JPH0566222A - Material analyzing method - Google Patents

Abstract

PURPOSE:To analyze many kinds of materials by one operation by mixing a univalent antibody, which is bonded with a detectable label material, with a sample, performing liquid chromatography for the mixture, and measuring the signal from the label material associated with eluted immune complex. CONSTITUTION:A univalent antibody is adjusted by treating immunoglobulin G and the like with enzyme such as pepsin and papain and/or reducing agent such as dithiothreitol and mercaptoethanol. The material is bonded to a label material such as light absorbing material, light emitting material and a fluorescence material, and a label antibody is obtained. When the mixed liquid is undergoes liquid chromatography, the isolated label antibody and the label antibody which forms the immune complex are separated in a column filled with gel with the elapse of time. The antibodies are eluted to the outside of the column. The detection of the label material is appropriately performed in accordance with the property of the label material. For example, when the label material is the material indicating the light absorption in a specified wavelength, the light absorption is measured at the specified wavelength. For the different label materials indicating the light absorptions at two different wavelengths, white light is used, the transmitted light through eluent is divided into two parts and the absorbances are measured at the same time.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for analyzing substances such as immunoglobulins and lipoproteins present in a sample using a monovalent antibody bound to a labeling substance and a liquid chromatography technique. More specifically, the present invention relates to a method for analyzing the amount of a specific substance in a sample in which various substances such as serum are mixed, which combines the specificity of an immune reaction with the convenience of liquid chromatography.

[0002]

2. Description of the Related Art Generally, in order to analyze trace substances such as proteins contained in biological samples such as serum and urine, EIA (En
enzyme immuno assay), RIA (Radio Immuno Assay)
The immunoassay and the liquid chromatography called are performed.

In the immunoassay, for example, a substance to be analyzed in a sample is first placed on a carrier by mixing a sample with an antibody (immobilized antibody) immobilized on a suitable carrier for the substance to be analyzed. Fix it. Next, an antibody to the substance to be analyzed (labeled antibody) bound to a detectable labeling substance is mixed therewith to form an immune complex of antibody-substance to be analyzed-labeled antibody on the carrier.
Then, the amount of the labeling substance in this immune complex is measured.

In the immunoassay method, when the labeled substance is not measured, it is analyzed that the substance to be analyzed does not exist in the sample, and when the labeled substance is measured, its amount is determined based on the result. It is possible to analyze (concentration).

On the other hand, in liquid chromatography, for example, a column packed with a gel, an ion exchange gel, a molecular sieve gel or the like having an affinity for a substance to be analyzed is used, and a sample is applied to the column, The eluate is measured, for example, by absorption of ultraviolet rays. In this method, when the expected elution signal is not measured, it is analyzed that the substance to be analyzed is not present in the sample, and when the elution signal is measured, the height or width of the peak, It is possible to analyze the amount (concentration) based on the area or the like.

[0006]

2. Description of the Related Art According to an immunoassay method such as EIA or RIA, it is possible to accurately determine the presence or absence of a substance to be analyzed and its amount (concentration). However, this method is directed to one substance. That is, in order to analyze various substances at the same time, it is necessary to use an antibody for each substance to be measured as an immobilized and labeled antibody, and for example, in the analysis of two types of substances, four or more types of substances should be used. The antibody must be specified, etc.
There are points to be improved.

In the immunoassay, a substance to be analyzed is immobilized on a carrier, a labeled substance antibody is mixed, and then B / F (bound: Bound / bound) for separating unbound labeled antibody is separated.
Free (Free) separation is required, and in order to minimize experimental error, the above B / F separation operation is strictly performed, and further non-specific adsorption of the labeled antibody to the carrier is performed. It must be prevented.

On the other hand, the analysis by liquid chromatography has a characteristic that the operation is simple and even if there are various substances to be analyzed, if they can be separated, they can be analyzed in a single operation. is doing. However, the sensitivity is low as compared with the immunoassay method, and when the sample contains many substances such as serum, the contaminants tend to reduce the analysis sensitivity. In particular, when a plurality of substances to be analyzed have similar properties, not only the amount (concentration) but also the presence or absence thereof is difficult to analyze. For example,
When using molecular sieve chromatography, separation of various substances to be analyzed becomes difficult if they have similar molecular weights.

[0009]

[Means for Solving the Problems] As a result of intensive investigations by the present inventors, a method for analyzing various substances by a simple one-time operation with respect to a biological sample containing many substances such as serum The present invention has been completed. That is, the present invention relates to a method in which a monovalent antibody against a substance to be analyzed bound to a detectable labeling substance is mixed with a sample and subjected to liquid chromatography to elute an immune complex between the substance to be analyzed and the antibody. Is a method for analyzing a substance, which comprises measuring a signal from a labeled substance related to. Hereinafter, the present invention will be described in detail.

The monovalent antibody used in the present invention is an antibody whose antibody titer against the antigen is monovalent, in other words,
It has one binding site with the antigen. The use of a monovalent antibody prevents the labeled antibody from binding to more than one substance to be analyzed to form a macromolecule. Moreover, since the monovalent antibody is a small molecule compared to a normal antibody, separation of the formed immune complex and the unreacted labeled antibody, which will be described later, becomes accurate.

Monovalent antibodies are, for example, immunoglobulin G
It can be prepared by treating (IgG) and the like with an enzyme such as pepsin and papain and / or a reducing agent such as dithiothreitol and mercaptoethanol. The same process may be performed when using an Ig belonging to another class.

In the present invention, the antibody may be monovalently converted prior to the immune reaction with the substance to be analyzed in the sample. That is, for example, when the substance to be analyzed in the sample is a substance which is difficult to be affected by the reducing agent or the like,
You may mix a sample and a bivalent antibody, and may add a reducing agent etc.

Antibodies can be used without limitation for Ig class or subclass. Here, it is preferable to reduce the difference in molecular weight between the substance to be analyzed and the immune complex (formed by binding of the substance to be analyzed and the antibody). For this purpose, for example, treatment with papain, pepsin, etc. It is preferable to use the antibody fragment obtained.

The antibody preferably has a high affinity for the substance to be analyzed in order to increase the sensitivity of the analysis. Furthermore, it is preferable that the antibody has homogeneous properties in order to achieve more sensitive measurement. For example, a monoclonal antibody and the like are particularly preferable because they have relatively high affinity, are easy to prepare, and have completely uniform properties. A polyclonal antibody against the substance to be analyzed can be prepared by immunizing a mammal such as mouse, goat, rabbit, or sheep with the substance. -The method of La et al. (Nature, 256 volumes, p495-, 1975
Year).

In the present invention, a monovalent antibody (labeled antibody) bound to a detectable labeling substance is used. Detectable labeling substances include, for example, light-absorbing substances that can detect light absorption or light emission at a specific wavelength by optical means, luminescent substances, fluorescent substances, radioisotopes that can be detected by radiation measurement, and themselves are directly Although it is undetectable (difficult to detect), it is possible to cause a change correlated with the amount thereof.
It is possible to cause other substances to emit light, etc.) or physiologically active substances such as enzymes (by acting on other substances, it is possible to convert the substances into fluorescent substances etc.). ..

Of these, fluorescent substances such as fluorescein and 4-methylumbelliferone, which do not require special preparation prior to the detection operation, have no fear of being exposed to radiation, have a small molecular weight, and can be easily optically detected, are preferable.

The binding between the monovalent antibody and the labeling substance may be carried out according to the usual method described in, for example, Ishikawa et al. (Enzyme-linked immunosorbent assay, 3rd edition, Medical Shoin).

The operation of mixing the labeled antibody with the sample may be simply adding the labeled antibody to the sample. However, for a sample in which the labeled antibody may lose its physiological activity or the like, it may be adjusted within a range in which the substance to be analyzed in the sample is not immunologically denatured.

The sample is not particularly limited, but if one example is described, in addition to biological fluids such as serum, urine and sweat, cell culture medium,
Examples include polluted liquids collected from rivers. The sample need not be subjected to any special pretreatment prior to the present invention, but if, for example, insoluble substance clumps or the like are suspended, they are subjected to centrifugation or membrane separation treatment to remove them. It is preferable to set it in order to simplify the subsequent operation.

After mixing the labeled antibody and the sample, before subjecting the mixed solution to liquid chromatography, it is advisable to allow a sufficient time for the labeled antibody and the substance to be analyzed in the sample to react. The time to leave can be appropriately determined depending on the temperature and the affinity of the antibody to be used. It can be carried out in a short time by using an antibody with high affinity, but in some cases it is not necessary to leave it There is also. According to the knowledge of the present inventor, when the mixed solution has a pH range of 4 to 10, it is sufficient to leave it for 1 minute to 24 hours under the condition of 0 to 60 ° C.

In the present invention, it is not always necessary to use one type of labeled antibody, and two or more types of labeled antibodies may be used simultaneously for two or more types of substances to be analyzed.
In this case, the same labeling substance can be used if the substance to be analyzed is separable in the subsequent liquid chromatography,
When it is difficult to separate them or there is a risk that they cannot be separated, two or more different labeling substances may be used.

At the stage where the above operation is completed, an immune complex containing a labeling substance is produced in the mixed solution unless the substance to be analyzed is not present in the sample.

Liquid chromatography in the present invention
Is not limited as long as it can separate the labeled antibody not complexed with the labeled antibody complexed with the substance to be analyzed. Here, liquid chromatography is
When there are two or more substances to be analyzed, it is preferable that they can be separated. For example, if the substances to be analyzed have different molecular weights, molecular sieve chromatography may be adopted, and if they differ in ion value, ion exchange chromatography may be adopted. More specifically, the substance to be analyzed is Ig
In the case of G and IgM, since they can be separated by utilizing the difference in their molecular weights, it is possible to exemplify the use of molecular sieve chromatography. When analyzing apoprotein and lipoprotein simultaneously, ion exchange chromatography
Alternatively, it can be exemplified to employ molecular sieve chromatography.

On the other hand, however, as described above,
When two or more substances to be analyzed are analyzed at the same time and they cannot be completely separated, etc., those labeled with different labeled substances should be used as the labeled antibodies for each, and then each labeled antibody should be used. It suffices to perform the detection operation for the labeling substance.

The gel used in the liquid chromatography described above may be a gel according to each purpose. That is, when molecular sieve chromatography is adopted, gel for molecular sieve or the like may be used. A hydrophilic gel that can prevent adsorption of protein components is preferably used. Further, the buffer solution for leveling the gel preferably has a pH similar to that of the mixed solution of the sample and the labeled antibody.

Generally, according to the method called high performance liquid chromatography, it is possible to realize more accurate separation in a shorter period of time as compared with ordinary liquid chromatography. Therefore, it is preferable to employ high performance liquid chromatography also in the present invention.

When the mixture is subjected to liquid chromatography, at least free labeled antibody and labeled antibody forming an immune complex are separated in the gel-filled column with the elapse of time, and eluted out of the column.

The detection of the labeling substance in the eluate may be appropriately performed according to the property of the labeling substance. For example, if the labeling substance is a substance that absorbs light at a specific wavelength, the absorbance at the specific wavelength may be measured. In the present invention, the detection operation may be performed after acquiring a fixed amount of the eluate, but in order to perform a highly sensitive analysis, it is preferable to perform the detection operation without directly collecting the eluate.

In the present invention, when a labeled antibody bound to two or more different labels is used for simultaneously analyzing two or more substances to be analyzed, one of the labels is used as described above. It suffices to detect the substance and simultaneously detect other labeled substances at the same time or later. For example, when different labeling substances each of which absorbs light at two different wavelengths are used, white light is used as the excitation light, and the light transmitted through the eluate is divided into two parts to simultaneously measure the absorbance at two different wavelengths. can do.

When a substance that cannot be optically detected directly, such as an enzyme, is used as the labeling substance, for example, the eluate may be once separated, and detection may be performed after adding the substrate or the like.

As a result of the detection of the labeled substance, if the signal related to the substance to be measured is not detected, that is, only the free labeled antibody is observed, it means that the substance to be measured is not present in the sample. Shown. Specifically,
For example, when molecular sieve chromatography is adopted, if no labeled substance is detected near the expected elution time, it indicates that the substance to be analyzed is not present in the sample.

On the other hand, when a signal related to the substance to be analyzed is detected, the result is compared with the result when the same operation is performed for a known amount of the substance to be analyzed, It is possible to know the amount (concentration) of the substance to be measured in the sample. The signal relating to the substance to be analyzed here is specifically the signal from the labeling substance relating to the immune complex formed from the substance and the labeled antibody. However, when it is possible to know the signal from the labeling substance associated with the immune complex by measuring the signal associated with the free labeled antibody, that is, when the total amount of the signal from the labeling substance used is clear, , Measuring the signal associated with free labeled antibody is the same as measuring the signal from the labeled substance associated with the immune complex. When the same operation is performed on a known amount of the substance to be analyzed, the same amount of the same labeled antibody is used.

[0033]

EXAMPLES Examples will be described below in order to explain the present invention in more detail, but the present invention is not limited to these examples.

Example 1 Analysis of Immunoglobulin G-1 Anti-mouse IgG goat antibody F (ab) conjugated with FITC
′) 2 (manufactured by IMMUNOSEARCH, hereinafter referred to as FITC-labeled antibody) was used to analyze the amount of IgG in the sample (purified mouse IgG or mouse serum). 5 μl of FITC-labeled antibody, 50 μl of sample and 1 μl of β-mercaptoethanol (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed and allowed to stand at room temperature for 2 hours. Also, at this time, for comparison, β-mercaptoethano-
The operation of leaving it unadded was also performed separately.

1 μl of this mixture was added to phosphate buffer (pH
Column packed with molecular sieve gel that was leveled with 7.0) (TOSO)
Co., Ltd., TSK gel G4000SW, inner diameter 0.75 cm x length 60 cm)
I went to After addition of the mixed solution, a phosphate buffer (pH 7.0) buffer was applied to the column.

Regarding the labeling substance in the eluate from the column,
The absorbance at 280 nm and the fluorescence intensity at 520 nm at an excitation wavelength of 490 nm were measured simultaneously. The results are shown in FIGS. 1 and 2.

From FIGS. 1 and 2, (1) purified mouse Ig
G or mouse serum itself has an absorbance but does not show fluorescence intensity. (2) Purified mouse IgG as a sample
Alternatively, when either β-mercaptoethanol was not added (when the labeled antibody is not a monovalent antibody), the signal from the labeled substance bound to mouse IgG and the release of either mouse serum were used. Incomplete separation of the signal from the labeled antibody and (3) the labeled antibody was monovalent when β-mercaptoethanol was added regardless of whether purified mouse IgG or mouse serum was used as the sample. It can be seen that the signal from the free labeled antibody can be clearly separated from the signal from the labeled antibody bound to mouse IgG because it is converted into a sex antibody.

From (1), it can be seen that there is no spontaneous emission of 520 nm fluorescence, and in this analysis, it is not necessary to make a correction in consideration of the background. From (2) and (3), when a monovalent antibody is used, the molecular weight of the labeled antibody becomes small, so that the distinction between the signal from the free labeled antibody and the signal from the immune complex is clarified. It can be seen that the analysis accuracy can be increased particularly in the analysis of low-molecular weight compounds having a molecular weight of about 150,000 daltons such as mouse IgG. When a monovalent antibody is used, the labeled antibody binds to two or more substances to be analyzed,
It is possible to eliminate the possibility of obtaining a signal from a higher-molecular-weight fraction than expected.

Example 2 Analysis of Immunoglobulin G-2 Anti-mouse IgG goat antibody (ab) conjugated with FITC
′) 2 (manufactured by IMMUNOSEARCH, hereinafter referred to as FITC-labeled antibody) is used to detect IgG in the sample (purified mouse IgG)
The amount was analyzed.

5 μl of FITC-labeled antibody, 50 μl of sample and 1 μl of β-mercaptoethanol (Wako Pure Chemical Industries, Ltd.)
Was mixed for 40, 80, 390 minutes or 3 days at room temperature.

1 μl of this mixture was added to phosphate buffer (pH
Column packed with molecular sieve gel that was leveled with 7.0) (TOSO)
Co., Ltd., TSK gel G4000SW, inner diameter 0.75 cm x length 60 cm)
I went to After addition of the mixed solution, a phosphate buffer (pH 7.0) buffer was applied to the column.

For the eluate from the column, the absorbance at 280 nm and the fluorescence intensity at 520 nm at an excitation wavelength of 490 nm were measured simultaneously. The results are shown in FIGS. 3 and 4.

From FIGS. 3 and 4, it can be seen that under the mixing conditions of the sample and the labeled antibody used in this example, a sufficient immune reaction occurs after 40 minutes of standing.

[0044]

According to the present invention, there is provided a method for analyzing various substances contained in a sample such as blood or serum at one time. That is, by preparing a monovalent labeled antibody for a substance to be analyzed in advance, mixing this with a sample, and subjecting it to liquid chromatography, it is possible to analyze various substances at once.

In the present invention, since a monovalent antibody is used as the labeled antibody, one labeled antibody binds to two or more substances to be analyzed, and as a result, three or more signals from the eluate from the column appear. It is also possible to more accurately quantify the substance to be analyzed. By using a monovalent antibody having a small molecular weight, particularly when molecular sieve chromatography is adopted as liquid chromatography,
The analysis method of the present invention realizes accurate analysis because the separation between the labeled antibody bound to the substance to be analyzed and the free labeled antibody is easily and clearly defined. The use of fragmented monovalent antibody allows for more accurate analysis. This is because the difference in molecular weight can be increased when the complex of the labeled antibody with the substance to be analyzed and the labeled antibody is compared.

Even when the molecular weights of various substances to be measured are similar, by using labeled antibodies bound to different labeling substances and detecting the labeling substances for each labeling substance, Note that it is possible to carry out an accurate analysis.

Regarding the standing time after the sample and the labeled antibody are mixed, generally, a monochrome sample having a high affinity is used.
The present invention is an excellent analytical method in terms of rapidity because it can be completed in a short time by using a null antibody. In addition, since a monoclonal antibody generally has high homogeneity, it is used to perform an operation on a known amount of a substance to be analyzed, and the result and the result of an operation on a sample containing an unknown amount of the substance to be analyzed. If compared, highly accurate analysis is possible.

According to the present invention, each of the immunoglobulin classes (IgG, IgA, IgD, IgE and IgM) can be analyzed, but it has a high affinity for a specific substance and It is also possible to analyze its subclass by using an antibody characterized by having low cross-reactivity to the substance. Further, for example, if a labeled antibody against a chain common to IgG and IgM is used, both can be analyzed simultaneously.

Conventionally, in the analysis of lipoproteins, separation and analysis of HDL, LDL and the like have been carried out by ion exchange chromatography or molecular sieve chromatography (edited by Ichiro Hara et al., Plasma lipoprotein, Kodansha Scientific, 1983). However, the apoprotein in it had to be analyzed separately (Denise Polacek et al., LIPID
S, Vol. 16, No. 2, p927, 1981). According to the present invention, it is possible to simultaneously analyze each of lipoprotein and apoprotein by using a labeled antibody against apoprotein.

[Brief description of drawings]

FIG. 1 shows the results of absorbance measurement at 280 nm in the results of Example 1. In the figure, a is mouse IgG
, B for mouse serum,
c is the result for the FITC-labeled antibody, d is mouse I
The results when gG and FITC-labeled antibody were mixed, e are the results when mouse serum and the FITC-labeled antibody were mixed, and f is the results when β-mercaptoethanol was added to the FITC-labeled antibody, g Shows the result when β-mercaptoethanol was added to the mixture of mouse IgG and FITC-labeled antibody, and h shows the result when β-mercaptoethanol was added to the mixture of mouse serum and FITC-labeled antibody. In the figure, βME indicates the elution peak of β-mercaptoethanol, and the arrow indicates the position where the sample was added to the column.

FIG. 2 shows the fluorescence intensity at 520 nm at an excitation of 490 nm among the results of Example 1. In the figure,
a is the result for mouse IgG, b is the result for mouse serum, c is the result for FITC-labeled antibody, d is the result when mouse IgG and FITC-labeled antibody are mixed, and e is the mouse serum. The result when FITC-labeled antibody was mixed, f is the result when β-mercaptoethanol was added to the FITC-labeled antibody, and g is mouse I.
The result when β-mercaptoethanol was added to the mixture of gG and FITC-labeled antibody, h is mouse serum and FIT
The results when β-mercaptoethanol was added to the mixture of C-labeled antibodies are shown. In the figure, A indicates the elution peak of the FITC-labeled antibody, AC indicates the elution peak of the immune complex containing the FITC-labeled antibody, and B indicates the FITC-labeled antibody fragmented by β-mercaptoethanol. The elution peak is shown, BC shows the elution peak of the immune complex containing the fragmented FITC-labeled antibody, and the arrow shows the position where the sample was added to the column.

FIG. 3 shows the results of absorbance measurement at 280 nm in the results of Example 2. In the figure, a is immediately after mixing the sample, b is 40 minutes after mixing the sample, c is 80 minutes after mixing the sample, and d is 390 after mixing the sample. In the case of minutes, e indicates the results of the case 3 days after mixing the samples. In the figure, βME indicates the elution peak of β-mercaptoethanol, and the arrow indicates the position where the sample was applied to the column.

FIG. 4 shows the fluorescence intensity at 520 nm at 490 nm in the results of Example 2. In the figure, a is immediately after mixing the samples, b is 40 minutes after mixing the samples, and c is 80 minutes after mixing the samples.
Shows the result in the case of 390 minutes after mixing a sample, and e shows the result in the case of 3 days after mixing a sample, respectively. B in the figure
Is FITC fragmented by β-mercaptoethanol
The elution peak of the labeled antibody is shown, and BC is the fragmented FI.
The elution peak of the immune complex containing the TC-labeled antibody is shown, and the arrow shows the position where the sample was added to the column.

Claims (7)

[Claims] 1. A monovalent antibody to a substance to be analyzed bound to a detectable labeling substance is mixed with a sample and subjected to liquid chromatography to elute an immune complex between the substance to be analyzed and the antibody. A method for analyzing a substance, which comprises measuring a signal from a labeled substance related to. 2. The method for analyzing a substance according to claim 1, further comprising an operation of comparing a measurement result obtained by performing the same operation on a sample containing a known amount of the substance to be measured with the obtained measurement result. .. 3. The method for analyzing a substance according to claim 1, wherein the substance to be analyzed is an immunoglobulin. 4. The substance to be analyzed is lipoprotein,
The method for analyzing a substance according to claim 1. 5. The method for analyzing a substance according to claim 1, wherein the liquid chromatography is molecular sieve chromatography. 6. The method for analyzing a substance according to claim 1, wherein the antibody is a monoclonal antibody. 7. The antibody is a fragmented monovalent antibody,
The method for analyzing a substance according to claim 1.