JP4288750B2 - Immunological measurement method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an immunoassay method for measuring a hapten compound.
[0002]
[Prior art]
As an enzyme immunoassay method for measuring a hapten compound, an enzyme immunoassay method (3rd edition) (edited by Eiji Ishikawa, Medical School, 1987) or the like already known is used. It can be measured by a method using a competitive reaction using a matrix in which an antibody is adsorbed on an insoluble carrier. However, such an enzyme immunological reaction is very useful as a method for measuring a hapten compound, but the incubation time of an antigen and an antibody is usually several hours, and it often takes a long time. It was. Therefore, as a method for promoting the antigen-antibody reaction, a method in which a water-soluble polymer such as polyethylene glycol, polyvinyl pyrrolidone, or polyglucosyl methacrylate is present in the antigen-antibody reaction solution is known.
However, since such water-soluble compounds have the effect of precipitating proteins as used as a non-selective fractionation method for proteins, these water-soluble polymer compounds are Although it has the effect of promoting the antigen-antibody reaction, there has been a problem of promoting non-specific adsorption reaction and the like.
[0003]
A lipid peroxidation secondary product (hereinafter sometimes abbreviated as “lipid peroxide”) is a product produced by peroxidation of a highly unsaturated fatty acid residue and further peroxidative decomposition. It is known that oxides are involved in various pathologies in living organisms and that their value is reduced in foods. As such lipid peroxides, substances such as malondialdehyde (abbreviation: MDA), 4-hydroxy-2-nonenal (abbreviation: HNE) or acrolein (abbreviation: ACR) are known. Since these substances are very reactive, they react with substances that can be modified in vivo (substances to be modified) to form lipid peroxides. Examples of such modified substances include amino acids, peptides, proteins, nucleic acids, and the like, and particularly proteins. When lipid peroxides are added to proteins, protein degradation occurs, and these proteins are described above. It contributes to pathological conditions in the living body or deterioration in food. Advanced glycation end product (hereinafter sometimes abbreviated as “AGE”) is a product that is finally produced by non-enzymatic reaction of aldehyde group of reducing sugar with amino acid, peptide or protein. (1) Fluorescence, (2) Browning, (3) Intramolecular / intermolecular crosslinking and (4) Biological recognition It has been reported that a substance having a high reactivity reacts with a substance that can be modified in a living body (modified substance) to form an AGE adduct. Examples of such substances include methylglyoxal (abbreviation: MG), and examples of the substance to be modified include proteins. When AGE is added to proteins, protein degradation occurs, and these proteins and in vivo The relationship with pathological conditions has been studied.
[0004]
As a result of diligent research on the modification reaction of such proteins, the present inventors have found that, for example, carbonyl compound-protein complexes represented by these include dihydropyridine structures, 2-hydroxy-5- (pentyltetrahydrofuran-4- Il) It was reported that a protein complex having histidine (Michael adduct structure), formyl dehydropiperidine structure, argypirimidine structure, etc. was generated, and a monoclonal antibody specific to the structure was developed [(1) FEBSLetters 359 Volume (1995) pp. 189-191, (2) Proc. Nacl. Acad. Sci. USA 91 (1994) 2616-2620, (3) Japan Oil Chemists' Society 47 (1998) 29-37, (4) Japan Agricultural Chemical Society 71 (1997) 311 Such).
[0005]
Therefore, these lipid peroxide addition compounds or AGE addition compounds can be used as an indicator of oxidation or saccharification stress in the living body, and deterioration in foods, so it is very difficult to measure their amounts. Is important to.
[0006]
As a method for measuring such a substance, JP-A-9-72907 discloses a method for measuring a carbonyl compound. However, the technique described above is a polyclonal antibody, and is not a method for measuring a modified structure of a biomolecule derived from a specific carbonyl compound with high specificity. For example, oxidative and glycation stress is strictly measured. It was not suitable for evaluation. Further, the disclosed technique is not intended to efficiently measure an antigen-antibody reaction in a short time. Furthermore, as an immunological measuring method, a sandwich type measuring method is known, but it is not suitable in principle for measuring a hapten-protein complex.
Therefore, it has been desired to develop a means for measuring a hapten compound simply, with high specificity and in a short time.
[0007]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for measuring a hapten compound simply, with high specificity, and in a short time. In particular, a carbonyl compound-protein complex, more specifically, a compound having a formyl dehydropiperidine structure or a compound having a 2-hydroxy-5- (pentyltetrahydrofuran-4-yl) histidine structure, etc. is easily and highly specific, And it is providing the method for measuring in a short time.
[0008]
[Means for Solving the Problems]
As a result of investigations in view of the above problems, the present inventors have found an immunological measurement method for measuring a hapten compound simply, with high specificity and in a short time, and completed the present invention. That is, the present invention includes the following [1] to [4].
[1] In an immunological measurement method for measuring a hapten compound,
By using an immune reaction container in which the hapten-protein complex is physically adsorbed by bringing the hapten-protein complex into contact with an insoluble carrier and precoated,
(1) The immune reaction container is incubated in this immune reaction container in the order of the monoclonal antibody to the sample and the hapten, or a reaction solution in which the monoclonal antibody to the sample and the hapten is reacted in another container in advance. Reacting a monoclonal antibody against the hapten-protein complex pre-coated on to form an antigen-antibody reaction complex;
(2) The monoclonal antibody reacted with the hapten-protein complex precoated on the immune reaction container by incubating the antibody labeled antibody reacting with the monoclonal antibody reacting with the hapten as an antigen and placing it in the immune reaction container. Reacting and binding the enzyme-labeled antibody to the antibody
And a method for immunologically measuring a hapten compound.
[0009]
[2] The immunological assay method described above, wherein the protein constituting the hapten-protein complex is casein or skim milk.
[0010]
[3] The immunoassay method described above, wherein the hapten compound to be measured is a carbonyl compound-protein complex.
[0011]
[4] The hapten compound to be measured is represented by the following formula (I)
[0012]
[Chemical 3]
[0013]
(Wherein R ¹ , R ² Is a protein residue, peptide residue, or R ¹ = R ² = H is represented. )
Or a compound having a formyldehydropiperidine structure represented by the following formula (II):
[0014]
[Formula 4]
[0015]
(Wherein R ¹ , R ² Is a protein residue, peptide residue, or R ¹ = R ² = H is represented. )
The above-mentioned immunological measuring method which is a compound which has 2-hydroxy-5-pentyltetrahydrofuran-4-yl) histidine structure shown by these.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the hapten compound means a compound which is not immunogenic but has reactivity with an antibody, and examples thereof include simple haptens and complex haptens.
Examples of the protein of the hapten-protein complex used in the present invention include various commonly used proteins. Specifically, for example, α-, β- or γ-casein (CAS), skim milk (SML), bovine serum albumin (BSA), ovalbumin (OVA), gelatin, limpet hemocyanin and the like can be mentioned. Preferably, α-, β- or γ-casein and skim milk are used.
The hapten-protein complex used in the present invention can be prepared by a general method capable of binding the above-described low molecular weight compound hapten to a protein. For example, when a compound capable of giving an epitope structure derived from a hapten compound (here, epitope means an antigenic determinant) on a protein reacts only by mixing itself with the protein, For example, low molecular weight compounds that form various aldehydes and Michael adducts can be obtained by reacting the low molecular weight compound and protein in an appropriate buffer solution or ion-exchanged water that does not interfere with the target low molecular weight compound and protein. Just do it. Examples of such a buffer include buffer solutions such as phosphate and carbonate.
When the low molecular weight compound is not water-soluble, it can be used by dissolving in a small amount of an organic solvent such as ethanol, methanol, dimethylformamide, dimethyl sulfoxide, or dioxane.
The pH of the buffer solution is not particularly limited as long as the reaction proceeds, but it is pH 6 to 11, preferably pH 7 to 9.
The concentration of the buffer is not particularly limited as long as the reaction proceeds, but it is 1 mM to 0.5 M, preferably 10 to 100 mM.
[0017]
In addition, when the low molecular weight compound itself is a hapten compound and the hapten compound does not react with a protein by itself, for example, a functional group reactive to the hapten compound is introduced to prepare a hapten-protein complex. A hapten may be bound to an amino group or a carboxyl group which is a reactive group of a protein, and these methods are already known. These reaction processes can be detected by the reduction rate of amino acids in the protein.
The reduction rate of amino acids can be determined, for example, by measuring amino acids in a sample before and after the reaction using an automatic amino acid analyzer. The reduction rate of amino acids varies depending on the modified product used, but it is desirable that the reduction rate of amino acids involved in the modification is reduced by 10% or more, preferably 20% or more.
[0018]
The reaction conditions that give such an amino acid reduction rate are, for example, preferably 4 to 45 ° C. for 1 to 96 hours, and more preferably 10 to 40 ° C. for 2 to 40 hours.
As described above, the hapten-protein complex of the present invention can be obtained by reacting a hapten compound and a protein.
[0019]
The immunological measurement method according to the present invention specifically reacts with a hapten compound, and since the uniformity of the antigen-antibody reaction is important, a monoclonal antibody against the hapten compound to be measured is necessary. A monoclonal antibody against the target hapten compound may be prepared by a known method. The monoclonal antibody against the hapten compound and the sample containing the measurement target are simultaneously mixed, or after the sample containing the monoclonal antibody against the hapten compound and the measurement target is mixed in advance, the hapten-protein complex as the measurement target is adsorbed. Reaction with an insoluble carrier, separation of BF, reaction with an enzyme-labeled antibody that reacts with a monoclonal antibody against a hapten as an antigen, and the concentration of the enzyme-labeled substance immobilized on the carrier is determined based on the enzyme-substrate reaction. This can be done by optical measurement.
[0020]
Here, the hapten-protein complex adsorption-insoluble carrier can be prepared by bringing the hapten-protein complex into contact with the insoluble carrier and physically adsorbing it. The physical adsorption method can be prepared, for example, by adding an appropriate amount of a hapten-protein complex solution to an insoluble carrier and allowing to stand, and physically adsorbing the hapten-protein complex to the insoluble carrier. The concentration of the hapten protein complex solution used for adsorbing such a hapten-protein complex to an insoluble carrier may be 1 ng / ml or more, but it is uneconomical to increase the antigen concentration more than necessary. Preferably, it is 0.1 μg / ml to 200 μg / ml. As the buffer solution, a buffer solution of phosphate, tris or carbonate can be used.
The pH of the buffer solution is not particularly limited as long as the adsorption reaction proceeds, but it is pH 6 to 11, preferably pH 7 to 9.
The concentration of the buffer is not particularly limited as long as the reaction proceeds, but it is 1 mM to 0.5 M, preferably 10 to 100 mM. The temperature at the time of adsorption is 0 to 55 ° C, preferably 4 to 40 ° C. The time required for adsorption may be several minutes.
[0021]
Such a hapten-protein complex adsorption insoluble carrier may be prepared in advance, or a hapten-protein complex adsorption insoluble carrier may be prepared immediately before the measurement. In addition, blocking treatment for suppressing non-specific reaction may be performed, but it is not particularly necessary because the hapten-protein complex simultaneously serves as a blocking agent.
[0022]
There are the following two methods for reacting the sample and the monoclonal antibody solution with the hapten-protein complex adsorption insoluble carrier.
The first method is a method in which a sample and a monoclonal antibody solution are dispensed and reacted with an insoluble carrier adsorbed to a hapten-protein complex.
The second method is a method in which a sample and a monoclonal antibody solution are reacted in a separate container in advance, and then this mixed solution is dispensed and reacted with a hapten-protein complex adsorption insoluble carrier.
Either the first method or the second method may be used.
The monoclonal antibody solution to be used may be any solution in which the monoclonal antibody is dissolved in a buffer solution that does not interfere with the antigen-antibody reaction. For example, a phosphate, Tris or carbonate buffer solution can be used. The pH of the buffer solution is not particularly limited as long as the antigen-antibody reaction proceeds, but it is pH 6 to 11, preferably pH 7 to 9. The concentration of the buffer is not particularly limited as long as the reaction proceeds, but it is 1 mM to 0.5 M, preferably 10 to 100 mM. The monoclonal antibody solution to be used preferably contains 0.5 to 10 mg / ml protein, and is not particularly limited as long as it is a protein that does not participate in the reaction, but bovine serum albumin, ovalbumin, casein, milk Examples include proteins. Furthermore, saccharides and surfactants may be included. Examples of saccharides include sucrose, glucose, trehalose and the like. The concentration of the surfactant is 0.1 to 10%, and the surfactant is polyoxyethylene sorbitan monolaurate ( Examples thereof include Tween 20 manufactured by Wako Pure Chemical Industries, Ltd., and may be contained at a concentration of 0.01 to 5%. The concentration of the monoclonal antibody to be used varies depending on each measurement system, hapten compound, etc., but may be any concentration as long as a standard curve in the target measurement range can be obtained, and is determined by performing a test. If it is too much, a target standard curve cannot be obtained, and 0.01 to 10 μg / ml is appropriate.
[0023]
Moreover, a carbonyl compound-protein complex is mentioned as said hapten compound. Specific examples include acetaldehyde-protein complex, 4-hydroxy-2-nonenal-protein complex, acrolein-protein complex, malondialdehyde-protein complex, methylglyoxal-protein complex, and the like. Furthermore, the hapten compounds to be measured include the following formula (I)
[0024]
[Chemical formula 5]
[0025]
Hormi indicated by Le Examples thereof include compounds having a dehydropiperidine structure. Where R in formula I ¹ , R ² Is a protein residue or peptide residue or R that binds to a lysine residue in the protein. ¹ = R ² = H is represented. R ¹ = R ² In the case of = H, that is, an amino acid residue of a lysine residue. In other words, the compound represented by the formula (I) is a protein, a peptide containing a lysine group. The It is a reaction product of amino acid of tide or lysine and acrolein. Or as a hapten compound to be further measured, the formula (II)
[0026]
[Chemical 6]
[0027]
The compound which has 2-hydroxy-5- (pentyltetrahydrofuran-4-yl) histidine structure shown by these is mentioned. Where R in formula I ¹ , R ² Is a protein residue or peptide residue or R that binds to a histidine residue in the protein. ¹ = R ² = H is represented. R ¹ = R ² In the case of = H, that is, an amino acid residue of a histidine residue. In other words, the compound represented by the formula (II) is a protein containing a histidine group. The It is a reaction product of amino acid of tide or histidine and 4-hydroxy-2-nonenal.
[0028]
The sample, the monoclonal antibody solution, and the hapten-protein complex adsorbing insoluble carrier are reacted, and then BF separation is performed. It is sufficient that the hapten-protein complex adsorbing insoluble carrier can be washed. Although it will not specifically limit if it is a buffer solution which does not carry out, the buffer solution containing surfactant is common, pH 6-9 containing 0.01-5% polyoxyethylene sorbitan monolauric acid ester (for example, Tween20). 10 to 100 mM phosphate buffer or Tris buffer is used.
[0029]
The enzyme-labeled antibody that reacts with the monoclonal antibody that reacts with the hapten compound as an antigen is not particularly limited as long as it reacts with the monoclonal antibody to be used. However, since the monoclonal antibody is generally derived from a mouse, it is a mouse immunoglobulin. It is an antibody against. The enzyme to be labeled is not limited as long as it is an enzyme used in a general enzyme immunoassay, but peroxidase, alkaline phosphatase, β-galactosidase and the like are suitable. Such an enzyme-labeled antibody may be produced by itself, but a commercially available one can be easily obtained. As the reaction solution, the obtained enzyme-labeled antibody solution can be appropriately diluted and used, as long as it is dissolved in a buffer solution that does not interfere with the antigen-antibody reaction and does not damage the enzyme. For example, a buffer solution such as phosphate, tris or carbonate can be used.
The pH of the buffer solution is not particularly limited as long as the antigen-antibody reaction proceeds, but it is pH 6 to 11, preferably pH 7 to 9. The concentration of the buffer is not particularly limited as long as the reaction proceeds, but it is 1 mM to 0.5 M, preferably 10 to 100 mM. The enzyme-labeled antibody reaction solution may contain 0.5 to 10 mg / ml of protein and is not particularly limited as long as it is a protein that does not participate in the reaction, but bovine serum albumin, ovalbumin, casein, milk protein Etc. are desirable. Furthermore, saccharides and surfactants may be included. Examples of the saccharides include sucrose, glucose, trehalose and the like. The concentration of the surfactant is 0.1 to 10%, and the surfactant is polyoxyethylene sorbitan monolaurate (Tween 20). Etc.) and may be contained at a concentration of 0.01 to 5%.
[0030]
As temperature conditions for performing the antigen-antibody reaction, the temperature is 1 to 55 ° C, preferably 4 to 40 ° C. If the immunological measurement method according to the present invention is used, the time required for measurement may be very short.
[0031]
After the BF separation, a color developing solution used for the enzyme reaction derived from the enzyme-labeled antibody used is added to cause color development, and the reaction is stopped with a reaction stop solution. Finally, the objective can be achieved by measuring the absorbance of the color developing solution. In that case, it is obtained using the standard curve and sample obtained by performing the same operation using a standard solution with a known concentration. By comparing the obtained absorbances, the concentration of the hapten compound in the sample can be measured. What is necessary is just to utilize what is already marketed for the apparatus used for these reaction liquids and a measurement.
[0032]
【The invention's effect】
As is clear from the above explanation, the hapten compound can be measured easily, with high specificity and in a short time by using the immunological measurement method of the present invention. Therefore, it is useful in clinical diagnosis, analysis, etc., and is expected to help elucidate the relevance of low molecular weight compounds to diseases and pathologies.
[0033]
【Example】
Hereinafter, the present invention will be specifically described by way of examples.
[Preparation Example 1] Preparation of hapten-protein complex
Immediately add a solution (concentration: 1 mg / ml) of 1 mM casein (abbreviation: CAS) or skim milk (abbreviation: SML) dissolved in 50 mM phosphate buffer (pH 7.2) to 2 mM acrolein (abbreviation: ACR). The mixture was reacted at 37 ° C. for 2 hours to prepare a hapten-protein complex, ie, ACR and CAS or ACR and SML conjugates (abbreviations: “ACR-CAS” and “ACR-SML”, respectively). When amino acids before and after the reaction were examined with an automatic amino acid analyzer [JEOL JLC-500, manufactured by JASCO Corporation], both ACR-CAS and ACR-SML may be abbreviated as lysine (hereinafter, “Lsy”). )) Decreased by more than 20%. Similarly, using 5 mM 4-hydroxy-2-nonenal (abbreviation: HNE), HNE and CAS or HNE and SML conjugates (abbreviation: “HNE-CAS” and “HNE-SML”, respectively) were prepared. did. When the obtained hapten-protein complex was analyzed by amino acid analysis, histidine (abbreviation: His) was reduced by 15% or more.
[0034]
[Preparation Example 2] Preparation of hapten-protein complex adsorption plate
The protein concentration of the ACR-CAS, ACR-SML, HNE-CAS and HNE-SML solutions obtained in Preparation Example 1 was diluted with ion exchange water to 10 μg / ml. 150 μl / well of this solution was dispensed onto a 96-well immunoplate (Nunk, F16, Maxisorp) and immobilized at room temperature. After immobilization, it was washed with 10 mM phosphate buffered saline (pH 7.4, abbreviation: PBS). In this way, an immune reaction container in which ACR-CAS, ACR-SML, HNE-CAS and HNE-SML were immobilized was prepared.
[0035]
[Test Example 1] ELISA test method 1
A compound having a formyl dehydropiperidine structure prepared at a concentration of 1 μg / ml in 10 mM PBS containing 0.1% casein first in the well of the immune reaction container prepared in Preparation Example 2, or 2- A monoclonal antibody solution (50 μl / well) specific for a compound having a hydroxy-5- (pentyltetrahydrofuran-4-yl) histidine structure was added and allowed to react at room temperature. After washing the wells, a rabbit antibody (Bio-Rad) against mouse antibody labeled with horseradish peroxidase was diluted 2000 times with PBS containing 0.05% Tween 20 (100 μl / well) (hereinafter “enzyme”). Abbreviated as “labeled antibody solution”) and allowed to react at room temperature for an arbitrary time. After washing the well, 0.1 M citrate-phosphate buffer solution (pH 5) (100 μl / well) containing O-phenylenediamine (0.4 mg / ml) and hydrogen peroxide (0.003%) (hereinafter, color development) Abbreviated as liquid) and allowed to react at room temperature for 15-20 minutes. The color reaction was stopped by adding 1 M sulfuric acid (50 μl / well) (hereinafter abbreviated as “reaction stop solution”), and the absorbance at 492 nm was measured with a microplate reader (Molecular Device, Spectramax 250). . All measurements were performed in duplicate. As a sample, a hapten-protein complex prepared by reacting 2 mM ACR or 5 mM HNE with human serum albumin according to Preparation Example 1 was added to PBS for 48 hours (externally dialyzed). The solution was changed every 12 hours) and the solution obtained by dialysis was diluted with PBS.
[0036]
[Test Example 2] ELISA test method 2
A compound having a formyl dehydropiperidine structure or 2-hydroxy-5- (prepared in PBS containing 0.1% casein in advance of a sample (120 μl / well) and 1 μg / ml was added to the well of the immune reaction container prepared in Preparation Example 2. (Pentyltetrahydrofuran-4-yl) A monoclonal antibody solution specific for a compound having a histidine structure (120 μl / well) was added in a polypropylene test tube and reacted at room temperature. After washing the well, an enzyme-labeled antibody solution (100 μl / well) was added and allowed to react at room temperature for an arbitrary time. After washing the well, a coloring solution (100 μl / well) was added and allowed to react at room temperature for 15-20 minutes. The color reaction was stopped by adding a reaction stop solution (50 μl / well), and the absorbance at 492 nm was measured with a microplate reader. All measurements were performed in duplicate. As a sample, a hapten-protein complex prepared by reacting 2 mM ACR or 5 mM HNE with human serum albumin according to Preparation Example 1 in PBS for 48 hours (excluding dialysis) The solution was changed every 12 hours) and the solution obtained by dialysis was diluted with PBS.
[0037]
[Example 1] Examination of immobilization time of hapten-protein complex to insoluble carrier
The immobilization time of the hapten-protein complex prepared in Preparation Example 1 on an insoluble carrier was examined. The immobilization of the hapten-protein complex on the insoluble carrier was in accordance with Preparation Example 2. The ELISA test was performed according to Test Example 1. The reaction time for the sample and the monoclonal antibody solution was 60 minutes, and the reaction time for the enzyme-labeled antibody was 60 minutes. The results are shown in FIG. In the figure, ACR-CAS shows the results of measurement using ACR-CAS as an immobilized antigen and human serum albumin added with ACR as a sample. Hereinafter, similarly, ACR-SML uses ACR-SML as an immobilized antigen, and human serum albumin added with ACR is used as a sample. HNE-CAS uses HNE-CAS as an immobilized antigen, and HNE The results obtained by measuring the added human serum albumin as a sample show the results of measuring HNE-SML using HNE-SML as an immobilized antigen and using human serum albumin added with HNE as a sample. The same applies to FIGS.
[0038]
As shown in FIG. 1, the immobilization time of the hapten-binding protein according to the present invention on the insoluble carrier became constant after 3 minutes. Therefore, it was shown that the hapten-binding protein according to the present invention adsorbs to an insoluble carrier in a very short time.
[0039]
[Example 2] Examination of reaction time after dispensing sample and monoclonal antibody solution into immune reaction container 1
Using the immunological measurement method of the present invention, the reaction time after dispensing the sample and the monoclonal antibody solution into the immune reaction container was examined. The experimental conditions were the same as in Test Example 1 except for the reaction time after dispensing the sample and the monoclonal antibody solution into the immune reaction container. The test was conducted with the hapten-protein complex immobilized on the immunoplate for 3 minutes and the enzyme-labeled antibody reaction time for 60 minutes. The results are shown in FIG.
[0040]
As shown in FIG. 2, according to the immunological measurement method of the present invention, the reaction time after dispensing the sample and the monoclonal antibody solution into the immune reaction container became constant after 10 minutes. Therefore, in the immunological measurement method of the present invention, it became clear that the reaction time after dispensing the sample and the monoclonal antibody solution into the immune reaction container may be about 10 minutes.
[0041]
[Example 3] Examination of sample incubation time 2
Using the immunological measurement method of the present invention, the reaction time between the reaction solution and the immune reaction container when the sample and the monoclonal antibody solution were reacted in separate containers was examined. The test was carried out in accordance with Test Example 2, in which the hapten-protein complex was immobilized on the insoluble carrier for 3 minutes, the sample and the monoclonal antibody solution were reacted in a separate container for 60 minutes, and the enzyme-labeled antibody reaction time was 60 Minutes. The results are shown in FIG.
[0042]
As is clear from the results, the reaction time of the sample and the reaction solution of the monoclonal antibody solution with the immune reaction container became constant after 3 minutes.
[0043]
[Example 4] Examination of sample incubation time 3
Using the immunological measurement method of the present invention, the time for reacting the sample and the monoclonal antibody solution in separate containers was examined. The test was conducted in accordance with Test Example 2, with a hapten-protein complex immobilization time of 3 minutes on the insoluble carrier, and a reaction solution obtained by reacting the sample and the monoclonal antibody solution in a separate container in an immune reaction container. The time was 3 minutes and the reaction time of the enzyme-labeled antibody was 60 minutes. The results are shown in FIG.
[0044]
[Comparative Example 1]
Using the immunological measurement method of the present invention, the time for reacting a polyclonal antibody as a sample in another container was examined. In Test Example 2, the test was performed in the same manner as in Example 4 except that the test was performed using a polyclonal antibody specific to the formyl dehydropiperidine structure instead of using a monoclonal antibody specific to the formyl dehydropiperidine structure. . The results are shown in FIG.
As is clear from the results of FIG. 4, it can be seen that the reaction is clearly constant in a short time when the monoclonal antibody is used as compared with the case where the polyclonal antibody is used as the sample.
[0045]
[Example 5]: Examination of reaction time of enzyme-labeled antibody solution
The reaction time of the enzyme-labeled antibody solution was examined using the immunological measurement method of the present invention. The test was performed in accordance with Test Example 2 in which the hapten-protein complex was immobilized on the insoluble carrier for 3 minutes, the sample monoclonal antibody was allowed to react in a polypropylene test tube for 3 minutes, and the reaction solution was immunoreacted. The test was conducted with a reaction time of 3 minutes after dispensing into the container. The results are shown in FIG.
As shown in FIG. 5, the reaction time of the enzyme-labeled antibody became constant at 7 minutes.
[0046]
Example 6: Preparation of standard curve by immunological measurement method-1
A standard curve was prepared using the immunological assay method of the present invention. As a standard, formyl dehydropiperidine-Nα-acetyllysine prepared by addition reaction of acrolein and Nα-acetyllysine was used. As the hapten-protein complex, ACR-CAS obtained in Preparation Example 1 was used. An immune reaction container was prepared according to Preparation Example 2, and the test was performed according to Test Example 2. In addition, the immobilization time of the hapten-protein complex on the insoluble carrier is 3 minutes, the reaction time of the sample and the monoclonal antibody in the polystyrene test tube is 3 minutes, and the reaction after dispensing the reaction solution into the immune reaction container The test was conducted with a time of 3 minutes and a reaction time of the enzyme-labeled antibody of 7 minutes.
The results are shown in FIG.
[0047]
[Comparative Example 2]; Preparation of standard curve by immunological measurement method-2
The immobilization time of the hapten-protein complex on the insoluble carrier, the reaction time of the sample and the monoclonal antibody in the polystyrene test tube, and the reaction time after dispensing the reaction solution into the immune reaction container are all 30 minutes (B) The test was conducted in the same manner as in Example 6 except that 60 minutes (C) and 120 minutes (D) were used, and a standard curve was obtained in the same manner. The results are shown in B, C and D of FIG.
[0048]
[Example 7]; Simultaneous reproducibility test -1
Using the immunological measurement method of the present invention, acrolein adducts in a sample were measured, and simultaneous reproducibility was examined. The test was performed under the same conditions as in Example 6, and human urine was used as a sample. The results are shown in Table 1 and Table 2A. Table 1 shows the results of Sample 1, and Table 2 shows the results of Sample 2.
[0049]
[Comparative Example 3]; Simultaneous reproducibility test-2
The acrolein adducts in the sample were measured and the simultaneous reproducibility was examined. The test was performed under the same conditions as in Comparative Example 2, and human urine was used as a sample. The results are shown in Tables 1 and 2. In the table, B to D indicate that measurement was performed by the methods B, C, and D shown in Comparative Example 2. Table 1 shows the results of Sample 1, and Table 2 shows the results of Sample 2.
[0050]
[Table 1]
[0051]
[Table 2]
[0052]
As is clear from the above results, from the results of FIG. 6, Table 1 and Table 2, according to the immunological measurement method of the present invention, the performance exactly the same as that of the immunological measurement method measured for a long time. An immunological measurement method can be provided. Accordingly, it is possible to provide an immunological measurement method capable of completing the measurement in a short time.
It can be seen that the examples of the present invention are immunological measurement methods that have high specificity and can obtain measurement results in a short time compared to the comparative examples.
Therefore, it is useful in research in the medical field and food field.
[Brief description of the drawings]
FIG. 1 is a graph showing the immobilization time of a hapten-protein complex.
FIG. 2 is a graph examining the reaction time after dispensing an immune reaction container of a sample of an immunological measurement method according to the present invention and a monoclonal antibody solution.
FIG. 3 shows the reaction time after dispensing the reaction solution of the sample and the monoclonal antibody solution into the immunological reaction container when the sample and the monoclonal antibody solution are reacted in another container of the immunological measurement method according to the present invention. It is the examined graph.
FIG. 4 is a graph showing the reaction time of a sample and an antibody solution when the sample and the antibody solution are reacted in another container of the immunological measurement method according to the present invention.
FIG. 5 is a graph showing the reaction time of an enzyme-labeled antibody solution in the immunological measurement method according to the present invention.
FIG. 6 is a graph showing a standard curve of an immunological measurement method according to the present invention.

Claims (1)

The hapten compound to be measured is represented by the following formula (I)
(Wherein R ¹ and R ² represent protein residues, peptide residues, or R ¹ = R ² = H), or a compound having a formyldehydropiperidine structure represented by the following formula (II)
(Wherein R ¹ and R ² represent a protein residue, a peptide residue, or R ¹ = R ² = H) 2-hydroxy-5- (pentyltetrahydrofuran-4-yl) histidine structure In an immunological measurement method for measuring a hapten compound which is a compound having a hapten- protein complex, the protein constituting the hapten-protein complex is casein or skim milk by contacting the hapten-protein complex with an insoluble carrier. Using an immune reaction container in which a protein complex is physically adsorbed and pre-coated, (1) In this order, a monoclonal antibody to the sample and the hapten compound is added to the immune reaction container in advance. Incubate the reaction mixture that has been incubated in the container. Reacting a monoclonal antibody against the hapten-protein complex precoated on the immune reaction container to form an antigen-antibody reaction complex, and (2) reacting with the monoclonal antibody that reacts with the hapten compound as an antigen. Reacting and binding the enzyme-labeled antibody to the monoclonal antibody reacted with the hapten-protein complex precoated on the immune reaction container by incubating the enzyme-labeled antibody in the immune reaction container. A method for immunological measurement of a hapten compound.