JP2762058B2 - Method for quantitative determination of carbonyl compound-protein complex - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for quantifying a carbonyl compound-protein complex.

[0002]

BACKGROUND OF THE INVENTION Proteins react with carbonyl compounds such as acetaldehyde for various reasons to form carbonyl compound-protein complexes. Such denaturation of proteins by chemical modification is directly linked to aging of plant seeds, aging of cells, deterioration of foods, and the like.
For example, in foods, it is known that a Maillard reaction that occurs non-enzymatically between a reducing sugar having a carbonyl group and a protein causes protein degradation, resulting in a reduction in food value. It is also known that a similar reaction occurs in aging of cells associated with diabetes, and a carbonyl compound-protein complex is formed. It is also known that a similar reaction occurs between acetaldehyde, which is an ethyl alcohol oxide, and a protein in alcohol dependence or alcohol poisoning, and an aldehyde-protein complex is formed.

[0003] Therefore, quantification of a carbonyl compound-protein complex can play an important role in the investigation of the quality of plant seeds and foods, the progress of diabetes, and the diagnosis of alcoholism and the like.

It is expected that an immunological analysis using an antibody will be effective as a method for quantifying such a carbonyl compound-protein complex. However, in the Maillard reaction, various types of reducing sugars are involved in the reaction, and the types of conjugates to be formed are also various. Therefore, it is difficult to prepare an antibody that can be applied to these various types of conjugates. for that reason,
Versatile analytical reagents or diagnostics have not yet been realized.

[0005] In view of the above circumstances, it is known in advance that the antibody exhibits toxicity, and an antibody is prepared using a complex of a volatile carbonyl compound and a protein, which is rich in membrane permeability and mobility, as an antigen. It seems quite reasonable to use this as an analytical or diagnostic reagent. From this viewpoint, in studies on alcoholism or alcoholism, attempts have been made to quantitatively analyze a complex of acetaldehyde and a protein by an antigen-antibody method. However, in this case, a so-called sandwich method using a purified polyclonal antibody is employed, but the sensitivity is reduced during the purification process. On the other hand, when a monoclonal antibody is used, not only a complicated procedure is required for preparing the monoclonal antibody, but also a highly sensitive monoclonal antibody has not been successfully prepared. Furthermore, there is a problem that it is extremely difficult to grasp the total amount of the complex formed from acetaldehyde and various proteins existing in the living body.

[0006]

The present invention has been made in view of the above circumstances, and can be applied to a wide range of quantification of a carbonyl compound-protein complex, does not require a complicated technique for preparing a monoclonal antibody, and An object of the present invention is to provide a method for quantifying a carbonyl compound-protein complex, which allows the total amount of a carbonyl compound-protein complex contained in a sample to be ascertained.

[0007] Such a method has the following utility in various fields. The first is the prediction of seed marching by quantifying the degree of aging of plant seeds, for which the importance of conservation as a genetic resource has been pointed out.

[0008] The second is to measure the degree of deterioration of the food itself. The third is the search for antigens in the onset of food allergy patients. Fourth, to establish a method for quantifying the degree of progression of alcoholism and alcoholism. Today, alcohol is sold at vending machines,
Because the price is also at a level that can be reached by young people,
Alcoholism, which is already becoming a social problem in developed countries, is expected to increase in Japan as well.
Therefore, it is desired to introduce a simple blood test related to this as one item of the medical examination. The present invention is intended to provide a simple analysis kit and a diagnostic kit that can meet such a demand.

[0009]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies in order to achieve the above-mentioned object. As a result, during the ELISA measurement, a special pre-coating was applied to the immunoassay container to obtain a polyclonal antibody. The present inventors have found that sufficiently high-precision measurement can be performed by using this method, and have reached the present invention.

That is, the method for quantifying a carbonyl compound-protein complex according to the present invention uses a polyclonal antibody as a primary antibody reagent when measuring the amount of a carbonyl compound-protein complex contained in a sample by ELISA. It is characterized in that a predetermined pre-coating is applied to the immunoassay container. This antibody reagent is prepared using, as an immunogen, for example, a carbonyl compound-protein complex mixture obtained by reacting acetaldehyde with an appropriate protein source. The precoating is performed by incubating a solution of a carbonyl compound-carrier protein complex obtained by reacting a predetermined concentration of acetaldehyde with an appropriate carrier protein in an immunoassay container.

More specifically, the assay method according to the present invention is characterized in that (a) it is known that it is toxic to an appropriate protein source such as bovine serum albumin in advance, and is rich in membrane permeability and mobility. Reacting a volatile carbonyl compound (eg, acetaldehyde) at a predetermined concentration to form a carbonyl compound-protein complex mixture, and preparing an immunogenic composition containing the complex mixture;
(B) immunization by administering the immunogenic composition to a mammal, and isolating an antibody against the carbonyl compound-protein complex from the serum of the immunized mammal; c) preparing an assay solution containing a substantial portion of the carbonyl compound-protein complex present in the analyte by subjecting the analyte to an appropriate treatment such as extraction or cell destruction; ,
(D) a step of preparing a reaction solution by adding a solution of the antibody adjusted to a predetermined concentration to the measurement solution; and (e) a step of preparing an appropriate carrier protein.
By reacting the volatile carbonyl compound (for example, acetaldehyde) at a predetermined concentration, a carbonyl compound-carrier protein complex is formed, and the solution of the carrier protein complex is accommodated in an immunoassay container and incubated, whereby the solution is incubated. Pre-coating the immunoassay container; and (f) containing the reaction solution in the pre-coated immunoassay container and incubating the same, so that the carbonyl compound-
Reacting the protein complex with the antibody to form an antigen-antibody complex, and binding the antigen-antibody complex to the carbonyl compound-carrier protein complex precoated on the immunoassay container; A) adding a solution of an enzyme-labeled secondary antibody to the antibody to the immunoassay container and incubating the secondary antibody with the antibody, and (h) binding the secondary antibody to the antibody. Adding a sex substrate solution to the enzyme bound to the secondary antibody and reacting the solution, and measuring the product of this enzyme reaction; and (i) using a standard solution having a known concentration of the carbonyl compound-protein complex. creating a standard curve by performing the steps a to i;
(J) determining the concentration of the carbonyl compound-protein complex contained in the measurement solution by comparing the measurement result in the step h with the standard curve obtained in the step i. It is characterized by having done.

[0012]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The embodiments of the present invention will be described below step by step. (1) Preparation of antibody In a 50 mM sodium carbonate buffer (pH 9.5), 1 mM of a volatile carbonyl compound (for example, acetaldehyde), 2 mg / ml of a suitable protein source (for example, BSA), and 10 mM of nNaCNBH ₃ Is reacted for 3 days at room temperature. The reaction mixture is then
Phosphate buffer containing saline and KCl (PBS; pH
Perform dialysis against 7.4). Preferably, 1.2 ml of Freund's complete adjuvant is mixed with the carbonyl compound-protein complex thus artificially synthesized, and the mixture is used as an antigen.

The above-prepared antigen is injected subcutaneously into rabbits at a rate of twice a week for two weeks, and the serum collected from the rabbit is examined for the antibody value against the antigen, and injected subcutaneously until the value reaches the maximum value. To continue. Normally,
Antibody levels reach a maximum after 5 to 6 subcutaneous injections. Thereafter, blood is collected from the immunized rabbit and the serum is separated. In some cases, a 33% ammonium sulfate precipitated portion is collected and dialyzed against PBS and used as an antibody. However, it has been found that for the purposes of the present invention, purification by affinity chromatography is unnecessary.

(2) Measurement of titer by ELISA method The measurement of the carbonyl compound-protein complex contained in the sample is performed by the following procedure using the ELISA method.

A) Pre-coating of the immunoplate:
1 mg / ml gelatin, 1 mM volatile carbonyl compound (specifically, acetaldehyde) and 10 mM Na
After reacting the mixture with CNBH ₃ for 3 days, the PB
Dialyze against S. The concentration of the protein complex thus obtained is adjusted to 0.1 mg / ml.

After pre-coating the immunoplate with 80 μl of the above-adjusted concentration for 1 hour, the solution is removed and washed with PBS. b) Preparation of measurement solution: The measurement solution containing the carbonyl compound-protein complex in the sample is prepared as follows.

First, when the sample is plant tissue, PBS
After extraction and centrifugation, the supernatant is used directly as a measurement solution. On the other hand, when the sample is blood, only erythrocytes are taken out by centrifugation, washed with PBS, and then lysed by adding water to use as a measurement solution.
In each case, the solution contains a carbonyl compound-protein complex.

Next, the previously prepared antibody is diluted to a certain multiple, and this diluted antibody solution is added to 2.5 to 10 mg / ml of the measurement solution prepared above. Further, the absolute amount of the measurement solution is adjusted to 20 mg / ml by using a PBS solution of ovalbumin protein. The measurement solution thus prepared is transferred to a sample tube, stirred, and stored at 4 ° C. overnight.

C) Measurement procedure 50% of the measurement solution (sample / antibody mixture) prepared above was used.
The μl is placed in the wells of the immunoplate precoated in a) and left at 25 ° C. for 30-60 minutes. Thereafter, the measurement solution was removed, and 0.05% Tween was used.
Wash 4 times with a PBS solution containing en20.

Next, anti-rabbit IgG (TAGO) as a secondary antibody to the rabbit antibody, labeled with alkaline phosphatase, was added and incubated for 60 minutes. Thereafter, p-nitrophenol phosphate, which is a substrate of alkaline phosphatase, is added for color development, and the absorbance at a wavelength of 405 nm is measured.

D) Calculation of Data In order to grasp the total amount of the carbonyl compound-protein complex, a method for preparing a standard line was devised. That is, in order to widely cover the concentration range of the carbonyl compound-protein complex, the concentration of the complex contained in the standard solution was set to 0.01 mM and 0.1
mM and 1 mM, by treating ovalbumin or hemoglobin albumin by the same means as described above,
Prepare standard lines for different protein complex concentration levels. Then, the relative binding ratio (RB) between the sample and the modified ovalbumin is calculated by the following formula, and the total amount of the carbonyl compound-protein complex present in the living body is calculated.

In this case, the background value (A
_In order to calculate _B ), ovalbumin at a high concentration of 2 mg / ml (10 mM) was added in the presence of 10 mM NaCNBH _3.
The absorbance resulting from the complex formed by treatment with the carbonyl compound of M) (in this case using acetaldehyde) is used. (A _M ) is the absorbance of 20 mg / ml ovalbumin itself. (A _S ) is the absorbance of the sample solution or ovalbumin-carbonyl compound complex.

BR = (A _M −A _S ) × 100 / (A _M −A _B ) In the above embodiment, the most important step characterizing the present invention is the pre-coating of the immunoplate.
In the present invention, this precoating technique substantially determines the measurement sensitivity, and enables the quantification of a carbonyl compound-protein complex having a low level in vivo.

In the present invention, in the pre-coating step, a complex in which a volatile carbonyl compound and a carrier protein are bonded is artificially synthesized and used for pre-coating. In such a case, there may be a carbonyl compound concentration (0.5 to 1.0 mM in the case of acetaldehyde concentration) appropriate for the in vivo level of the test substance, and an optimal carrier protein concentration (up to 16 mg / ml) corresponding thereto. Do you get it. To obtain such an optimal concentration,
After dialysis, it may be concentrated by an ultrafilter. Also, it was found that gelatin was most suitable as the carrier protein.

According to such a preferred embodiment, the background can be suppressed to a low value of 0.04 to 0.05 at the time of color development using p-nitrophenol phosphate, and the measurement accuracy can be increased. As a result, it became possible to determine the total amount of proteins modified with various carbonyl compounds, that is, the total amount of carbonyl compound-protein complexes, from a low concentration.

[0026]

Embodiments of the present invention will be described below. In addition,
The following examples are in accordance with the preferred embodiments of the present invention described above, and used acetaldehyde as the carbonyl compound and gelatin as the carrier protein in the precoating.

Example 1: Preparation of a standard line A standard line was prepared as described in the embodiment (2) d) of the invention. At this time, standard solutions containing a complex in which 2 mg / ml ovalbumin and 1 mM, 0.1 mM and 0.01 mM acetaldehyde were bound in the presence of 10 mM NaCNBH ₃ were prepared, and these standard solutions were prepared. Was used to create a standard straight line. The result is shown in FIG. In the figure, ○ represents a standard straight line with an acetaldehyde concentration of 1 mM,
Δ indicates a standard straight line at an acetaldehyde concentration of 0.1 mM, and □ indicates a standard straight line at an acetaldehyde concentration of 0.01 mM.

Example 2: Relationship between reduced vigor of lettuce seeds treated with gaseous aldehyde and content of carbonyl compound-protein complex Lettuce seeds are treated with gaseous aldehyde at a predetermined concentration, and the treated seeds are extracted with PBS. After centrifugation, the supernatant was used as a measurement solution. Using this measurement solution, the total amount of the carbonyl compound-protein complex was measured according to the method described in the embodiment of the invention.

The germination status of the seeds treated as described above was examined, and the vitality of the seeds was measured as a percentage of the germinated seeds. The result is shown in FIG. The base axis in the figure indicates the relative humidity (RH) of the atmosphere in which the seeds were subjected to the acetaldehyde treatment. It can be seen that the higher the relative humidity, the higher the metabolic activity of the seed, and as a result, the higher the degree of protein modification by acetaldehyde. O-Al means an ovalbumin complex prepared with 1 mM acetaldehyde.

From these results, it can be seen that the measured value of the total amount of the carbonyl compound-protein complex according to the method of the present invention well reflects the vitality of the seed. This indicates that the results obtained by the measurement method according to the present invention are sufficiently accurate to be able to be used as an appropriate index of degeneration and deterioration of seeds.

Example 3: Relationship between the vitality of various naturally aged seeds and the content of the carbonyl compound-protein complex In the pea, lettuce, burdock, soybean and carrot seeds, the reduced vitality and the carbonyl The relationship with the content of the compound-protein complex was examined. The seed sample used had a storage period of 1 to 17 years after harvest.

Regarding the seed vitality, as in the case of Example 2,
It was measured in% of germinated seeds. Also, carbonyl compounds
-Regarding the content of the protein complex, as in Example 1, seeds were extracted with PBS, centrifuged, and the supernatant was used as a measurement solution. The result of the measurement is shown in FIG. In the figure,

[0033]

[Table 1]

From these results, it is understood that the measurement method of the present invention can be sufficiently used as an indicator of aging of naturally deteriorated seeds. Example 4 Application to Diagnosis of Alcoholism Blood was collected from healthy alcohol-independent (-), mildly alcoholic (+) and severely alcoholic (++) patients. From the blood, only red blood cells were taken out by centrifugation, washed with PBS, added with water and lysed, and used as a measurement solution.

Using this measurement solution, the total amount of the carbonyl compound-protein complex was measured by applying the method described in the section of the embodiment of the invention. Table 1 shows the results. Table 1 Alcohol carbonyl compound-protein complex complex Dependent total amount (O-Al (mg) / protein (g))-0.26 + 4.67 ++ 18.68 From the above results, the method of the present invention It turns out that the measured value is extremely useful as an index for diagnosing the presence or absence of alcohol dependence and the degree thereof. This result also shows that the accuracy of the measuring method according to the invention is significantly higher.

[0036]

As described in detail above, according to the present invention,
Highly accurate total amount of carbonyl compound-protein complex using polyclonal antibodies that can be prepared relatively easily without using monoclonal antibodies that require complicated preparation steps by adopting the described pre-coating process And can be easily detected. The use of a polyclonal antibody is beneficial not only in terms of simple operation but also in terms of measuring the total amount of the carbonyl compound-protein complex.

Thus, the measuring method according to the present invention can predict seed renewal by quantifying the degree of aging of plant seeds,
It is useful for measuring the degree of deterioration of the food itself, searching for antigens at the onset of food allergy patients, and quantifying the progress of alcoholism and alcoholism. Moreover, since the operation is simple, the present invention is extremely useful in realizing a diagnostic kit for such measurement or diagnosis.

[Brief description of the drawings]

FIG. 1 is a standard straight line created for performing a measurement according to the present invention.

FIG. 2 is a graph showing the relationship between the decrease in vitality and the content of a carbonyl compound-protein complex measured by the method of the present invention in lettuce seeds treated with gaseous aldehyde.

FIG. 3 is a view showing the relationship between the vitality of various naturally aged seeds and the content of the carbonyl compound-protein complex measured by the method of the present invention.

Claims (5)

(57) [Claims] 1. A method for quantifying a carbonyl compound-protein complex, comprising: (a) a predetermined concentration which is known to exhibit toxicity to an appropriate protein source and which is rich in membrane permeability and mobility; By reacting the volatile carbonyl compound of
Form a carbonyl compound-protein complex mixture,
Preparing an immunogenic composition containing the complex mixture; and (b) immunizing by administering the immunogenic composition to a mammal, and immunizing the mammal from the serum of the immunized mammal. Separating an antibody against the carbonyl compound-protein complex, and (c) performing an appropriate treatment such as extraction or cell destruction on the analyte, thereby obtaining the carbonyl compound-protein present in the analyte. Preparing a measurement solution containing a substantial part of the complex; and (d) preparing a reaction solution by adding a solution of the antibody adjusted to a predetermined concentration to the measurement solution. (E) reacting a predetermined concentration of the volatile carbonyl compound with a suitable carrier protein,
(C) pre-coating the immunoassay container by forming a carbonyl compound-carrier protein complex, housing the solution of the carrier protein complex in an immunoassay container, and incubating the immunoassay container; By containing and incubating the reaction solution in an immunoassay container, the carbonyl compound-protein complex in the reaction solution is reacted with the antibody to form an antigen-antibody complex, and the antigen-antibody complex is formed. Binding to the carbonyl compound-carrier protein complex precoated on the immunoassay container, and (g) adding a solution of an enzyme-labeled secondary antibody to the antibody to the immunoassay container. By incubating to bind the secondary antibody to the antibody, h) adding a sex substrate solution for the enzyme bound to the secondary antibody to cause a reaction, and measuring the product of the enzyme reaction; and (i) a standard solution having a known concentration of the carbonyl compound-protein complex. And a step of preparing a standard curve by performing the above steps a to i, and (j) comparing the measurement result in the step h with the standard curve obtained in the step i to obtain the standard curve. Determining the concentration of the carbonyl compound-protein complex contained in the solution. 2. The method according to claim 1, wherein the predetermined concentration of the volatile carbonyl compound which has been previously shown to be toxic and which is rich in membrane permeability and mobility is acetaldehyde. 3. The method according to claim 1, wherein the suitable carrier protein in step e is gelatin. 4. The method according to claim 1, wherein the suitable protein source in step a is bovine serum albumin (BSA). 5. The method according to claim 1, wherein the predetermined concentration of the volatile carbonyl compound in the step (e) is a low concentration of 0.5 to 1 mM which is close to the concentration in the living body. One way.