JP2011037791A - Antibody-luciferyl peptide complex, method for measuring sample using the same, and reagent for measuring sample, and method for preparing the measuring reagent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring samples, applicable to wide variety of samples in a simple manner. <P>SOLUTION: An antibody-luciferyl peptide complex in which a luciferyl peptide compound is bonded to an antibody which specifically couples with the sample is used. The luciferyl peptide compound is a condensed compound in which a peptide is bonded to firefly D-luciferin or a D-luciferin derivative. The antibody-luciferyl peptide complex and a test substance containing the sample are put together in a solution to substitute the sample with the luciferyl peptide to dissociate the luciferyl peptide and then luciferin cut out from the dissociated luciferyl peptide is measured using the luciferase reaction. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention uses a complex of a luciferyl peptide compound in which a peptide is bound to luciferin or a luciferin derivative and an antibody, and a substance that can specifically bind to the antibody by luminescence analysis using the complex. The present invention relates to a measurement method for a test substance that can be measured as a substance, a measurement reagent for the test substance, and a preparation method for the measurement reagent.

  Firefly luminescence, famous as bioluminescence, is due to the reaction of the firefly luciferin-firefly luciferase luminescence system (firefly bioluminescence system), and the luminescence substrate firefly luciferin emits light by firefly luciferase in the presence of ATP and magnesium ions. It emits light when converted to oxyluciferin, the body. The above firefly bioluminescence system has a very high emission yield and can be measured with high sensitivity. Therefore, it has already been applied to hygiene inspection using ATP detection and in vivo monitoring. The application examination that becomes is awaited.

  As a conventional technique related to measurement using a bioluminescent system, a system lacking any of the elements necessary for the luminescent reaction (luminescent substrate, ATP, luminescent enzyme, etc.) is created, and when the target phenomenon occurs, There is a system that is configured to emit light by supplementing the deficient elements, and the target phenomenon can be monitored by the generated light emission. Specifically, for example, a measurement system using a compound in which galactose is bound to the phenolic hydroxyl group of luciferin using galactosidase activity as a target is commercially available. When galactosidase acts on this compound, the bond between galactose and luciferin is cleaved to release luciferin, and the released luciferin emits light by luciferase, thereby detecting and visualizing the presence of target galactosidase activity (for example, , See Patent Document 1). The target enzyme is not limited to galactosidase, and another enzyme activity can be detected by preparing a measurement compound to which an appropriate substance is bound according to the action of the target enzyme and using it.

  In addition, as a method for quantifying antigens, haptens or antibodies, an antigen-, hapten- or antibody-complex is formed by combining these with an enzyme that liberates luciferin from a luciferin derivative, such as amylase or amidase. It is described that the above-mentioned enzyme is detected (that is, a ligand is detected) by causing the luciferin released by the enzyme when the luciferin derivative acts on the complex to emit light using the luciferase contained in the measurement reagent. (For example, refer to Patent Document 2). As the luciferin derivatives used in this method, several luminescent substrate-bound amino acid compounds in which the phenolic hydroxyl group or carboxyl group of luciferin is bound to an amino acid have been presented.

  In the field of diagnosis, various methods have recently been developed as methods for measuring trace substances, and immunoanalysis / measurement methods using antigen-antibody reactions are widely used as highly sensitive measurement methods. An ELISA (Enzyme-linked immunosorbent assay) is conventionally used as a measurement method widely used in biochemical tests and the like, and is classified into a direct adsorption method, a sandwich method, a competitive method, etc. depending on the protein quantification form. In particular, the sandwich method is widely used.

In the sandwich method, an antibody against a test substance is immobilized on a plate or the like in advance, and this solid-phased antibody is supplemented with an antigen as a test substance, and then this is reacted with a labeled antibody (detection antibody). This is a method for detecting an antigen bound to. This method is generally highly sensitive because it uses the reaction specificity for the test substance. However, it is necessary to repeat the addition of the sample to the plate and the washing, and the work for that is complicated and time-consuming, so that there is a problem that the suitability for rapid measurement is low.
In the sandwich method, it is necessary to prepare two types of antibodies having different specific recognition sites for the same antigen. Moreover, the two types of antibodies need to be screened to find an appropriate combination of antibodies in consideration of the position of the recognition site, etc., so that each can function sufficiently when both are used in the measurement system. However, depending on the target substance (antigen), it may be difficult to find an appropriate antibody combination. In addition, since it is difficult to label the antibody of the target substance, a secondary antibody for detecting the antibody may be necessary, which is accompanied by a problem of high cost.
When a set of antibodies applicable to the sandwich method cannot be found, a measurement method by a competitive method is often selected. When this method is used, the test substance can be measured by immunoassay even if the antibody set required by the sandwich method cannot be obtained. However, when this method is performed, in order to obtain a sufficiently accurate measurement result, complicated operations such as sample dilution, concentration, washing, and separation are often required.

On the other hand, several methods have been disclosed as immunoassays that do not require washing or separation operations. For example, a method using fluorescence energy transfer that occurs between two adjacent fluorescent materials is known (see, for example, Patent Document 3 and Non-Patent Document 1). Fluorescence energy transfer is a phenomenon in which the fluorescence emitted when one fluorescent material is excited excites another fluorescent material to generate longer wavelength fluorescence. In this method, one of a pair of fluorescent substances capable of inducing fluorescence energy transfer is bound to a first binding partner that can specifically bind to an object to be measured, and an affinity for the first binding partner is obtained. Each of the other fluorescent substances is bound to a second binding partner having a label, and then the antigen-antibody reaction between the labeled first binding partner and the second binding partner is allowed to compete with the analyte to be measured. This is a method of measuring a measurement object using a change in the amount of fluorescence energy transfer caused thereby as an index.
However, since this method uses fluorescence as an index, it cannot be applied to a system containing a fluorescence quencher, and there is a limit to signal amplification. Furthermore, it cannot be measured with a normal fluorometer or the like, and requires a special and expensive instrument as an analyzer, so that it is difficult to use as a general-purpose method.

As another method, a method using complement has been reported (for example, see Patent Document 4). Complement is active when bound to a complex when an antigen-antibody complex is formed. In this method, a specimen containing an antigen (test substance) is added to an antibody solution, and then complement is added to bind the previously formed antigen-antibody complex and complement. Using the enzyme activity of the bound complement as an index, the amount of the antigen-antibody complex, ie, the amount of the test substance is measured.
In this method, it is not necessary to prepare two types of antibodies having different specific recognition sites for one antigen, and only one type may be prepared. However, the method using complement is a useful method only when the antibody-antibody complex has an antibody structure that can activate the complement by binding to the complement of the antigen-antibody complex. It is difficult to apply to a wide range of systems such as ligand / receptor reaction, and its usefulness must be limited.

As another method using an antigen labeled with an enzyme, a technique called EMIT (Enzyme Multiplied Immunoassay Technique) has been reported. This is a method that utilizes the phenomenon that the activity of an enzyme decreases due to steric hindrance when an antibody binds to an antigen that has been labeled with an enzyme in advance. When a specimen containing an antigen (test substance) is added, The antigen in the sample solution reacts competitively with the antibody, and the antigen concentration in the sample can be measured based on the residual enzyme activity.
However, EMIT is sufficiently accurate unless it is limited to competitive assays, or unless a system in which the enzyme activity is reduced when a substance such as an antibody is bound to an antigen is constructed. There is a problem that the type of the substance to be detected and the reaction system are narrow and limited, such as inability to perform measurement.

An enzyme channeling immunoassay method has been reported as a method using an enzyme-labeled antibody, which is different from EMIT and does not require a separation step (see, for example, Non-Patent Document 2). This uses two types of enzymes such that the product of one enzyme serves as a substrate for another enzyme, and constructs a reaction system that develops color when the two types of enzymes come close to each other via an antigen in a gel. It is the analysis method characterized by this. Specifically, the product of the first enzyme (the first product) becomes a substrate for the second enzyme, and a pigment is produced as the product of the second enzyme to enable visual judgment. There is established a reaction system in which a dye is generated only when the first enzyme and the second enzyme are close to each other in the gel. Therefore, in order to prevent the first product from reacting with the second enzyme without limitation, the second enzyme is immobilized in the gel, and the first product convertase is present on the solution side. Thus, the reaction field is substantially separated between the gel and the solution.
Furthermore, regarding an enzyme channeling immunoassay method, a method of using an enzyme that decomposes a reaction intermediate called a scavenger enzyme is also disclosed in order to suppress a coloring reaction that occurs when two kinds of enzymes are not close to each other in a gel. However, this method requires immobilization of the second enzyme in the gel, and requires an operation and time for that purpose. In addition, the antibody that forms a complex with the first enzyme has a disadvantage that it takes a long time to react with the antigen immobilized in the gel. Such an analysis time problem arises in a non-competitive system, that is, a system in which an antigen and an antibody complexed with a first enzyme need to react simultaneously or sequentially with a second antibody in a gel. , Will be more prominent. In addition, because the gel and solution are contained in the system, there is a high possibility that noise such as scattering will enter the analysis system, sampling is not uniform during automation, and control of the suction pressure with a normal nozzle is difficult. There are substantially many problems, such as being. Regarding antibodies to be used, it is necessary to prepare two types of antibodies having different specific recognition sites for one antigen.

Japanese Patent Laid-Open No. 11-332593 JP-T 63-501571 JP-A-60-233555 JP-A-6-94710

Herman et al., Fluorescence Microscopy of Living Cells in Culture- Part B, Academic Press, New York, pp 220-245 (1989) D.J.Litman et al., Analytical Biochemistry 106, 223-229 (1980)

As described above, in the measurement method using the antigen-antibody reaction, it is not necessary to prepare two types of antibodies having different specific recognition sites for the antigen, without requiring a plurality of complicated washing steps, There is a demand for a method for measuring a test substance that can more easily measure the test substance and has a wide application range.
An object of the present invention is to provide a method for measuring a test substance having a wide range of application, which can more easily measure a test substance, omitting a complicated washing step in the measurement method using an antigen-antibody reaction.

In order to solve the above-mentioned problems, the present inventors have conceived that a luciferyl peptide obtained by condensing firefly (D-) luciferin or a derivative thereof and a peptide is used as a labeling peptide for detection. As a result of diligent investigations on the application of biotin, we used bioluminescence to detect and measure a test substance having the amino acid sequence of the epitope recognized by the antibody, using a complex in which the luciferyl peptide and the antibody were combined in a replaceable state. The present invention has been completed by finding out what can be done. That is, the present invention relates to the following.
According to one aspect of the present invention, an antibody-luciferyl peptide complex includes a luciferyl peptide compound obtained by condensing firefly D-luciferin or a D-luciferin derivative with a peptide, an antibody capable of specifically binding to a test substance, and And the peptide portion of the luciferyl peptide compound is bound to the antibody so that the luciferyl peptide compound can be displaced by the test substance.
According to another aspect of the present invention, a method for measuring a test substance includes preparing the complex, supplying a specimen to the complex, and including the luciferyl peptide compound of the complex in the specimen. A luciferyl peptide compound is produced by substitution with a test substance, firefly D-luciferin or a D-luciferin derivative is generated from the resulting luciferyl peptide compound, subjected to luminescence reaction, and the test substance is measured by detecting the luminescence The gist is to do.
Furthermore, according to one aspect of the present invention, a test reagent for a test substance has the above complex as a luminescence detection element for test substance measurement.
The measurement reagent for the test substance may further include an enzyme capable of releasing firefly D-luciferin or a D-luciferin derivative from the luciferyl peptide compound, ATP, magnesium ion, and firefly luciferase.
According to another aspect of the present invention, a method for preparing a measurement reagent includes the steps of preparing an antibody that can specifically bind to a test substance, and the epitope of the test substance that the antibody specifically recognizes or A step of preparing a peptide having the amino acid sequence of the mimotope, a step of preparing the luciferyl peptide compound by condensing the peptide with firefly D-luciferin or a D-luciferin derivative, and the luciferyl peptide compound and the antibody. And a step of preparing a complex to be bound.

  According to the present invention, a complicated operation step associated with a measurement method using an antigen-antibody reaction, that is, repeated operations of sample addition and washing can be omitted, and a test substance can be measured more easily. Moreover, according to the present invention, it is not necessary to prepare two types of antibodies having different recognition sites for a test substance, and a measurement method with a wide application range can be provided. Furthermore, since it can be detected using a general-purpose emission spectrometer, it is advantageous in that a special and expensive instrument is not required.

It is a conceptual diagram which shows one Embodiment of the measuring method of this invention. It is a figure which shows the residual rate of the epitope peptide and modified peptide which remained in the antibody, without being substituted with an antigen, when an antigen (CRP) is added with various amounts with respect to the composite_body | complex of an antibody, an epitope peptide, and a modified peptide. . It is a figure which shows the relationship between the amount of CRP and the light-emission quantity when CRP is measured with the measuring method of this invention.

In an antigen-antibody reaction, an antibody is known not to recognize the whole antigen but to recognize and bind to a specific site of the antigen, and this antibody-binding portion is called an epitope (antigen recognition site). Therefore, the substance to which the antibody reacts reacts not only with the antigen itself but also with a substance having the same amino acid sequence as the epitope of the antigen. In some cases, an antibody reacts with an amino acid sequence that is not identical to an epitope but has a similar amino acid sequence, and a peptide that has an amino acid sequence similar to such an epitope and reacts with an antibody is called a mimotope peptide.
A luciferyl peptide compound (hereinafter simply referred to as luciferyl peptide), which is a compound obtained by binding firefly luciferin or a luciferin derivative and a peptide, can be configured as an epitope or mimotope capable of antigen-antibody reaction of the peptide portion. The luciferyl peptide thus configured is considered to be recognized and bound by the antibody to form a complex. When the complex luciferyl peptide is in a competitive relationship with the antigenic substance, it can be dissociated from the antibody by being replaced with the antigenic substance when in close proximity to the antigenic substance. When luciferin is liberated from the dissociated luciferyl peptide, luminescence can be detected using luciferase. That is, the antigen substance is indirectly detected by luminescence based on the competitive substitution reaction in the complex, and the luciferyl peptide acts as a labeled peptide. Based on such an idea, the present invention applies a luminescence detection system using a luciferyl peptide to an antigen-antibody reaction system to detect luminescence of an antigen substance or an antigen-like substance (that is, a substance having an amino acid sequence of an epitope). And realize the measurement. In other words, an antibody capable of specifically recognizing and binding a predetermined site contained in a test substance (detection target) as an epitope is bound to an epitope peptide or a luciferyl peptide having its mimotope peptide as a peptide component. Using the complex (antibody-luciferyl peptide complex), a measurement reagent is constituted. When the antibody-luciferyl peptide complex and the specimen containing the test substance coexist in the solution, the difference between the antibody-luciferyl peptide and the antibody-test substance due to the difference in affinity. The luciferyl peptide is dissociated and the luciferyl peptide is dissociated, the luciferin is released from the dissociated luciferyl peptide, and the amount of luciferin is measured as the amount of light detected using an enzyme reaction with luciferase to replace the complex. Indirect measurement of the bound test substance can be easily performed. Hereinafter, the present invention will be described in detail.

(Test substance)
The test substance that can be measured using the measurement method of the present invention is not particularly limited as long as it contains an epitope peptide that can be specifically recognized and bound by an antibody as a constituent part. Can be targeted. Therefore, a substance containing in its molecule an epitope amino acid sequence that can be obtained from a database or the like described later can be used as a test substance. Further, as described later, since it is possible to produce an antibody that recognizes an arbitrary amino acid sequence as an epitope, for any peptide or protein, a certain site in the amino acid sequence is set as an epitope, By producing an antibody to be recognized, the peptide or protein can be used as a test substance.
Using the sample that may contain the test substance as a specimen, the test substance contained in the specimen is indirectly measured. The specimen is preferably in a liquid state at the time of measurement. For example, liquids collected from living bodies including body fluids such as blood, urine, spinal fluid, saliva, sputum, and cell suspension can be exemplified. When the specimen is not liquid, it can be used in the measurement method of the present invention after being made into a liquid state using means such as miniaturization, grinding or solubilization, and the liquefaction method can be arbitrarily selected. The measurement method of the present invention can be used for detection of the presence or absence of a test substance in a specimen, quantification of a test substance, etc. without any particular limitation on its purpose. Further, as immunoassay, it can be used for analysis in clinical tests, food tests, environmental tests and the like.

(Epitope and mimotope)
Epitopes have been identified in various natural proteins. For example, in the case of C reactive protein (CRP), the amino acid sequence of the epitope <YLGGPFSPNVLN (Tyr-Leu-Gly-Gly-Pro-Phe-Ser-Pro-Asn-Val-Leu-Asn) is known. (See, for example, Non-Patent Document 3 below). In addition, epitopes of HCV virus-derived proteins, HIV virus-derived proteins, mite allergens and the like have also been identified (see, for example, Patent Document 5 and Patent Document 6 below). Information on such various epitopes is published on databases such as Immune epitope database analysis resource (http: www.iedb.org).
(Patent Document 5) Japanese Translation of PCT International Publication No. 2001-500723 (Patent Document 6) Japanese Patent Application Publication No. 5-508837 (Non-Patent Document 3) THE JOURNAL OF BIOLOGICAL CHEMISTRY 25095-25102,2005
(Non-Patent Document 4) Molecular Immunology 1999; 36: 53-60
A peptide having the same amino acid sequence as such a known epitope site, or a peptide having a partially different similar amino acid sequence obtained by modifying (deleting, adding or substituting) a part thereof, luciferin or a derivative thereof A luciferyl peptide can be prepared by condensing with the luciferyl peptide. The obtained luciferyl peptide is used as a labeled peptide that can bind to an antibody that recognizes an antigen having the above-described epitope (test substance) based on the affinity with an antibody described below. There may be a peptide that can bind to an antibody even if it has an amino acid sequence that does not show similarity to the epitope, and such a peptide is also an antibody-luciferyl constituting the measurement reagent of the present invention. It can be used for the preparation of peptide conjugates. The method using the sequence information of the epitope peptide and its modified peptide is very advantageous as one means for efficiently preparing an optimal measurement reagent, but is not limited thereto.

(antibody)
The antibody used in the measurement method of the present invention can be used without particular limitation as long as it is an antibody that can specifically recognize and bind to an epitope of a test substance. A monoclonal antibody, a polyclonal antibody, a single chain antibody, a human The antibody may be any of a synthetic antibody, a bispecific antibody, a synthetic antibody, an antibody fragment such as a Fab, Fv or scFv fragment, or a chemically modified derivative thereof, whether artificial or synthetic. An antibody specific for a specific substance can be prepared by using various known methods using a natural protein or the like. For example, in the case of a monoclonal antibody, it can be prepared by a method including fusing spleen cells derived from an immunized mammal with mouse myeloma cells (see, for example, Non-Patent Document 5 and Non-Patent Document 6 below).
(Non-Patent Document 5) Kohler and Milstein, Nature 256 (1975), 495
(Non-patent document 6) Galfre, Meth. Enzymol. 73 (1981), 3
Moreover, at present, it is easy to synthesize peptides from amino acids according to preset amino acid sequences due to advances in synthesis reaction technology and development of peptide synthesizers, and any natural or artificial peptides (high molecular weight peptides) Including proteins). It is also known that antibodies can be induced by immunizing the synthetic peptide. Therefore, by using these methods, various antibodies that recognize any amino acid sequence as an epitope can be produced. Therefore, by preparing an antibody that recognizes a part of the amino acid sequence of the substance to be measured as an epitope and preparing this antibody, a luciferyl peptide-antibody complex that can be used for measurement using this substance as a test substance is prepared. be able to.

(D-luciferin and its derivatives)
D-luciferin and D-luciferin derivatives emit light by being converted to oxyluciferin, which is a luminescent material, by firefly luciferase in the presence of ATP and magnesium ions. Therefore, the amount of luminescence is measured using an appropriate luminescence measuring device. Can be quantified. Therefore, the amount of the test substance substituted with the luciferyl peptide can be calculated based on the luminescence amount of D-luciferin.
The D-luciferin or D-luciferin derivative used in the present invention is not particularly limited as long as it causes a luminescence reaction by the above firefly luciferase. Examples of the D-luciferin derivative include those in which the hydroxy group of D-luciferin is substituted with an amino group, and those in which a sulfur atom is substituted with an oxygen atom, as described in Patent Document 7 below. .
(Patent Document 7) JP-A-2006-219381

(Luciferyl peptide and its production)
The peptide part of the luciferyl peptide has the same amino acid sequence as the epitope of the test substance, or a similar amino acid sequence in which the peptide part is partially modified (deletion, addition or substitution of one to several amino acids) (Amino acid sequence of mimotope). Therefore, the epitope peptide or mimotope peptide of the test substance, or a peptide having any of these as a part in the molecule can be prepared according to a known peptide synthesis method and used as a raw material for the synthesis of luciferyl peptide. Moreover, it is not limited to the above, What is necessary is just to complete the peptide part containing the amino acid sequence of an epitope or a mimotope finally by combining with D-luciferin or its derivative (s). As peptide synthesis methods, peptide formation using a condensing agent, active esterification method, mixed acid anhydride method, ligation, solid phase synthesis method, and the like are known, and can be appropriately selected from these, and are commercially available. This may be done automatically using a peptide synthesizer.

  As one form of the luciferyl peptide used in the measurement method of the present invention, the α-position amino group serving as the amino terminus of the peptide chain and the carboxyl group of D-luciferin or D-luciferin derivative are bonded by dehydration condensation, and these are amides And D-luciferyl peptide linked through a bond. D-luciferin is known to emit light as an active substance when the carboxyl group is converted to AMP, but the D-luciferin portion of the above D-luciferyl peptide has an amide bond at the carboxyl group site. Since it is blocked, it does not emit light unless it is cleaved from the peptide. The same applies when a D-luciferin derivative is used instead of D-luciferin. This amide can be produced by a reaction using a carbodiimide method according to a conventional method. Specifically, for example, the amide can be produced with reference to the operation of Example 2 of the present application. Therefore, a luciferyl peptide used as a labeled peptide can be obtained by preparing a peptide portion of a luciferyl peptide by a known peptide synthesis method and amidating it with D-luciferin or a derivative thereof as a raw material.

(Affinity of peptide with antibody)
As described above, a peptide having the same or similar amino acid sequence as the epitope of the test substance (epitope peptide, mimotope peptide or peptide having a part thereof) binds to firefly D-luciferin or D-luciferin derivative Thus, D-luciferyl peptide is prepared, and this is used as a labeled peptide for measuring a test substance to prepare a measurement reagent. In the present invention, the luciferyl peptide has an affinity for the antibody to such an extent that it can bind to an antibody that specifically recognizes the test substance as a labeled peptide to form an antibody-luciferyl peptide complex. Therefore, as one form for having affinity with such an antibody, the amino acid sequence of the peptide portion of the luciferyl peptide is an epitope, that is, the antibody is an antigen (test substance). Some contain the same amino acid sequence as the recognition site.
In the measurement method of the present invention, when the test substance is detected, when the antibody-luciferyl peptide complex comes into contact with the test substance, the luciferyl peptide of the antibody-luciferyl peptide complex is the test substance. It is necessary for the luciferyl peptide to be dissociated by substitution. Therefore, the affinity between the antibody and the luciferyl peptide is sufficiently high to form a complex, but is low compared to the affinity between the antibody and the test substance, and the substitution occurs favorably under the measurement conditions. is required. In the present invention, in order to obtain a luciferyl peptide suitable for such a labeled peptide, the affinity between various peptides and antibodies can be evaluated and selected based on this. The affinity between the peptide and the antibody is not the same as the affinity between the luciferyl peptide obtained from the peptide and the antibody, but is useful for predicting suitability.
The affinity between a luciferyl peptide whose peptide part is an epitope peptide and an antibody can be different from the affinity between an antigen (test substance) having the epitope and an antibody, but the difference is the above-mentioned complex If a suitable substitution on the body is not obtained, a part of the amino acid sequence is modified (added, deleted or deleted) instead of the epitope peptide as necessary in order to realize the preferred substitution. Substituted) peptides with similar sequences can be applied. What modification is appropriate depends on the amino acid sequence of the peptide to be used, the antigen, the structure of the antibody, and various measurement conditions, but a mimotope peptide that can react with the antibody can be appropriately selected and used.

  When determining peptides to be used in the measurement method of the present invention based on epitope information having a specific amino acid sequence, various modified peptides can be prepared based on the amino acid sequence of the epitope, and their suitability can be compared and evaluated. it can. For example, each peptide is examined for the possibility of binding to an antibody and the possibility of substitution with a test substance, and after selecting a peptide capable of binding and substitution, a luciferyl peptide is prepared for the selected peptide and bound to the antibody. And the replacement can be confirmed again. Alternatively, in the comparative evaluation at the peptide stage, only the possibility of binding with the antibody may be examined, and the possibility of substitution may be examined in the state of the luciferyl peptide. The evaluation of suitability may be performed using the measurement method of the present invention itself. In this case, if a suitable substitution on the complex is obtained, the amount of luminescence detected depending on the antigen concentration increases. Conversely, if a suitable substitution is not obtained, substitution with the antigen does not occur and no luminescence is detected. Further, as in Example 1, biotin is added to the epitope peptide, and when the epitope peptide is labeled using a complex of streptavidin and alkaline phosphatase that binds to biotin, the absorbance is measured by a chromogenic method. Since it can be detected, it is possible to screen whether a peptide having a modified amino acid sequence is effective for this measurement. If the epitope peptide is effective for this measurement, when the labeled epitope peptide is bound to the antibody, the absorbance is sufficiently higher than the background, and if a suitable substitution with the antigen is obtained, Accordingly, substitution occurs and the absorbance decreases. This method using biotin-streptavidin binding requires washing and separation steps, but is useful for screening because biotinylated peptides can be easily obtained.

(Preparation of antibody-luciferyl peptide complex)
The antibody-luciferyl peptide complex can be bound by the affinity between the antibody and the luciferyl peptide by reacting the luciferyl peptide and the antibody in an appropriate buffer to prepare a complex. The buffer used is not particularly limited, and examples thereof include phosphate buffer, glycine-sodium hydroxide buffer, Tris-hydrochloric acid buffer, ammonium chloride-ammonia buffer, Good's buffer, and the like. The concentration and pH of the buffer solution are not particularly limited and can be appropriately set and used as necessary, but the concentration of 10 mM to 800 mM and the range of pH 4 to 10 are usually used. The reaction may be performed at 20 ° C. to 45 ° C. for 1 minute to 4 hours, preferably at 25 ° C. to 37 ° C. for 1 minute to 1 hour.
The antibody-luciferyl peptide complex used in the measurement method of the present invention is stable in any of the above-mentioned buffers and can be stored and distributed. Therefore, it is not necessary to prepare at the time of use for each measurement.

(Immobilization of antibody-luciferyl peptide complex)
In the measurement method of the present invention, the above-described complex bound to an appropriate carrier and immobilized can also be used. Immobilization of the complex is useful in that the luciferyl peptide that was added excessively to the antibody and did not form a complex can be separated and removed from the complex to reduce the excess background. is there. The material of the carrier is not particularly limited, and examples thereof include polyethylene, polystyrene, polypropylene, polyvinyl chloride, glass, ceramic, agarose, and the like, and enzyme immunity such as beads, plates, test tubes, and latex made of such materials. Those usually used for the measurement method can be appropriately selected and used. Among various carriers, in particular, when a complex is bound to a polystyrene 96-well microplate, it is useful in that multiple samples can be measured simultaneously. In addition, as shown in Example 1 described later, a microplate in which protein G that recognizes the Fc portion of an antibody or protein A is immobilized on a 96-well polystyrene microplate is also commercially available. When used, it is more useful because it increases the amount of complex that can be bound on the support. When the antibody-luciferyl peptide complex is immobilized on the carrier as described above, it can be stored and distributed easily because it is stable in either a dry state or a liquid state.

(Dissociation of luciferin from D-luciferyl peptide)
In the measurement method of the present invention, it is important to release the luciferyl peptide D-luciferin dissociated from the antibody in order to enable detection of luminescence. This release can be achieved by using an enzyme. Examples of degrading enzymes used to release D-luciferin from luciferyl peptides include esterases, peptidases, phosphatases, lipases, acetylesterases, nucleotidases, glucosidases, amylases, amidases, acylases, proteases, etc. It is not limited to. In particular, in the case of a luciferyl peptide obtained by condensing the carboxyl group of luciferin and the amino group of the peptide, examples of the degrading enzyme for cleaving the bond include carboxypeptidase, aminopeptidase, aminoacylase, and protease. However, it is not limited to these. The luciferyl peptide bound to the antibody is stable and does not undergo cleavage by an enzyme such as peptidase, or at least is less susceptible to cleavage as compared to the dissociated luciferyl peptide. The luciferyl peptide in the complex that has not caused the reaction does not affect the luminescence detection. The cleavage reaction using an enzyme can proceed in a buffer solution, such as a phosphate buffer solution, glycine-sodium hydroxide buffer solution, Tris-hydrochloric acid buffer solution, ammonium chloride-ammonia buffer solution, Good's buffer solution, etc. These various buffer solutions can be used as a reaction system. Specifically, in a buffer solution having a concentration of 10 mM to 800 mM and a pH of 4 to 10, the temperature is 20 ° C. to 45 ° C. for 1 minute to 4 hours, and preferably the temperature is 25 ° C. to 37 ° C. for 1 minute to 1 hour. When the enzyme reaction is carried out to a certain extent, luciferin is preferably dissociated from the luciferyl peptide.

(Luciferase and luciferase reaction)
In the measurement method of the present invention, it is important to finally detect the released D-luciferin by a luciferase luminescence reaction. As for luciferase, not only fireflies but also enzymes that emit light using firefly luciferin can be used regardless of their origin, whether they are naturally derived, recombinant, or even introduced with mutations.
The measurement system for emitting D-luciferin is preferably a buffer solution having a concentration of 10 mM to 800 mM and a pH of 4 to 10. Examples of the buffer solution include phosphate buffer, glycine-sodium hydroxide buffer, Tris- Hydrochloric acid buffer, ammonium chloride-ammonia buffer, Good's buffer, etc. are mentioned. The luciferase concentration is adjusted to 0.1 μg / ml or more, preferably 1 to 1000 μg / ml. Further, ATP and magnesium ions are required for light emission, and the ATP concentration and the magnesium ion concentration are each 0.001 mM or more, preferably 1 to 1000 mM.
Since the luminescence reaction of D-luciferin and the D-luciferin release reaction of the luciferyl peptide proceed in the same system, the reaction proceeds continuously when the conditions are optimized so that the measurement system also serves as the reaction system described above. It is convenient for. In order to smoothly perform the enzyme reaction and the luminescence reaction, various additives may be added to the measurement system. Examples of such additives include stabilizers, surfactants, activators, and the like. For example, as a luciferase stabilizer, dithiothreitol (0.1-10 mM), EDTA (0.1-10 mM), BSA (0.01-10%), and the like can be used. As the agent, Tricine buffer or HEPES buffer (20 to 200 mM) can be used.

(Luminescence detection)
The measurement method of the present invention is a method based on detection of a luciferin-luciferase luminescence reaction, and the luciferin-luciferase luminescence reaction can be detected using a general-purpose luminescence detection device, so an expensive and large excitation light irradiation device is required. Instead, a general-purpose light emission detection device may be used.

(Measurement of test substance using the method of the present invention)
An embodiment of the measurement method of the present invention will be specifically described with reference to FIG.
First, an aqueous liquid (buffer solution as described above) is used as a measurement system, and an antibody-luciferyl peptide complex is present in the measurement system. The luciferyl peptide of this complex contains in its peptide part a peptide (epitope peptide or mimotope peptide) that is identical or partly modified with the epitope sequence in which the antibody recognizes the test substance. A bond that can be dissociated and replaced is formed. Here, when an antigen having an epitope that is a test substance (= original binding antigen) is supplied, the affinity between the test substance and the antibody is greater than the affinity between the antibody and the luciferyl peptide that was bound earlier. Since the sexuality is stronger, the luciferyl peptide dissociates from the antibody and the test substance binds to the antibody, and as a result, the luciferyl peptide and the test substance are replaced. Next, a cleaving enzyme (peptidase or the like) in the measurement system acts on the dissociated luciferyl peptide to dissociate luciferin. At this time, the luciferyl peptide that forms a complex with the antibody without being substituted with the test substance is not cleaved by the enzyme, so that luciferin does not dissociate or at least the dissociated luciferyl peptide Less susceptible to cutting. Therefore, the luciferyl peptide in the complex that did not undergo the substitution reaction has substantially no effect on the measurement. Furthermore, luminescence reaction by firefly luciferase occurs in the presence of ATP and magnesium ions using the produced luciferin as a substrate, and luciferin can be quantified by the amount of luminescence produced, and based on this, the test substance bound to the antibody is quantified. Is possible.

  The reaction conditions of the measurement method of the present invention are not particularly limited as long as each step of the above reaction suitably proceeds, and when the test substance is added, the luciferyl peptide bound to the antibody and The pH and temperature suitable for good substitution with the test substance and the pH and temperature suitable for cleaving the dissociated luciferyl peptide with an enzyme are set. If necessary, the conditions of each step can be made different and advanced step by step. However, if conditions common to both steps can be set, it is more preferable because they proceed continuously. For example, in a buffer solution having a concentration of 10 mM to 800 mM and pH 4 to 10, the reaction proceeds at 5 ° C. to 60 ° C. for 1 minute to 4 hours, preferably 5 ° C. to 45 ° C. for 1 minute to 1 hour. can do. As the buffer solution, for example, various buffer solutions such as phosphate buffer solution, glycine-sodium hydroxide buffer solution, Tris-hydrochloric acid buffer solution, ammonium chloride-ammonia buffer solution, Good's buffer solution and the like can be used.

In the method of the present invention, it is not necessary to repeat the operation of washing / separating non-adsorbed antigens, antibodies, etc. when measuring the test substance, and the measurement can be carried out by adding the reagents sequentially. Therefore, the test substance can be measured based on immunoassay more easily than the conventional method that requires repetition of these operations.
In addition, only one type of antibody may be used, and it is not necessary to prepare a set of two types of antibodies with different recognition sites for one substance to be detected as in the sandwich ELISA method. Cost reduction can be achieved. Furthermore, because it is physically small, it is not suitable for the simultaneous binding of two types of antibodies with different recognition sites. For this reason, substances (antigens) that could not be measured by sandwich ELISA are also measured. It becomes possible to make it a target.
Further, since the luminescence reaction of luciferase is used as the detection method, a general-purpose luminometer or the like may be used as the luminescence measuring device, and no special and expensive equipment is required. Furthermore, since detection can be performed by simply adding reagents sequentially, measurement can be automated easily.

(Measurement reagent for test substance)
A measurement reagent for a test substance can be prepared by combining the above-described elements used in the measurement method of the present invention. Specifically, the measurement reagent for the test substance includes (a) an antibody-luciferyl peptide complex, (b) an enzyme capable of releasing luciferin from the luciferyl peptide, (c) ATP, (d) magnesium ion, and (E) Firefly luciferase as a constituent component. The antibody-luciferyl peptide complex comprises (a1) an antibody that can specifically bind to a test substance and (a2) a D-luciferyl peptide that binds displaceably to the antibody, and the D-luciferyl peptide Is a compound in which a peptide having the same or modified amino acid sequence as an epitope (antigen recognition site) of a test substance that is specifically recognized by the antibody is bound to firefly D-luciferin or a D-luciferin derivative.
The concentration of each constituent component is not particularly limited as long as the test substance can be accurately measured, and can be optimized as appropriate. For example, (a) antibody-luciferyl peptide complex: 0.1 ng / ml or more, preferably 1 ng / ml to 100 mg / ml, (b) enzyme capable of dissociating luciferin from luciferyl peptide: 0.1 U / ml or more (C) ATP: 0.001 mM or more, preferably 0.005 to 100 mM, (d) Magnesium ion: 0.001 mM or more, preferably 1-1000 mM, e) Firefly luciferase: 0.1 μg / ml or more, preferably 1-1000 μg / ml.
These reagents may contain a preservative, a buffering agent, and the like, and it can be selected whether all of them are stored in separate containers or a plurality of components are stored together in the same container. In addition, a test substance measurement kit including a container containing these reagents, a measurement method instruction, and, if necessary, standard test substance standards can be prepared.
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the description described below does not limit the scope of the present invention.

(C-reactive protein epitope peptide production and affinity modification)
C reactive protein (hereinafter referred to as CRP) is one type of plasma protein component. Normally, only a very small amount (1 μg / ml or less) is found in the blood of normal humans, whereas blood is present during inflammatory diseases such as bacterial infections and viral infections, or tissue disintegrating diseases such as myocardial infarction. Medium concentrations are known to increase rapidly. Using this phenomenon, the CRP test is useful for diagnosing inflammatory diseases or tissue disintegrating diseases, determining the degree of serious injury, observing the course, and determining the prognosis. Using this C-reactive protein as a model, an epitope peptide was prepared as follows.

As an epitope sequence of CRP, Tyr-Leu-Gly-Gly-Pro-Phe-Ser-Pro-Asn-Val-Leu-Asn (hereinafter referred to as STD) is known. Based on this sequence information, an epitope peptide and a modified peptide having the same amino acid sequence or an amino acid sequence in which one or more amino acids are modified (added, deleted or substituted) are prepared, and each is combined with an antibody to prepare an antibody -Peptide complexes were prepared, and further, whether or not a substitution reaction with the antigen was caused when the antigen (CRP) was added was examined to evaluate the suitability of each peptide.
The biotinylated epitope peptide was prepared using Peptide.Ab (SIGMA) service. In addition, nine types of modified epitope peptides with amino acids added or deleted to modify affinity with antibodies were synthesized as shown in Table 1 below.
A Tris-HCl (50 mM, pH 7.0) buffer solution containing 1% BSA (bovine serum albumin; Wako Pure Chemical Industries, Ltd., catalog number: 014-15134), 0.05% Tween 20 and 150 mM sodium chloride. Prepared. Using this, a solution dissolved in the above Tris-HCl buffer was prepared so that the concentration of the anti-CRP polyclonal antibody (BETHYL LABORATORIES, INC, catalog number: A80-125A) was 14.6 μg / ml. Each synthesized peptide was prepared by dissolving it in the above Tris-HCl buffer so as to be 1 μg / ml. Furthermore, a solution prepared by dissolving streptavidinated alkaline phosphatase (catalog number 199-12031 manufactured by Wako Pure Chemical Industries, Ltd.) for labeling the peptide in the above Tris-HCl buffer so as to have a concentration of 10 μg / ml was prepared.
An antibody solution (100 μl), a peptide solution (100 μl), and a streptavidinated alkaline phosphatase solution (100 μl) were placed in a microtube and allowed to stand at room temperature for 1 hour to perform a binding reaction. In addition, a microplate on which protein G is immobilized (Reactive Bind Protein G coated microplate; PIERCE, catalog number: 15131) is mixed with Tris-HCl (50 mM, pH 7.5) containing 0.05% Tween 20 and 150 mM sodium chloride. 0) Washing was performed 3 times with 200 μl of buffer solution, and then 200 μl of Tris-HCl (50 mM, pH 7.0) buffer solution containing 150 mM sodium chloride containing 2% BSA was reacted for 30 minutes to perform a blocking reaction. . Subsequently, 100 μl of this solution was placed in the above-mentioned microplate to immobilize the antibody. Therefore, when the antibody-peptide-labeled enzyme complex is produced, it is immobilized on the microplate. After fixation, washing was performed 3 times with a Tris-HCl (50 mM, pH 7.0) buffer containing 0.05% Tween 20 and 150 mM sodium chloride.

  Approximate concentrations of CRP (C-reactive protein; Life Diagnostics, inc, catalog number: 8000) were added to Tris-HCl (50 mM, pH 7.0) containing 1% BSA, 0.05% Tween 20 and 150 mM sodium chloride. ) CRP solutions having different concentrations were prepared by dissolving in a buffer solution. Using CRP solution, whether or not the peptide was dissociated from the complex was examined as follows. At this time, 100 μl of the CRP solution was added to the microplate on which the antibody-peptide-labeled enzyme complex was immobilized, and left at room temperature for 1 hour to perform a substitution reaction. Thereafter, washing was performed three times with a Tris-HCl (50 mM, pH 7.0) buffer solution containing 0.05% Tween 20 and 150 mM sodium chloride.

As a measuring solution for coloring alkaline phosphatase, which is a labeling enzyme, p-nitrophenyl phosphate is added to a 1.0 M diethanolamine solution adjusted to pH 9.8 containing 0.5 mM magnesium chloride so as to have a final concentration of 10 mM. A prepared solution was prepared. Add 200 μl of the solution to the microplate before and after adding the above CRP solution, measure the absorbance at a wavelength of 405 nm every 20 seconds with a microplate reader (Molecular Devices, SPECTRAmax), and change the absorbance per minute. The amount was calculated. When the peptide bound to the labeling enzyme and the antibody are bound, the amount of change in absorbance in the microplate before adding the CRP solution becomes high. In addition, the greater the amount of peptide bound to the label enzyme bound to the antibody without being replaced by CRP, the higher the amount of change in absorbance after adding the CRP solution, that is, the higher the residual ratio of the peptide in the antibody. . Table 1 shows the results of evaluating the binding and substitution of each peptide and antibody. When the change in absorbance was 0.5 mAbs / min or more, it was determined that the peptide and the antibody were bound. The substitution was determined to be a substitution when the change in absorbance after addition of CRP decreased.

  Using the values calculated when CRP solutions with different concentrations were added, the ratio of the change in absorbance when the amount of change in absorbance without addition of CRP was 100 was used as the residual ratio of the epitope peptide and modified peptide. The relationship between the amount and the residual ratio of the peptide is shown in FIG. That is, at the CRP addition amount of 0 mg / dL, the peptide has all bound to the antibody and no substitution has occurred, so the change in absorbance is the highest. When an epitope peptide (STD) whose amino acid sequence was not altered was used, the change in absorbance was constant regardless of the amount of CRP added. On the other hand, when CRP is added in a system using a peptide having a modified amino acid sequence: Phe-Tyr-Leu-Gly-Gly-Pro-Phe-Ser-Pro-Asn-Val-Leu (hereinafter referred to as I174F), the affinity It was confirmed that the substance that binds to the antibody was replaced with CRP due to the difference in sex, and the amount of change in absorbance decreased as the amount of CRP added increased. This indicates that the modified peptide and the CRP epitope have undergone substitution. That is, under the current measurement conditions, when a peptide whose epitope sequence was not modified was used, substitution between the peptide and the CRP epitope did not occur, while the amino acid sequence of the epitope was modified to change the affinity. When the peptide was used, it was confirmed that the change in the absorbance change was decreased according to the amount of CRP added, that is, the substitution according to the amount of the test substance added occurred.

(Production of luciferyl peptide)
D-luciferin (8.5 mg, 0.03 mmol), 1-hydroxybenzotriazole (4.1 mg, 0.03 mmol), and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (5.8 mg) N, N-diisopropylethylamine (10 μl, 0.1 mmol) was added to 0.3 ml of an anhydrous DMF solution containing 0.03 mmol) while stirring on an ice bath and stirred for 5 minutes. Next, anhydrous containing peptide 1174F (2 mg, 1.53 × 10 ⁻³ mmol) having a modified sequence of CRP epitope: Phe-Tyr-Leu-Gly-Gly-Pro-Phe-Ser-Pro-Asn-Val-Leu 0.3 ml of DMF solution was added dropwise to the luciferin solution over 1 minute on an ice bath, stirred at room temperature for 2 hours, and luciferyl peptide represented by the following structural formula (wherein R is α- Peptide 1174F condensing at the amino group is shown). The reaction solution was concentrated under reduced pressure, and partitioned with 4 ml of ethyl acetate and 4 ml of pure water. The ethyl acetate phase was separated and concentrated by centrifugation.

The luciferyl peptide was purified by high performance liquid chromatography (HPLC). The purification conditions were a reverse phase column (Kanto Chemical Co., Mightysil PR-18 GP (H), 250 × 4.6 (5 μm)) and a gradient using CH ₃ CN solvent containing 0.05% TFA. The function was used to increase the CH ₃ CN concentration from 10% to 90% in 20 minutes. The flow rate was 1 ml / min, the column temperature was 20 ° C., and the detection was performed at wavelengths of 254 and 330 nm.

(Immobilization of luciferyl peptide-antibody complex to carrier)
In a Tris-HCl (50 mM, pH 7.0) buffer solution containing 1% BSA (bovine serum albumin; Wako Pure Chemical Industries, Ltd., catalog number: 014-15134), 0.05% Tween 20 and 150 mM sodium chloride. Anti-CRP polyclonal antibody (BETHYL LABORATORIES, INC, catalog number: A80-125A) was dissolved to a concentration of 14.6 μg / ml. Using the same Tris-HCl buffer, a luciferyl peptide solution in which the luciferyl peptide prepared in Example 2 was dissolved to a concentration of 1 μg / ml was prepared. An antibody solution (100 μl) and a luciferyl peptide solution (100 μl) were placed in a microtube and allowed to stand at room temperature for 1 hour for a binding reaction. In addition, a microplate on which protein G is immobilized (Reactive Bind Protein G coated microplate; PIERCE, catalog number: 15131) is mixed with Tris-HCl (50 mM, pH 7.5) containing 0.05% Tween 20 and 150 mM sodium chloride. 0) Washing was performed 3 times with 200 μl of buffer solution, and then 200 μl of Tris-HCl (50 mM, pH 7.0) buffer solution containing 150 mM sodium chloride containing 2% BSA was reacted for 30 minutes for blocking. Subsequently, 100 μl of the antibody-luciferyl peptide complex solution described above was added to the microplate described above, and the antibody-luciferyl peptide complex was immobilized on the microplate. After fixation, the plate was washed 3 times with Tris-HCl (50 mM, pH 7.0) buffer containing 0.05% Tween 20 and 150 mM sodium chloride and dried.

(Measurement of CRP concentration using luciferyl peptide-antibody complex)
Tris-HCl (50 mM, pH 7.0) buffer containing 1% BSA, 0.05% Tween 20 and 150 mM sodium chloride in a known concentration of CRP (C-reactive protein; Life Diagnostics, inc, catalog number) : 8000) at different concentrations were prepared. For each concentration, 100 μl of the CRP solution was placed on the microplate on which the above-mentioned luciferyl peptide-antibody complex was immobilized, and reacted at room temperature for 1 hour to perform a substitution reaction.

  Carboxypeptidase A (SIGMA, catalog number: C9268) and D-Aminoacylase Amano (Wako Pure Chemical Industries, Ltd.) were added to a Tris-HCl (50 mM, pH 7.0) buffer containing 0.05% Tween 20 and 150 mM sodium chloride. Catalog Number: 329-61061) were dissolved to concentrations of 0.6 mg / ml and 2.5 mg / ml, respectively. 200 μl of this enzyme solution was placed in a microtube, 50 μl of the solution after the above substitution reaction was added, and a luciferyl peptide cleavage reaction was performed at 37 ° C. for 1 hour.

To luciferase in Trien-NaOH (50 mM, pH 7.8) containing 4.8 mM ATP, 12 mM MgSO ₄ , 1 mM EDTA, 6 g / l glycerol, 1.2 g / l BSA and 0.1 mM mercaptoethanol. Was dissolved to a concentration of 50 mg / ml, and this was used as a luminescence measuring solution.

  50 μl of the above enzyme-cleaved solution was placed in a 96-well white microplate, and luminescence was measured as follows using a luminometer (Belto Lud Luminometer, LB96V). The luminescence measurement solution was added using the automatic injection function of the luminometer so that the solution after enzymatic cleavage reacted with 100 μl of the luminescence measurement solution immediately before the start of measurement. The measured value is a light emission amount obtained by integration for 3 seconds. The measurement results are shown in FIG.

  As shown in FIG. 3, the CRP content is increased to 0.5 mg / dl, 2.1 mg / dl, and 5.3 mg / dl compared to the amount of luminescence when a solution containing no CRP is measured by this measurement method. Was measured, the amount of luminescence increased according to the CRP concentration. Therefore, it was confirmed that the test substance (CRP) can be measured by this measurement method. In addition, in this measurement, after the addition of the non-test substance, there was no washing or separation operation, and the measurement was simple.

(Preparation of test substance measurement reagent)
A measurement reagent for a test substance composed of (a) to (c) having the following composition was prepared.
(A) Luciferyl peptide-antibody complex solution: luciferyl peptide having CRP epitope-modified sequence (Phe-Tyr-Leu-Gly-Gly-Pro-Phe-Ser-Pro-Asn-Val-Leu) and anti-CRP polyclonal 0.1 mg / ml luciferyl peptide-antibody complex conjugated with an antibody (BETHYL LABORATORIES, INC, catalog number: A80-125A) according to Example 3, 1% BSA (bovine serum albumin; Wako Pure Chemical Industries, Ltd.) Catalog Number: 014-15134), 0.05% Tween 20, 150 mM sodium chloride and Tris-HCl (50 mM, pH 7.0).
(B) Luciferyl peptide-cleaving enzyme solution: 0.6 mg / ml Carboxypeptidase A (SIGMA, catalog number: C9268), 2.5 mg / ml D-Aminoacylase Amano (Wako Pure Chemical Industries, catalog number: 329) -61061), 0.05% Tween 20, 150 mM sodium chloride and Tris-HCl (50 mM, pH 7.0).
(C) Luminescence measurement solution: 4.8 mM ATP, 12 mM MgSO ₄ , 1 mM EDTA, 6 g / l glycerol, 1.2 g / l BSA, 0.1 mM mercaptoethanol, Trien-NaOH (50 mM, pH 7.8) and 50 mg / ml Luciferase.
According to Example 3, when a solution containing 3.0 mg / ml of CRP was used as a sample and measurement using CRP as a test substance was performed using the above-described measurement reagent, the test substance could be measured without problems. .

Claims (12)

  A complex of a luciferyl peptide compound in which firefly D-luciferin or a D-luciferin derivative is condensed with a peptide and an antibody capable of specifically binding to a test substance, wherein the peptide portion of the luciferyl peptide compound is A complex characterized in that the luciferyl peptide compound binds to the antibody in a replaceable manner by a test substance.   The complex according to claim 1, wherein the affinity of the peptide portion of the luciferyl peptide compound with the antibody is smaller than the affinity of the test substance with the antibody. The luciferyl peptide compound is a condensed compound of firefly D-luciferin represented by the following general formula (wherein R represents the peptide bonded to the α-amino group). Complex.
  4. The peptide according to claim 1, wherein the peptide portion of the luciferyl peptide compound is a peptide having the same amino acid sequence as the epitope of the test substance specifically recognized by the antibody or a modified peptide thereof. Complex.   A test substance measurement reagent comprising the complex according to any one of claims 1 to 4 as a luminescence detection element for test substance measurement.   The test substance according to claim 5, further comprising an enzyme capable of dissociating firefly D-luciferin or a D-luciferin derivative from the luciferyl peptide compound, ATP, magnesium ion, and firefly luciferase. Measuring reagent. A composite according to any one of claims 1 to 4 is prepared,
Supplying a specimen to the complex, substituting the luciferyl peptide compound of the complex with a test substance contained in the specimen to generate a luciferyl peptide compound;
A firefly D-luciferin or a D-luciferin derivative is produced from the resulting luciferyl peptide compound to cause a luminescence reaction,
A method for measuring a test substance, comprising measuring the test substance by detecting the luminescence.   The method for measuring a test substance according to claim 7, wherein the complex is immobilized on a solid phase carrier and is in contact with the supplied sample in a liquid.   Release of the firefly D-luciferin or D-luciferin derivative from the luciferyl peptide compound is performed by esterase, carboxypeptidase, arylsulfatase, alkaline and acid phosphatase, lipase, acetylesterase, nucleotidase, kininase, glucosidase, amylase, amidase 9. The method for measuring a test substance according to claim 7 or 8, wherein at least one selected from the group consisting of aminopeptidase, aminoacylase and protease is used. Preparing an antibody capable of specifically binding to a test substance;
Preparing a peptide having the amino acid sequence of the epitope of the test substance or the mimotope of the test substance specifically recognized by the antibody;
Condensing the peptide with firefly D-luciferin or a D-luciferin derivative to prepare a luciferyl peptide compound;
And a step of preparing a complex in which the luciferyl peptide compound and the antibody bind to each other.   The method for preparing a measurement reagent according to claim 10, further comprising a step of confirming that the luciferyl peptide compound of the complex is replaced with a test substance. The step of preparing the peptide comprises:
Preparing one or more kinds of epitope peptides of the test substance and modified peptides thereof;
The method for preparing a measurement reagent according to claim 10 or 11, further comprising a step of selecting a peptide capable of binding to the antibody from the epitope peptide and the modified peptide.