Method of detecting lutein and method of producing Anti-lutein antibodies

Abstract

To provide methods of producing anti-lutein antibodies and detecting lutein by immunoassay, and to provide a kit therefor.SOLUTION: A method of the present invention for detecting lutein by immunoassay comprises a step of bringing a sample containing lutein with an anti-lutein antibody to form an antigen-antibody reaction product containing a complex of the lutein in the sample and the antibody.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for detecting lutein and a kit therefor, as well as a method for producing an anti-lutein antibody. [Background technology]

[0002] Carotenoids known to exist in human blood include β-carotene, α-carotene, β-cryptoxanthin, lycopene, lutein, and zeaxanthin. Among these, lutein and zeaxanthin are known to accumulate specifically in the retina.

[0003] Age-related macular degeneration (AMD) is a serious disease that can lead to blindness, but lutein intake is expected to be effective in preventing it. In recent years, many functional foods have been labeled with lutein as a functional ingredient. Lutein analysis is usually performed using high-performance liquid chromatography (HPLC), and the Japanese Agricultural Standards (JAS0008) also specifies HPLC as a method for quantifying lutein in spinach (Non-Patent Document 1). However, measurement using HPLC requires specialized knowledge and skill in instrumental analysis, making it difficult to measure lutein.

[0004] On the other hand, immunoassays such as enzyme-linked immunosorbent assays (ELISAs) are easier to perform than HPLC, but require antibodies specific to the target compound. Generating antibodies against low-molecular-weight compounds is difficult, and no antibodies specific to lutein have been reported. Non-Patent Document 2 describes an antibody against fucoxanthin, but does not mention its use in immunoassays, nor does it describe antibodies against other carotenoids. Furthermore, Non-Patent Document 3 lists "hydrophilicity" as the number one criterion for selecting antigen peptides when generating antibodies. Non-Patent Document 4 states that many antigen sequences are believed to be long hydrophilic portions present on the surface of natural proteins. Therefore, it has been thought that antibodies against hydrophobic molecules are more difficult to generate than those against hydrophilic molecules. Non-Patent Document 5 states that the calculated octanol / water partition coefficient (ClogP) of lutein is 14.82, indicating that lutein is a highly hydrophobic compound. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Japanese Agricultural Standards JAS0008, Ministry of Agriculture, Forestry and Fisheries, established on January 31, 2019, https: / / www.maff.go.jp / j / jas / jas_kikaku / attach / pdf / kokujikaisei-75.pdf [Non-patent document 2] Mari Yamashita, Grant-in-Aid for Scientific Research, Research Report, Project Number 26660094, May 14, 2016 [Non-patent document 3] "Procedure for designing antigen peptides," [online], Japan Bio Services, Internet, URL: https: / / www.jbios.co.jp / antibody / peptidedesign.html [Non-patent document 4] "Custom Polyclonal Antibody Production: Technical Information," section "Peptide Sequence Selection Method for Peptide Immunization," [online], Merck KGaA, Internet, URL: https: / / www.sigmaaldrich.com / JP / ja / products / protein-biology / antibodies / custom-antibodies / polyclonal-antibody / technical-information#a07 [Non-patent document 5] J. Nutr., 127: 1699S-1709S, 1997 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for producing an anti-lutein antibody and detecting lutein by immunological assay, and a kit therefor. [Means for solving the problem]

[0007] As a result of extensive research aimed at solving the above problems, the present inventors have succeeded in producing an anti-lutein antibody that can be used in immunoassays, thereby completing the present invention. That is, the present invention provides the following methods for detecting lutein by immunoassay, a kit for detecting lutein by immunoassay, and a method for producing an anti-lutein antibody. [1] A method for detecting lutein by immunoassay, comprising: A method comprising the step of contacting a sample containing lutein with an anti-lutein antibody to form an antigen-antibody reaction product comprising a complex of lutein in the sample and the antibody. [2] The method according to [1] above, wherein the lutein is detected by a competitive method. [3] applying the antigen-antibody reaction product to a substrate having lutein immobilized on its surface to allow the unreacted antibody that has not formed the complex to bind to the lutein on the substrate; detecting the antibody bound to the lutein on the substrate; The method according to [1] above, further comprising: [4] The antibody is immobilized on a substrate before contact with the sample; contacting the sample with the antibody in the presence of labeled lutein; The method according to [1] above, wherein the labeled lutein bound to the antibody is detected after the formation of the antigen-antibody reaction product. [5] The method according to any one of [1] to [4] above, wherein the immunological assay is an enzyme-linked immunosorbent assay (ELISA) or a dot blot assay. [6] A kit for detecting lutein by immunoassay, comprising an anti-lutein antibody. [7] The kit according to [6], further comprising a substrate having lutein immobilized on its surface, or a substrate having the antibody immobilized on its surface. [8] The kit according to [6], wherein the immunological assay is carried out by a competitive method. [9] The kit according to any one of [6] to [8] above, wherein the immunological measurement method is ELISA or dot blot method.

[10] A method for producing an anti-lutein antibody, comprising: providing lutein as an antigen, the lutein being bound to a carrier protein via one hydroxyl group; immunizing a non-human animal with the antigen; A method comprising:

[11] The method according to

[10] , wherein the step of preparing the antigen comprises a step of binding lutein to a carrier protein via a succinic acid linker.

[12] The step of preparing an antigen comprises: preparing an active ester of lutein in which one hydroxyl group is succinylated (monosuccinylated lutein); reacting the active ester with a carrier protein; The method according to

[10] above, comprising:

[13] The step of preparing an activated ester comprises: succinylating lutein to obtain a first reaction product comprising the monosuccinylated lutein; reacting the monosuccinylated lutein with a carboxylic acid activator to obtain a second reaction product comprising the activated ester; optionally, purifying the activated ester; The method according to

[10] above, comprising:

[14] The method according to any one of

[10] to

[13] above, wherein the carrier protein comprises bovine thyroglobulin and / or bovine serum albumin. [Effects of the Invention]

[0008] According to the present invention, lutein can be easily detected by immunological assay using an anti-lutein antibody. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows the results of titration of anti-lutein antibodies in the serum of immunized animals. [Figure 2] 1 shows the results of lutein detection by competitive ELISA. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will now be described in further detail. The present invention relates to a method for detecting lutein. Lutein has the following structure: [ka] Lutein is a carotenoid having the following structure. Generally, to generate antibodies against a low molecular weight compound, it is necessary to chemically modify the low molecular weight compound and form a complex with a carrier protein through the chemical modification to prepare an antigen for immunization. However, lutein has only two hydroxyl groups that can be chemically modified. Furthermore, chemical modification of both of these hydroxyl groups reduces the structure characteristic of lutein, which may result in cross-reactivity with other carotenoids. Therefore, only one hydroxyl group is available for chemical modification, and there is a concern that immunogenicity may not be sufficiently enhanced.

[0011] Furthermore, as can be seen from the above structure, lutein is highly hydrophobic among carotenoids, with a ClogP of 14.82 (Non-Patent Document 5). Fucoxanthin, for which antibodies have been reported to be produced, has a ClogP of 11.40 (Non-Patent Document 5), making lutein more hydrophobic than fucoxanthin. Astaxanthin has a ClogP of 13.27 (Non-Patent Document 5), a value close to that of lutein. However, given that no antibodies against astaxanthin have yet been reported, it was thought that producing antibodies against the more hydrophobic lutein would be extremely difficult. However, surprisingly, it was discovered that an antigen prepared by chemically modifying one of the hydroxyl groups of lutein could be used to produce an anti-lutein antibody that could be used in immunoassays.

[0012] The anti-lutein antibody may be a whole antibody molecule that binds to lutein or a fragment containing its antigen recognition site (e.g., Fab or F(ab')2), and may be a polyclonal or monoclonal antibody. The anti-lutein antibody can be produced by any method commonly used in the art. For example, the antibody may be produced by immunizing a non-human animal with lutein bound to a carrier protein via a succinic acid linker as an antigen. A more detailed example of the production method will be described later as another embodiment of the present invention.

[0013] The detection method of the present invention is a method for detecting lutein by immunoassay. The term "immunoassay" as used herein refers to a specific detection and measurement method that utilizes an antigen-antibody reaction between an antibody and a target substance. The immunoassay is not particularly limited, and may be, for example, a competitive method, an enzyme-linked immunosorbent assay (ELISA), a dot blot assay, or the like.

[0014] The detection method of the present invention includes a step of contacting a sample containing lutein with an anti-lutein antibody to form an antigen-antibody reaction product containing a complex between the lutein in the sample and the antibody. The sample is not particularly limited as long as it is applicable to immunoassays, and may be, for example, a body fluid such as plasma, urine, breast milk, or semen, or a homogenate of a vegetable such as spinach, or an extract thereof.

[0015] In one embodiment, the detection method of the present invention may further include the steps of applying the antigen-antibody reaction product to a substrate having lutein immobilized on its surface to allow the unreacted antibody that has not formed the complex to bind to the lutein on the substrate, and detecting the antibody that has bound to the lutein on the substrate. When the lutein concentration in the sample is high, the amount of unreacted antibody that forms the complex decreases, resulting in a decrease in the signal derived from the antibody that has bound to the lutein on the substrate. When the lutein concentration in the sample is low, the amount of unreacted antibody that has not formed the complex increases, resulting in an increase in the signal derived from the antibody that has bound to the lutein on the substrate.

[0016] The signal derived from the antibody is not particularly limited, but may be derived from a label of a labeled secondary antibody against the anti-lutein antibody, or from a label pre-bound to the anti-lutein antibody. Labels commonly used in the art can be used as labels bound to the secondary antibody or the anti-lutein antibody without particular limitation. For example, the label may be an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), a fluorescent dye such as fluorescein, a fluorescent protein such as phycoerythrin, or biotin. When the label is biotin, the biotin is detected using labeled streptavidin or the like. Labels other than biotin from the above-mentioned examples can be used as the label for streptavidin.

[0017] In one embodiment, in the detection method of the present invention, the antibody may be immobilized on a substrate before contact with the sample. In this case, the sample and the antibody may be contacted in the presence of labeled lutein, and the labeled lutein bound to the antibody may be detected after the formation of the antigen-antibody reaction product. When the lutein concentration in the sample is high, a large number of complexes are formed, the number of antibodies to which the labeled lutein can bind is reduced, and the signal derived from the labeled lutein is reduced. When the lutein concentration in the sample is low, the complexes are not formed as much, the number of antibodies to which the labeled lutein can bind is increased, and the signal derived from the labeled lutein is increased. The label bound to the labeled lutein can be any label commonly used in the art without any particular limitation, and for example, the labels exemplified above as antibody labels can be similarly used.

[0018] The signal can be appropriately detected by a method commonly used in the art. For example, when the label is an enzyme, a color-developing reagent such as a substrate of the enzyme may be applied, and the resulting color may be detected using a microplate reader or the like. When the label is a fluorescent dye or a fluorescent protein, the fluorescence thereof may be detected using a microplate reader or the like.

[0019] The substrate on which lutein or the antibody is immobilized can be any substrate commonly used in the art, without any particular limitations. For example, the substrate may be a microplate such as an ELISA microplate, or a membrane such as a nitrocellulose membrane or a polyvinylidene fluoride (PVDF) membrane.

[0020] The detection method of the present invention may further include any steps commonly used in the art, such as a blocking step and a washing step, as long as the steps do not impair the object of the present invention.

[0021] In another aspect, the present invention relates to a kit for detecting lutein by immunoassay, comprising an anti-lutein antibody. Specific aspects of the anti-lutein antibody contained in the kit of the present invention and specific aspects of the immunoassay targeted by the kit of the present invention are as described above in relation to the detection method of the present invention.

[0022] In one embodiment, the kit of the present invention may further include a substrate having lutein immobilized on its surface. In this case, a sample containing lutein may be contacted with an anti-lutein antibody to form an antigen-antibody reaction product containing a complex between the lutein in the sample and the antibody, and the antigen-antibody reaction product may then be applied to the substrate to allow the unreacted antibody that has not formed the complex to bind to the lutein on the substrate, and the antibody bound to the lutein on the substrate may then be detected.

[0023] In one embodiment, the kit of the present invention may further comprise a substrate on which the antibody is immobilized. In this case, a sample containing lutein may be contacted with the antibody in the presence of labeled lutein to form an antigen-antibody reaction product containing a complex of lutein in the sample and the antibody, and then the labeled lutein bound to the antibody may be detected. That is, the kit of the present invention may further comprise labeled lutein.

[0024] The kit of the present invention may further contain any reagent commonly used in the art, such as a buffer solution, a blocking agent, a labeled secondary antibody, and a color-developing reagent, as long as it does not impair the objective of the present invention.

[0025] In another aspect, the present invention relates to a method for producing an anti-lutein antibody. The method of the present invention includes the steps of preparing lutein bound to a carrier protein via a succinic acid linker as an antigen and immunizing a non-human animal with the antigen. The non-human animal can be any animal commonly used in the art without any particular limitations, and may include, for example, rabbits, rats, mice, and goats.

[0026] The antigen can be prepared by any method commonly used in the art, for example, by binding lutein to a carrier protein via a succinic acid linker. More specifically, the antigen can be prepared by succinylating lutein to obtain a first reaction product containing lutein with one hydroxyl group succinylated (monosuccinylated lutein), reacting the monosuccinylated lutein with a carboxylic acid activator to obtain a second reaction product containing an active ester of the monosuccinylated lutein, and then reacting the active ester of the monosuccinylated lutein with a carrier protein.

[0027] The carboxylic acid activator may be any carboxylic acid activator commonly used in the art without any particular limitation, and may include, for example, N-hydroxysuccinimide, sulfated N-hydroxysuccinimide, etc. The carrier protein may be any carboxylic acid activator commonly used in the art without any particular limitation, and may include, for example, bovine thyroglobulin, bovine serum albumin, etc.

[0028] The first reaction product may contain two types of monosuccinylated lutein and disuccinylated lutein in which two hydroxyl groups are succinylated. However, in order to obtain an antibody with low cross-reactivity with other carotenoids, i.e., an antibody with high specificity to lutein, it is preferable to leave the structure characteristic of lutein in the antigen. Therefore, the purity of the active ester of monosuccinylated lutein when reacted with the carrier protein may be increased by increasing the molar ratio of the monosuccinylated lutein to the disuccinylated lutein in the first reaction product, or by increasing the molar ratio of the active ester of monosuccinylated lutein to the active ester of disuccinylated lutein in the second reaction product. The method for increasing the purity is not particularly limited, and for example, in order to increase the molar ratio of the monosuccinylated lutein to the disuccinylated lutein in the first reaction product, the amount of succinic anhydride added during the succinylation reaction may be set to be equal to that of lutein, so that theoretically only one hydroxyl group is reacted with the succinylation reaction.Furthermore, in order to increase the molar ratio of the monosuccinylated lutein active ester to the disuccinylated lutein active ester in the second reaction product, for example, the monosuccinylated lutein may be reacted with the carboxylic acid activator, and then the disuccinylated lutein active ester and the monosuccinylated lutein active ester may be separated by silica gel column chromatography.

[0029] The production method of the present invention may further include any steps commonly used in the art, such as a step of obtaining serum from the non-human immunized animal, a step of measuring the antibody titer in the serum, a step of producing hybridomas, and a step of purifying the antibody, as long as the steps do not impair the objective of the present invention.

[0030] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to these examples. [Example]

[0031] 1. Preparation of anti-lutein antibodies (1) Chemical modification of lutein Lutein was succinylated by reaction with succinic anhydride in pyridine and 4-dimethylamino pyridine (DMAP). The resulting succinylated product was a mixture of succinyl groups introduced at one or both of the two hydroxyl groups of lutein. N-hydroxysuccinimide (NHS), N,N'-diisopropylcarbodiimide, and 4-dimethylaminopyridine were added to the succinylated product in dichloromethane and reacted at room temperature for 12 hours to obtain the NHS esters of succinylated lutein. These NHS esters contained active esters A and B derived from monosuccinylated lutein, in which one of the two hydroxyl groups of lutein was succinylated, and active ester C derived from disuccinylated lutein, in which both hydroxyl groups of lutein were succinylated. The NHS esters were fractionated by silica gel column chromatography, and each fraction was analyzed by mass spectrometry to identify the fractions containing active esters A and B. These fractions were then pooled to enrich the relative amounts of active esters A and B. It was confirmed by conventional NMR that the active esters A and B were present in a ratio of 1:1 in the mixture (active ester AB) in which the relative amounts of active esters A and B were increased.

[0032] [ka]

[0033] (2) Immunity The activated ester AB was mixed with bovine thyroglobulin in an appropriate ratio to prepare an antigen. Rabbits were immunized with this antigen by standard methods, and after 8 weeks, whole blood was collected to obtain serum (antiserum).

[0034] (3) Measurement of antibody titer The titer of anti-lutein antibodies in the collected serum was measured by ELISA. Specifically, a 0.1% by mass bovine fetal serum albumin (BSA) solution was mixed with the active ester AB (50 ng / 50 μL), and 50 μL of this mixture was added to a 96-well plate for immobilizing the antigen, immobilizing lutein and BSA. A lutein-free BSA solution was added to a control 96-well plate, immobilizing only BSA. Separately, serum collected from the immunized rabbit was diluted 100-fold with phosphate-buffered saline (PBS) containing 1% BSA and 0.05% Tween 20, and then further diluted two-fold to prepare a series of diluted serum. The plate was washed by standard methods, and 50 μL of the diluted serum was added to each well and incubated at 37°C for 30 minutes. The plate was washed in a conventional manner, and color was developed in a conventional manner using HRP-labeled goat Fab' against rabbit IgG and o-phenylenediamine, and the absorbance at 490 nm was measured.

[0035] As a result, it was found that the serum collected from the immunized rabbits contained antibodies that specifically bound to the lutein-immobilized plate, i.e., anti-lutein antibodies, as shown in Figure 1. The absorbance cutoff value was set at 0.2 or higher, and the antibody titer was determined to be 25,600.

[0036] (4) Specific purification of antibodies The activated ester AB and BSA were mixed in an appropriate ratio to prepare an antigen column (10 mL volume) by a conventional method. Then, the serum (68.8 mL) obtained in the above item (2) was passed through this antigen column, and the eluate (11.988 mL) was recovered from the column by a conventional method. The antibody contained in this eluate was used as an anti-lutein antibody in the subsequent tests.

[0037] (5) Measurement of cross-reactivity The protein concentration of the anti-lutein antibody was quantified by measuring absorbance at 280 nm and diluted to a concentration of 2 μg / mL. 50 μL of the anti-lutein antibody solution was added to a reaction plate. 50 μL of a carotenoid solution (diluted to 100 μg / mL to 3.2 μg / mL) or a control 0.1% BSA solution listed in Table 1 below was added, followed by preincubation overnight at 4°C. ELISA was then performed using a lutein-immobilized plate in the same manner as in (3) above, except that the preincubated anti-lutein antibody solution was used instead of the diluted serum.

[0038] As a result, when pre-incubated with lutein, a decrease in color development was observed depending on the lutein concentration, but when pre-incubated with other carotenoids, no concentration-dependent decrease in color development was observed.

[0039] The binding inhibition rate was calculated as the percentage of absorbance decreased by preincubation with a carotenoid-containing solution, based on the absorbance of the control preincubated with a BSA solution. For example, the absorbance of the control was 1.044, and the absorbance after preincubation with 50 μg / mL lutein was 0.492. In this case, the binding inhibition rate was (1.044-0.492) / 1.044×100=53.0% The calculation results when a 50 μg / mL carotenoid solution was used are summarized in Table 1.

[0040] [Table 1]

[0041] The carotenoid concentration of 50 μg / mL during preincubation was sufficient to inhibit 53.0% of antibody binding in the case of lutein, but not in the case of other carotenoids. Therefore, the anti-lutein antibody produced in this example was considered to have low cross-reactivity with other carotenoids.

[0042] 2. Detection of lutein by competitive ELISA The protein concentration of the anti-lutein antibody was adjusted to 0.3 mg / mL, and 50 μL of each solution was added to the reaction plate. Serially diluted lutein solutions starting from 500 μg / mL or a 0.1% BSA control solution were prepared as test samples. 60 μL of each solution was added to the reaction plate and preincubated at 37°C for 30 minutes. Then, plates with immobilized lutein or BSA were prepared as described in Section 1(3) above. After washing by standard methods, 50 μL of the preincubated anti-lutein antibody solution was added to each well and incubated at 37°C for 60 minutes. The plates were washed by standard methods, and color development was performed using HRP-labeled goat Fab' against rabbit IgG and TMB (3,3',5,5'-tetramethylbenzidine) by standard methods. The absorbance at 450 nm was measured. The measurement results are shown in Figure 2, and the binding inhibition rates calculated as described in Section 1(5) above are shown in Table 2.

[0043] [Table 2]

[0044] As shown in Figure 2 and Table 2, the binding of anti-lutein antibody to immobilized lutein was inhibited depending on the lutein concentration during preincubation. Therefore, lutein could be detected by competitive ELISA.

[0045] From the above, it was found that an anti-lutein antibody that can be used for immunoassays could be produced and that it could be used to easily detect lutein.

Claims

1. A method for detecting lutein by immunoassay, comprising: A method comprising the step of contacting a sample containing lutein with an anti-lutein antibody to form an antigen-antibody reaction product comprising a complex of lutein in the sample and the antibody.

2. The method of claim 1, wherein the lutein is detected by a competitive method.

3. applying the antigen-antibody reaction product to a substrate having lutein immobilized on its surface to allow the unreacted antibody that has not formed a complex to bind to the lutein on the substrate; detecting the antibody bound to the lutein on the substrate; The method of claim 1 further comprising:

4. the antibody is immobilized on a substrate prior to contact with the sample; contacting the sample with the antibody in the presence of labeled lutein; The method of claim 1, wherein the labeled lutein bound to the antibody is detected after the formation of the antigen-antibody reaction product.

5. The method according to any one of claims 1 to 4, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA) or a dot blot assay.

6. A kit for detecting lutein by immunoassay, comprising an anti-lutein antibody.

7. The kit of claim 6 , further comprising a substrate on which lutein is immobilized or a substrate on which the antibody is immobilized.

8. The kit according to claim 6, wherein the immunoassay is carried out by a competitive method.

9. The kit according to any one of claims 6 to 8, wherein the immunological assay is an ELISA or dot blot method.

10. A method for producing an anti-lutein antibody, comprising: providing lutein as an antigen, the lutein being bound to a carrier protein via one hydroxyl group; immunizing a non-human animal with the antigen; A method comprising:

11. 11. The method of claim 10, wherein the step of providing the antigen comprises conjugating lutein to a carrier protein via a succinic acid linker.

12. The step of providing the antigen comprises: preparing an active ester of lutein in which one hydroxyl group is succinylated (monosuccinylated lutein); reacting the active ester with a carrier protein; The method of claim 10, comprising:

13. providing the activated ester, succinylating lutein to obtain a first reaction product comprising the monosuccinylated lutein; reacting the monosuccinylated lutein with a carboxylic acid activator to obtain a second reaction product comprising the activated ester; optionally, purifying the activated ester; The method of claim 10, comprising:

14. The method of any one of claims 10 to 13, wherein the carrier protein comprises bovine thyroglobulin and / or bovine serum albumin.

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