such as serum, plasma, saliva, urine, culture supernatants, cell extracts, and industrial waste water. The incubation conditions are not particularly limited as long as they are generally applicable to antibody-antigen reactions, and the temperature conditions may be set to, for example, 1 to 30 ℃, preferably 18 to 25 ℃, and the reaction time may be set to, for example, 5 to 180 minutes, preferably 60 to 120 minutes. The solution after completion of the incubation may be directly subjected to the following step (b) without a step such as washing. This is a large feature of the antigen concentration determination and detection method of the present invention.

In the above-mentioned measurement method [ I ] or the step (b) of the measurement method [ II ] of the present invention, the fluorescence intensity of the fluorescent dye in the solution can be measured by irradiating the solution prepared in the above-mentioned step (a1-1) or (a1-2) with excitation light. The fluorescence measuring apparatus used for the measurement is not particularly limited, and preferable examples thereof include: MF20/FluoroPoint-Light (manufactured by Olympus), FMBIO-III (manufactured by Hitachi software engineering Co., Ltd.), and the like. In the measurement, as a negative control of the solution prepared in the above step (a1-1), the following solutions are preferably measured: 1) a solution containing no antibody light chain variable region polypeptide but only an antibody heavy chain variable region polypeptide labeled with a fluorescent dye; 2) a solution containing only the antibody heavy chain variable region polypeptide labeled with a fluorescent dye and the test substance without the antibody light chain variable region polypeptide; 3) a solution containing an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide labeled with a fluorescent dye, but not containing a test substance; 4) a solution containing an antibody light chain variable region polypeptide, an unlabeled antibody heavy chain variable region polypeptide, and a test substance; etc., as negative controls for the solution prepared by the above step (a1-2), the following solutions are preferably measured: 1) a solution containing only the antibody light chain variable region polypeptide labeled with a fluorescent dye, without the antibody heavy chain variable region polypeptide; 2) a solution containing only the antibody light chain variable region polypeptide labeled with a fluorescent dye and the test substance without the antibody heavy chain variable region polypeptide; 3) a solution containing an antibody heavy chain variable region polypeptide and an antibody light chain variable region polypeptide labeled with a fluorescent dye, but not containing a test substance; 4) a solution containing an antibody heavy chain variable region polypeptide, an unlabeled antibody light chain variable region polypeptide, and a test substance; and the like.

In the above-mentioned method [ I ] or method [ II ] of the present invention, in the step (c), the amount of the antigen contained in the test substance can be calculated from the fluorescence intensity measurement value obtained in the step (b). That is, since the concentration of the antigen in the solution prepared in the above step (a1-1) or (a1-2) has a positive correlation with the fluorescence intensity measured in the step (b), the amount of the antigen contained in the test substance can be determined by measuring the fluorescence intensity when the test substance containing the antigen at a known concentration is used, creating a calibration curve showing the relationship between the antigen concentration and the fluorescence intensity, and calculating the antigen concentration corresponding to the measured value of the fluorescence intensity when the test substance containing the antigen at an unknown concentration is used from the calibration curve. The term "calculating the amount of antigen" in step (c) also includes a case where the amount of antigen is automatically calculated by a conversion equation or the like set in advance based on a standard curve.

In the above-described non-human animal detection method [ I ] of the present invention, in the step (a2-1), the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide labeled with a fluorescent dye are administered to the subject non-human animal, and a complex of the three is formed in the subject non-human animal, the antibody light chain variable region polypeptide, the antibody heavy chain variable region polypeptide labeled with a fluorescent dye, and the antigen specifically recognized by the antibody. In the non-human animal detection method [ II ] (step (a 2-2)), the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide labeled with a fluorescent dye are administered to the subject non-human animal systemically or locally to form a complex of the three, i.e., the antibody heavy chain variable region polypeptide, the antibody light chain variable region polypeptide labeled with a fluorescent dye, and the antigen specifically recognized by the antibody, in the subject non-human animal. The subject non-human animal subject is not particularly limited as long as it is an animal other than human, and examples thereof include: mouse, rat, hamster, monkey, pig, and the like. The "administration" method may be appropriately selected from parenteral topical administration methods such as intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, implant, and coating, and oral administration methods.

In the above-described non-human animal detection method [ I ] or non-human animal detection method [ II ] of the present invention, in the step (b), fluorescence of a fluorescent dye in the non-human animal subject to which either one of the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide labeled with a fluorescent dye was administered in the above-described step (a2-1) or (a2-2) is detected noninvasively while the individual is kept, or fluorescence of a fluorescent dye in a tissue or cell collected from the non-human animal subject is detected. The "detection" method is not particularly limited as long as it is a method capable of irradiating an individual, tissue or cell of a non-human animal subject with excitation light to detect fluorescence of a fluorescent dye in two or three dimensions. In the detection, it is preferable to simultaneously create an image showing the structure of an individual, tissue, or cell of the non-human animal subject to be tested using an endoscope, X-ray, CT, MRI, ultrasound, microscope, or the like.

In the above-mentioned non-human animal detection method [ I ] or non-human animal detection method [ II ] of the present invention, in the step (c), the antigen in the non-human animal subject to be tested is visualized based on the detection result of fluorescence of the fluorescent dye obtained in the step (b). That is, since the amount of the antigen in the test non-human animal subject has a positive correlation with the fluorescence intensity of the fluorescence detected in step (b), the localization (position) of the antigen can be known by comparing the image data showing the structure of the individual, tissue, or cell of the test non-human animal subject with the two-dimensional or three-dimensional image of the fluorescence detected in step (b). For example, in the case of detection using an endoscope, the localization of an antigen in a tissue can be known by irradiating the tissue of a non-human animal subject with visible light to produce a visible light image that can be observed, bringing either of the labeled antibody light chain variable region polypeptide and the labeled antibody heavy chain variable region polypeptide into contact with the tissue by coating or the like, then irradiating the tissue with excitation light for the labeled fluorescent dye to produce a fluorescent image, and comparing the visible light image with the fluorescent image.

In the step (a3-1) of the in vitro detection method [ I ] of the present invention, the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide labeled with a fluorescent dye are incubated in vitro with an antigen in a subject to form a complex of the three, i.e., the antibody light chain variable region polypeptide, the antibody heavy chain variable region polypeptide labeled with a fluorescent dye, and the antigen specifically recognized by the antibody. In the step (a3-2) of the in vitro detection method [ II ], the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide labeled with a fluorescent dye are incubated in vitro with an antigen in a subject to form a complex of the three, i.e., the antibody heavy chain variable region polypeptide, the antibody light chain variable region polypeptide labeled with a fluorescent dye, and the antigen specifically recognized by the antibody. As the above subject, there can be mentioned: there are a possibility that cultured cells, tissue sections, tissues or cells collected from a living body, and cell extracts blotted on nitrocellulose membranes, PVDF membranes, and the like may be contained as target antigens to be measured. The incubation conditions are not particularly limited as long as they are generally applicable to antibody-antigen reactions, and the temperature conditions may be set to, for example, 1 to 30 ℃, preferably 18 to 25 ℃, and the reaction time may be set to, for example, 5 to 180 minutes, preferably 60 to 120 minutes. The solution after completion of the incubation may be directly subjected to the following step (b) without a step such as washing. This is a large feature of the antigen concentration determination and detection method of the present invention.

In the in vitro detection method [ I ] or the in vitro detection method [ II ], in the step (b), the fluorescence of the fluorescent dye in the subject incubated with the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide, either of which is labeled with the fluorescent dye in the step (a3-1) or (a3-2), is detected two-dimensionally or three-dimensionally. Examples of the "detection" method include: fluorescence microscopes, fluorescence image analyzers, and the like.

In the step (c) of the in vitro detection method [ I ] or the in vitro detection method [ II ], the antigen in the subject can be visualized on the basis of the result of detection of fluorescence of the fluorescent dye obtained in the step (b). That is, since the amount of the antigen in the subject has a positive correlation with the fluorescence intensity of the fluorescence detected in step (b), the location (position) of the antigen can be known based on the two-dimensional or three-dimensional image of the fluorescence detected in step (b).

The present invention is explained in more detail specifically in the examples shown below. The following examples are examples for illustrating the present invention, and the technical scope of the present invention is not limited by these examples.

Example 1

1. Establishment of homogeneous fluorescence immunoassay method Using anti-BGP antibody-derived VH and VL

(construction of anti-BGP antibody V region Gene expression vector)

ProX containing amber codon is added to the N-terminus of the DNA sequence encoding the heavy chain variable region (VH; SEQ ID NO: 1) or light chain variable region (VL: SEQ ID NO: 2) of an antibody against human osteocalcin (human glutamic acid Protein, human Bone Gla Protein; BGP)^TMThe DNA sequence of the tag (MSKQIEVNXSNET (X is a fluorescent-labeled amino acid); SEQ ID NO: 3) was incorporated into the NcoI and HindIII sites of pIVX2.3d vector (manufactured by Roche diagnostics). The constructed expression vector has ProX added to the N-terminus of the inserted VH or VL^TMA tag (MSKQIEVNXSNET (X is a fluorescent-labeled amino acid); SEQ ID NO: 3) and a His-tag attached to the C-terminus. Schematically shown in FIG. 1 are CR 110-labeled anti-BGP antibody light chain variable region polypeptide (CR110-VL), TAMRA-labeled anti-BGP antibody heavy chain variable region polypeptide (TAMRA-VH), and complexes thereof (CR 110-VL/TAMRA-VH). Wild-type VH and VL expression vectors were made with a substitution of a fluorescently labeled amino acid residue for a phenylalanine residue by replacing the TAG codon with a TTT codon in the same manner.

Further, the following vectors were also prepared: mutant VL (W33F, W36F, W47F, W106F) expression vectors obtained by replacing 4 tryptophan codons (TGG; Trp33, Trp36, Trp47, Trp106) contained in the VH gene with phenylalanine codons (TTT), respectively, were labeled with ProXAn additional spacer VH expression vector having a spacer (GGGSGGGS; SEQ ID NO: 4) between the tag and the N-terminus of VH gene, a single chain antibody (scFv) expression vector obtained by binding VH gene to VL gene using a linker (LVTVSSGGGGSGGGGSGGGGS, SEQ ID NO: 5; or GGGGSGGGGSGGGGS, SEQ ID NO: 9), and a method of producing the same in ProX^TMA single chain antibody (scFv) expression vector having a spacer (GGGS, GGGSGGGS, SEQ ID NO: 4; or GGGSGGGSGGGS, SEQ ID NO: 10) sequence between the tag and the N-terminus of the scFv, and an scFv expression vector having MX (X is a fluorescent-labeled amino acid) at the N-terminus.

(preparation of protein labeling anti-BGP antibody V region)

Using RTS100 e.coli disulfide protein expression kit (e.coli disulfide kit) (manufactured by roche diagnostics), a fluorescently labeled amino acid was introduced into the V-region protein N-terminal region using a cell-free translation system. To the reaction solution (50. mu.L) were added 7. mu.L of the amino acid mixture, 1. mu.L of methionine, 7. mu.L of the reaction mixture, 25. mu.L of the activated E.coli lysate, 5. mu.L of plasmid DNA (500ng), and 5. mu.L of the fluorescence-labeled succinyl-aminoacyl tRNA (0.8 nmol). Fluorescently labeled aminoacyl tRNA (TAMRA-X-AF-amber suppressor tRNA, CR 110-X-AF-amber suppressor tRNA and ATTO 655-X-AF-amber suppressor tRNA) for making fluorescently labeled proteins protein functionalized cloverleverdirect using a protein that introduces an unnatural amino acid at a site^TMtRNA reagent (プロテインエクスプレス). The reaction mixture was reacted at 20 ℃ and 600rpm for 2 hours, and then further reacted at 4 ℃ for 16 hours. After completion of the reaction, 1. mu.L of the reaction solution was subjected to SDS-PAGE (15%) and protein expression was observed by a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi software engineering Co., Ltd.). Furthermore, western blotting was performed using a His-tag antibody to confirm that the target protein was synthesized.

The synthesized V-domain protein was purified by using His-Spin Trap column (GE ヘルスケア). To the above reaction solution (50. mu.L), a washing solution (20mM phosphate buffer solution (pH7.4)/0.5M NaCl/60mM imidazole/0.1% polyoxyethylene (23) lauryl ether) was added to make 400. mu.L, and applied to a His-Spin Trap column. After incubation for 15 minutes at room temperature, three washes were performed. Next, 200. mu.L of an eluent (20mM phosphate buffer solution (pH7.4)/0.5M NaCl/0.5M imidazole/0.1% polyoxyethylene (23) lauryl ether) was eluted twice. Further, the eluate was buffer-exchanged and concentrated with PBS (+0.05% Tween 20) using an Ultrafree-0.5 centrifuge tube (manufactured by ミリポア Co.). The concentration of the purified sample was determined by SDS-PAGE and FCS (MF 20; manufactured by Olympus).

Example 2

(measurement of fluorescence Spectroscopy)

The TAMRA-labeled anti-BGP antibody VH protein and CR 110-labeled anti-BGP antibody VL protein (1. mu.g/mL, 30. mu.L, respectively) prepared in example 1 and a 7-residue C-terminal peptide of BGP (RRFYGPV; SEQ ID NO: 8) as an antigen were prepared to a total of 200. mu.L using PBS (+0.05% Tween 20). After leaving at 25 ℃ for 90 minutes, the resultant was subjected to fluorescence spectrometry using a fluorescence spectrophotometer (FluoroMax-4; manufactured by ホリバ & ジヨバンイボン Co.). The excitation wavelength was set at 490nm for the mixture of CR110-VL and TAMRA-VH, and 550nm for TAMRA-VH. The fluorescence intensity ratio I of the mixture of CR110-VL and TAMRA-VH was calculated_A/I_D。I_AAnd I_DFluorescence intensities at 575nm and 525nm, respectively. By fluorescence intensity ratio (I)_A/I_D) Or the fluorescence intensity at the maximum fluorescence wavelength, the dissociation constant (Kd) value is calculated by curve fitting. At this time, an S-type dose-effect model of GraphpadPrism (manufactured by Graphpad) was used as statistical analysis software. FIG. 2 shows the results obtained by reacting CR110-VL with TAMRA-VH in the presence of BGP peptide at different concentrations and measuring the fluorescence spectrum using excitation light at 490nm, FIG. 3 shows the results obtained by reacting CR110-VL with TAMRA-VH in the presence of BGP peptide at different concentrations and measuring the changes in fluorescence intensity at 525nm and 575nm, and FIG. 4 shows the results obtained by reacting CR110-VL with TAMRA-VH in the presence of BGP peptide at different concentrations and analyzing the changes in the ratio of fluorescence intensities at 525nm and 575nm (F575/F525). TAMRA-tagged anti-BGP antibody scFv proteins (2. mu.g/mL, 25. mu.g/mL) with or without spacers were labeled with PBS (+0.05% Tween 20, 0.2% BSA)μ L) and BGP peptide as an antigen were prepared into a total of 200 μ L of sample. Then, the sample was left at 25 ℃ for 70 minutes, and then fluorescence spectroscopy was performed using a fluorescence spectrophotometer (FluoroMax-4; manufactured by ホリバ & ジヨバンイボン) to calculate the fluorescence intensity by curve fitting. At this time, an S-type dose-effect model of ImageJ software (http:// rsbweb. nih. gov/ij /) was used as statistical analysis software. The measurement was carried out under the conditions that the excitation wavelength was 550nm and the measurement wavelength was 580 nm.

(fluorescence intensity distribution analysis method)

The fluorescently labeled VH protein and the fluorescently labeled VL protein (1. mu.g/mL, 7.5. mu.L, respectively) or the fluorescently labeled scFv (1. mu.g/mL, 7.5. mu.L) prepared in example 1 together with the BGP peptide were prepared to 50. mu.L using PBS (+0.05% Tween 20), added to 384-well glass-bottom microplates (manufactured by Olympus Co., Ltd.), and incubated at 25 ℃ for 90 minutes. Measurement by Fluorescence Intensity Distribution Analysis (FIDA) was carried out at 25 ℃ using MF20/FluoroPoint-Light (manufactured by Olympus). TAMRA and ATTO655 were excited with 543nm and 633nm lasers, respectively. Data was acquired for 10 seconds for each assay, and 10 assays were performed for each sample. From the measurement values, the mean value and standard deviation were calculated.

Example 3

(evaluation of binding Activity of TAMRA-VH and CR110-VL to BGP peptide)

First, an attempt was made to establish an antibody/antigen binding activity evaluation system using Fluorescence Resonance Energy Transfer (FRET). CR110 and TAMRA were used as donor and acceptor, respectively, in FRET assays. The fluorescence of the donor (CR110) and the absorption of the acceptor (TAMRA) are sufficiently overlapped, and thus can be used as a FRET pair. Aligning factor (k)²) At 2/3, the Foster (フエルスタ -) distance (R) is calculated₀) Is composed ofThis value is suitable for detecting intermolecular interactions of proteins. In the absence ofIn the case of antigen, since the interaction between VL and VH is weak, FRET from CR110 to TAMRA does not occur, whereas in the case of antigen, VH, VL and antigen form a three-component complex, and as a result, FRET from CR110 to TAMRA is predicted to occur.

To confirm whether or not FRET from CR110 to TAMRA is detected when CR110-VL is bound to TAMRA-VH by an antigen, the following experiment was performed. CR110-VL was incubated with TAMRA-VH or unlabeled VH together with different concentrations of BGP antigen peptide (110000ng), and the change in fluorescence intensity of CR110 was analyzed by Fluorescence Intensity Distribution Analysis (FIDA). In the measurement, 488nm laser is used as excitation light, and a fluorescence filter of 510-560 nm is used for detection. The results are shown in FIG. 5. When CR110-VL is reacted with TAMRA-VH, the fluorescence intensity of CR110 decreases depending on the concentration of BGP antigen peptide. From these results, it was confirmed that CR110-VL and TAMRA-VH form a complex by binding of the antigen peptide, and thereby cause the expected FRET from CR110 to TAMRA. On the other hand, it was surprising that in the case of reacting CR110-VL with unlabeled VH, the fluorescence intensity of CR110 increased depending on the concentration of BGP antigen peptide. From this result, the following possibility is presumed: VL acts as a quencher on CR110, and in the case where CR110-VL alone is present, the fluorescence of CR110 is quenched by VL, but in the case where CR110-VL, VH and the antigen peptide form a complex of the three, the quenching effect is released.

In order to confirm whether the quenching effect by VL was observed even in VH, the following experiment was performed. TAMRA-VH and CR110-VL or unlabeled VL were incubated together with different concentrations of BGP antigen peptide (1-10000 ng), and the change in fluorescence intensity of TAMRA was analyzed by Fluorescence Intensity Distribution Analysis (FIDA). In the measurement, a 543nm laser is used as an excitation light, and a 560 to 620nm fluorescence filter is used for detection. The results are shown in FIG. 6. When TAMRA-VH is reacted with CR110-VL, the fluorescence intensity of TAMRA increases depending on the concentration of BGP antigen peptide. Similarly, when TAMRA-VH is reacted with unlabeled VL, the fluorescence intensity of TAMRA-VH increases depending on the concentration of BGP antigen peptide. The following possibilities are presumed from the above results: in the same manner as in VL, VH also acts as a quencher for TAMRA, and in the case where TAMRA-VH is present alone, the fluorescence of TAMRA is quenched, but in the case where TAMRA-VH, VL and the antigen peptide form a three-part complex, the quenching effect is released. The above results suggest that VH and VL have a quenching effect on the fluorescent dye, and that this effect is released by the formation of a complex of VH/VL/antigen (fig. 9).

Example 4

(establishment of homogeneous fluorescence immunoassay method of antigen concentration utilizing quenching phenomenon)

The present inventors conducted the following experiment in consideration of whether a new immunoassay method can be established by utilizing the quenching phenomenon as clarified in example 3. FIG. 7 shows the results of the reaction of TAMRA-VH with BGP peptide at different concentrations in the presence or absence of unlabeled VL and the measurement of the fluorescence intensity using a 543nm He-Ne laser. In the presence of VL, the fluorescence intensity of TAMRA-VH increased depending on the concentration of BGP peptide. On the other hand, in the absence of VL, the fluorescence intensity of TAMRA-VH remained low regardless of the concentration of BGP peptide reacted with it. Furthermore, as a result of analyzing the ratio of fluorescence intensities of TAMRA-VH in the presence/absence of VL (+ VL/-VL), the dissociation constant Kd was 1.2X 10^-7[M](FIG. 8). The above results show that a novel homogeneous fluorescence immunoassay method utilizing the quenching phenomenon caused by VH and VL proteins can be established.

Example 5

(study of quenching Effect by Trp in VH)

From the results of example 3, it was shown that for the titration of antigen, the increase in fluorescence of TAMRA was higher than the decrease in fluorescence of CR 110. TAMRA is a Rhodamine (Rhodamine) dye, and studies to date report that the Rhodamine dye is quenched (quenched) with an amino acid such as tryptophan (Trp). Therefore, the present inventors speculate that the Trp residue present in VH participates in the quenching of TAMRA, thereby establishing the following hypothesis: in the case where TAMRA-VH is present alone, the fluorescence of TAMRA is quenched by the Trp residue present in the vicinity thereof, but the quenching is released by the complex formation of TAMRA-VH with VL and antigen, which changes the mutual position between TAMRA/Trp. As shown in fig. 9, VH has 4 Trp residues (Trp33, Trp36, Trp47, Trp 106). In the analysis using the predictive molecular model, Trp33, Trp36 and Trp106 were predicted to participate in hydrophobic interactions with VL and Trp33 was predicted to participate in interactions with BGP peptides. To examine whether these Trp residues have an effect on quenching, the following experiment was performed using 4 mutant VH types in which Trp was replaced with Phe.

Wild-type or mutant anti-BGP antibody heavy chain variable region polypeptides (W33F, W36F, W47F, W106F) and VL were reacted in the presence of different concentrations of BGP peptide, and fluorescence intensity was measured using a 543nm He-Ne laser. The results are shown in fig. 10 and table 1. As a result of measuring the fluorescence intensity of the mutant fluorescently labeled VH alone, W106F and W36F showed increases in fluorescence of 31% and 29%, respectively, as compared with the Wild Type (WT). W47F showed an 11% increase in fluorescence. On the other hand, W33F showed a 9% decrease in fluorescence. From the above results, it was revealed that mainly Trp36, Trp47 and Trp106 were involved in fluorescence quenching of TAMRA. In addition, W33F, W36F, and W106F showed 1.5-fold, 1.3-fold, and 1.5-fold increases in fluorescence depending on the antigen concentration by reacting with VL and BGP peptides, respectively. The above results suggest that Trp33, Trp36 and Trp106 are partially involved in quenching, because the release of antigen-dependent quenching is reduced by mutation of Trp to Phe. On the other hand, in the case of reacting W47F with BGP peptide and VL, no increase in fluorescence was observed at all. As a result of the analysis of diffusion time (diffusion time) by FCS measurement (fig. 11), it was found that Trp47 in VH is essential for binding between the antibody and the antigen because the binding activity of the antibody is lost by mutation of Trp 47.

From the above results, it was confirmed from the two points of the increase in fluorescence of the fluorescent-labeled VH alone and the decrease in the amount of increase in fluorescence depending on the antigen concentration that Trp33 and Trp106 are trps important for quenching. Furthermore, the association of Trp47 with antigen concentration-dependent quenching is not clear, but it is known to be a very important site for antigen-mediated complex formation of VH and VL. It should be noted that Trp106 in the amino acid sequence of the VH described above corresponds to position 103 in the numbering system of the Kabat database (Kabat, E.et al, "Sequences of proteins, 5th edn.", U.S. department of Health and Human Service, Public Service, National Institute of Health, Washington, DC, 1991.).

[ Table 1]

TABLE 1 relative fluorescence quenching of TAMRA-labeled VH proteins

*1(I_O) = fluorescence intensity without addition of VL and BGP peptide (fluorescence intensity of fluorescent marker VH alone)

*2(I_H) = fluorescence intensity when VL is added but BGP peptide is not added

*3(I_HP) = fluorescence intensity when VL and BGP peptide were added (in this case, concentration of BGP peptide was 10000ng/mL)

4 fluorescence increase = each I_OFluorescence intensity of (2) relative to the I of WT_OFluorescence intensity of (expressed as I)_WT) In a ratio of

5 fluorescence change = increase in fluorescence depending on antigen concentration

6 dissociation constant

Example 6

(Study on quenching Effect of oxazine fluorescent dye by addition to spacer

The effect of the fluorochrome and the spacer on the sensitivity of the homogeneous fluoroimmunoassay method was investigated. The quenching efficiency varies greatly depending on the type of the fluorescent dye, and it has been reported that compared with rhodamine-based dyes,oxazine (Oxazin) based pigments are more efficiently quenched. Thus, the inventors made the use ofThe experiment was carried out in the same manner as in example 4 using ATTO655-VH as a labeling substance for an oxazine fluorescent dye ATTO 655. Further, ATTO655-VH (+ spacer) in which GGGSGGGS (SEQ ID NO: 4) was added as a spacer between ATTO655 and VH was prepared, and the effect of the presence or absence of the spacer on the quenching effect was examined. Fig. 12 shows a model for predicting the three-dimensional structure of a complex of a fluorescently labeled anti-BGP antibody heavy chain variable region polypeptide (Fluorescent labeled BGP-VH) and an anti-BGP antibody light chain variable region polypeptide (BGP-VL) with a spacer attached (Fluorescent labeled BGP-VH/VL).

The results of the same experiment as in example 4 (FIG. 13) using ATTO655-VH with or without the addition of a spacer were that in either case, the fluorescence intensity of ATTO655-VH increased depending on the concentration of BGP peptide. Furthermore, the fluorescence intensity obtained from ATTO655-VH without spacer was 3-fold higher than that obtained using TAMRA-VH. Further, it was found that the fluorescence intensity was increased by about 2 times by adding the spacer region as compared with the case where the spacer region was not added. It is considered that addition of the GGGS spacer brings the distance between Trp as a quencher and a fluorochrome close to each other, thereby affecting such increase in fluorescence intensity.

Furthermore, the result of analyzing the ratio of fluorescence intensities (+ VL/-VL) of ATTO655-VH in the presence/absence of VL (FIG. 14) was that, in the absence of an additional spacer, the dissociation constant Kd was 8.4X 10^-8[M]With the addition of a spacer, the dissociation constant Kd is 1.8X 10^-7[M]. The higher the dissociation constant, the higher the sensitivity of the assay systemTherefore, the above results mean that a measurement system with higher sensitivity can be established by providing a spacer between the fluorescent dye and VH.

Example 7

(establishment of homogeneous fluoroimmunoassay method Using Single-chain antibody)

The following experiment was performed in order to establish a homogeneous fluorescence immunoassay method using a single-chain antibody (scFv) in which VH and VL are bound by a linker composed of an amino acid sequence represented by sequence No. 5 or 9. Fig. 15 shows a model for predicting the three-dimensional structure of a fluorescently labeled single-chain antibody obtained by binding the fluorescently labeled antibody heavy chain variable region polypeptide of the present invention to the antibody light chain variable region polypeptide, and fig. 16 shows the one-dimensional structure of the fluorescently labeled single-chain antibody. Similarly to the case of using peptide fragments of VH and VL, respectively, an increase in fluorescence intensity depending on the concentration of BGP peptide was also observed in the case of using fluorescence-labeled scFv. FIGS. 17 to 22 show the results of labeling the anti-BGP antibody scFv with ATTO655 and the results of labeling the anti-BGP antibody scFv with TAMRA and the linker of SEQ ID NO. 9, respectively, and FIG. 23 shows the results of labeling the anti-BGP antibody scFv with TAMRA and the linker of SEQ ID NO. 5, respectively.

Example 8

(fluorescence immunoassay in human plasma)

TAMRA-labeled anti-BGP antibody scFv proteins (2. mu.g/mL, 6.25. mu.L) were prepared in total with BGP peptide as antigen to 50. mu.L using PBS (+0.05% Tween 20, 0.2% BSA) to form a sample containing 50% human plasma. Then, the plate was left at 25 ℃ for 90 minutes and then observed with a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi software engineering Co., Ltd.). The measurement was carried out under the conditions that the excitation wavelength was 532nm and the measurement wavelength was 580 nm. The results are shown in FIG. 24. An increase in fluorescence intensity dependent on BGP peptide concentration was also observed in samples containing 50% human plasma.

Example 9

2. Establishment of homogeneous fluorescence immunoassay method Using anti-BPA antibody-derived VH and VL

(construction of V region Gene expression vector derived from anti-BPA antibody)

ProX comprising amber codon added to N-terminus of gene encoding VH (SEQ ID NO: 6) of anti-bisphenol A (BPA) antibody^TMThe DNA sequence of the tag (MSKQIEVNXSNET (X is a fluorescent-labeled amino acid); SEQ ID NO: 3) was incorporated into the NcoI and HindIII sites of pIVX2.3d vector (manufactured by Roche diagnostics). The constructed expression vector was constructed such that a ProX comprising an amber codon was added to the N-terminus of the inserted VH or VL^TMA tag (MSKQIEVNXSNET (X is a fluorescent-labeled amino acid); SEQ ID NO: 3) and a His-tag attached to the C-terminus. In addition, ProX was attached to the N-terminus of the gene encoding VL (SEQ ID NO: 7) of the anti-BPA antibody in the same manner^TMThe DNA sequence obtained by substituting F for amino acid X of the tag was recombined into the NcoI and HindIII sites of pIVEX2.3d vector (manufactured by Roche diagnostics). The constructed expression vector was constructed so that ProX was added to the N-terminus of the inserted VL^TMThe sequence obtained by substituting F for amino acid X in the tag was designed so that a His-tag was attached to the C-terminus. Further, the following vectors were also prepared: a single chain antibody (scFv) expression vector obtained by binding VH gene and VL gene using linker (GGGGSGGGGSGGGS; SEQ ID NO: 9) in ProX^TM3 single chain antibody (scFv) expression vectors having a spacer (GGGSGGGS, SEQ ID NO: 4; GGGSGGGSGGGS, SEQ ID NO: 10; or GGGSGGGSGGGSGGGSGGGS, SEQ ID NO: 11) between the tag and the N-terminus of the scFv.

(preparation of fluorescent-labeled protein Using cell-free translation System)

Using RTS100 E.coli disulfide protein expression kit (Roche diagnostics), a fluorescently labeled amino acid was introduced into the N-terminal region of the V-region protein using a cell-free translation system. To the reaction solution (50. mu.L) were added 7. mu.L of the amino acid mixture, 1. mu.L of methionine, 7. mu.L of the reaction mixture, 25. mu.L of the activated E.coli lysate, 5. mu.L of plasmid DNA (500ng), and 5. mu.L of ATTO 655-X-AF-amber suppressor tRNA (0.8 nmol). For making fluorescent-marked eggsWhite ATTO 655-X-AF-amber suppressor tRNA protein functionalized CloverDirect with site directed introduction of unnatural amino acids^TMtRNA reagent (プロテインエクスプレス). The reaction mixture was reacted at 20 ℃ and 600rpm for 2 hours, and then at 4 ℃ for 16 hours. After completion of the reaction, 1. mu.L of the reaction solution was subjected to SDS-PAGE (15%) and protein expression was observed by a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi software engineering Co., Ltd.). Furthermore, western blotting was performed using a His-tag antibody to confirm that the target protein was synthesized.

Subsequently, the synthesized V-domain protein was purified using a His Spin Trap column (GE ヘルスケア). To the above reaction solution (50. mu.L), a washing solution (20mM phosphate buffer solution (pH7.4)/0.5M NaCl/60mM imidazole/0.1% polyoxyethylene (23) lauryl ether) was added to make 400. mu.L, and applied to a His-Spin Trap column. After incubation for 15 minutes at room temperature, three washes were performed. Next, 200. mu.L of an eluent (20mM phosphate buffer solution (pH7.4)/0.5M NaCl/0.5M imidazole/0.1% polyoxyethylene (23) lauryl ether) was eluted twice. Furthermore, the eluate was concentrated while being exchanged with PBS (+0.05% Tween 20) using an Ultrafree-0.5 centrifuge tube (manufactured by ミリポア Co.). The concentration of the sample was determined by SDS-PAGE and FCS (MF 20; manufactured by Olympus).

Example 10

(measurement of fluorescence Spectroscopy)

The ATTO 655-labeled anti-BPA antibody VH protein and the unlabeled anti-BPA antibody VL protein (1. mu.g/mL and 7.5. mu.L, respectively) prepared in example 9 and BPA as an antigen were prepared into a 10% MeOH in PBS (+0.05% Tween 20) solution in a total amount of 50. mu.L, and after standing at 25 ℃ for 90 minutes, they were observed by a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi software engineering Co., Ltd.). The measurement was carried out under the conditions that the excitation wavelength was 635nm and the measurement wavelength was 670 nm. The dissociation constant (Kd) values were calculated by curve fitting of fluorescence measurements. At this time, an S-type dose-effect model of Graphpad Prism (manufactured by Graphpad corporation) was used as statistical analysis software.

TAMRA-labeled anti-BPA antibody scFv proteins (2. mu.g/mL, 25. mu.L) with or without a spacer and BPA as antigen were prepared with PBS (+0.05% Tween 20, 0.2% BSA, 1% MeOH) into a total of 200. mu.L of sample. Then, the sample was left at 25 ℃ for 10 minutes, and then fluorescence spectroscopy was performed using a fluorescence spectrophotometer (FluoroMax-4; manufactured by ホリバ & ジヨバンイボン) to calculate the fluorescence intensity by curve fitting. At this time, an S-type dose-effect model of ImageJ software (http:// rsbweb. nih. gov/ij /) was used as statistical analysis software. The measurement was carried out under the conditions that the excitation wavelength was 550nm and the measurement wavelength was 580 nm.

Example 11

(establishment of homogeneous fluorescence immunoassay method Using V-region protein derived from labeled anti-BPA antibody)

To establish a homogeneous fluorescence immunoassay method using ATTO655-VH and VL without fluorescent labeling, the following experiment was performed. ATTO655-VH was reacted with different concentrations of BPA in the presence or absence of unlabeled VL and the fluorescence intensity was measured (FIG. 25). In the presence of VL, the fluorescence intensity of ATTO655-VH increased depending on the concentration of BPA. On the other hand, in the absence of VL, the fluorescence intensity of ATTO655-VH remained low regardless of the concentration of BPA reacted with. Furthermore, as a result of analyzing the ratio of the fluorescence intensities of ATTO655-VH in the presence/absence of VL (+ VL/-VL), the dissociation constant (Kd) was 2.4X 10^-8[M](FIG. 26). In addition, the following experiment was performed in order to establish a fluorescence immunoassay method using a single chain antibody (scFv) in the same manner as in example 7. In the case of using TAMRA labeled anti-BPA antibody scFv, an increase in fluorescence intensity depending on BPA concentration was also observed as in the case of using peptide fragments of VH and VL, respectively (fig. 27).

Example 12

3. Establishment of homogeneous fluorescence immunoassay method Using Single-chain antibodies derived from various antibodies

(construction of V region Gene expression vector derived from anti-HEL antibody, anti-estradiol antibody, anti-SA antibody)

Will have a ProX comprising an amber codon at the N-terminus of the DNA sequence of a Single chain antibody (scFv)^TMA tag (MSKQIEVNXSNET (X is a fluorescent-labeled amino acid); SEQ ID NO: 3) having a His-tag at the C-terminus and a ProX^TMThe DNA sequence having a spacer (GGGSGGGS, SEQ ID NO: 4) between the tag and the N-terminus of the scFv was recombined into the NcoI and HindIII sites of pIVEX2.3d vector (manufactured by Roche diagnostics), thereby constructing an expression vector. The DNA sequence of each single chain antibody (scFv) is shown below: the DNA sequence of the anti-hen egg-white lysozyme (HEL) antibody scFv is a sequence formed by sequentially combining VH (sequence number 12) and VL (sequence number 13) of an anti-HEL antibody by using a linker sequence (GGGGSGGGGSGGS, sequence number 9); the DNA sequence of the anti-estradiol (estradiol) antibody scFv is a sequence in which VH (SEQ ID NO: 14) and VL (SEQ ID NO: 15) of the anti-estradiol antibody are sequentially combined by using a linker sequence (GGGGSGGGGSGGGS; SEQ ID NO: 9); the DNA sequence of scFv of the anti-SA (serum albumin ) antibody is a sequence in which VH (seq id No. 16) and VL (seq id No. 17) of the anti-SA antibody are sequentially bound to each other by a linker sequence (GGGGSGGGGSGGGGS, seq id No. 9).

(preparation of fluorescent-labeled anti-HEL antibody, anti-estradiol antibody, anti-SA antibody V region protein)

Using RTS100 E.coli disulfide protein expression kit (Roche diagnostics), a fluorescently labeled amino acid was introduced into the N-terminal region of the V-region protein using a cell-free translation system. To the reaction solution (50. mu.L) were added 7. mu.L of the amino acid mixture, 1. mu.L of methionine, 7. mu.L of the reaction mixture, 25. mu.L of the activated E.coli lysate, 5. mu.L of plasmid DNA (500ng), and 5. mu.L of the fluorescence-labeled succinyl-aminoacyl tRNA (0.8 nmol). Fluorescently labeled aminoacyl tRNA (TAMRA-X-AF-amber suppressor tRNA) for making fluorescently labeled proteins using protein-functionalized clover direct with site-directed introduction of unnatural amino acids^TMtRNA reagent (プロテインエクスプレス). The reaction solution was reacted at 20 ℃ and 600rpm for 2 hours, and then further reacted at 4 ℃ for 16 hours. After completion of the reaction, 1. mu.L of the reaction solution was subjected to SDS-PAGE (15%) and protein expression was observed by a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi software engineering Co., Ltd.). Furthermore, western blotting was performed using a His-tag antibody to confirm that the target protein was synthesized.

The synthesized V-domain protein was purified by using His-Spin Trap column (GE ヘルスケア). To the above reaction solution (50. mu.L), a washing solution (20mM phosphate buffer solution (pH7.4)/0.5M NaCl/60mM imidazole/0.1% polyoxyethylene (23) lauryl ether) was added to make 400. mu.L, and applied to a His-Spin Trap column. After incubation for 15 minutes at room temperature, three washes were performed. Next, 200. mu.L of an eluent (20mM phosphate buffer solution (pH7.4)/0.5M NaCl/0.5M imidazole/0.1% polyoxyethylene (23) lauryl ether) was eluted twice. Further, the eluate was buffer-exchanged and concentrated with PBS (+0.05% Tween 20) using an Ultrafree-0.5 centrifuge tube (manufactured by ミリポア Co.). The concentration of the purified sample was determined by SDS-PAGE and FCS (MF 20; manufactured by Olympus).

Example 13

(measurement of fluorescence Spectroscopy)

TAMRA-labeled anti-HEL antibody scFv proteins (2. mu.g/mL, 25. mu.L) were prepared with HEL protein as an antigen into a total of 200. mu.L of sample using PBS (+0.05% Tween 20, 1% BSA). TAMRA-labeled anti-estradiol antibody scFv protein (2. mu.g/mL, 25. mu.L) and estradiol as an antigen were prepared into a total of 200. mu.L of sample using PBS (+0.05% Tween 20, 1% BSA). TAMRA-labeled anti-SA antibody scFv protein (2. mu.g/mL, 25. mu.L) and BSA (bovine serum albumin) or HSA (human serum albumin) as an antigen were prepared into a total of 200. mu.L of a sample using PBS (+0.05% Tween 20, 0.2% gelatin). Then, the sample was left at 25 ℃ for 5 minutes, and then fluorescence spectroscopy was performed using a fluorescence spectrophotometer (FluoroMax-4; manufactured by ホリバ & ジヨバンイボン) to calculate the fluorescence intensity by curve fitting. At this time, an S-type dose-effect model of ImageJ software (http:// rsbweb. nih. gov/ij /) was used as statistical analysis software. When the measurement was performed under the conditions of an excitation wavelength of 550nm and a measurement wavelength of 580nm, an increase in fluorescence intensity depending on the antigen concentration was observed (FIGS. 28 to 30). As described above, it is known that the fluorescence immunoassay method can be carried out using a fluorescence-labeled single-chain antibody (scFv) of each type of antibody.

Example 14

4. Conservation of Trp residues in the VH of mouse antibodies

As shown in example 4, fig. 9 and table 1, it can be seen that: in the numbering system of the Kabat database, Trp at positions 33, 36 and 106 in the amino acid sequence of VH plays an important role in quenching of fluorescent dye labeling the anti-BGP antibody, and Trp at position 47 is essential for binding the antibody (VH and VL) to the antigen (note that Trp106 in the amino acid sequence of VH corresponds to position 103 in the numbering system of the Kabat database). Therefore, it was confirmed whether these tryptophan residues are conserved in the VH region of mouse antibodies other than the anti-BGP antibody. Analysis of amino acid residue distribution of mouse antibodies an Abysis database (team of Drew C.R. Martin Phd.; http:// www.bioinf.org.uk/abs/index. html) was used. Further, the residue numbers of the respective Antibody residues in the above databases obtained according to Kabat Sequence notation can be investigated by AbCheck (team of Drew C.R. Martin, A.C.R. accessing the Kabat Antibody Sequence by Computer PROTECTins: Structure, Function and Genetics,25(1996), 130-. As a result, as shown in FIGS. 31 to 35, the conservation rates of 4 Trp residues in the VH region of the mouse antibody were 40% for Trp33, 98% for Trp36, 94% for Trp47 and 95% for Trp 103. The above results indicate that the 4 Trp residues of VH, which are important in carrying out homogeneous fluorescence immunoassay, are conserved in most mouse antibody VH.

The results obtained by numbering the positions of tryptophan residues contained in the VH (sequence number 1) and VL (sequence number 2) of the anti-BGP antibody, the VH (sequence number 6) and VL (sequence number 7) of the anti-BPA antibody, and the results obtained by numbering the positions of tryptophan residues contained in the anti-BGP antibody scFv, the anti-BPA antibody scFv, the anti-HEL antibody scFv, the anti-SA antibody scFv, and the anti-estradiol antibody scFv according to the Kabat sequence notation are shown in table 2, and the results obtained by numbering the positions of tryptophan residues contained in the anti-BGP antibody scFv, the anti-BPA antibody scFv, the anti-HEL antibody scFv, the anti-SA antibody scFv, and the anti.

[ Table 2]

[ Table 3]

Industrial applicability

According to the present invention, a homogeneous fluoroimmunoassay method using an antibody (fragment) labeled with a fluorescent dye and using the release of quenching of the fluorescent dye as an indicator can be provided. The homogeneous fluorescence immunoassay method of the present invention does not require immobilization or washing of an antibody or an antigen, and can measure the concentration of a target substance by directly monitoring the fluorescence intensity of a mixed solution in which the antibody and a test substance are mixed, and therefore, it is predicted that a low-molecular compound can be detected more easily and rapidly. In addition, since the Trp residues of the VH region of the antibody that affect quenching are conserved among many kinds of antibodies, the homogeneous fluorescence immunoassay method of the present invention can be used for measurement of various antigen concentrations.

Claims (12)

1. A kit for measuring and detecting an antigen concentration, comprising an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide, wherein either one of the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide is labeled with a fluorescent dye, and the fluorescent dye is quenched in a state of being labeled on the antibody heavy chain variable region polypeptide or the antibody light chain variable region polypeptide, wherein quenching is released when the antibody light chain variable region polypeptide and the antibody light chain variable region polypeptide form a complex with an antigen, whereby the fluorescence intensity is increased, and the measurement of the antigen concentration or the visualization of the antigen can be performed using as an index that there is a positive correlation between the antigen concentration in a liquid phase and the fluorescence intensity of the fluorescent dye.

2. The antigen concentration measurement and detection kit according to claim 1, wherein the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide are combined into a single chain antibody.

3. The antigen concentration measurement and detection kit according to claim 1 or 2, wherein the fluorescent dye is a rhodamine-based fluorescent dye orOxazine fluorescent pigments.

4. The antigen concentration measurement and detection kit according to claim 3, wherein the fluorescent dye is CR110, TAMRA or ATTO 655.

5. The antigen concentration measurement and detection kit according to claim 1 or 2, wherein the antibody heavy chain variable region polypeptide comprises a polypeptide consisting of the amino acid sequence represented by sequence number 1, and the antibody light chain variable region polypeptide comprises a polypeptide consisting of the amino acid sequence represented by sequence number 2.

6. The antigen concentration measurement and detection kit according to claim 1 or 2, wherein the antibody heavy chain variable region polypeptide comprises a polypeptide consisting of the amino acid sequence represented by SEQ ID No. 6, and the antibody light chain variable region polypeptide comprises a polypeptide consisting of the amino acid sequence represented by SEQ ID No. 7.

7. A method for measuring and detecting the concentration of an antigen, comprising the following steps (a) to (c) in this order:

(a) an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide labeled with a fluorescent dye which is quenched in a state of being labeled to the antibody heavy chain variable region polypeptide, or an antibody heavy chain variable region polypeptide and an antibody light chain variable region polypeptide labeled with a fluorescent dye which is quenched in a state of being labeled to the antibody light chain variable region polypeptide

(a1) In liquid phase with the antigen in the test substance, or

(a2) Contacting with an antigen in a subject non-human animal subject administered with an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide labeled with a fluorescent dye, or an antibody heavy chain variable region polypeptide and an antibody light chain variable region polypeptide labeled with a fluorescent dye, or

(a3) Contacting an antigen in a subject in vitro;

(b) in the case of (a1), the fluorescence intensity of the fluorescent dye is measured,

detecting fluorescence of the fluorescent dye in the cases of (a2) and (a 3);

(c) the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide are released from quenching when they form a complex with an antigen, and the fluorescence intensity is increased, and the amount of the antigen contained in the test substance is calculated in the case of (a1) and the antigen contained in the test subject is visualized in the cases of (a2) and (a3) using as an index the fact that the concentration of the antigen in the liquid phase has a positive correlation with the fluorescence intensity of the fluorescent dye.

8. The method for measuring and detecting the antigen concentration according to claim 7, wherein the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide are combined into a single chain antibody.

9. The method for measuring and detecting the concentration of the antigen according to claim 7 or 8, wherein the fluorescent dye is a rhodamine-based fluorescent dye orOxazine fluorescent pigments.

10. The method for measuring and detecting the concentration of an antigen according to claim 9, wherein the fluorescent dye is CR110, TAMRA or ATTO 655.

11. The method for measuring and detecting the concentration of an antigen according to claim 7 or 8, wherein the antibody heavy chain variable region polypeptide comprises a polypeptide consisting of the amino acid sequence represented by SEQ ID No. 1, and the antibody light chain variable region polypeptide comprises a polypeptide consisting of the amino acid sequence represented by SEQ ID No. 2.

12. The method for measuring and detecting the concentration of an antigen according to claim 7 or 8, wherein the antibody heavy chain variable region polypeptide comprises a polypeptide consisting of the amino acid sequence represented by SEQ ID No. 6, and the antibody light chain variable region polypeptide comprises a polypeptide consisting of the amino acid sequence represented by SEQ ID No. 7.