JPH04142460A - Detecting method for human hemoglobin - Google Patents

Abstract

PURPOSE:To increase sensitivity without being affected by color and without requiring washing and amplification reaction by employing the sandwich method used for EIA as a principle to increase sensitivity with a gold colloid antibody. CONSTITUTION:An anti-human hemoglobin polyclonal antibody 2mg/ml is spotted 1mul on a 1cm-square nitrocellulose film, it is adsorbed all night at the room temperature, it is blocked with PBS containing 1%-BSA, then it is dried at the room temperature to form an antibody adsorbing film. The 1cm-square filter paper is stuck to the back side of the antibody adsorbing film, and it is put on a glass plate. 0, 0.1, 0.5, 1, 10mug/ml-human hemoglobin solutions 0.03ml are added and mixed to the anti-human hemoglobin monoclonal antibody-sensing gold colloid liquid 0.3ml set with the absorbance at 540nm to 50.D, 0.2ml of it is dripped on the antibody adsorbing nitrocellulose film and reacted for 5min, then the coloring of purple gold colloid grains is observed. It is judged as positive (-) if the coloring of the gold colloid grains is noticed, and it is judged as negative (-) if no coloring is noticed.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for detecting hemoglobin in feces. Detection of hemoglobin in feces indicates gastrointestinal cancer,
It is important for diagnosing diseases that involve bleeding such as ulcers.

(Prior art) There are two methods for detecting hemoglobin in feces: one uses the peroxidase-like activity of hemoglobin, and the other uses antigen-antibody reaction. However, the former method only detects all the hemoglobin that bleeds from the digestive tract. It also detects hemoglobin in foods such as fish and meat, so dietary restrictions are necessary. On the other hand, the method using antigen-antibody reaction does not require dietary restrictions, and since it does not react with denatured hemoglobin, it detects only bleeding from the large intestine and rectum.

The method using this antigen-antibody reaction has the following prior application. JP-A-2-141665 uses a monoclonal antibody against one type of human hemoglobin, and is a measurement method based on the principle of color change due to aggregation.

Japanese Patent Publication No. 61-228351. Japanese Patent Publication No. 58-73983,
JP-A-56-106154 is all based on the EIA method. JP-A-59-125044 is a latex method.

(Problems to be Solved by the Invention) However, the conventional EIA method described above requires a washing operation to remove unreacted enzyme-labeled antibodies, and has a fatal flaw in terms of simplicity.

Furthermore, in order to measure the amount of labeled enzyme that has reacted, it is necessary to react with a substrate, requiring multiple constituent reagents and making the reaction complicated.

Furthermore, although the latex agglutination reaction is excellent in simplicity, the reaction changes over time, so the determination must be made while observing a predetermined reaction time. Moreover, it had drawbacks such as requiring skill to make such judgments.

The method using a colloidal gold-labeled antibody does not require the above-mentioned plurality of constituent reagents, is extremely simple, and has the advantage that the final determination does not require any skill. However, the above-mentioned conventional example, JP-A-2-141665, is based on a color change based on an agglutination method using one type of monoclonal antibody, so the measurement sensitivity is low and the color of the sample is affected by the fact that the sample is stool. It had drawbacks such as being easy to use.

(Means for solving the problem) This method increases sensitivity and is hardly affected by color. Furthermore, unlike EIA, there is no need for washing, and there is no need for amplification reactions such as reactions with substrates to develop color or increase sensitivity. - If it is desired to increase the sensitivity of the layer, this can be achieved by using antibodies to be immobilized and colloidal gold-labeled antibodies that recognize different epitopes. For example, hemoglobin is a tetramer in which two molecules each of the a and β subunits are bonded, and a combination of an antibody that recognizes α and an antibody that recognizes β is considered. Furthermore, the types of antibodies may be a combination of monoclonal antibodies and polyclonal antibodies. With the above, almost clinically satisfactory sensitivity can be obtained. In order to further increase the sensitivity, the material for immobilizing the antibody is a porous membrane, and an absorber is attached to the back side of the membrane.The degree of capture of the reaction product of hemoglobin and colloidal gold-labeled antibody to the immobilized antibody is reduced by the membrane. The chance of reaction increases when the light passes through the film, which further increases the sensitivity.

Example 1 Preparation of gold colloid Add 1O% chloroauric acid solution (
1 mfl of HAu (j2.4 HzO) is added with stirring, and after 1 minute, 5 mQ of 2% sodium citrate solution is added, and after further stirring for 20 minutes, the mixture is cooled to 30°C. 0 after cooling
．． pF17. with 1M potassium carbonate solution. o, add 1 mQ of 1% PE020.000 as a stabilizer,
After stirring for a minute, it was filtered through a 0-22 μm Millipore filter.

Example 2 Anti-human hemoglobin monoclonal antibody, ) ISU-1
1O (Japan Biotest Institute) in 10mM HEPE
S% pH 7, 1T diluted to a concentration of 200 tt g/+no. 3 mQ of this solution and 30 mff of the gold colloid solution prepared in Example 1 are placed in a centrifuge tube and stirred thoroughly. Then, Al buffer (10mM HEPES, 0.3
M D-mannitol, 0.05% PEG20000,
Add 3.3 m12 of 0.1% BSA pH 7,1),
After an hour, stir and centrifuge at 10°C and 9000 PPM for 10 minutes, collect the supernatant into another centrifuge tube, and
Centrifuge for 30 minutes at 000 PPM. The upper groove was removed, and 30 mQ of A1 buffer was added to the precipitate to homogeneously suspend it, followed by further centrifugation at 10° C. and 15,000 PPM for 30 minutes. The same operation was carried out twice, and Al/'-/Fur 10+nQ was added to the resulting precipitate.

(2) Preparation of antibody-bound membrane anti-human hemoglobin rabbit voriclonal antibody (DAK
U PATTS) or the anti-human hemoglobin monoclonal antibody used in Example 2 (1), MSU-110, or the anti-human hemoglobin monoclonal antibody with a different epitope from this monoclonal antibody, MSU-107 (Japan Biotest Institute). PBS to 1mg/+nQ
(0.9% NaCQ, 10mM phosphate buffer y-P
diluted with 1 cm x 1 cm of nitrocellulose membrane (Bi.

A 3μQ plot was made on a Rad company. After air-drying, it was immersed in PBS containing 1% BSA at 37°C for 1 hour, and then air-dried.

Example 3 Human hemoglobin detection sensitivity test ■Human hemoglobin was dissolved in ^1 buffer to a concentration of 0.0.5.1.
0.3.0.5.0. Prepare human hemoglobin solutions of io, 0 μg/m (2).
Mixed with Q. Human hemoglobin final concentration is 0.0.05
．． 0.1.0.3.0.5.1.0 μg/i (2).

The antibody-bound membrane prepared in Example 2 (2) was immersed in this for 5 minutes, then taken out and the coloration of the area where the antibody was plotted was observed. When coloring is observed, it is judged as positive (+), and when no coloring is observed, it is judged as negative (-); as a result, when the antibody bound to the membrane is a rabbit polyclonal antibody, the detection limit concentration of human hemoglobin is the final Concentration 0.1 μg
/mQ.

When the antibody bound to the membrane was a monoclonal antibody that recognized a different epitope from the colloidal gold-labeled anti-human hemoglobin monoclonal antibody, the lower detection limit concentration of human hemoglobin was a final concentration of 3 μg/mQ.

When the antibody bound to the membrane was the same as the colloidal gold-labeled anti-human hemoglobin monoclonal antibody, the lower detection limit concentration of human hemoglobin was a final concentration of 0.5 μg/mQ.

On the other hand, when the color tone change method was measured according to the example of JP-A-2-141665, the measurement sensitivity was 1 to 2 μg/
It was mQ.

These are summarized in the table below.

Table measurement method Hemoglobin sandwich method Monoclonal Ab 0°1 cm square of nitrocellulose H (Bio-Rad,
2 mg/ml of anti-human hemoglobin voriclonal antibody (DAKOPATTS,
:L-de No., A118) was spotted, adsorbed overnight at room temperature, blocked with PBS containing 1% BSA, and dried at room temperature to prepare an antibody-adsorbed membrane.

On the back side of the antibody adsorption membrane, place 1 1 1 IC square filter paper (Whatman).

AA disk) was glued and placed on a glass plate.

The absorbance at 540 nm is 50. 0.0.1.0.5.1.0.0.1.0.5.1. IOμg/m
0.03 mI2 of each human hemoglobin solution of Q was added and mixed, and this 0.2 no2 solution was dropped onto the antibody-adsorbed nitrocellulose membrane, and after reacting for 5 minutes, the purple colloidal gold particles were observed. Similarly, experiments were also conducted using an antibody-adsorbed membrane without adhering filter paper.

Positive (+) if coloring of colloidal gold particles is observed;
The case where no test was observed was determined to be negative (-), and the lower limit of measurement was 0.01 μg/mQ as shown in the following table.

Final concentration of human hemoglobin (μg/va12) 0 0.0
1 0.05 0.1 0.5 1 Glue the filter paper
− + + +10+ Antibody adsorption membrane Antibody adsorption membrane that does not adhere to filter paper+ 10+ (Effects of the invention) The present invention can detect minute amounts of human hemoglobin in feces, so it can be used to treat diseases that involve bleeding such as cancer and ulcers. Extremely useful for diagnosis.

Claims (2)

[Claims] (1) A first antibody labeled with colloidal gold against human hemoglobin is reacted with a solid layer bound with a second antibody against human hemoglobin that recognizes the same or different epitope as the first antibody, and an immunological agglutination reaction is performed. How to detect human hemoglobin. (2) The method according to claim (1), which comprises immobilizing the second antibody on a permeable membrane and adhering a water-absorbing agent to the back surface thereof.