JP3004162B2 - Hemoglobin detection system - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological hemoglobin detection system using an anti-hemoglobin antibody, and more particularly, to an immunological system capable of detecting hemoglobin existing at a low concentration with high sensitivity. It relates to a hemoglobin detection system.

[0002]

BACKGROUND OF THE INVENTION Gastrointestinal bleeding occurs in a wide variety of diseases,
It is well known that it is important as an early symptom particularly in gastrointestinal malignancies. In recent years, as a method for examining these diseases, a method for detecting blood components in feces, particularly hemoglobin, caused by gastrointestinal bleeding has been widely used.

As a method for detecting such hemoglobin, for example, immunological detection methods such as one-dimensional immunodiffusion method, reverse passive hemagglutination method, latex agglutination method, colloidal gold agglutination method, enzyme immunoassay method and radioimmunoassay method are used. It has been known.

In the above immunological detection method, hemoglobin, which is a substance to be detected, is usually subjected to a test together with feces, which is an inactivating factor, so that the inactivation of antigenic determinants of hemoglobin progresses with temperature and time. And as a result,
With the conventional detection method, the detection sensitivity is significantly reduced.
In particular, when hemoglobin is at a low concentration, the inactivation is remarkable, and it is difficult to detect in a low concentration range, which is diagnostically significant.

[0005] However, in the actual test of blood components in feces (fecal occult blood test), the subject himself collects stool at home or the like,
It may be left for several days before being submitted to the laboratory, and may be left for several hours before the test due to work.In such a state, the antigenic determinant of hemoglobin Deactivation occurs, which is not preferable.

Further, as a practical problem, for example, in a typical example in which a subject himself / herself takes a stool twice at home or the like for two days, storing the sample in a refrigerator until it is submitted to a testing institution requires food and feces. As a result of hating the coexistence of these, there is virtually no expectation. In addition, fecal samples mailed from distant subjects are usually tested for more than one day after shipment, especially in high summer temperatures, where hemoglobin denaturation or degradation occurs, and Is reported to be negative [Kenji Fujiyoshi and Mariko Koyama, "Basic and Clinical", Basic Examination of Immunofecal Occult Blood Reagent, 23 (15), 6097-6101
(1989)].

[0007] For the purpose of preventing such inactivation of hemoglobin, for example, bovine serum albumin and saccharides have been added, but these methods have not been sufficiently effective [Hando]. Shigetomo, Hoshina Hoshina, Akemi Usui, Yasushi Yamazaki and Seijiro Senna, "Medical Examination", Examination of Fecal Lysate Used for Immunological Fecal Occult Blood Test. 3rd Report: Stabilizing hemoglobin 42 (9), 1523 -152
8 (1993)].

[0008]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned drawbacks of the prior art, and an object of the present invention is to provide hemoglobin in a sample, particularly at a low concentration under high temperature conditions in summer. An object of the present invention is to provide a system capable of detecting hemoglobin with high sensitivity and accuracy by preventing inactivation of the existing hemoglobin until it is subjected to an inspection.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies in order to suppress the inactivation of hemoglobin in a subject such as feces, and as a result, if sodium fluoride is present in the subject, Hemoglobin inactivation is prevented, and as a result,
Found that the presence of hemoglobin could be detected correctly,
The present invention has been completed.

That is, the present invention provides a hemoglobin detection system for detecting hemoglobin in a subject using an anti-hemoglobin antibody, characterized in that sodium fluoride is present in the system. .

In the present invention, the analyte and sodium fluoride coexist in the detection system to suppress the inactivation of hemoglobin in the analyte. Sodium fluoride may be present.

As an example of a method for causing sodium fluoride to be present in a hemoglobin detection system, a method of adding and dissolving sodium fluoride to a base solution for dissolving hemoglobin, which is a substance to be detected, can be mentioned.

The amount of sodium fluoride added to the base solution may be such that the concentration is 0.05 to 16% by weight, and preferably about 1 to 2% by weight. If the added concentration of sodium fluoride is too low, the effect of suppressing the denaturation of hemoglobin becomes small, and hemoglobin cannot be detected accurately. On the other hand, 4-1
Of course, the effect can be seen even with the addition of about 6% by weight, but the effect of suppressing the denaturation of hemoglobin tends to be smaller than the optimum concentration. At a higher concentration, sodium fluoride dissolves in the buffer solution. The disadvantage is that it becomes difficult. In order to dissolve a high concentration of sodium fluoride, which is close to 16%, in water, it is preferable to make the liquid slightly alkaline (for example, about pH 8.0).

Examples of the base solution to be used include a phosphate buffer, a glycine buffer, a Tris-HCl buffer, an ammonia buffer, a borate buffer and the like. The pH of these base solutions (buffers) is 5 to 10, preferably 6.5.
８8.5.

It is preferable to add a salt having a physiological salt concentration to the base solution.
For example, it is preferable to add 0.05 to 0.5% by weight of sodium azide. Further, it is also preferable to add bovine serum albumin at a concentration of 0.05 to 2% by weight as a protein stabilizer such as an antibody.

The method of adding sodium fluoride is not particularly limited, but when the above buffer is used as a base solution, it is preferable to dissolve the buffer so as to have a concentration of 16% by weight or less.

In addition, there is an inspection method using a gel as a special form of the base solution. In this case, for example, after adding sodium fluoride to the above-mentioned buffer solution so that the final concentration in the detection system is within the above range, To this, commercially available agarose is added at about 2% by weight, and the reagent may be prepared in a gel form.

As another example of the method for causing sodium fluoride to be present in the hemoglobin detection system, there is a method in which sodium fluoride is previously applied to a test device such as a sample collection tool or a test solution container. .

When a container having a bottom is used as a container for detecting hemoglobin, the material of the container body is not particularly limited, and plastics and glass such as polyethylene, polypropylene and polyethylene terephthalate can be used. Can be applied according to the amount of the test solution contained in the container so that the amount of sodium fluoride in the test solution remains at the above-mentioned concentration while storage is required. As an example of a method of applying sodium fluoride to a container, a solution or a gel of a predetermined concentration or a higher concentration is previously charged in the container, for example, the inner surface of the container is coated, and after being supported on a suitable carrier, For example, is put into a container.

Further, in the case of an inspection method using a stick-shaped member such as a sag or a bar-shaped object for collecting feces, sodium fluoride can be previously applied to the stick-shaped member. As a method of applying sodium fluoride to the stick-shaped member, for example, sodium fluoride is added to the base solution so as to have the above-described concentration, and the tip of a feces sampling line or the like is immersed in this solution. A method of leaving it overnight and then drying it at room temperature to coat the surface.

Furthermore, in the case of the inspection method using the sheet-like member such as a filter paper or cellulose film, a method to keep applying the sodium fluoride in the sheet. In this case, the sheet-like member may be coated, impregnated, or the like so as to have the above-described concentration at the time of use, and may be stored and used.

In the present invention, in order to prevent toxic sodium fluoride from flowing out as a liquid, it is more effective to give it as a gel or a solid than to give it as a liquid. In this case, the solid or, if necessary, gel-form sodium fluoride may be used by dissolving it in a base solution separately prepared as needed. At least a part of the components other than water of the base liquid may be provided in a solid or gel form, and an aqueous solution or water containing the remaining components may be provided.

To carry out the detection method of the present invention, an immunological detection method using an anti-hemoglobin antibody can be adopted.

Hereinafter, the present invention will be described with reference to a detection system utilizing a latex agglutination method as a typical example conventionally known. However, it goes without saying that the present invention can be used for various immunodetection systems other than the latex agglutination method, and can also be applied when the hemoglobin-inactivating substance is other than feces.

Firstly, the human hemoglobin A ₀ purified as an antigen, a rabbit, after immunized animals such as goats, bled to obtain an anti-human hemoglobin antibody was purified. This antibody is mixed with a polystyrene latex (eg, 0.3 μm in particle size) at neutral pH and allowed to undergo an adsorption reaction for several hours, followed by centrifugation and purification with a buffer solution containing bovine serum albumin and salt, and the like. Obtain sensitized latex.

Next, the latex reagent and the liquid to be detected are stirred and mixed on a glass plate, and the presence of human hemoglobin can be qualitatively detected by an aggregation image of the latex after several minutes.

The present inventors use various inorganic salts such as sodium iodide, potassium iodide, potassium bromide, sodium chloride, etc. and preservatives such as bread in addition to sodium fluoride as a substance for preventing deactivation of hemoglobin. Sodium propionate and diisopropyl fluorophosphate, which is a type of protease inhibitor, were also studied.However, none of them was confirmed to be effective, and literatures (Shigetomo Hanto, Hizuru Hoshina, Akemi Usui, Yasushi Yamazaki and Senna Seijiro, "Medical Examination", Examination of Fecal Lysate Used for Immunological Fecal Occult Blood Test. 3rd Report: Hemoglobin Stabilizer 4
2 (9), 1523-1528 (1993)], only sodium fluoride, whose efficacy was not recognized, was recognized as effective.

[0028]

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

EXAMPLE 1 10 ml of 5% carboxylated polystyrene latex
In, of 1 mg / ml 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide 10ml was added and reacted with stirring for 20 min, 0.01 mol / l-boric
Acid buffer pH 8.0 (hereinafter sometimes abbreviated as purified solution)
Centrifugation and purification twice (after removing the supernatant after centrifugation ).
Add 0 ml of the purified solution, stir to homogeneity, and centrifuge to remove the supernatant. Finally, 10 ml of the purified solution was added for dispersion dilution).

An anti-human hemoglobin A0 antibody (rabbit IgG, concentration 5 mg) prepared by immunizing rabbits with purified human hemoglobin AO in 10 ml of this latex (5% concentration)
/ Ml), and the mixture was reacted with gentle stirring for 5 hours. Further, 33 ml of 0.01 mol / l-borate buffer (pH 8.0) containing 0.1% bovine serum albumin was added.
1 and centrifuged and purified. After centrifugation, the supernatant was removed, and 50 ml of a purified solution was added to disperse and dilute to obtain 50 ml of an anti-human hemoglobin antibody-sensitized latex reagent having a latex concentration of 1%.

Next, a 0.2 mol / l ammonia / ammonium chloride buffer (pH 8.0) contains 0.1% sodium azide, 0.1% bovine serum albumin, and 0.9% sodium chloride. An aqueous solution was prepared from the resulting solution and used as an ammonia buffer. In 100 ml of this ammonia buffer, 0.5 g of stool with negative hemoglobin of a healthy person was dissolved to obtain a stool solution.

[0032] Then, in the stool lysis solution 100 ml, was dissolved a commercially available human hemoglobin A ₀ 2 mg, and 4 mg / g flights solution (Sample 1), below, and bye-bye diluted with stool lysis solution, human hemoglobin A ₀ 2 mg / g fecal solution (sample 2) and human hemoglobin A ₀ 1 mg / g fecal solution (sample 3). Sodium fluoride was dissolved in each of Sample 1, Sample 2 and Sample 3 so as to have a concentration of 1%, respectively, and incubated at 37 ° C. for 6 days (test method).

On the other hand, Sample 1, Sample 2 and Sample 3 containing no sodium fluoride were similarly incubated at 37 ° C. for 6 days (comparative method). Every day, 80 µl of these solutions and 20 µl of the above-mentioned latex reagent were mixed on a slide glass serum reaction plate, and the mixture was stirred. After 5 minutes, the aggregation image was visually observed. Table 1 shows the results.

[0034] However, −… no aggregation +… aggregation

From the above results, in the conventional method not using sodium fluoride, when incubated at 37 ° C.
Hemoglobin can be detected only for about 1 to 3 days,
According to the method of the present invention using sodium fluoride, it was found that, even when the temperature was continuously maintained at 37 ° C., the period of time during which high-sensitivity detection was possible was extended by at least two days.

Example 2 A commercially available 5 ml glass plain spitz having a capacity of 5 ml was prepared, and 250 μl of a methanol solution of 80 mg / ml sodium fluoride was evenly applied to the inner wall surface of the pipette tip with a pipette tip. Coated. Into the stool inspection container thus obtained, Sample 1, Sample 2 and Sample 3 prepared in the same manner as in
After inverting and mixing, the mixture was incubated at 37 ° C. in the same manner as in Example 1.

On the other hand, as a comparative example , a plain spitz having a capacity of 5 ml made of glass to which sodium fluoride was not applied was used as a stool inspection container, and a sample was taken and incubated as described above. As a result, the same results as in Table 1 were obtained. That is, it was found that the expiration date for detection with high sensitivity was extended by 2 days even at 37 ° C. as compared with the conventional method.

[0038]

As described above in detail, the sodium fluoride used in the present invention can suppress the inactivation of hemoglobin in the liquid to be detected over time, so that it can be used for a considerable period even in a high temperature in summer. The presence of hemoglobin can be detected with high sensitivity. Therefore, according to the hemoglobin detection system of the present invention, it is possible to detect hemoglobin co-existing with an inactivated substance such as feces, without being very concerned about the inactivation of hemoglobin by an inactivated substance such as feces or the rise in temperature. Can,
For example, the presence of gastrointestinal bleeding, occult blood, and the like can be accurately detected. that's all

Claims (6)

(57) [Claims] 1. A hemoglobin detection reagent for detecting hemoglobin in a subject using an anti-hemoglobin antibody, wherein sodium fluoride is present in the reagent. 2. The method according to claim 1, wherein sodium fluoride is contained in the test solution at 0.05%.
The hemoglobin detection reagent according to claim 1, which is present at a concentration of 16% by weight. 3. The method according to claim 1, wherein sodium fluoride is added to the hemoglobin detection base solution.
The reagent for detecting hemoglobin according to the above. 4. A kit for detecting hemoglobin , comprising the reagent for detecting hemoglobin according to claim 1 or 2, wherein the container for subject is provided with sodium fluoride in advance. 5. A hemoglobin detection kit comprising the hemoglobin detection reagent according to claim 1 or 2, wherein sodium fluoride is previously provided to the sample collection member. 6. A hemoglobin detection method for detecting hemoglobin in a subject using an anti-hemoglobin antibody, wherein sodium fluoride is present in the sample.