JP2010266352A - Immune sensor for detecting organic nitrate-based explosive and method of manufacturing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immune sensor with a monoclonal antibody having selective binding capacity to a target organic nitrate-based explosive as the probe, and to provide a method of manufacturing the same. <P>SOLUTION: As an antibody having binding capacity to an organic nitrate-based explosive which does not exhibit immunogenicity, an antibody having cross reactivity to the organic nitrate-based explosives is selected from a group of antibodies to low-molecular compounds having a structure similar to a structure characteristic of the organic-nitrate explosive. The antibody having the cross reactivity is immobilized to a transducer to form the immune sensor. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an immunosensor for detecting explosives using a monoclonal antibody that specifically binds to explosives, particularly organic nitrate explosives, and a method for producing the same.

  As one of the organic nitrate-based explosives, 1,3,5,7-tetranitroperhydro-1,3,5,7-tetrazocine (1,3,5,7-tetranitroperhydro-1,3,5, 7-tetrazocine (hereinafter referred to as HMX) is known and used in military explosives and explosion terrorism. This compound has the structural formula shown below.

  For this reason, there is a need for a technique that can measure the explosives for explosives management and anti-terrorism measures. As a technique for detecting this explosive material, a high performance liquid chromatography method has been conventionally used (Non-Patent Document 1). However, due to the complexity of the operation, anyone could not measure easily. Moreover, the measurement cost per time was high, and the measurement time required several minutes or more. There is also a technique for measuring explosives using a mass spectrometer (Patent Document 1). However, there is a problem that a substance having a molecular weight similar to an explosive is erroneously measured as an explosive. For example, one component of a certain type of perfume has a molecular weight of about 300, so that it is erroneously detected as HMX. In addition, although an immunoassay method using an antigen-antibody reaction is considered promising as a simple measurement method, an antibody that shows an immune reaction against HMX could not be produced, and therefore an immunoassay method can be performed. could not. The reason for this is that no suitable antigen used for antibody production was found. That is, it was a problem to produce an immunosensor that can produce this antibody and detect HMX using this antibody.

JP 2008-262922 A

Industrial & Engineering Chemistry Research, Volume 45, 2006, 2954-2961 Chemistry Letter Volume 35, Number 10, 2006, pp. 1126-1127 Indian Journal of Chemistry, Volume 43B, (2004) 432-436

  However, the production of the antibody has the following problems.

  First, no suitable antigen was found. For the production of antibodies, an antigen that can be recognized by a mammal is necessary. However, since the molecular weight of HMX is as small as 296.2, the mammal could not recognize it as an antigen.

  On the other hand, as a method for recognizing an antigen having a small molecular weight, there is a method in which a protein is bound to the antigen to increase the apparent molecular weight (Non-patent Document 2; Chemistry Letter Volume 35, Number 10, 2006, 1126). 1127).

  However, since HMX does not have a pendant group for binding to a protein, the apparent antigen could not be enlarged using this method. For this reason, the antibody of HMX could not be produced.

  The present invention has been made in view of the above reasons. That is, an object of the present invention is to immunize a mammal using an HMX analog that can be bound to a protein as an antigenic determinant as an antigen, obtain a monoclonal antibody, and provide an immunosensor using this antibody. is there. More specifically, a mouse is immunized with 3,7-dinitro- [1,3,5,7] tetrazocan-1,5-diamine (hereinafter referred to as HMXAR) conjugated with a protein as an antigen, and is specific to HMX. And a method for immobilizing the monoclonal antibody on a working electrode functioning as an immunosensor.

  The immunosensor of the present invention comprises a compound having a structure represented by the following formula (I): 1,3,5,7-tetranitroperhydro-1,3,5,7-tetrazocine (1,3,5,7 An immunosensor for detecting explosives, comprising a monoclonal antibody having a binding ability to -tetranitroperhydro-1,3,5,7-tetrazocine) and a transducer having the monoclonal antibody immobilized on the surface thereof.

Formula (I)
The method for producing an immunosensor for detecting explosives according to the present invention comprises a compound having a structure represented by the following formula (I): 1,3,5,7-tetranitroperhydro-1,3,5,7-tetrazocine An immunosensor for detecting explosives comprising a monoclonal antibody having a binding ability to (1,3,5,7-tetranitroperhydro-1,3,5,7-tetrazocine) and a transducer having the monoclonal antibody immobilized on the surface thereof A manufacturing method of

Formula (I)
A monoclonal antibody against a low molecular weight compound having a structure similar to the characteristic structure in the compound represented by the formula (I), and having a cross-reactivity with the compound represented by the formula (I), other than human Producing a monoclonal antibody derived from a mammal of
Immobilizing the prepared monoclonal antibody on the transducer,
The step of producing a monoclonal antibody derived from a mammal other than human, at least,
Using the modified protein obtained by binding a low molecular weight compound having a structure similar to the characteristic structure of the compound represented by the formula (I) on a carrier protein as an immunogen, Immunizing;
After establishing immunity using the modified protein as an immunogen, collecting spleen cells from the immunized mammal other than the immunized mammal, and producing a monoclonal antibody-producing hybridoma cell from the collected spleen cells;
From the group of monoclonal antibodies produced by the produced antibody-producing hybridoma cells, an antigen-antibody reaction using a monoclonal antibody having cross-reactivity with the compound represented by the formula (I) as an antigen, and the compound represented by the formula (I) as an antigen Is a method for producing an immunosensor for detecting explosives, characterized by including a step of sorting.

  According to the present invention, since a monoclonal antibody capable of binding to a low molecular weight compound of an organic nitrate explosive that does not have a reactive group to be bound to a carrier protein can be created, immunity using the monoclonal antibody as a probe A sensor can be provided. As a result, the concentration of 1,3,5,7-tetranitroperhydro-1,3,5,7-tetrazocine, one of the explosives, in the solution can be accurately determined by an immune antibody reaction using the immunosensor. Can be measured.

It is sectional drawing of the immunosensor of embodiment of this invention. It is a graph which shows the evaluation result of the antibody obtained in Example 1 of this invention. It is a graph which shows the measurement result of Example 1 of this invention.

  The immunosensor for detecting explosives has at least a monoclonal antibody capable of binding to an organic nitrate-based explosive compound, and a transducer on which the monoclonal antibody is fixed on the surface.

  Below, the monoclonal antibody which has the binding ability with respect to the compound of the organic nitrate type explosive used for the immunosensor of this invention, and its manufacturing method are demonstrated in detail.

  As a means to create antibodies against low molecular weight organic compounds, if the target low molecular weight organic compound alone does not show immunogenicity, the modified carrier obtained by binding the target low molecular weight organic compound onto the carrier protein There is a technique of using a protein as an immunogen (Non-patent Document 2: Chemistry Letters, Vol. 35, No. 10, p. 1126-1127 (2006)).

Specifically, the low molecular weight organic compound has a reactive functional group such as an amino group (—NH ₂ ), a hydroxyl group (—OH), a sulfanyl group (—SH), or a carboxyl group (—COOH). In such a case, the reactive functional group can be used to link an organic compound having another reactive functional group covalently directly or via a crosslinking agent. The carrier protein has a three-dimensional structure composed of a peptide chain in which a plurality of amino acid residues are linked, but on its surface, an amino acid residue having a reactive functional group on the side chain There are several. For example, an amino group of a lysine residue or a sulfanyl group of a cysteine residue. Therefore, a low molecular weight organic compound having a reactive functional group using a reactive functional group on the side chain of an amino acid residue present on the surface of a carrier protein, having a three-dimensional structure. Can be combined. The modified carrier protein, which is modified on the surface by a low molecular weight organic compound, is a non-natural protein molecule and is recognized as a foreign substance that is different from the endogenous protein molecule of the mammal itself. Is frequent. In particular, a site that has been modified due to a low molecular weight organic compound is highly immunogenic. When the modified carrier protein surface modified with a low molecular weight organic compound exhibits immunogenicity, when the mammal is immunized with the modified carrier protein, the modified carrier Specific antibodies against proteins are created.

  There may be a plurality of sites (antigenic determinants) that exert immunogenicity on the surface of the modified carrier protein. In that case, multiple types of antibodies specific to each of the plurality of immunogenic sites (antigenic determinants) are created. Among the multiple types of antibodies that are specific to modified carriers and proteins that have been created, there are antibodies that use the low molecular weight organic compounds themselves used for their modification as sites that exhibit immunogenicity (antigenic determinants) The frequency of doing is high. By screening based on the binding ability to the low molecular weight organic compound itself used for modification, an antibody that uses the low molecular weight organic compound itself used for modification as an immunogenic site (antigenic determinant) It is possible to sort.

  However, if the mammal to be immunized already has an antibody that exhibits high cross-reactivity with the antigenic determinant present on the surface of the modified carrier protein, the antigenic determination that exhibits this cross-reactivity is determined. Creation of new antibodies against the group does not occur. That is, when an immune reaction to the modified carrier protein is possible using the high cross-reactivity exhibited by the antibody already held by the mammal to be immunized, the antigen determinant exhibiting this cross-reactivity, Creation of new antibodies does not occur.

  Furthermore, even when the modified site functions as a site that exhibits immunogenicity (antigenic determinant), the antibody specific for the antigenic determinant has a low molecular weight used for the modification. There are many cases where the binding ability to the organic compound itself is not high. In other words, using the technique of binding the target low molecular weight organic compound on the carrier protein and using the resulting modified carrier protein as an immunogen, it is specific to the low molecular weight organic compound itself used for modification. Whether a specific antibody can be created depends on the following factors. Specifically, it depends on the three-dimensional structure of the target low molecular weight organic compound itself, the combination with the carrier protein to be used, the binding form to the carrier protein, the selection of the modification site, and other factors. . With regard to such factors, the binding ability of the antibody to be created has an influence depending on whether or not an appropriate combination can be selected.

  On the other hand, when the compound of the organic nitrate explosive according to the present invention does not have a reactive functional group in the molecule, the target low molecular weight organic compound is bound on the carrier protein, The technique of using the obtained modified carrier protein as an immunogen cannot be applied. Of course, the organic nitrate explosive compound according to the present invention is a low molecular weight organic compound and does not exhibit immunogenicity alone.

  Therefore, in the present invention, a large number of antibodies against low molecular weight compounds having a structure similar to the characteristic structure in the target organic nitrate explosive compound are created, and the low molecular weight having a structure having the similarities is created. A method of selecting an antibody having cross-reactivity with the target organic nitrate explosive compound among a large number of antibodies against the compound is adopted.

  As described above, a monoclonal antibody can be obtained using a modified protein obtained by binding a low molecular weight compound having a structure similar to that of the target organic nitrate explosive compound on the carrier protein. A manufacturing method will be described below.

  First, non-human mammals using a modified protein formed by binding a low molecular weight compound having a structure similar to the characteristic structure and similarity of the target organic nitrate explosive compound on a carrier protein. Immunize. Subsequently, after establishing immunity using the modified protein as an immunogen, spleen cells are collected from the immunized mammal other than the immunized mammal, and monoclonal antibody-producing hybridoma cells are prepared from the collected spleen cells. Furthermore, from the group of monoclonal antibodies produced by the produced antibody-producing hybridoma cells, a monoclonal antibody having cross-reactivity with the compound of the target organic nitrate explosive, the compound represented by the above formula (I) as an antigen, Sort by antigen-antibody reaction. A monoclonal antibody can be produced by the above steps.

  The measurement method used for the immunosensor of the present invention is not particularly limited as long as it is a measurement method capable of detecting the presence / absence or quantitative change due to the antibody immune reaction between the monoclonal antibody possessed by the immunosensor and the target substance. For example, it can be used in an electrochemical measurement method such as a voltammetry method, a quartz crystal microbalance measurement method, a magnetic measurement method, or a measurement method using surface plasmon resonance. As the voltammetry method, it can be used for a square wave voltammetry method and the like.

  A known material can be used for the transducer used in the immunosensor, and a material suitable for a measurement method using the immunosensor can be appropriately selected. For example, in the case of using a measuring device using the surface plasmon resonance method or a measuring device using a crystal oscillator microbalance method, a material suitable for those methods can be selected.

  As a method for immobilizing the monoclonal antibody to the transducer, known methods such as a covalent bond method, an ion bond method, a physical adsorption method, and a crosslinking method can be used. An ion binding method or the like that can be immobilized under mild conditions that are not allowed is preferable. Alternatively, the surface of the transducer can be immobilized by preliminarily treating the surface with an immobilized protein and then contacting with a solution containing an antibody. As a specific immobilized protein, a protein containing electrochemically active amino acids such as tryptophan or tyrosine may be used, and streptavidin, protein A, protein G and the like are preferably used.

(First embodiment)
Hereinafter, as a compound of the target organic nitrate explosive compound, a compound having a structure represented by the following formula (I): 1,3,5,7-tetranitroperhydro-1,3,5,7-tetrazocine ( 1,3,5,7-tetranitroperhydro-1,3,5,7-tetrazocine (hereinafter also referred to as HMX) is taken as an example to describe the present invention more specifically.

Formula (I)
The compound represented by the formula (I) does not retain a reactive functional group and cannot be bound to a carrier protein by a crosslinking reaction or the like. Therefore, the reactive functional group has a structure similar to the characteristic structure of the compound represented by formula (I) having a reactive functional group for binding to the carrier protein, and is bound to the carrier protein. It is necessary to select a low molecular weight compound having an as an antigenic determinant. As such a compound, a compound having a structure represented by the following formula (II): 3,7-dinitro- [1,3,5,7] tetrazocan-1,5-diamine (3,7-dinitro- [1 , 3, 5, 7] tetrazokan-1,5-diamine :; hereinafter also referred to as HMXAR).

Formula (II)
In the present invention, a reactive functional group present on the surface of a carrier protein is utilized by utilizing an amino group (—NH ₂ ), which is a reactive functional group present in the molecule of a compound having the structure represented by formula (II). For example, by forming an amide bond (—CO—NH—) with a carboxyl group (—COOH), a form to be bonded to the surface of the carrier protein can be selected. In addition, the compound represented by the formula (II) can be bound to the surface of the carrier protein via a crosslinking agent. As the cross-linking agent, glutaraldehyde, bis [sulfosuccinimidyl] subleate, or the like can be used.

  At that time, the carrier protein binds the target low molecular weight organic compound onto the carrier protein, and uses the resulting modified carrier protein as an immunogen (Non-patent Document 2: Chemistry Letters, Vol. 35, No. 10, p. 1126-1127 (2006)), various carrier proteins already used can be used. As the carrier protein, bovine serum albumin (Bovine Serum Albumin: BSA), bovine thyroglobulin, keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin) and the like can be suitably used.

  When a target low molecular weight organic compound is bound on a carrier protein and the resulting modified carrier protein is used as an immunogen, a specific antibody against the modified carrier protein is used in an immunized mammal. Is created. In this case, since the carrier protein itself to be used generally has immunogenicity, in addition to the antibody specific to the modified site in the modified carrier protein, it is specific to the antigenic determinant of the carrier protein itself. New antibodies are also created.

  Considering this point, it is preferable that the mixing ratio of unmodified carrier / protein is low with respect to the modified carrier / protein used for immunization. Of course, it is more preferable to use a modified carrier protein that does not contain unmodified carrier protein.

  On the other hand, on the carrier protein to be used, there are generally a plurality of sites (modifiable sites) where the target low molecular weight organic compound can be bound and modified. Each of these modifiable sites has a reactive functional group, but generally there is a difference in reactivity. Therefore, the binding of the low molecular weight organic compound of interest proceeds preferentially from the highly reactive modifiable site, and the low molecular weight organic compound of interest is consumed. , The efficiency with which the low molecular weight organic compound of interest is bound tends to be further reduced. In order to achieve binding of the low molecular weight organic compound of interest to all of the plurality of modifiable sites present on the carrier protein, the amount of the low molecular weight organic compound of interest used in the reaction must be multiple. It is desirable to select a considerably excessive amount relative to the total number of modifiable sites.

  The immunization may be performed by, for example, administering a solution containing an effective amount of a modified carrier protein, to which the low molecular weight organic compound of the subject is bound, by injection to a mammal to be immunized. desirable. As the administration form by injection, subcutaneous injection, intradermal injection, intravenous injection, or intraperitoneal administration can be used. The solution is usually administered by subcutaneous injection. At this time, it is preferable to add various adjuvants to a solution containing an effective amount of the modified carrier protein. Usually, adjuvants that have been conventionally used for immunization can be used as adjuvants. Available adjuvants include Freund's complete adjuvant, water-in-oil-in-water emulsion, oil-in-water emulsion, liposome, aluminum hydroxide gel, and silica adjuvant. Freund's complete adjuvant is widely used and can be suitably used in the present invention. For example, during the first immunization (sensitization), it is preferable to use Freund's complete adjuvant as the adjuvant.

  In the immunization, booster immunization is performed when a predetermined period has elapsed after the first immunization (sensitization). In this additional immunization, it is preferable to add various adjuvants to a solution containing an effective amount of the modified carrier protein. For example, it is preferable to use Freund's complete adjuvant as the adjuvant at the time of booster immunization, but a considerable effect can be obtained by using Freund's incomplete adjuvant.

  After the first immunization operation (sensitization), the booster immunization to be performed is preferably performed a plurality of times. As for the interval, it is desirable to perform booster immunization at the time when the antibody concentration in the blood pressure is maximized due to the immune response to the previous immunization (sensitization) and the antibody concentration is decreasing. The number of days until the antibody concentration in blood pressure reaches the maximum after the last immunization (sensitization) usually depends on the metabolic rate of the immunogen used in the body. Thus, the booster interval depends on the type of immunogen used, the type of immunized animal of interest, and its health condition. When a small animal such as a mouse is used as an immunized animal, a form in which booster immunization is performed after the first immunization (sensitization), for example, 2 weeks, 4 weeks, 6 weeks, or 8 weeks can be selected.

  Scientifically, the type of mammal to be immunized is not limited, but is selected from mammals other than humans from an ethical viewpoint. In order to create hybridoma cells that produce monoclonal antibodies, mice, rats, goats, and the like can be selected as mammals other than humans that can be used for mammals to be immunized.

  As a mammal to be immunized, a mammal that already holds an antibody showing cross-reactivity with the modified carrier protein is not preferable. That is, it is generally preferable that the mammal to be immunized is an individual who has no acquired immunity. In view of the above requirements, it is preferable to use mammals grown after birth in an environment that is essentially free from exposure to various immunogenic substances. Alternatively, it is preferable to use a mammal having a short period until it grows to the extent that it can be subjected to immunization after giving birth. In consideration of these conditions, it is more preferable to use small pedigree mammals used for medical research. Specifically, mice, rats, rabbits and the like that are used for the creation of various novel antibodies are preferable, and in particular, mice or rats, and more preferably mice are used.

  Prior to immunization, the non-human mammal used as an immunized animal is used to produce the modified carrier protein, the immunogenicity of the carrier protein itself, and a specific antibody against the carrier protein It is desirable to investigate in advance the immunization conditions for which is created. A method in which a low molecular weight organic compound of interest is bound to the above carrier protein and the resulting modified carrier protein is used as an immunogen (Non-patent Document 2: Chemistry Letters, Vol. 35, No. 10, p. 1126-1127 (2006)), regarding the various carrier proteins already used, the above matters have already been described for mice, rats, rabbits, etc., which are used for the creation of various novel antibodies. It has been investigated and reports are available.

  In addition, a method of binding a target low molecular weight organic compound on the carrier protein and using the resulting modified carrier protein as an immunogen (Non-patent Document 2: Chemistry Letters, Vol. 35, No. 10, p.1126-1127 (2006)), referring to the successful examples already reported, the immunization of modified carrier proteins for mice, rats, rabbits, etc., which are used for the creation of various new antibodies It is also possible to estimate the effective amount with considerable accuracy.

  It is desirable to select the procedure of immunization for mice, rats, rabbits, etc., which are used to create various novel antibodies, in accordance with the procedures used in the successful examples already reported.

  When a mouse or rat is selected as the mammal to be immunized, the age at which the first immunization operation (sensitization) is performed is selected in consideration of the number of subsequent immunizations and the interval thereof. Specifically, it is necessary to verify that an antibody specific for the immunogen is present in the blood of the immunized animal after completion of multiple boosters. Therefore, it is preferable to select an age at which the first immunization operation (sensitization) is performed so that the immunized animal does not reach an age at which the ability to produce antibodies decreases at the time of completing multiple boosters. . When the period until the end of multiple boosts after the first immunization (sensitization) is selected to be about 8 weeks, in mice or rats, the age at which the first immunization (sensitization) is performed is: It is preferable to select the range of 10 to 15 weeks of age, and it is usually more preferable to select the range of about 12 weeks of age. It is known that mice or rats have the ability to produce sufficient antibodies when they reach about 12 weeks of age, and the ability to create new antibodies is the highest.

  The compound (HMXAR) having a structure represented by the formula (II) is a known compound, and its synthesis method has already been reported in the literature (Non-Patent Document 3; Indian Journal of Chemistry, Volume 43B, (2004) 432-436).

  Subsequently, the synthesized HMXAR is bound to a carrier protein. The carrier protein is preferably bovine serum albumin (BSA) or keyhole limpet hemocyanin. The solvent for dissolving HMXAR and carrier protein is not particularly limited, but dimethyl sulfoxide and boric acid can be preferably used. When boric acid is used, the pH is preferably neutral to slightly alkaline, and usually about 8.5. This is because the pH buffering function of boric acid is exhibited and HMXAR can be dissolved efficiently.

  Subsequently, glutaraldehyde is used to bind HMXAR and protein. Glutaraldehyde is used as a cross-linking agent and forms a cross-linked structure between the amino group of HMXAR and the amino group, sulfanyl group, etc. of protein. It is. Further, as another crosslinking method, bis [sulfosuccinimidyl] sublite (hereinafter referred to as BS3) may be used. The amount of the cross-linking agent glutaraldehyde is preferably selected in the range of 5 to 20 molecules per molecule of the compound having the structure represented by the formula (II).

  In the present invention, a modified carrier protein obtained by binding a target low molecular weight organic compound on a carrier protein is used as an immunogen to be used for the above-described immunization. Among the multiple types of antibodies specific to the modified carrier / protein newly created by immunization, high response to the low molecular weight organic compound itself that is not bound to the carrier / protein used. First of all, it is verified that an antibody showing sex is actually present.

  After completion of the above-mentioned last round of booster immunization, when a significant increase in the blood concentration of an antibody specific for the modified carrier protein used as an immunogen is found, blood is collected from the immunized animal and collected. Antiserum is prepared from the collected blood. Among the multiple antibodies specific to the modified carrier protein contained in the antiserum, high reactivity with the low molecular weight organic compound itself that is not bound to the carrier protein used It is verified that an antibody showing is actually present.

  That is, the presence or absence of a polyclonal antibody having the low molecular weight organic compound itself as an antigenic determinant is verified. An enzyme immunoassay (ELISA method) is preferably used as a means for verifying the presence of an antibody that specifically binds to a specific antigenic determinant in antisera containing multiple types of antibodies. The The enzyme immunoassay (ELISA method) uses a specific reactivity of an antibody to a specific antigenic determinant, and therefore has high selectivity, particularly for a specific antigenic determinant contained in the antiserum. When the antibody concentration is unknown, the antibody titer can be easily evaluated.

  In the present invention, in order to detect an antibody that is not bound to the carrier protein used and shows high reactivity with the low molecular weight organic compound itself, it is used in an enzyme immunoassay (ELISA method). As an antigen, another kind of modified carrier protein in which the low molecular weight organic compound is bound to the surface of another kind of carrier protein is used. Of course, it is necessary that the different type of carrier protein itself does not react with a plurality of types of antibodies specific to the modified carrier protein.

  The carrier protein used for the preparation of the immunogen and the other kind of carrier protein used for the preparation of the antigen used in the enzyme immunoassay (ELISA method) are preferably used for the preparation of the immunogen. It is preferable to select a combination of two different carrier proteins from the group of carrier proteins.

  In the carrier protein combination, antigenic determinants of the carrier protein itself are usually different, and multiple types of antibodies specific to the modified carrier protein react with the different carrier protein itself. The possibility of showing sex can be eliminated. Further, in the combination of the two different carrier proteins described above, it is very unlikely that the local structure (partial amino acid sequence) of the site capable of binding the low molecular weight organic compound substantially matches. Therefore, in the above-mentioned combination, another type of modified carrier protein in which the low molecular weight organic compound is bound to the surface of another type of carrier protein among a plurality of types of antibodies specific to the modified carrier protein of the immunogen. An antibody that shows binding ability to can be regarded as an antibody that binds to the low molecular weight organic compound itself.

  In particular, bovine serum albumin, which is widely used as a blocking protein, is selected as another type of carrier protein used for the production of antigens used in the enzyme immunoassay (ELISA method). As the carrier protein to be used, it is more preferable to select a general-purpose carrier protein other than bovine serum albumin, such as Keyhole Limpet Hemocyanin. When bovine serum albumin is selected as another type of carrier protein used for the production of a modified carrier / protein type antigen used in the enzyme immunoassay (ELISA method), the modified carrier / protein type antigen The phenomenon of non-selective binding of antibody molecules to the bovine serum albumin moiety is also eliminated. Further, the modified carrier protein type antigen using bovine serum albumin as a carrier protein can be immobilized on an ELISA plate at a high density.

  After verifying that the obtained antiserum contains an antibody having reactivity to the low molecular weight organic compound itself, which is used to produce an immunogen, the reactivity to the low molecular weight organic compound itself It is verified whether or not a polyclonal antibody having a cross-reactivity with a target organic nitric acid-based explosive compound, for example, a compound (HMX) having a structure represented by the formula (I) itself.

  In the present invention, the verification of the cross-reactivity of the antibody preferably uses a competitive reaction of the two antigens with the antibody.

  The target organic nitrate explosive compound, for example, the compound having the structure shown in formula (I) (HMX) itself is a low molecular weight organic compound, and when an antigen-antibody reaction with an antibody is performed, its binding is This is considered to be achieved by one of the complementarity determining sites of the antibody molecule. In addition, when a low molecular weight organic compound having a similar structure used for the production of a modified carrier / protein used for the above-described immunization is also subjected to an antigen-antibody reaction with an antibody, its binding is caused by an antibody molecule. It is thought that this is achieved by one of the complementarity determining sites.

  Therefore, when an antibody has cross-reactivity, the complementarity determining site of the antibody molecule involved in binding to a low molecular weight organic compound having a similar structure, which is used to produce a modified carrier protein of an immunogen Is highly likely to coincide with the complementarity-determining site of the antibody molecule involved in the binding of the target organic nitrate explosive compound, for example, the compound (HMX) represented by formula (I). In this case, when the target organic nitrate explosive compound, for example, the compound (HMX) represented by the formula (I), binds to the complementarity determining site of the antibody molecule, the modified carrier protein of the immunogen is produced. It inhibits the binding of low molecular weight organic compounds with similar structures that are utilized. By utilizing this phenomenon of competitive inhibition, it is possible to verify whether or not a specific complementarity determining site of the antibody molecule exhibits cross-reactivity.

  Specifically, the low molecular weight compound having the similar structure used for the verification of the reactivity to the low molecular weight organic compound itself having a similar structure used for the production of the modified carrier protein of the immunogen described above. Another type of modified carrier protein having an organic compound bound to the surface of another type of carrier protein is immobilized on an ELISA plate. On the other hand, the target organic nitrate explosive compound, for example, the compound (HMX) represented by the formula (I), together with the antiserum containing the polyclonal antibody in the reaction solution in which the antigen-antibody reaction is carried out in the ELISA method, Dissolve.

  When the competitive reaction proceeds, the cross-reactive antibody present in the reaction solution is a target organic nitrate explosive compound, for example, the compound (HMX) represented by formula (I) and an antigen antibody. As a result of the reaction, the amount of antibody molecules immobilized is reduced through an antigen-antibody reaction with the modified carrier / protein type antigen immobilized on the plate. Due to this competitive reaction, the decrease in the amount of the immobilized antibody through the antigen-antibody reaction with the modified carrier protein type antigen immobilized on the plate is determined by enzyme immunoassay (ELISA method). Detect by applying.

  By using this technique, the polyclonal antibody against the low molecular weight organic compound itself having a similar structure used in the production of the modified carrier protein of the above-mentioned immunogen contained in the antiserum, It can be verified that an organic nitric acid-based explosive compound, for example, an antibody having cross-reactivity with the compound (HMX) represented by the formula (I) is included.

  In other words, the antiserum verified as described above includes a polyclonal antibody having a binding ability to a target organic nitrate explosive compound, for example, the compound (HMX) represented by the formula (I).

  Antibody-producing cell groups of immunized animals that have been verified to produce cross-reactive antibodies with the target organic nitrate explosive compound, for example, the compound (HMX) represented by formula (I) The antibody-producing cell group and myeloma-derived cell line cells are fused to produce a group of hybridoma cells.

  Usually, a spleen cell group is prepared by removing the spleen from the immunized animal that has been verified as described above. This spleen cell group and myeloma-derived cell line cells are fused to produce a group of hybridoma cells.

  The myeloma-derived cell line used for the above-mentioned cell fusion needs to be compatible with the spleen cell derived from the immunized animal to be fused. The proliferation ability of hybridoma cells created by cell fusion depends on the myeloma-derived cell line used for cell fusion, and it is preferable to use a myeloma-derived cell line with excellent proliferation ability. .

  For example, when a mouse is selected as the immunized animal, the myeloma-derived cell line used for cell fusion is a mouse myeloma-derived cell line such as P3X63 Ag8.653, P3X63Ag8U, Sp2 / O Ag14, FO.1, S194 / 5. XX0 BU. 1 or the like is preferably used. In particular, the use of the cell line P3X63Ag8U has a high proliferation ability of the hybridoma cells to be created, and the antibody molecules produced by the hybridoma cells are all antibodies that have been properly assembled, and antibodies that have not been assembled yet More preferred because it does not contain molecular fragments.

  For example, when a rat is selected as the immunized animal, the myeloma-derived cell line used for cell fusion is the rat myeloma-derived cell line 210, RCY3. Ag1.2.3, YB2 / 0, etc. are mentioned.

  Examples of the cell fusion technique for creating the hybridoma cell include a polyethylene glycol method, a method using Sendai virus, and a method using an electric current. The polyethylene glycol method is more suitable for the present invention because it has low cytotoxicity, is easy to fuse, and particularly has high reproducibility. That is, in the present invention, among a group of hybridoma cells that produce a specific monoclonal antibody against a modified carrier protein of an immunogen, a target organic nitrate explosive compound, for example, a compound represented by the formula (I) It is necessary to select hybridoma cells that produce monoclonal antibodies having cross-reactivity to (HMX). Since the frequency of inclusion of hybridoma cells producing the above-mentioned monoclonal antibody having cross-reactivity among a group of hybridoma cells to be created is never high, the number of cell lines (number of populations) of a group of hybridoma cells to be screened Need to be larger. Therefore, it is preferable to select a cell fusion method with higher reproducibility, and the polyethylene glycol method meets the above-mentioned requirements.

  The created group of hybridoma cells are dispersed, dispensed into a microplate, and grown under known culture conditions appropriately selected according to the utilized myeloma cell line. For a group of hybridoma cell lines established by the culture described above, for the monoclonal antibody produced by each hybridoma cell line, the desired organic nitrate explosive compound, for example, a compound represented by formula (I) Whether the antibody is cross-reactive with (HMX) is verified.

  For a group of hybridoma cell lines established by culture, the culture supernatant of each hybridoma cell line is collected. The culture supernatant of each hybridoma cell line contains a monoclonal antibody produced by the cell line.

  Monoclonal antibodies contained in the culture supernatant of each hybridoma cell line have reactivity to low molecular weight organic compounds themselves with similar structures used for the production of modified carrier proteins used for immunization. First, it is verified whether it has or not.

  The verification is based on an enzyme immunoassay (ELISA method) in which a low molecular weight organic compound having a similar structure is bound to the surface of another type of carrier protein and another type of modified carrier protein is used as an antigen. Verification methods can be used. The specific measurement method is in principle the same as the verification regarding the reactivity of the polyclonal antibody contained in the antiserum.

  By this verification, a monoclonal antibody having reactivity to a low molecular weight organic compound itself having a similar structure, used for production of a modified carrier protein used for immunization, from a group of hybridoma cell lines. A cell line of hybridoma cells that produces is selected. With respect to the group of cell lines of hybridoma cells selected by this primary screening, the monoclonal antibody produced by the cell line of hybridoma cells is a compound of the target organic nitrate explosive, for example, a compound represented by formula (I) It is verified whether or not the antibody exhibits cross-reactivity to (HMX).

  The verification regarding the cross-reactivity can be performed by applying the same method in principle as the verification regarding the cross-reactivity of the polyclonal antibody contained in the antiserum.

  By verifying the cross-reactivity with the target organic nitrate explosive compound, for example, the compound (HMX) represented by the formula (I) (secondary screening), the target organic nitrate explosive compound, for example, A cell line of a hybridoma cell that produces a monoclonal antibody exhibiting cross-reactivity with the compound (HMX) represented by the formula (I) is selected. The type of monoclonal antibody selected depends on the antibody type specificity exhibited by the anti-Ig antibody used in the enzyme immunoassay (ELISA method).

  By using the cell line of the hybridoma cell selected by this secondary screening, a monoclonal antibody having binding ability to the target organic nitrate explosive compound, for example, the compound (HMX) represented by the formula (I), is obtained. Can be produced.

  The selected hybridoma cell line can be cultured in vitro, the culture supernatant can be collected, and the contained monoclonal antibody can be purified. Further, when the selected hybridoma cell line is inoculated into the abdominal cavity of a mammal other than a human used for immunization, it grows in the abdominal cavity and accumulates the monoclonal antibody produced in the ascites. Thereafter, the ascites can be collected and the contained monoclonal antibody can be purified.

  For purification of monoclonal antibodies contained in ascites or culture supernatant, for example, DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation method, PEG fractionation method, ethanol fractionation method, etc. Purification to a purity of Desirable purity is 95% or more, more preferably 98% or more. For example, a target organic nitrate explosive compound, for example, a monoclonal antibody having a binding ability to the compound (HMX) represented by the formula (I) is used to detect the organic nitrate explosive, or a concentration measuring device. When used for the above, if an antibody having reactivity to contaminants is mixed, the accuracy is reduced. Considering this point, it is desirable to carry out purification to the above purity.

  Next, the immunosensor of the present invention produced using the HMX monoclonal antibody obtained by the above description will be described in detail with reference to FIG.

  A carbon electrode 2 is manufactured on the insulating substrate 1, and the monoclonal antibody 3 obtained on the carbon electrode is fixed and connected to an electrochemical measurement device (not shown).

  The insulating substrate 1 is not particularly limited as long as it is glass, quartz, or transparent plastics, but glass having high strength and transparency is preferably used.

  The carbon of the carbon electrode 2 is formed on the insulating substrate 1 using carbon paper, graphite carbon, carbon nanotubes, carbon nanohorns, fullerenes, carbon powder, or the like as appropriate using a binder.

  For immobilization of the monoclonal antibody 3, the carbon electrode 2 may be preliminarily treated with the immobilized protein, and then a solution containing the antibody may be contacted. The immobilized protein has a function for immobilizing the monoclonal antibody 3 on the carbon electrode 2. As a specific immobilized protein, a protein containing electrochemically active amino acids such as tryptophan or tyrosine may be used, and streptavidin, protein A, protein G and the like are preferably used. Needless to say, if the above-mentioned electrochemically active amino acid is contained in the measurement target substance, these amino acids may not be contained in the immobilized protein. Moreover, even if it is not contained in both the substance to be measured and the immobilized protein, the solution used at the time of measurement may contain an electrochemically active amino acid. These selections can be appropriately changed depending on the substance to be measured. If necessary, the monoclonal antibody 3 may be immobilized on the carbon electrode 2 and then coated with a polymer material such as polyvinyl alcohol to improve the retention of the monoclonal antibody 3.

  Then, it mounts on the flexible board | substrate with which wiring was given, and it connects with an electrochemical measuring apparatus, and measures an immune reaction. The immune reaction is preferably carried out by the square wave voltammetry method with high measurement sensitivity. This measurement makes it possible to detect organic peroxides in the solution.

  The immunosensor for detecting explosives and the manufacturing method thereof in the first embodiment of the present invention are as follows.

  The immunosensor for detecting explosives in the first embodiment of the present invention is a compound having the structure represented by the formula (I): 1,3,5,7-tetranitroperhydro-1,3,5,7 An immunosensor having a monoclonal antibody capable of binding to tetrazocine (1,3,5,7-tetranitroperhydro-1,3,5,7-tetrazocine) and a transducer having the monoclonal antibody immobilized on the surface thereof.

  The monoclonal antibody having the ability to bind to the compound represented by the formula (I) is a monoclonal antibody against a low molecular weight compound having a structure similar to the characteristic structure of the compound represented by the formula (I). An antibody having cross-reactivity with the compound shown in (I) can be preferably used.

  Further, as a low molecular weight compound having a structure similar to the characteristic structure in the compound represented by the formula (I), a compound having the structure represented by the formula (II): 3,7-dinitro- [1, 3,5,7] tetrazocan-1,5-diamine (3,7-dinitro- [1,3,5,7] tetrazokan-1,5-diamine) can be used.

  In addition, as a monoclonal antibody capable of binding to the compound represented by the formula (I), a low molecular weight compound having a structure similar to the characteristic structure of the compound represented by the formula (I) is used on a carrier protein. A monoclonal antibody against the low molecular weight compound, which is created by immunizing a mammal other than a human with a modified protein bound to the immunogen, and is cross-reactive with the compound represented by the formula (I) Can be used. As mammals other than humans, mice and the like can be used.

  Further, in a modified protein obtained by binding the low molecular weight compound on a carrier protein, Keyhole Limpet Hemocyanin can be selected as the carrier protein.

  The transducer in the immunosensor can be used as an electrode that functions as at least a working electrode. The transducer functioning as a working electrode can be formed on an insulating substrate. A platinum electrode functioning as a counter electrode and a silver / silver chloride electrode functioning as a reference electrode can be further provided on the insulating substrate. A carbon electrode can be used as the electrode.

The method for producing an immunosensor for detecting explosives in the first embodiment of the present invention comprises a compound having a structure represented by the formula (I): 1,3,5,7-tetranitroperhydro-1,3, Explosives having a monoclonal antibody capable of binding to 5,7-tetrazocine (1,3,5,7-tetranitroperhydro-1,3,5,7-tetrazocine) and a transducer having the monoclonal antibody immobilized on the surface thereof It is a manufacturing method of the immunosensor for a detection. In this method, the following (i) and (ii)
It has the process of.
(I) a monoclonal antibody against a low molecular weight compound having a structure similar to the characteristic structure of the compound represented by the formula (I), and having a cross-reactivity to the compound represented by the formula (I) And a step of producing a monoclonal antibody derived from a mammal other than a human.
(Ii) A step of fixing the produced monoclonal antibody on the transducer.

The step of producing the monoclonal antibody of (i) can further include at least steps (i-1) to (i-3).
(I-1) Using the modified protein obtained by binding a low molecular weight compound having a structure similar to the characteristic structure and similarity of the compound represented by the formula (I) on a carrier protein as an immunogen, the human Immunizing a mammal other than.
(I-2) After establishing immunity using the modified protein as an immunogen, spleen cells are collected from the immunized mammal other than the immunized mammal, and monoclonal antibody-producing hybridoma cells are prepared from the collected spleen cells. Process.
(I-3) From the group of monoclonal antibodies produced by the produced antibody-producing hybridoma cells, a monoclonal antibody having cross-reactivity with the compound represented by the above formula (I) is used, and the compound represented by the above formula (I) is used as an antigen. And selecting by antigen-antibody reaction.

  As a low molecular weight compound having a structure similar to the characteristic structure of the compound represented by the formula (I), a compound having the structure represented by the formula (II): 3,7-dinitro- [1,3, 5,7] tetrazokan-1,5-diamine (3,7-dinitro- [1,3,5,7] tetrazokan-1,5-diamine) can be used. As the non-human mammal, a mouse or the like can be used.

  Further, in a modified protein obtained by binding the low molecular weight compound on a carrier protein, Keyhole Limpet Hemocyanin can be selected as the carrier protein.

(Deposit of hybridoma strain)
As a hybridoma cell producing a monoclonal antibody capable of binding to the compound (HMX) represented by the formula (I) according to the present invention, the mAb-H004 hybridoma cell line described in the Examples is an independent government based on the Budapest Treaty. It is deposited internationally at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Chuo 6-1, Higashi 1-chome, Tsukuba, Ibaraki, Japan, postal code 305-8656) (Receipt date; May 12, 2009, Receipt number; FERM ABP-11127).

  The above hybridoma cell line is a hybridoma cell line created and selected by the procedure disclosed in the examples described later.

  The antibody having the ability to bind to the compound related to the organic nitrate explosive according to the present invention and the production method thereof will be specifically described with reference to examples, but the technical scope of the present invention is not limited by the examples. Absent.

Example 1
The compound (HMX) represented by the above formula (I) was selected as the target organic nitrate explosive compound. 3,7-dinitro- [1,3,5,7] tetrazocan-1,5-diamine represented by the following formula (II) having a structure similar to that of the compound (HMX) represented by the formula (I) ( HMXAR) was selected as the antigenic determinant.

Formula (II)
<Synthesis of Compound having Structure Shown by Formula (II)>
The compound (HMXAR) represented by the formula (II) is a known compound, and its synthesis method has already been reported in the literature (Non-Patent Document 3: Indian Journal of Chemistry, Volume 43B, (2004) 432-436).

  According to the synthesis method described in the above-mentioned document, ether and lithium aluminum hydroxide are added to the compound (HMX) represented by formula (I) (manufactured by AccuStandard), reacted at 30-40 ° C. overnight, and water is added. The reaction was stopped and synthesized. When it measured using the liquid high performance chromatography (LC-MS) provided with the mass spectrometer, it confirmed that HMXAR was synthesize | combined.

<Preparation of Modified Carrier Protein for Immunogen Modified with Compound (HMXAR) Represented by Formula (II)>
Subsequently, 2 ml of DMSO solution containing 10 mg of keyhole limpet hemocyanin (manufactured by Sigma Aldrich Japan) was mixed with 1.8 ml of DMSO solution containing 3 mg of HMXAR, and then 16 μl of 25% glutaraldehyde was added. Then, the mixture was allowed to stand at room temperature for 1 hour, and then 0.1 ml of 1M glycine was added, followed by PBS dialysis. This solution is used as a GA crosslinking solution.

  Subsequently, 1.8 ml of DMSO solution containing 3 mg of HMXAR was mixed with 2 ml of DMSO solution containing 10 mg of keyhole limpet hemocyanin (manufactured by Sigma Aldrich Japan), and then 10 mg of BS3 (manufactured by Techno Chemical Co., Ltd.). ) Containing 0.5 ml of solution was added. Then, the mixture was allowed to stand at room temperature for 1 hour, and then 0.1 ml of 1M glycine was added, followed by PBS dialysis. This solution is designated as BS3 solution.

<Immunomanipulation using modified immunogen carrier protein>
Mice (SLC: C57BL / 6) were sensitized with the above-mentioned two modified immunogen carriers and proteins. The solution composition for injection per mouse was 0.075 ml of GA solution, 0.075 ml of BS3 solution, and 0.15 ml of Freund's complete adjuvant (Funakoshi). This solution was injected subcutaneously into 12-week-old mice (SLC: C57BL / 6). Injections were performed on the first day, day 22, day 35, and day 49. On day 66, blood was collected from the immunized mice.

<Verification of cross-reactivity of polyclonal antibody contained in the obtained antiserum>
Antiserum was obtained from the selected blood, and the competitive reaction of HMX against the polyclonal antibody in the serum was measured by ELISA.

  For measurement, HMXAR bound to bovine serum albumin (BSA) was prepared and immobilized on a measurement plate. The binding conditions were 1.8 ml DMSO solution containing 3 mg HMXAR, 0.1 ml solution containing 30 mg bovine serum dissolved in pure water, 0.9 ml 0.2 M carbonate buffer (pH 9.5), and 10 mg. DMSO solution containing 0.5 ml of BS3 was added, reacted at room temperature for 5 hours, and 0.6 ml of 1M glycine was added to stop the reaction. This obtained solution was used as a HMXAR-BSA solution.

  The HMXAR-BSA solution diluted 1000-fold was immobilized on a plate for ELISA measurement by the natural adsorption method, and 50 μl of HMX (AQ Standard Co.) solution having a final concentration of 100 ppm and anti-dilute diluted 100-fold. 50 μl of serum was put on a plate and reacted at room temperature for 2 hours.

  After completion of the reaction, the plate was washed 3 times with PBS, and 50 μl of an anti-mouse IgG-POD labeled antibody (Funakoshi) diluted 2000 times was added and allowed to react at room temperature for 1 hour. Subsequently, the plate was washed 4 times with PBS, 50 μl of ELISA peroxidase substrate (TMBZ, manufactured by Funakoshi) was added, and the reaction was stopped with 1N sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.). Then, the absorbance at 450 nm was measured.

<Preparation of hybridoma cells>
By this screening, spleen cells were prepared by extracting spleen cells from mice on the 66th day after the initial sensitization that was confirmed to produce a polyclonal antibody showing cross-reactivity with HMX.

The prepared mouse spleen cells and P3-X63-Ag8-U mouse myeloma cells in a ratio of 5: 1 cells in RPMI1640 medium (Invitrogen), 50% polyethylene glycol (sum) In the presence of Kogyo Pharmaceutical Co., Ltd.), the cells were mixed at 37 ° C. for 2 minutes to cause cell fusion. After the cell fusion treatment, the obtained hybridoma cells are suspended in a HAT medium (20% fetal calf serum) and then dispensed into a microplate, and half of the HAT medium is renewed once every four days. The medium was replaced with HT medium (10% fetal calf serum). The culture conditions were 37 ° C. and 5% CO ₂ .

  The HAT medium is obtained by adding an appropriate amount of HAT supplement (manufactured by Invitrogen) to RPMI1640 medium. In this example, 20 μl of HAT supplement was added per 1 ml of RPMI 1640 medium.

  The HT medium is obtained by adding an appropriate amount of HT supplement (manufactured by Invitrogen) to RPMI 1640 medium. In this example, 20 μl of HT supplement was added per 1 ml of RPMI 1640 medium.

  Under the above culture conditions, the hybridoma cells dispensed on the microplate were cultured for 2 weeks to establish hybridoma cell lines, respectively. After culturing for 2 weeks, the presence or absence of the antibody in the culture supernatant was confirmed by the ELISA method in the same manner as the confirmation of the polyclonal antibody in the serum.

  As a result, it was confirmed that the four plates exhibited a competitive reaction against 100 ppm of HMX, and it was confirmed that the HMX monoclonal antibody was obtained. FIG. 2 shows data obtained by measuring the absorbance of monoclonal antibodies obtained from four strains obtained from hybridomas. Since the absorbance of HMX decreases with respect to the absorbance of the buffer (denoted as Borate Buffer in the graph), it reflects that these antibodies are bound to HMX. The antibody used for the immunosensor was mAb-H003.

<Manufacture of immunosensors>
Subsequently, a manufacturing method of the immunosensor will be described below.

  First, the carbon electrode was manufactured as follows.

  A glass substrate of 10 × 10 × 0.7 mm was prepared, and ultrasonic cleaning was performed for 5 minutes in a nitric acid-hydrogen peroxide solution. Next, carbon paper of the same size (Toray Industries, TGP-H-120) was bonded to a glass substrate with a silicone resin (SE9186 manufactured by Toray Dow Corning Silicone) to produce a carbon electrode. .

  Subsequently, the above electrode was immersed in 0.2 ml of a solution containing 1 mg of mAb-H003 antibody for 1 hour. Then, it is immersed in 3 mM 1,3-diaminobenzene (manufactured by Aldrich Co., Ltd., USA, containing pH 7.4 phosphate buffer and 0.1 M sodium chloride), and 0 to 0.8 V is 2 mV. The antibody of mAb-H003 was immobilized by sweeping 100 times at / s and applying at 0.65 V for 5 hours. Subsequently, it was immersed in 1 w / v% polyvinyl alcohol for 1 hour to coat the surface of the antibody with polyvinyl alcohol.

  Subsequently, it was mounted on a wired flexible board. Subsequently, the wires were connected by wire bonding, and the connected portions were sealed with silicone resin.

<Measurement of HMX by square wave voltammentary method>
Using this carbon electrode as a working electrode, the concentration of HMX was measured by the square wave voltammetry method using a glass reference electrode and a platinum counter electrode. The measurement conditions are a 0.1-1.2V sweep range, a pulse potential of 40 mV, a frequency of 4 Hz, and a step potential of 10 mV.

  First, these electrodes were immersed in a phosphate buffer (0.01 M, pH 6.8) containing no HMX and left for several minutes. And it swept once on the above-mentioned conditions, and the base current was measured.

  Subsequently, HMX as a final concentration was added to the phosphate buffer so as to have a final concentration of 300 ppm, and it was swept once under the conditions described above, and the response current was measured. The results are shown in FIG.

  As a result, it was found that the HMX immunosensor according to the present invention produced a clear response current with respect to HMX, and was able to detect 300 ppm of HMX. Moreover, it was shown that only the HMX can be accurately detected without showing any response current for perfume A and perfume B as similar substances of HMX. The response time was about 10 seconds.

  On the other hand, as a comparative example, surface plasmon resonance method (described as SPR method in the graph), crystal oscillator microbalance method (described as QCM method in the graph), high-performance liquid chromatographic method (described as HPLC method), and ionization Mass spectrometers (mass spectrometry methods) equipped with an ion source using corona discharge were compared. The measurement of the SPR method and the QCM method was performed according to a normal measurement method using an indirect competition method. The HPLP method and the mass spectrometry method were performed in accordance with a normal measurement method.

  As a result, in the SPR method, the QCM method, and the HPLC method, 300 ppm of HMX was detected as 305 ppm, 303 ppm, and 300 ppm, and there was no reaction with perfume A and perfume B. The response time was about 10 seconds for the SPR method and the QCM method, about 3 minutes for the HPLC method, and about 6 seconds for the mass spectrometry method. Further, it was shown that the mass spectrometry method detected as 280 ppm, and that perfume A and perfume B were erroneously detected as HMX. The results of these measurement methods are summarized in Table 2.

  Therefore, it was found that the HMX sensor of the present invention has a high measurement accuracy and is an HMX detection method that does not erroneously detect.

  The immunosensor having a transducer in which a monoclonal antibody having binding ability is fixed to the organic nitrate explosive according to the present invention can be used for detection of the organic nitrate explosive.

1 Insulating substrate 2 Carbon electrode 3 Monoclonal antibody

  Regarding the compound (HMXAR) represented by the formula (I) according to the present invention, the hybridoma cell line of mAb-H004 disclosed in the Examples is based on the Budapest Treaty. Kokusai is deposited internationally at Tsukuba, Higashi 1-chome, Central, Ibaraki Prefecture, Japan (postal code 305-8565) (Receipt date; May 12, 2009, Receipt number FERM ABP-11127).

Claims (19)

Compounds having the structure shown in the following formula (I): 1,3,5,7-tetranitroperhydro-1,3,5,7-tetrazocine (1,3,5,7-tetranitroperhydro-1,3, An immunosensor for detecting explosives, comprising a monoclonal antibody having a binding ability to 5,7-tetrazocine) and a transducer having the monoclonal antibody immobilized on the surface thereof.

Formula (I) A monoclonal antibody having binding ability to the compound represented by the formula (I) is:
A monoclonal antibody against a low molecular weight compound having a structure similar to the characteristic structure of the compound represented by the formula (I), and having a cross-reactivity to the compound represented by the formula (I) The immunosensor for detecting explosives according to claim 1. A low molecular weight compound having a structure having a characteristic structure and similarity in the compound represented by the formula (I),
Compounds having the structure represented by the following formula (II): 3,7-dinitro- [1,3,5,7] tetrazocan-1,5-diamine (3,7-dinitro- [1,3,5,7 ] The immunosensor for detecting explosives according to claim 2, characterized in that it is tetrazokan-1,5-diamine).

Formula (II) A monoclonal antibody having binding ability to the compound represented by the formula (I) is:
Immunize mammals other than humans with a modified protein formed by binding a low molecular weight compound having a structure similar to the characteristic structure of the compound represented by the formula (I) onto a carrier protein. The explosion according to claim 2 or 3, wherein the explosion is a monoclonal antibody against the low molecular weight compound and is an antibody having cross-reactivity with the compound represented by the formula (I). Immunosensor for detecting objects. The immunosensor for detecting explosives according to claim 4, wherein the mammal other than a human is a mouse. 6. The modified protein obtained by binding the low molecular weight compound on a carrier protein, wherein keyhole limpet hemocyanin is selected as the carrier protein. Immunosensor for detection of explosives. A monoclonal antibody having binding ability to the compound represented by the formula (I) is:
The immunosensor for detecting explosives according to any one of claims 1 to 6, which is a monoclonal antibody produced by a hybridoma cell line (FERM ABP-11127).   The immunosensor for detecting explosives according to any one of claims 1 to 7, wherein the transducer is an electrode that functions as at least a working electrode.   The immunosensor for detecting explosives according to claim 8, wherein the transducer functioning as the working electrode is formed on an insulating substrate.   The immunosensor for detecting explosives according to claim 9, further comprising a platinum electrode functioning as a counter electrode and a silver / silver chloride electrode functioning as a reference electrode on the insulating substrate.   The immunosensor for detecting an explosive according to any one of claims 8 to 10, wherein the electrode is a carbon electrode.   The immunosensor for detecting explosives according to any one of claims 8 to 11, wherein a square wave voltammetry method is used.   The immunosensor for detecting explosives according to any one of claims 1 to 7, wherein the transducer is a transducer of a measuring apparatus using a surface plasmon resonance method.   The immunosensor for detecting explosives according to any one of claims 1 to 7, wherein the transducer is a transducer of a measuring apparatus using a crystal oscillator microbalance system. Compounds having the structure shown in the following formula (I): 1,3,5,7-tetranitroperhydro-1,3,5,7-tetrazocine (1,3,5,7-tetranitroperhydro-1,3, 5,7-tetrazocine), a method for producing an immunosensor for detecting explosives, comprising a monoclonal antibody having a binding ability, and a transducer having the monoclonal antibody immobilized on its surface,

Formula (I)
A monoclonal antibody against a low molecular weight compound having a structure similar to the characteristic structure in the compound represented by the formula (I), and having a cross-reactivity with the compound represented by the formula (I), other than human Producing a monoclonal antibody derived from a mammal of
Immobilizing the prepared monoclonal antibody on the transducer,
The step of producing a monoclonal antibody derived from a mammal other than human, at least,
Using the modified protein obtained by binding a low molecular weight compound having a structure similar to the characteristic structure of the compound represented by the formula (I) on a carrier protein as an immunogen, Immunizing;
After establishing immunity using the modified protein as an immunogen, collecting spleen cells from the immunized mammal other than the immunized mammal, and producing a monoclonal antibody-producing hybridoma cell from the collected spleen cells;
From the group of monoclonal antibodies produced by the produced antibody-producing hybridoma cells, an antigen-antibody reaction using a monoclonal antibody having cross-reactivity with the compound represented by the formula (I) as an antigen, and the compound represented by the formula (I) as an antigen The manufacturing method of the immunosensor for explosives detection characterized by including the process of sorting by. A low molecular weight compound having a structure having a characteristic structure and similarity in the compound represented by the formula (I),
Compounds having the structure represented by the following formula (II): 3,7-dinitro- [1,3,5,7] tetrazocan-1,5-diamine (3,7-dinitro- [1,3,5,7 ] The method for producing an immunosensor for detecting explosives according to claim 15, wherein tetrazokan-1,5-diamine).

Formula (II) The method for producing an immunosensor for detecting explosives according to claim 15 or 16, wherein the mammal other than a human is a mouse. The modified protein formed by binding the low molecular weight compound on a carrier protein, wherein as the carrier protein, keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin) is selected. A method for producing an immunosensor for detecting an explosive according to claim 1. As an antibody capable of binding to the compound represented by the formula (I),
The method for producing an immunosensor for detecting an explosive according to any one of claims 15 to 18, wherein a monoclonal antibody produced by a hybridoma cell line (FERM ABP-11127) is selected.

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