JP3481894B2 - Measurement method using immunological chromatographic method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring body fluid components using immunological chromatography used in clinical tests.

[0002]

2. Description of the Related Art Kits for measuring body fluid components using chromatography are generally used for urine sugar test papers and urine protein test papers. These are used to color a specific component by a chemical reaction to visually check its color tone. The method of reading was electro-optical.

Taking a urine sugar test paper strip as an example, this is a system in which the degree of color development due to the urine to be inspected is visually compared with a separately printed stepwise color tone table to try to determine the approximate density.
These automatic reading devices have already been put on the market and used for clinical examinations.

[0004] In addition, recently, a measuring method using an immunological chromatographic method has prevailed for measuring an object to be measured which cannot be detected by a chemical reaction, for example, an ultratrace amount of protein component or virus, and many kits have already been released. For example, a pregnancy determination reagent, an LH hormone measuring reagent, a colorectal cancer (fecal occult blood reaction) reagent, a hepatitis virus (HbsAg, HbsAb, HCAb, etc.) reagent, and a chlamydia detection reagent have been put on the market. The assay method using the immunological chromatographic method has already been put into practical use because the principle and the like have already been disclosed. In this measurement method, when a sample (4) such as blood, serum, or urine is injected into a predetermined sample injection part (2) as shown in FIG. 5, the sample moves according to the principle of paper chromatography. During this development, colloidal gold-labeled antibody and dye-labeled latex antibody (5) are installed in Category A, and the specimen (4) moves and comes into contact with the labeled antibody (5) to form an antigen-antibody complex. It It was common for this complex to migrate further and be immunologically complemented by Section B (6) and Section C (7), respectively.

Further, as these labeled antibodies (5) of the immunological chromatographic system, two types of labeled antibodies were used if there were two measurement items. These are used for measurement of different measurement items in sections B and C, respectively. These were methods in which the rest of the labeled antibody moved further and was captured in Section D. As a matter of course, the labeled antibody captured in Category B changes depending on the concentration of the test sample. The remaining labeled antibody naturally changes depending on the concentration of the test sample, and the concentration of Category D also changes.

[0006] Up to now, the commercially available immunological chromatographic assay methods generally refer to Section D as the control line, which simply means that the test sample has moved to the end of the chromatographic system. It was just to show.

FIG. 5 illustrates a pregnancy diagnostic drug, which is a conventional immunological chromatographic assay, as an example. Colloidal gold-labeled anti-human hCG mouse antibody (5) is movably installed in Section A, anti-human hCG mouse antibody (6) is immobilized in Section B, and anti-mouse goat antibody ( 7) is fixed.

When a urine sample (4) of a pregnant patient is injected into a predetermined injection part (2), the sample (4) moves according to the principle of paper chromatography. The hCG in this urine was bound to the colloidal gold-labeled antibody (5) in the section A during the development, and this complex further moved and was immobilized in the section B.
It is captured by the G mouse antibody (6) (recognizes the human hCG side). The remaining labeled antibody (5) moves further and is caught by the anti-mouse goat antibody (7) in Section C (recognizing the labeled antibody side). When there is no hCG in urine, the labeled antibody passes through without binding to Section B and is captured in Section C.
Therefore, if the pregnancy test is positive, lines will appear in categories B and C,
If negative, the line appears only in Category C. In this conventional method, the gold colloid-labeled antibody captured in Category C changes depending on the amount of hCG in the sample. The color tone of so-called category C is not constant.

References Takeshi Maruo et al .: High-sensitivity hCG detection kit, basic examination and clinical application of HCG test pack plus, obstetrics and gynecology 58 (8) 1423, 1991 Patent publication, Japanese patent publication 7-46107 Published patent publication , JP-A-10-48212

[0010]

The immunological chromatographic method is generally a qualitative measurement method. The present invention relates to a new method in which this qualitative method can be used as a method in which a change in concentration is known to some extent or as a quantitative method. It is said that the immunological chromatographic method has poor quantitativeness.
The reason why the quantitativeness is poor is that even if the color tone that appears optically is read, the background substrate is slightly colored with the substance on the sample and the substrate itself is non-uniform The reason is that it was not good.

Since the sample moves on the support, it is inevitable that part of the sample or part of the labeled antibody remains on the support. Even if the color tone of the measurement result is optically read, the background changes for each sample, so that accurate measurement cannot be performed.

An object of the present invention is to provide a method capable of more simply and accurately measuring without being influenced by the color tone of the background of the support, which is different for each specimen.

[0013]

In the present invention, taking into consideration the non-uniformity of carriers (supports) and the difference in background, a calibration curve is formed on the same membrane surface for each inspection or for each same test strip. Has a section C or a section C and D and a section B which is a place for capturing an unknown sample, and the colloidal gold labeled antibody or the blue latex labeled antibody (specifically binds to the section B and the section C or the sections C and D respectively ( Hereinafter, these are simply referred to as labeled antibodies) are previously set in the section A. In the section C or the sections C and D, an antigen or antibody adjusted so that a certain concentration, which has a close relationship with an unknown sample, appears by an antigen-antibody reaction is installed.

In the present invention, in addition to the labeled antibody that binds only to the sections C or C and D, respectively, another labeled antibody that binds only to the unknown sample is installed in combination to obtain the color tone of the section B and the section C. Alternatively, the colors are arranged so that the color tones of the sections C and D are different from each other. (Figure 1)

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION There are roughly two types of embodiments of the invention. One is the measurement by an automatic device, and the color tone of one (Category C) line or two (Category C and D) lines having a certain color tone and the measured line (Category B) is read by an optical automatic device, respectively. We tried to quantify the concentration of the sample by comparing them.

The other is the case where the above-mentioned division C line having a certain color tone is visually read, and naturally this cannot be a number. Therefore, an embodiment will be described for the case of having one section C line (FIG. 2).

By comparing the color tone of the section C line where a certain density appears and the measured line of the section B with the naked eye, at least four comparison judgments of "absent", "light", "same", and "dark" (20) Is easy.

FIG. 3 shows the recording paper. According to this 3
The three color tones and the inspection date (19) are respectively set as the X axis and the Y axis, and the white circles are simply painted black according to the measurement results. One graph (22) can be obtained by connecting the black circles on the recording paper with a line. This graph generally represents a state in which the concentrations of substances such as proteins, sugars, and inorganic substances in human body fluids, so-called blood and urine, are constantly changing.

[0019]

Example 1 In this example (FIG. 1), two kinds of labeled antibodies of Category A were blended, and a total amount of the labeled antibody was captured in Category C so that a predetermined amount of premixed color tone appears. In this example, a method for quantifying prostate specific antigen (PSA), which is a cancer marker that needs to be quantified, will be described. The colloidal gold-labeled anti-human PSA rabbit antibody (11) and the colloidal gold-labeled anti-goat antibody (12) are movably installed in Category A, and the anti-human PSA monoclonal mouse antibody (13) is immobilized in Category B. In section C, an anti-rabbit IgG goat antibody (14) was immobilized. The amount of colloidal gold-labeled anti-goat antibody (12) in Category A is
The color tone of the gold colloid captured in Category C was adjusted to be exactly 4 ng / mL in terms of PSA concentration. These were measured with a reflection type optical densitometer. The results are shown in Fig. 4.
The concentration Xc of the unknown samples 1, 2, and 3 in FIG. 4 is expressed by the following calculation formula.

Xc = reading of Category B of unknown specimen × 4.0 (ng / m
L) ÷ Category C reading From this calculation formula, the measurement result of an unknown sample is 1 4.7 ng / mL 2 7.1 ng / mL 3 9.1 ng / mL. If a section in which a color tone equivalent to another density value is obtained is set in the section D, the measurement value of the unknown sample can be easily obtained from the sections C and D by the following formula. The concentration X _CD of the unknown sample when the value of Category C is 4 ng / mL and the value of Category D is 10 ng / mL is given by the following formula.

X _CD = (4 × reading of Category B of unknown specimen −10
× reading of section C + 4 × reading of section D) ÷ (reading of section D −
(Category C reading)

[0022]

[Example 2] In the case of Example 1, an attempt was made to optically read the emerging line and measure the concentration of the unknown sample from the result. In this example, a method for obtaining an approximate measurement value of an unknown sample when the determination of the result is performed visually will be described. This example describes the measurement of luteinizing hormone (LH).

In FIG. 2, the colloidal gold-labeled anti-human LH mouse antibody (16) and the colloidal gold-labeled anti-rabbit antibody (15) are installed in Category A, and the anti-human LH mouse antibody (17) is classified in Category B. It was fixed and in Category C anti-rabbit goat antibody (1
8) fixed. In addition, the amount of colloidal gold-labeled anti-rabbit antibody in Category A is such that the color of the gold colloid captured in Category C is LH.
It was adjusted to a concentration of just 20 mIU / mL. In this example, luteinizing hormone (L
It is applied to a diagnostic agent for treating infertility by utilizing the fact that H) rapidly rises just before the start of ovulation. This hormone is generally the most stable and concentrated in the urine in the morning. Therefore, continue the test once a day for at least 4 to 7 days from several days before the scheduled ovulation date. The results are recorded in the results table in Figure 3.

Visually seeing the line of the section C and the line of the section B, as shown in the criterion of 20 in FIG.
"Same" and "dark" are compared, and the corresponding white circles are filled with black and recorded. A line graph can be obtained by connecting the black circles on this recording paper with lines as shown in FIG.
By reading this graph, it can be read by anyone that the peak of May 4 is when this hormone is most secreted, which is clinically the time when it is most likely to become pregnant.

[0025]

EFFECT OF THE INVENTION At present, it is said that the measurement of a cancer marker must be a quantitative method because it is a serious concern how to determine whether cancer is normal or normal and how the value decreases during treatment. . At small and medium-sized hospitals and practitioners, the only way to quantitatively measure this measurement item was to use an expensive immunoassay device or outsource it to a test center. Also, in the case of measuring trace amounts of hormones and hepatitis virus, they were also dependent on the introduction of dedicated measuring instruments and inspection centers.

The fact that the immunological chromatographic system can be used quantitatively or semi-quantitatively according to the present invention is effective in the following aspects. 1. The highly sensitive immunological chromatographic method is now available as a quantitative or semi-quantitative method. 2. Cancer markers can be quantified by the present invention. 3. Since most of the reagents are solid-phased, it has become possible to reduce the size and cost of measuring equipment. 4. This measurement method can now be used by bedside, small and medium-sized hospitals, and practitioners. 5. Depending on the measurement item, the reagent to be measured can be stored at room temperature, making it simple and easy to handle. 6. Depending on the measurement item, it was judged by visual inspection, not by measurement with a device, and as in Example 2 of the present invention, a reading device became unnecessary. 7. Depending on the measurement item, the patient himself / herself can perform an examination and can judge the result as in Example 2 of the present invention. 8. For handling after measurement, refer to what is called serum,
Since whole blood, urine, feces and the like are adsorbed by the adsorption pad, they are relatively hygienic without leaking to the outside. As described above, according to the present invention, the effect can further advance the development of this field in the future.

[Brief description of drawings]

[FIG. 1] “Explanatory drawing of Example 1” (A) State before inspection (B) State after inspection

[FIG. 2] “Explanatory drawing of Example 2” (A) State before inspection (B) State after inspection

[Fig. 3] "Example of visual measurement according to Example 2"

[Fig. 4] "Example of quantification by optical densitometer according to Example 1"

[Fig. 5] "Explanation diagram of conventional immunological chromatography method" (A) State before inspection (B) State after inspection

[Explanation of symbols]

1 Support 2 Specimen injection part 3 Specimen injection pipette 4 hCG 5 in human urine 5 Colloidal gold-labeled anti-human hCG mouse antibody 6 Anti-human hCG mouse antibody 7 Anti-mouse goat antibody 8 Adsorption filter paper (adsorption pad) 9 Expansion left Sample urine 10 hCG-conjugated gold colloid labeled anti-human hCG mouse antibody bound to Category B 11 gold colloid labeled human PSA rabbit antibody 12 gold colloid labeled anti-goat antibody 13 anti-human PSA monoclonal mouse antibody 14 anti-rabbit IgG goat antibody 15 gold colloid labeled Anti-rabbit antibody (20 mIU / mL) 16 Colloidal gold-labeled anti-human LH mouse antibody 17 Anti-human LH mouse antibody 18 Anti-rabbit goat antibody 19 Test date column 20 Judgment standard 21 Record column 22 Graph

Claims (6)

(57) [Claims] 1. A plurality of labeled antibodies such as a gold colloid-labeled antibody and a dye-labeled latex antibody are arranged in a dry state in a first section A on a support such as a cellulose acetate membrane or a nitrocellulose membrane so as to be movable. Cage, the first
The first antibody that specifically binds to the labeled antibody is immobilized in the next section B of the chromatographic development, and the second and third antibodies that specifically bind to the second and third labeled antibodies are In the support carrier fixed to section C or section D, the injected analyte binds to the labeled antibody chromatographically. The color tone of the sections B, C and D appearing at that time on the same support carrier is measured by an optical measuring means, and the concentration of the sample of the section B is measured from the calibration curve with reference to the color tones of the sections C and D. Method using the immunological chromatographic method described above. 2. A plurality of labeled antibodies include one or more labeled antibodies that bind to a measurement target (measurement item) and one or more labeled antibodies not directly related to the measurement target. A method for measuring using an immunological chromatographic method according to claim 1. 3. The color tone of the sections C and D is adjusted in advance so as to be a constant color tone defined by the antibody amounts of the second and third labeled antibodies. Measurement method using the immunological chromatographic method of 2. 4. An antibody that specifically binds to the second and third labeled antibodies is fixed to each of the sections C and D, and the amount of the antibody is adjusted to have a predetermined constant color tone. 3. The measuring method using the immunological chromatographic method according to claim 1 or 2. 5. An antibody that specifically binds to the second labeled antibody is immobilized in the sections C and D, and the amount of the antibody is adjusted so as to have a predetermined constant color tone ratio. 3. A measuring method using the immunological chromatographic method according to claim 1 or 2. 6. The color tone of the section C or D is arranged so as to have a certain density of the section B so that the color tone of the section B relating to an unknown sample is visually darker than the color tone of the section C or D. A measurement method using an immunological chromatographic method, which is characterized by determining whether or not the protein is thin and recording it to measure the approximate concentration of a trace amount of protein.