JP2001153868A - Immunoassay for casein phosphopeptide (CPP) - Google Patents

Abstract

(57) [Summary] [Problem] To provide a simple and accurate method for measuring CPP in food. SOLUTION: It has been found that CPP in food can be simply and accurately measured by using an immunological measurement method using an anti-CPP antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an antibody that specifically recognizes casein phosphopeptide (hereinafter, referred to as “CPP”) and an immunoassay for CPP using the antibody.

[0002]

2. Description of the Related Art CPP is a phosphopeptide obtained by allowing trypsin to act on milk casein, and is obtained by separating and purifying only the phosphorus-containing peptide portion (Kenkichi Yokoyama, Kunio Yamauchi: "Milk Sogo Encyclopedia") Asakura Shoten: p520-523,1992). This CPP has a solubilizing effect on minerals such as calcium and iron, and is attracting attention as a substance that promotes the absorption of these minerals, and is widely used in the field of foods. Therefore, measuring the amount of CPP contained in these products is not only important in terms of quality assurance and component management, but also an indispensable technique for studying the functionality of CPP. However, as a method for measuring CPP in food, CPP is fractionated by high performance liquid chromatography (HPLC),
At present, it is measured as the sum of peaks considered to be PP (Hirayama, M. et al .: Biosci. Biotech.
Biochem., 56 (7): 1126-1127, 1992). In the case of this method, there is no confirmation that the peak obtained by fractionation is really CPP, and it is difficult to specifically measure CPP in a food containing a large amount of similar substances, and technical problems remain. Have been.

[0003]

DISCLOSURE OF THE INVENTION The present invention relates to a method for producing C in foods.
It is an object to provide a method for simply and accurately measuring PP.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have made it possible to easily and accurately measure CPP in foods by using an immunoassay using an anti-CPP antibody. Thus, the present invention has been completed.

That is, the present invention provides (1) an immunoassay for casein phosphopeptide characterized by using an anti-casein phosphopeptide antibody, and (2) a casein phosphopeptide insolubilized on a solid phase (immobilized on a solid phase). Casein phosphopeptide), a certain amount of an anti-casein phosphopeptide antibody is added together with the sample containing the casein phosphopeptide to be measured, and a competition reaction between the immobilized casein phosphopeptide and the casein phosphopeptide in the sample with the antibody is performed. After forming an immune complex composed of CPP and an anti-casein phosphopeptide antibody on the solid phase, separating the immune complex (bound; B) on the solid phase from the liquid layer part (free; F), The amount of casein phosphopeptide to be measured is determined by measuring the signal of the antibody on the phase, the immunoassay of (1), (3) After reacting a sample containing casein phosphopeptide to be measured with a certain amount of an enzyme-labeled anti-casein phosphopeptide antibody in advance, adding the reaction solution to the immobilized casein phosphopeptide, (4) A second antibody against the anti-casein phosphopeptide antibody is added, and the signal of the second antibody is measured (2) or (3). (5) The signal is an enzyme label (2). Or (4) the immunoassay according to (1) to (5), wherein the anti-casein phosphopeptide antibody is a polyclonal antibody; and (7) the casein phosphopeptide immobilized on a solid phase. And a kit for measuring casein phosphopeptide comprising a reagent containing an anti-casein phosphopeptide antibody.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In a preferred embodiment of the immunoassay for CPP of the present invention (hereinafter referred to as "EIA"), a preferred embodiment is a CPP based on a competitive reaction between an immobilized CPP and a CPP in a sample for an anti-CPP antibody in a reaction system. EIA is provided. For this EIA, purified or unpurified CPP is bound to a support (solid phase) and insolubilized (hereinafter referred to as “solid-phased CPP”).
). The binding of the CPP to the insoluble carrier can be performed physically (adsorption) or chemically. In order to prevent non-specific adsorption of the antibody to the solid phase, the solid phase is blocked with bovine serum albumin (BSA) or the like. Test solution containing an unknown concentration of CPP (hereinafter referred to as "specimen")
Is labeled with a CPP-specific labeled antibody (hereinafter “anti-CPP antibody”).
Or simply referred to as an antibody). At this time, the antibody titer is checked in advance, and an antibody titer diluted to 10 ³ to 10 ⁵ is used. The reaction conditions should be appropriately set according to the CPP content of the sample, and for example, CPP is 0.1 to 10 μg
In the case of / ml, the standard is about 30 minutes at room temperature. The CPP in the sample is compared with the immobilized CP for binding to the labeled antibody.
Compete with P. After removing the unbound part by washing,
The signal of the labeled antibody bound to the solid phase is measured. A standard curve (hereinafter referred to as "calibration curve") in which the amount of CPP in the sample has been prepared in advance using a standard solution containing a known concentration of CPP.
From the above, the CPP content in the sample is determined.

[0007] If the amount of CPP in the sample is high, the labeled antibody will not bind to the solid phase. Conversely, CPP in the sample
If the amount is low or absent, much of the labeled antibody will bind to the solid phase. To determine the amount of CPP, the sample is diluted with the assay buffer so that it falls within the measurement range of the calibration curve. The degree of purity required for the CPP used for immobilization is determined by the specificity of the antibody. Polyclonal antisera will contain foreign antibodies that bind to and give a signal to impure CPP.
Due to their specificity, in the case of impure CPPs, monoclonal antibodies or affinity purified polyclonal antibodies are used.

One problem encountered in this EIA is that the labeled antibody may preferentially bind to CPP on the solid phase. The quantification of this EIA depends on two avidities: the avidity for the immobilized CPP and the antibody avidity for the CPP in the sample. These avidities may not be the same. Antibodies may bind divalently to these antigens because CPPs on the solid phase are locally present at high concentrations. These types of bivalent interactions cannot occur in solution when the antigen is monovalent. Many, but not all, immobilized CPPs will promote bivalent binding. Problems caused by differences in avidity can be found by comparing standard curves with known concentrations of CPP solution. If these EIAs give unusual results, several solutions can be used. First, try to lower the CPP concentration on the solid phase. Thus, the local concentration is reduced and the chance of divalent binding is reduced. Second, a labeled Fab fragment is used. Third, the analyte and the labeled antibody are pre-mixed and incubated for 30 minutes to 1 hour before being added to the solid phase.

In the third method, a second antibody against the anti-CPP antibody is used, and the signal of the second antibody is measured.

C used in the EIA of the CPP of the present invention
Antibodies that specifically recognize PP include antisera (polyclonal antibodies) and monoclonal antibodies. Polyclonal antibodies have the bonus effect of "increased affinity (avidity)" and "specificity" (P. Tijssen,
It is suitable for use in the EIA of the present invention because it is translated by Eiji Ishikawa, “Enzyme Immunoassay, Biochemical Experimental Method 11”, Tokyo Kagaku Dojin, 1989, pp. 54-56).

[0011] CPP used as an immunogen in preparing an anti-CPP antibody includes all the casein degradation products including the CPP fraction obtained by allowing casein to act on trypsin.
For example, alpha _s1 - casein, alpha _s2 - casein, obtained from β- casein, alpha _s1 - casein _{-7P (f43-79), α s2 -CN} -4P
(f46-70), α _s2- CN-4P (f1-32), β-casein-4P (f1-2
8), etc. (Kototada Ono: Japanese Journal of Dairy
and Food Science. 43 (4), 1994).

Each of these CPPs has a low molecular weight (5,000 or less) and, when used alone, has low immunogenicity, and is conjugated to a carrier protein to immunize animals. CPP
Many methods for binding proteins to carrier proteins are described in Eiji Ishikawa et al. (Eds.), “Enzyme immunoassay”, Medical Shoin, 1982, p128; Eiji Ishikawa, “Ultrasensitive enzyme immunoassay”, Academic Press Center, 1993, p41; Harlow and Lan
e: Antibodies [Antibodies: A Laboratory Manual ed.
by Harlow and Lane (Cold Spring Harbor Laboratory
Press: p319, 1988)], and those skilled in the art can prepare a complex of CPP and a carrier protein with reference to these documents.

When an animal is immunized with this complex as an immunogen to obtain an anti-CPP antibody, the characteristics of the polyclonal antibody differ depending on the animal species, strain, individual, breeding environment, and immunization method, and its prediction is extremely difficult. . Therefore, although it is difficult to select an immunized animal, a rabbit is tried first, and if that is not possible, a guinea pig, rat, mouse, chicken, etc. is tried to obtain a polyclonal antibody having as high a titer, affinity and specificity as possible. Regarding the preparation and purification of anti-CPP polyclonal antibodies, there are a number of documents relating to the preparation of polyclonal antibodies [for example, “Molecular Immunology III Antigen / Antibody / Complement (New Chemistry Laboratory Course 12)”, edited by The Japanese Biochemical Society, p1-31 , Tokyo Kagaku Doujin (1992); “Measuring Method Using Biological Activity (New Basic Biochemical Experimental Method 6)”, Masami Muramatsu
Et al. (Eds.), Maruzen, p109; Shinobu Okai, “Anti-Peptide Antibody Experimental Protocol”, Cell Engineering Separate Volume, p48, Shujunsha (1994); Harl
ow and Lane: Antibodies [Antibodies: A Laboratory
Manual ed.by Harlow and Lane (Cold Spring Harbor L
aboratory Press: p53, 1988)], and those skilled in the art can carry out the above with reference to these documents.

On the other hand, in the case of a monoclonal antibody, a mouse (sometimes a rat) is used as an immunized animal. Theoretically, the same antibody was used in the cell fusion method of Keller and Milstein (G. Koeller and Milstein, Nature, 256, 49
5-497 (1975)), and can be collected indefinitely from the culture solution of the hybridoma prepared. Regarding the preparation and purification of monoclonal antibodies specific to CPP, there are a number of documents relating to the preparation of monoclonal antibodies [for example, Tatsuo Iwasaki,
Et al., “Monoclonal Antibody”, Editorial Kodansha Scientific, Kodansha (1984); Nao Matsuhashi, Hiroshi Nakamura, Tsutomu Sugiura, “Molecular Immunology III Antigen / Antibody / Complement (New Chemistry Laboratory Course 12)”,
Edited by The Biochemical Society of Japan, p1-31, Tokyo Kagaku Doujin (1992); Shinobu Okai, “Anti-Peptide Antibody Experimental Protocol”, Cell Engineering Separate Volume, p48-74, Shujunsha (1994)] If it can be produced. Antibodies used in the present invention include F (ab ′) ₂ obtained by digesting IgG with pepsin, Fab ′ obtained by reducing F (ab ′) _2, and Fab obtained by digesting antibodies with papain. Antibody fragments can be used.

[0015] Enzyme labeling methods for antibodies are known [for example, Ha
Antibodies edited by rlow and Lane [Antibodies: A Labor
atory Manual ed.by Harlow and Lane (Cold Spring Ha
rborLaboratory Press: p319 (1988); Eiji Ishikawa, “Ultrasensitive enzyme immunoassay”, Gakkai Shuppan Center, p41 (199)
3)], and can be implemented by those skilled in the art with reference to these documents.

Examples of the support include polymers such as polyvinyl chloride, nitrocellulose, polystyrene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, agarose, and polysaccharide, as well as paper, glass, metal, and the like. These combinations can be mentioned. The shape of the support may be, for example, various shapes such as a plate, a sphere, a fiber, a particle, a bar, a disk, a container, and a test tube. As supports, polyvinyl chloride and nitrocellulose are most commonly used. Polyvinyl chloride is
Usually used as a microtiter plate. This microplate is suitable for the EIA of the present invention.

As the signal, an enzyme label is suitable. Examples of the enzyme include peroxidase, alkaline phosphatase, β-D-galactosidase and the like. When an enzyme is used as a labeling substance, it is conceivable that the binding is likely to be suppressed stereoscopically in the antigen-antibody reaction. In such a case, a labeling substance other than the enzyme, for example, a fluorescent substance, a radioactive substance, etc. can be used [for example, Hisashi Tanaka, Yo Yokoyama (eds.), “Development of Drugs (10) Diagnostics, Hirokawa Shoten (Heisei Year)"].

Examples of the fluorescent substance include fluorescein isothiocyanate and phycobiliprotein, and examples of the radioactive substance include ¹²⁵ I, ¹³¹ I and ¹⁴ I.
C, ³ H and the like can be used.

When the labeling substance is an enzyme, the binding method between the enzyme and the anti-CPP antibody can be a conventional method such as a glutaraldehyde method, a periodate method, or a maleimide method.
For example, the reaction can be carried out by reacting a maleimidated antibody or an antibody fragment with an SH-modified enzyme in a solution. Maleimidation of an antibody or antibody fragment can be performed, for example, by using succinimidyl-4- (N-maleimidomethyl) cyclohexane carbonate, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane carbonate, or succinimidyl-methmaleimidobenzoate (hereinafter abbreviated as MBS). ), Succinimidyl-6-maleimidohexanoate and the like. Introduction of an SH group into an enzyme can be performed by a known method ("Enzyme Immunoassay", 2nd edition, Eiji Ishikawa et al.
Medical Shoin 1982). For example, the reaction is carried out by reacting an enzyme with S-acetylmercaptosuccinic anhydride or N-succinimidyl-3- (2-pyridylthio) propionate.

An enzyme reaction is carried out by adding a substrate for the enzyme. For example, when using peroxidase, hydrogen peroxide is used as a substrate, and 2,2 is used as a color former.
2'-Azinodi- (3-ethylbenzthiazolinesulfonic acid) ammonium salt, 5-aminosalicylic acid, o-phenylenediamine, 4-aminoantipyrine, 3,3 ', 5,5'-tetramethylbenzidine, or the like is used. Further, 3- (p-hydroxyphenyl) propionic acid or the like can be used as a fluorescent substrate. When alkaline phosphatase is used as the enzyme, o-nitrophenyl phosphate, 3- (2′-
Spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1,2-dioxetane and the like, and when β-D-galactosidase is used as an enzyme, fluorescein di (β-D-galactopyrase) is used as a substrate. Noside) or 4-methylumbellifericyl-β
-D-galactopyranoside and the like are used in combination.

Another preferred embodiment of the EIA of CPP of the present invention provides an EIA of CPP by radioimmunoassay (RIA), fluorescence immunoassay, immunoagglutination, or the like.
Furthermore, the present invention includes a kit for measuring CPP in a solution comprising an anti-CPP antibody or a labeled anti-CPP antibody and CPP. Such a kit can include a secondary antibody for detecting an anti-CPP antibody and a reagent for detecting a labeling substance.

Preferably, the kit comprises a CPP and an anti-CPP
It consists of methods known to those skilled in the art for detecting the formation of an immune complex with an antibody, for example an ELISA.

C to be measured in the method of the present invention
The solution containing PP is a food, a medicine, or the like to which a decomposition product of CPP or casein is added. In addition, body fluids of humans or animals that have taken casein can also be measured. Further, even if the measurement target is a solid, it can be measured by dissolving it in an appropriate solution.

[0024]

The present invention will be described below with reference to examples and test examples, but the present invention is not limited to these examples.

Example 1 Preparation of Anti-CPP Polyclonal Antibody (1) Binding of CPP to Carrier Protein CPP used was CPP-III, a purified CPP manufactured by Meiji Seika. 20 mg of KLH was weighed and dissolved in 200 μl of 50% glycerol-0.01 M phosphate buffered saline (hereinafter abbreviated as PBS) (pH 6.0). 100 μl of dimethylsulfoxide (hereinafter “DMSO”) is added to this solution.
(Abbreviated as) and added with 3 mg of MBS.
The reaction was performed for 0 minutes. Further, after removing insolubles, gel filtration was performed using a G-25 column, and the protein fraction (KLH-
MBS). On the other hand, 10 mg of CPP-III was weighed and 1 ml of PBS (pH 6.0), 5 μl of 5N-NaOH,
It was dissolved in a mixed solution of 1 ml of DMSO. This CPP-II
The solution I and the KLH-MBS solution were mixed and reacted at room temperature for 1 hour. The solution thus obtained was added to PBS (pH 7.2) 2
L was dialyzed to obtain a CPP-III-KLH complex. (2) Preparation of anti-CPP polyclonal antibody 200 μg of the above CPP-III-KLH complex and Freund C
Emulsions with omplete Adjuvant were administered intradermally to rabbits. Two weeks later, the CPP-III-KLH complex 2
An emulsion of 00 μg and Freund Incomplete Adjuvant was administered intradermally. Further, the same intradermal administration was performed three times at 10-day intervals. One week after the final administration, blood was collected and used as serum, and the antibody titer was measured by enzyme immunoassay. That is, using a goat anti-rabbit IgG antibody labeled with horseradish peroxidase (hereinafter abbreviated as “HRP”) as a secondary antibody using CPP-III as an antigen, the antibody titer against CPP-III is determined to be 3000 times. It was confirmed. afterwards,
Partial blood was collected to obtain serum, which was purified by a protein A-Sepharose column. That is, a protein A-Sepharose column (gel volume) equilibrated with PBS (pH 7.2)
1 ml), 2 ml of serum diluted 10-fold with PBS (pH 7.2) was flowed, and after washing, the antibody was eluted with 0.2 M glycine-hydrochloric acid (pH 2.5) and dialyzed against PBS (pH 7.2). Thus, a purified antibody was obtained.

Test Example 1 CPP by Competition Method EIA
Of CP dissolved in PBS (pH 7.2) at a concentration of 100 μg / ml
Transfer the P-III solution to an EIA plate (MaxiSor, NUNC)
p) Dispensed 100 μl per well and incubated at room temperature for 1 hour. Next, the CPP-III solution is removed, and 1
% Bovine serum albumin (hereinafter abbreviated as BSA)-50 mM
Tris-HCl buffered saline (pH 8.2) (hereinafter “TB
S ") was dispensed in 200 μl aliquots and incubated at room temperature for 1 hour. Next, CPP-III (standard substance) was added to 0.1%.
TBS 50 contained in a concentration range of 16 to 20 μg / ml
1% BS containing μl and purified anti-CPP polyclonal antibody
50 μl of the A-TBS solution was mixed in advance, 50 μl thereof was added to the wells from which 1% BSA-TBS had been removed, and the mixture was incubated at room temperature for 1 hour. Next, after the solution in the well was removed, the well was washed with 0.1% Tween 80-PBS. Then dilute the enzyme-labeled antibody (20 mM Phospha
te Buffer, 0.15M NaCl, 0.5% BSA, 0.2
% Casein, 0.01% Microcide I, 0.8mg / ml ABTS, 0.06mg /
50 μl of HRP-labeled goat anti-rabbit immunoglobulin antibody (Cappel) diluted 4000-fold with ml uric acid, pH 7.2)
Was added and incubated at room temperature for 1 hour. Next, after removing the labeled antibody solution, the substrate was washed with 0.1% Tween 80-PBS, and a substrate color solution (DAKO TMB + Substrate-Chromoge) was used.
n No.S1599) was dispensed at 100 μl each. After the reaction for 15 minutes, 100 μl of a 2 N aqueous sulfuric acid solution was added to stop the reaction.

Next, the absorbance of this solution at a wavelength of 450 nm was measured using a spectrophotometer. This absorbance was plotted against a standard substance concentration of 0.16 to 20 μg / ml to obtain a calibration curve. This calibration curve is shown in FIG. From FIG. 1, when the measurement method of the present invention is used, 0.16 to 20 μg / m
It can be seen that the measurement can be accurately performed in the range of l.

Test Example 2 Measurement of CPP-III in Solution by EIA in the Presence of Casein at Various Concentrations Casein was determined to be 0, 1, 10, 100, 1000, and 2
A solution contained at a concentration of 000 μg / ml was measured with the EIA of the present invention to prepare a calibration curve. The results are shown in FIG. CP
Linearity is observed when P is in the concentration range of 0.1 to 10 μg / ml. That is, it was shown that the EIA of the present invention can quantify CPP even in the presence of casein.

[Test Example 3] CP in milk by EIA
Measurement of P-III The amount of CPP in milk was measured for sterilized milk to which CPP-III was added at a concentration of 250 μg / ml. Based on the method shown in Example 2, the prepared milk without CPP-III was added to T
CPP-III (standard substance) was dissolved in a solution diluted 200-fold with BS to prepare a standard solution for preparing a calibration curve, and a sample sample was similarly diluted 200-fold with TBS and measured. As a result, it was measured to be 254 μg / ml.

[0030]

According to the present invention, it has been clarified that CPP in a solution can be measured simply and accurately. In addition, for example, it is possible to accurately measure even a solution containing a large amount of a similar substance of CPP such as in milk, which is effective not only for quality control of foods and pharmaceuticals containing CPP but also for CPP.
In basic research on PP, it is thought that it is also effective in observing its pharmacokinetics.

[Brief description of the drawings]

FIG. 1 shows a calibration curve of CPP-III.

FIG. 2 shows the results of measuring CPP in the presence of various concentrations of casein.

Claims (7)

[Claims] 1. An immunoassay for casein phosphopeptide, which comprises using an anti-casein phosphopeptide antibody. 2. A casein phosphopeptide immobilized on a solid phase (immobilized casein phosphopeptide), a sample containing the casein phosphopeptide to be measured, and a fixed amount of an anti-casein phosphopeptide antibody are added thereto. A competitive reaction between the immobilized casein phosphopeptide and the casein phosphopeptide in the sample forms an immune complex composed of CPP and an anti-casein phosphopeptide antibody on the solid phase, and the immune complex (bound; B ) And the liquid phase (free; F), and then measuring the signal of the antibody on the solid phase to determine the amount of casein phosphopeptide to be measured. Law. 3. The method according to claim 2, wherein a sample containing the casein phosphopeptide to be measured is reacted with a predetermined amount of an enzyme-labeled anti-casein phosphopeptide antibody in advance, and the reaction solution is added to the immobilized casein phosphopeptide. Immunological assay. 4. The immunoassay according to claim 2, wherein a second antibody against the anti-casein phosphopeptide antibody is added, and the signal of the second antibody is measured. 5. The immunoassay according to claim 2, wherein the signal is an enzyme label. 6. The immunoassay according to claim 1, wherein the anti-casein phosphopeptide antibody is a polyclonal antibody. 7. A kit for measuring casein phosphopeptide, comprising a casein phosphopeptide insolubilized on a solid phase and a reagent comprising an anti-casein phosphopeptide antibody.