JPH0829429A - Hemoglobin measuring method - Google Patents

Abstract

PURPOSE:To accurately measure hemoglobin by containing iron transport protein in a hemoglobin-containing sample. CONSTITUTION:As iron transport protein, iron transport protein of a mammal such as man and bovine having hemoglobin decomposition restraining action is applicable, and blood transferrin, milk lactoferrin or altered ones thereof by genetic engineering is used. The iron transport protein to be contained in a hemoglobin-containing sample is desirably to be used at a weight ratio of 0.0008-one million to hemoglobin 1, and as the hemoglobin-containing sample, dejection, blood, urine and the like are listed. Measurement is conducted with a test sample containing the iron transport protein in the state of being dissolved in a buffering solution such as a phosphate buffer. In this case, pH of the buffering solution is desirably within a range of 6.5-8.5. Consequently, decomposition of the hemoglobin in the hemoglobin-containing sample, for example, dejection is restrained and highly accurate hemoglobin measurement can be conducted.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for suppressing the degradation of hemoglobin in a hemoglobin-containing sample such as a fecal occult blood test. By applying the degradation suppressing method to a clinical test method that requires preservation of a hemoglobin-containing sample, an accurate method for measuring hemoglobin is provided.

[0002]

2. Description of the Related Art As a method for testing gastrointestinal cancer such as colorectal cancer, the occult blood component (hemoglobin) in the feces caused by bleeding from the digestive tract is measured by an immunoassay using an anti-hemoglobin antibody. The way is done. In these clinical test methods, a test sample containing hemoglobin is often left for several days before measurement, and hemoglobin in the sample is decomposed during the storage period, so that hemoglobin may not be accurately measured.

General antibacterial agents such as thimerosal, chlorhexidine and sodium azide are used for the purpose of suppressing the decomposition of hemoglobin in the test sample. Further, a method of adding animal serum to a test solution (JP-A-4-145366), a method of adding a nitrogen-containing heterocyclic compound to the test solution (JP-A-60-35270), and iron protoporphyrin Addition method (JP-A-5-281227) and animal hemoglobin addition method (JP-A-2-2)
96149) is known, but a method using an iron transport protein is not known.

[0004]

With the spread of screening for colorectal cancer using mail-tested stool samples, it has been desired to develop a method for suppressing the degradation of hemoglobin in the test sample, which is stronger than known methods. By applying the degradation suppressing method of the present invention to a clinical test method that requires preservation of a hemoglobin-containing sample, a clinical test method that enables accurate measurement of hemoglobin is provided.

[0005]

The present invention relates to a method for suppressing the degradation of hemoglobin, characterized in that a hemoglobin-containing sample contains an iron transport protein. Furthermore, the present invention can provide a method for measuring hemoglobin, characterized in that the test sample contains an iron transport protein by applying the method for suppressing the degradation of hemoglobin.

[0006] The iron transport protein may be any mammalian iron transport protein such as human, bovine, porcine, equine, mouse, rabbit, sheep and goat, which has an inhibitory action on hemoglobin degradation, and blood transferrin. ,
Lactoferrin in milk, uteroferin in uterine fluid, or those modified by genetic engineering or the like is used. The iron transport protein contained in the hemoglobin-containing sample is
It is used in a weight ratio of 0.00002 to 2,000,000 to 1 hemoglobin, but 0.00 to 1 hemoglobin
It is preferably used in a weight ratio of 08 to 1,000,000. When a purified transferrin preparation having a purity of 95% or more and an iron content of 90% or more is used as the iron transport protein, the concentration in the sample is 0.5 to 2000 μg / ml, preferably 20.
It may be contained in an amount of ˜1000 μg / ml.

The hemoglobin-containing sample may be any sample as long as it contains hemoglobin, and examples thereof include feces, blood, urine and sputum.

The test sample is usually dissolved in a buffer or the like and used for the measurement of hemoglobin. As the buffer solution, a phosphate buffer solution, a borate buffer solution, an imidazole buffer solution, a glycine buffer solution, a Tris-hydrochloric acid buffer solution or the like is used, and these buffer solutions have a pH of 6.0 to 9.0, preferably 6.5.
The range of 8.5 is preferable, and the buffer solution may contain one or more kinds of sodium chloride, antibiotics, antibacterial agents such as sodium azide, lactic acid or albumin.

Examples of antibiotics include penicillin, acetylspiramycin, minocycline, etc., and examples of albumin include animal albumin of humans, rabbits, sheep, goats, horses, cows, pigs, etc., ovalbumin, etc.

To enhance the effects of iron transport proteins,
Antibiotics, albumin, sodium azide, in buffer
When lactic acid or the like is added, antibiotics 0.005 to 0.5% by weight, preferably 0.01 to 0.2% by weight, albumin 0.05 to 5% by weight, preferably 0.1 to 2% by weight, Sodium azide is added at a concentration of 0.05 to 0.5% by weight, preferably 0.1 to 0.3% by weight, and lactic acid 0.1 to 15% by weight, preferably 0.5 to 5.0% by weight. These additives were added to the hemoglobin in the sample in a weight ratio to hemoglobin 1 of 0.002 to 500 of antibiotics.
The ratio of albumin is 0.02 to 50 million, sodium azide is 0.025 to 5 million, and lactic acid is 0.04 to 150 million. The storage condition of the sample using the buffer solution is 0 to 45 ° C, preferably 4 to 10 ° C.

The method for measuring hemoglobin in the present invention may be any method for detecting or quantifying hemoglobin, and examples thereof include an immunological measurement method using an anti-hemoglobin antibody.

[0012] As an immunological assay for hemoglobin,
Any immunoassay method using an anti-hemoglobin antibody may be used, for example, a simple immunodiffusion method in which an anti-hemoglobin antibody and hemoglobin in a test sample are reacted on an agar plate, a double immunodiffusion method. Methods such as immunodiffusion method, reverse passive hemagglutination method using animal blood cells sensitized with anti-hemoglobin antibody, enzyme immunoassay method using anti-hemoglobin antibody labeled with enzyme, latex aggregation using latex particles sensitized with anti-hemoglobin antibody Method, latex aggregation inhibition method, gold colloid aggregation method using gold colloid particles sensitized with anti-hemoglobin antibody, and the like.

An immunological assay method using the latex agglutination method will be exemplified below.

Ordinary Polyclonal Antibody Production Method (Experimental Biology Course 14, "Immune Biology", edited by Shigeru Muramatsu, 19)
In 1985, published by Maruzen), animals capable of producing antibodies such as rabbits, sheep, and goats using human hemoglobin as an antigen are immunized with the antigen, and then blood is collected. After blood collection, a commonly used purification method (for example, salting out with ammonium sulfate, DEA
Purification by E-cellulose (IgG fractionation) to obtain anti-human hemoglobin antibody. The anti-human hemoglobin antibody was prepared by a conventional method (monoclonal and cancer, edited by Akira Taniuchi et al., 1985, published by Science Forum; monoclonal antibody, edited by Tatsuo Iwasaki et al., 1984, published by Kodansha Scientific). You may use a monoclonal antibody,
A commercially available anti-human hemoglobin antibody may be used.

The obtained antibody is latex particles (0.1 to 0) by a conventional method such as a physical adsorption method or a covalent binding method [Protein Chemistry 1 (amino acids, peptides), edited by Shiro Akahori et al., 1969, Kyoritsu Shuppan]. .6 μm) and centrifuged and washed with a buffer containing bovine serum albumin or the like to prepare an anti-human hemoglobin antibody-sensitized latex reagent.

Using the latex reagent thus obtained, a method for determining an agglutination image on a slide plate by a slide plate method, and the speed of the agglutination reaction can be measured optically or electrically by a spectrophotometer, a turbidimeter or the like. Human hemoglobin is measured by a method of quantitative measurement.

Examples of the present invention will be shown below.

[0018]

【Example】

Example 1 (1) Preparation of anti-human hemoglobin antibody-sensitized latex reagent 0.2 M containing 2 mg of polyclonal anti-human hemoglobin antibody prepared by immunizing rabbits with human hemoglobin
0.2M Tris-hydrochloric acid buffer solution (pH 8.2) in which 2 ml of Tris-hydrochloric acid buffer solution (pH 8.2) and 1% polystyrene latex [particle size 0.35 μm; manufactured by Kyowa Hakko Kogyo Co., Ltd.] were suspended.
2) 2 ml was mixed and stirred at 4 ° C. for 24 hours to adsorb the antibody to the latex. Then, the latex was washed with the above Tris-HCl buffer using a centrifuge, and 0.5% bovine serum albumin (BS
The anti-human hemoglobin antibody-sensitized latex reagent was prepared by suspending it in a 0.2 M Tris-HCl buffer (pH 8.2) containing A).

(2) Fecal dissolution buffer As a fecal dissolution buffer, sodium azide 0.1%,
The bovine transferrin described in Table 1 (manufactured by Seikagaku Corporation, purity 95) was added to 0.05M borate buffer (pH 8.0) containing 0.2% BSA and 0.85% sodium chloride.
% Or more, iron content 90% or more], and the concentration is 0.
Fecal lysis buffers of 5, 20, 100 and 1000 μg / ml were prepared.

(3) Fecal lysis buffer for control 0 As a control of the stool lysis buffer prepared in (2), 0.1% sodium azide, 0.2% BSA, and 0.85% sodium chloride were added. 0.05 M borate buffer (pH 8.
0) was prepared.

(4) Immediate detection operation The fecal lysis buffer prepared in (2) or the control stool lysis buffer prepared in (3) was dissolved by changing the concentration of human hemoglobin. Was dissolved at a concentration of 6 mg / ml. 100 μl of this test liquid and 25 μl of the latex reagent were dropped on a slide plate, and both liquids were mixed with a stir bar to spread the slide plate in a circle. After gently moving the slide plate back and forth and right and left for 3 minutes, an agglutination image of the latex reagent was observed. The results are shown in Table 1.

[0022]

[Table 1]

In the immediate detection operation shown in Table 1, both the method of the present invention and the control method could measure up to 125 ng / ml of human hemoglobin.

(Detection after storage for 7 days) Human hemoglobin was dissolved in the fecal lysis buffer prepared in (2) or in the control fecal lysis buffer prepared in (3) at different concentrations, and then dissolved in a healthy person. Was dissolved at a concentration of 6 mg / ml. The obtained solution was stored at 23 ° C. for 7 days, and then the latex reagent was added dropwise by the same method as in (4) to observe an aggregation image.
The results are shown in Table 2.

[0025]

[Table 2]

According to Table 2, the method of the present invention had a high sensitivity even when stored at 23 ° C. for 7 days and suppressed the decomposition of human hemoglobin.

(6) Detection after storage at high temperature Human hemoglobin was dissolved in the fecal lysis buffer prepared in (2) or in the control fecal lysis buffer prepared in (3) at different concentrations, and then dissolved in a healthy person. Was dissolved at a concentration of 6 mg / ml. The obtained solution was stored at 37 ° C. for 1 day, and then the latex reagent was added dropwise by the same method as in (4) to observe an aggregation image. The results are shown in Table 3.

[0028]

[Table 3]

According to Table 3, the method of the present invention has a high sensitivity even at a high temperature of 37 ° C. and suppresses the decomposition of human hemoglobin.

Example 2 A solution of 0.01% bovine transferrin, 0.2% BSA or 0.1% sodium azide in 0.05M borate buffer (pH 8.0), or no solution. The hemoglobin solution was treated at 23 ° C. in the same manner as in Example 1 except that the added solution was used as the fecal dissolution buffer.
The hemoglobin after storage for 7 days was measured. The results are shown in Table 4.

[0031]

[Table 4]

According to Table 4, the method of the present invention has the highest sensitivity and suppresses the degradation of human hemoglobin.

[0033]

INDUSTRIAL APPLICABILITY The present invention suppresses the decomposition of hemoglobin in feces and enables highly accurate measurement of hemoglobin in a clinical test method used for diagnosing colorectal cancer.

Claims (2)

[Claims] 1. A method for suppressing the degradation of hemoglobin, characterized in that the hemoglobin-containing sample contains an iron transport protein. 2. A method for measuring hemoglobin in a test sample, wherein the sample contains an iron transport protein.