JP2007183187A - SPECIFIC QUANTIFICATION METHOD OF beta-CASEIN PHOSPHOPETIDE (beta-CPP) USING IMMUNOLOGICAL TECHNIQUE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for specifically and simply measuring CPP added to food, such as dairy products or the like and β-CPP in a CPP composite, and to provide a rapid and economical immunological assay method of high sensitivity to social needs, capable of being utilized routinely in the analysis of food components. <P>SOLUTION: An immunological method using a monoclonal antibody (mAb 1A5) with respect to β-CPP and a sample preparation method suitable for a measuring system are combined, to simply, specifically and accurately measure β-CPP in food. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an immunoassay using a monoclonal antibody that specifically recognizes β-casein phosphopeptide (hereinafter referred to as “β-CPP”) among casein phosphopeptides.

It is known that casein, which is a milk protein, is decomposed by digestive enzymes to produce many physiologically active peptides such as opioid peptides. Casein phosphopeptide (hereinafter referred to as “CPP”) is a phosphorylated peptide produced by hydrolysis of αs1-casein, αs2-casein, and β-casein in vitro and in vivo. As a structural feature, it has two glutamic acids as a common motif following three consecutive phosphorylated serines. These motifs are known to interact with minerals such as calcium, iron, and zinc to enhance the use of living bodies (Non-patent Document 1). Furthermore, CPP and amorphous calcium phosphate (Amorphous Calcium Phosphate; CPP-ACP) complex has been scientifically proven to have the ability to remineralize teeth (Non-patent Document 2, Patent Document 1) The added product is a food for specified health that promotes remineralization.
Identification of CPP is performed by using mass analysis after separation by anion exchange HPLC or reverse phase HPLC (Non-patent Documents 3 and 4). However, it takes time to accurately determine the attribution of the peak and the like, and an expensive device that requires complicated operation is also required. Furthermore, when adding to dairy products, the amount of the compound can be quantified because similar substances coexist, so that ordinary immunological methods cannot be used as they are. An attempt has been made to quantify the fate of a specific peptide during the cheese ripening period using an anti-peptide polyclonal antibody (Non-patent Document 5). However, in order to ensure the specificity strictly, it is generally considered that a monoclonal antibody is more desirable than a polyclonal antibody. Furthermore, in this report, measurement is performed after freeze-drying and degreasing as pretreatment, but in the case of adding a milk-derived material to a dairy product, a preparation method that efficiently removes the existing analogs. Ingenuity is also required.
CPP is known to take various calcium phosphate phases depending on pH, calcium and phosphate concentrations (Patent Document 1). Therefore, at present, it is not easy to directly quantify CPP complexes such as CPP-ACP as they are. Furthermore, although there is a report that a complex of CPP and calcium phosphate is separated by gel filtration HPLC (Non-patent Document 6), it is not sufficient for use as a quantitative means. In addition, CPP-ACP has a particularly low coating efficiency on ELISA plates, and the usual indirect ELISA method cannot be applied.
Special table 2002-500626 Takanori Kasai (2003). Casein phosphopeptide: Foods Food Ingredients J. Jpn 208, 167-173. Reynolds E. C (1998). Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: A review.SCD Special Care in Densitry 18, 8-16. Miquel E, Alegria A, Barbera R and Farre R (2005) .Speciation analysis of calcium, iron, and zinc in casein phosphopeptide fractions from toddler milk-based formula by anion exchange and reversed-phase high-performance liquid chromatography-mass spectrometry / flame atomic-absorption spectroscopy. Anal Bioanal Chem 381, 1082- 1088. Ferranti P, Barone F, Chianese L, Addeo F, Scaloni A, Pellegrino L, and Resmini P (1997) .Phosphopeptides from Grana Padano cheese: nature, origin and changes during ripening.J. Of Dairy Research 64, 601-615. Pizzano R, Nicolai MA, Padovano P, Ferranti P, Barone F, and Addeo F (2000) .Immunochemical evaluation of bovine β-casein and its 1-28 phosphopeptide in cheese during ripening.J. Agric. Food Chem 48, 4555- 4560. Aoki T, Nakano T, Iwashita T, Sugimoto Y, Ibrahim HR, Toba Y, Aoe S and Nakajima I (1998). Preparation and characterization of micellar calcium phosphate-casein phosphopeptide complex.J Nutr Sci Vitaminol. 44, 447-456.

  It is an object of the present invention to provide a method for specifically and simply and accurately measuring CPP added to foods such as dairy products and β-CPP in CPP complexes. Furthermore, it is to provide an immunological assay for the social needs that are rapid, economical, accurate and sensitive and routinely available in the analysis of food ingredients.

As a result of earnest research, the present inventors have combined β-CPP in food with a simple and specific method by combining an immunological method with a monoclonal antibody against mAb (mAb 1A5) and a sample preparation method suitable for the measurement system. And the present invention was completed.
That is, the present invention
[1] Quantitative determination of β-casein phosphopeptide and / or β-casein phosphopeptide complex by immunoassay using a monoclonal antibody (mAb 1A5) against the phosphorylated peptide of SEQ ID NO: 1,
[2] The quantification method according to the above [1] by an immunological assay comprising the following steps:
(1) Step of adding acid to sample and denaturing / removing protein (2) Step of removing acid by solid phase extraction (3) Purified sample eluted from solid phase for phosphorylated peptide of SEQ ID NO: 1 Immunological measurements are performed using a monoclonal antibody (mAb 1A5).
[3] The method according to [1] or [2], wherein the β-casein phosphopeptide and / or β-casein phosphopeptide complex in the dairy product is quantified,
Consists of.

  By using the method of the present invention, the substance to be measured can be specifically quantified even when similar substances coexist in the product. Furthermore, there is an advantage in that it does not require expensive measuring equipment, special skills / knowledge, and is easy to operate.

Hereinafter, the present invention will be described in detail.
CPP used in the present invention refers to a casein phosphopeptide obtained by hydrolyzing casein with trypsin, and the production method is described in JP-A-56-123922, JP-A-59-159792, JP-A-59. -159793, JP-A-2-257854, and the like.
The casein phosphopeptide complex refers to a complex formed of a casein trypsin degradation product and an inorganic salt such as calcium and phosphoric acid, and derivatives thereof, and can be exemplified by JP-T 2002-500626.
The antibody used in the present invention can be exemplified by a monoclonal antibody against a partial sequence of β-casein described in SEQ ID NO: 1 (produced according to the 49th Annual Meeting of the Japan Society of Nutrition and Food Science p126 3A-3a, hereinafter referred to as mAb 1A5). .

  A dairy product sample containing CPP and CPP complex is subjected to acid treatment such as trichloroacetic acid, and then denatures and removes macromolecular proteins derived from casein, whey and the like by centrifugation. Next, the CPP-containing fraction that removes the acid by solid phase extraction and is bound to the solid matrix is eluted and dried. A calibration curve sample is prepared by dissolving CPP and CPP complex (standard product) in a solution having the same composition as the product, and pretreatment under the same conditions as the dairy product sample can offset the recovery factor. If a product that has the same composition as the product and does not contain the measurement target (placebo) cannot be used, it can be indirectly calculated from a calibration curve created by adding a known amount of CPP and CPP complex to the product. It is. Furthermore, in order to determine the CPP and CPP complex content present in the placebo, a calibration curve obtained by preparing a calibration curve sample with an appropriate solvent such as PBS or physiological saline can be used. As dairy products, it can be applied to raw milk, processed milk, cheese, yogurt, ice cream and the like.

Immunological quantification was performed by competitive Enzyme-linked immunosorbent assay (ELISA) (Hornbeck P. Enzyme-Linked Immunosorbent Assays. In Current Protocols in Immunology; Coligan, JE, Kruisbeek, AM, Margulies, DH, Shevach, EM Strober, W., Ed .; John Wiley & Sons, Inc, 1991; 2.1.1-2.1.22.).
A pretreated dairy sample or calibration curve sample containing CPP and CPP complex is reacted with a monoclonal antibody (preincubation). The 96-well plate for measurement is coated (solid phase) with CPPIII (Meiji Seika) and then blocked with bovine serum albumin (BSA) to prevent non-specific adsorption of the subsequent reaction. Transfer the pre-incubated dairy sample or calibration curve sample and monoclonal antibody mixture to the blocked assay plate. Β-CPP in dairy samples or calibration curve samples competes with β-CPP in immobilized CPPIII for binding to monoclonal antibodies. Subsequently, unreacted substances are washed and removed, and measurement is performed by a color reaction using a secondary antibody that is enzyme-labeled with a monoclonal antibody bound to β-CPP immobilized on a solid phase. CPP and CPP complex content in the dairy product sample is obtained from the approximate curve obtained from the calibration curve sample. Since the calibration curve sample is prepared by adding a known amount of CPP and CPP complex, the amount of CPP and CPP complex contained in the product is determined. Furthermore, the amount of β-CPP contained in the product can also be calculated by applying the above-mentioned competitive ELISA method to CPP and CPP complex bulk material using purified β-CPP as a standard substance.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by this.

[Example 1] Measurement of process cheese and CPP content added to process cheese New Zealand Gouda Process Cheese and New Zealand Gouda Process Cheese with 2% CPP were used as test materials. Distilled water was added to each cheese in a mortar to prepare a 10% (w / v) uniform aqueous solution. Furthermore, the following three dairy product samples were prepared, including those obtained by adding CPP to the obtained 10% (w / v) New Zealand Gouda processed cheese aqueous solution at 2 mg / ml.
1) New Zealand Gouda Process Cheese 10% solution
2) New Zealand Gouda Process Cheese 10% solution with 2% CPP (CPP: 2mg / ml)
3) 10% New Zealand Gouda Process Cheese with CPP (2mg / ml) Sample pre-treatment was performed according to the procedure described below.
Take 0.3 ml of dairy sample in a plastic tube, add 1.2 ml of 15% trichloroacetic acid (TCA) and then 1.5 ml of PBS while stirring with a vortex mixer, and centrifuge to remove acid-denatured polymer proteins such as casein (17000 xg 6 min, 4 ° C.). A 1.2 ml solid phase extraction column (Waters OASIS ^TM ) equilibrated with 2 ml of 100% methanol and 2 ml of HPLC distilled water (containing 0.1% TFA) was added to 1.2 ml of the collected supernatant and 1.2 ml of an equal volume of PBS. HLB1cc (30 mg)) was applied. TCA and non-adsorbed substances in the column were washed and removed sequentially with 2 ml of distilled water (containing 0.1% TFA) and 2 ml of 5% methanol (containing 0.1% TFA), and then CPP contained in 2 ml of 50% acetonitrile (containing 0.1% TFA). The fraction was eluted. The obtained CPP-containing fraction was dried using a centrifugal concentrator. As a calibration curve sample, CPPIII (standard substance) was dissolved in a phosphate buffer solution (PBS).
The competitive ELISA method was performed according to the procedure described below.
A pretreated dairy product sample was dissolved in 0.5 ml of PBS to prepare a sample stock solution. All sample stock solutions were further diluted 10-fold with PBS. The calibration curve sample was prepared by dissolving and diluting CPPIII (standard substance) with PBS to 6.25-100 μg / ml. Preincubation of dairy samples and calibration curve samples in U-bottom 96-well plates (FALCON # 351177) at a ratio of 1: 1 (50 μl: 50 μl) to mAb 1A5 (2 μg / ml) diluted in 2% BSA / PBS (37 ° C, 1.5 hours). A 96-well plate (Nunc MaxiSorp) for measurement was coated with CPPIII (50 μg / ml) dissolved in PBS at 100 μl / well at 37 ° C. for 2 hours or more, and then blocked with 1% BSA / PBS at 37 ° C. for 1 hour. The sample pre-incubated on the blocking measurement plate and the reaction product of mAb 1A5 were transferred at 50 μl / well and reacted at 37 ° C. for 1 hour. Subsequently, a secondary antibody (6000 times diluted with 1% BSA / PBS) was detected in order to detect mAb 1A5 bound to β-CPP in CPPIII coated on the plate by removing the unreacted competitor with a washing solution. 100 μl / well of peroxidase-labeled rabbit anti-mouse immunoglobulin antibody (Rockland) was added and incubated at 37 ° C. for 1 hour. Next, the unreacted secondary antibody was removed by a washing solution, and then a substrate coloring solution (DAKO ^™ TMB + SUBSTRATE-CHROMOGEN) was dispensed at 100 μl / well. After a color reaction at room temperature for 5 minutes, 100 μl / well of 1N sulfuric acid aqueous solution was added to stop the reaction, and absorbance (OD 450 nm) was measured using a plate reader. The amount of CPP in the dairy product sample was calculated from a calibration curve prepared by plotting the absorbance corresponding to each CPP concentration of the calibration curve sample on x (logarithmic) and y-axis, respectively. A calibration curve was prepared for each measurement, and data measured on the same plate as the dairy product sample was used.
A calibration curve obtained by carrying out the competitive ELISA method for a calibration curve sample prepared with a CPP concentration of 6.25-100 μg / ml at a common ratio of 2 is shown in FIG.
Next, the absorbance of each cheese sample was applied to the approximate function obtained from the calibration curve and multiplied by the dilution factor to obtain the amount of CPP blended (Table 1).

  This method combines a pretreatment consisting of acid treatment and solid-phase extraction with a competitive ELISA method using a monoclonal antibody specific for β-CPP. The amount could be specifically quantified.

[Example 2] Examination of epitope region of monoclonal antibody (mAb 1A5) Peptide 1 to 16 residues (β-CPP (f1-16)), Peptide 20 to 25 residues (β The reactivity of -CPP (f20-25)) and β-CPP (f1-25) with mAb 1A5 was examined by competitive ELISA (FIG. 2).
mAb 1A5 reacts strongly with β-CPP (f1-25) but not with β-CPP (f1-16) and β-CPP (f20-25), so the epitope is from peptide 17 to 19 It was suggested that the region contains a phosphorylated serine cluster of residues.

Example 3 Effect of Serine Residue Dephosphorylation on Monoclonal Antibody (mAb 1A5) Reactivity In order to confirm that serine residue phosphorylation is important for the reaction with an antibody, CPP 0.2- Dephosphorylated with 0.6N NaOH at 37 ° C for 3 hours (B. Jiang and Y. Mine (2000). Preparation of novel functional oligophosphopeptides from hen egg yolk phosvitin. J. Agric. Food. Chem. 48, 990- 994.). Subsequently, after neutralizing with hydrochloric acid, the phosphate group released by solid phase extraction was removed, and the dephosphorylated CPP-containing fraction was recovered. The control group neutralized immediately after the start of NaOH treatment to stop the dephosphorylation reaction. The Ames method was used to measure the amount of P contained in CPP (BN Ames (1966) Assay of Inorganic Phosphate, Total Phosphate. Methods in Enzymology VIII, pp. 115-118). Although the amount of P in the CPP decreased in proportion to the treatment concentration of NaOH (Fig. 3), it did not affect the peptide content (Fig. 4). The CPP content in the NaOH-treated sample measured by competitive ELISA using mAb 1A5 decreased in proportion to the progress of dephosphorylation (FIG. 5). The above results support that mAb 1A5 recognizes a region containing CPP, particularly a phosphorylated serine residue sequence.

[Example 4] Specificity of monoclonal antibody (mAb 1A5) reaction Trypsin digestion of bovine casein yields four major phosphorylated peptides (Table 2).

Bovine α and β caseins were trypsinized to prepare phosphorylated peptides, and then four types of phosphorylated peptides were purified by HPLC (EC Reynolds, PF Riley and NJ Adamson (1994). A selective precipitation purification procedure for multiple phosphoseryl- containing peptides and methods for their identification. Anal. Biochem. 217, 277-284.). The peptide specificity of mAb 1A5 was examined by competitive ELISA (FIG. 6).
As shown in FIG. 6, among the four major phosphorylated peptides generated by trypsin digestion of bovine casein, mAb 1A5 showed β-CPP-specific reactivity in the competitive ELISA assay system constructed this time.

[Example 5] Measurement of CPP-ACP content blended in dairy product (trial product) 1 g of CPP-ACP base material (Cadbury Schweppes) used for trial production as a calibration curve sample was weighed, and 100 ml volumetric flask with distilled water After being dissolved in (10 mg / ml), 2.5 ml was dispensed into a tube and dried by a centrifugal concentrator (25 mg / tube). Subsequently, 5 ml of milk was added to 25 mg of CPP-ACP drug substance and dissolved sufficiently (5 mg / ml), and then serially diluted with milk so that the final concentrations were 1, 2 and 3 mg / ml. The concentration of CPP-ACP in milk without CPP-ACP was 0 mg / ml.
The sample preparation method was performed according to the procedure described below.
Take 0.3 ml of dairy sample in a plastic tube, add 1.2 ml of 15% trichloroacetic acid (TCA) and then 1.5 ml of PBS while stirring with a vortex mixer, and centrifuge to remove acid-denatured casein and high molecular weight protein from whey (17000 xg, 6 min, 4 ° C). A 1.2 ml solid phase extraction column (Waters OASIS ^TM ) equilibrated with 2 ml of 100% methanol and 2 ml of HPLC distilled water (containing 0.1% TFA) was added to 1.2 ml of the collected supernatant and 1.2 ml of an equal volume of PBS. HLB1cc (30 mg)) was applied. TCA and non-adsorbed materials in the column were washed and removed with 2 ml of distilled water (containing 0.1% TFA) and 2 ml of 5% methanol (containing 0.1% TFA), and then CPP contained in 2 ml of 50% acetonitrile (containing 0.1% TFA). The fraction was eluted. The obtained CPP-containing fraction was dried using a centrifugal concentrator. The calibration curve sample was also pretreated in the same manner as the dairy product sample.
The competitive ELISA method was performed according to the procedure described below.
A pretreated dairy product sample and a calibration curve sample were dissolved in 0.5 ml of PBS to obtain a specimen stock solution. All specimen stock solutions were further diluted 5-fold with PBS, and a U-bottom 96-well plate (FALCON # 351177) with a ratio of 1: 1 (50 μl: 50 μl) to mAb 1A5 (2 μg / ml) diluted with 2% BSA / PBS. ) Was preincubated (37 ° C., 1.5 hours). A 96-well plate (Nunc MaxiSorp) for measurement was coated with CPPIII (50 μg / ml) dissolved in PBS at 100 μl / well at 37 ° C. for 2 hours or more, and then blocked with 1% BSA / PBS at 37 ° C. for 1 hour. The preincubated specimen and mAb 1A5 reaction product were transferred to a blocking measurement plate at 50 μl / well and reacted at 37 ° C. for 1 hour. Subsequently, the unreacted unreacted product was removed with a washing solution, and a secondary antibody diluted 6000 times with 1% BSA / PBS to detect mAb 1A5 bound to β-CPP in CPPIII coated on the plate ( 100 μl / well of peroxidase-labeled rabbit anti-mouse immunoglobulin antibody (Rockland) was added and incubated at 37 ° C. for 1 hour. Next, the unreacted secondary antibody was removed with a washing solution, and then a substrate coloring solution (DAKO ^™ TMB + SUBSTRATE-CHROMOGEN) was dispensed at 100 μl / well. After a color reaction at room temperature for 5 minutes, 100 μl / well of 1N sulfuric acid aqueous solution was added to stop the reaction, and absorbance (OD 450 nm) was measured using a plate reader. The amount of CPP-ACP in the dairy product sample was calculated from the calibration curve prepared by plotting the absorbance corresponding to each CPP-ACP concentration of the calibration curve sample on the x and y axes, respectively. A calibration curve was prepared for each measurement, and data measured on the same plate as the dairy sample was used.
FIG. 7 shows calibration curves obtained by performing pretreatment and competitive ELISA for calibration curve samples prepared with CPP-ACP concentrations of 0, 1, 2, and 3 mg / ml. A linear equation was applied as an approximate function of the calibration curve.
Next, the absorbance of the dairy sample (n = 3) was applied to the approximate function obtained from the calibration curve to obtain the CPP-ACP blending amount (Table 3).

[Example 6] Measurement of β-CPP content contained in unit CPP-ACP amount
FIG. 8 shows a calibration curve obtained by performing the competitive ELISA method for a calibration curve sample for β-CPP measurement prepared at a concentration of 0 to 10 μg / ml. An exponential curve was fitted as an approximate function of the calibration curve.
Next, the absorbance corresponding to each concentration of CPP-ACP was applied to the approximate function obtained from the calibration curve to obtain the converted value to β-CPP, and the β-CPP content contained in the unit CPP-ACP amount was obtained. (Table 4).

From Table 4, the β-CPP content in 1 mg of CPP-ACP drug substance was 0.109 mg.
The dairy sample contains 2.67 mg / ml of CPP-ACP (from Example 5),
Since the β-CPP content in 1 mg of CPP-ACP is 0.1090 mg (from Example 6), the amount of β-CPP compounded in the dairy product sample is 0.29 (= 2.67 × 0.109) mg / ml.

  The present invention can measure CPP and CPP complex easily and with high accuracy and specificity without using special equipment or expensive equipment. In particular, it is suitable for the measurement of the amount of CPP, CPP complex and β-CPP contained in a solution containing a large amount of substances similar to CPP and CPP complex to be blended such as dairy products. As a use, it can be applied to the standard and quality control of products in which CPP and CPP composite materials are blended with food or medicine. Furthermore, it is useful as an analytical tool for basic research such as pharmacokinetics and pharmacological studies.

It is a graph which shows the calibration curve of CPPIII (standard substance). 2 is a graph showing the reactivity of a monoclonal antibody (mAb 1A5) with β-CPP and chemically synthesized constituent regions. It is a graph which shows the concentration-dependent dephosphorylation reaction by NaOH processing. It is a graph which shows the influence on the protein amount accompanying a dephosphorylation reaction. It is a graph which shows that the reactivity with a monoclonal antibody falls as a dephosphorylation reaction advances. FIG. 5 is a graph showing specificity of β-CPP among monoclonal antibodies (mAb 1A5) among phosphorylated peptides derived from casein. It is a graph which shows the calibration curve of CPP-ACP (standard substance). It is a graph which shows the analytical curve of (beta) -CPP (standard substance).

Claims (3)

A method for quantifying β-casein phosphopeptide and / or β-casein phosphopeptide complex by immunoassay using a monoclonal antibody (mAb 1A5) against the phosphorylated peptide described in SEQ ID NO: 1. The quantification method according to claim 1, which comprises an immunological assay comprising the following steps.
(1) Step of adding acid to sample and denaturing / removing protein (2) Step of removing acid by solid phase extraction (3) Purified sample eluted from solid phase for phosphorylated peptide of SEQ ID NO: 1 Immunological measurements are performed using a monoclonal antibody (mAb 1A5). The method according to claim 1 or 2, wherein the β-casein phosphopeptide and / or β-casein phosphopeptide complex in the dairy product is quantified.

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