JPH0772154A - Method for measuring hemoglobin - Google Patents

Abstract

PURPOSE:To restrain hemoglobin from being decomposed and to precisely measure the hemoglobin which has been preserved for a long time by a method wherein a non-penicilin-based antibiotic is made to coexist with a sample which contains the hemoglobin. CONSTITUTION:A cephalosporin-based antibiotic such as cephamycin A, cephalosporin or the like or an aminoglucocide-based antibiotic such as streptomycin A, dehydrostreptomycin or the like is used as a non-penicilin-based antibiotic which is made to coexist. A dejection, blood, urine or the like is used as a sample which contains hemoglobin, and the sample is used to measure the hemoglobin in a state that the non-penicilin-based antibiotic has been dissolved in a buffer solution or the like. The amount to be added of the non- penicilin-based antibiotic is to be set in such a way that the concentration of 0.005 to 0.5wt.%, preferably 0.01 to 0.2wt.%, is obtained in the buffer solution. After a sample solution has been preserved for 1 to 30 days at 4 to 50 deg.C, it can be used to measure the hemoglobin.

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, which is useful in a clinical test method that requires long-term storage of a hemoglobin-containing sample. By applying the degradation suppressing method to a clinical test method that requires long-term storage 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, since the stool sample containing hemoglobin is stored for a long period of time, hemoglobin in the sample may be decomposed during the storage period, and thus hemoglobin may not be accurately measured. A method of adding penicillin for the purpose of suppressing the decomposition of hemoglobin in feces has been disclosed (JP-A-59-125064), but the effect of suppressing the decomposition of hemoglobin by this method is not always sufficient.

[0003]

With the spread of screening for colorectal cancer by mail-tested stool samples, it has been desired to develop a method for suppressing the degradation of hemoglobin in stored samples. By applying the degradation suppressing method to a clinical test method for storing a hemoglobin-containing sample for a long period of time, a clinical test method capable of accurately measuring hemoglobin is provided.

[0004]

The present invention relates to a method for suppressing the degradation of hemoglobin, which comprises allowing a non-penicillin antibiotic to coexist in a hemoglobin-containing sample. Furthermore, according to the present invention, the method for suppressing the degradation of hemoglobin is applied,
It is possible to provide a method for measuring hemoglobin, which comprises allowing a non-penicillin antibiotic to coexist in a test sample.

Examples of the non-penicillin antibiotics used in the present invention include cephamycin A, cephamycin B, cephamycin C, cephalosporins, cephalothin, cephalonium, cephalexin, cephaladin,
Cephalosporin antibiotics such as hetasporin, cephapirin, loracarbef, streptomycin A, streptomycin B, dehydrostreptomycin, oxystreptomycin, kanamycin, kasugamycin, gentamicin A, gentamicin C, lincomycin, bleomycin, mannoside hydroxy streptomycin, etc. Aminoglycoside antibiotics, nucleosides such as polyoxin A, polyoxin B, polyoxin C, polyoxin D, polyoxin E, polyoxin F, polyoxin G, polyoxin H, polyoxin J, polyoxin K, polyoxin L, polyoxin M, tubercidin and formycin B Antibiotics, ansamycin, refamycin, refamycin B, refamycin L, reamycin Ansamycin antibiotics such as amycin S, rifamycin V, rifamycin Y, rifampicin, polymyxin, grammycin A, grammycin B, grammycin C, viomycin, bacitracin, actinomycin, tyrosidin A, tyrosidine B, tyrosidine C, tyrosidine D, tyrosidine S. , Polypeptide antibiotics such as staphylamycin, and other ticloheximide, ticloserin, sarcomycin A, spectinomycin, chloramphenicol, mitomycin, blasticin S, fumagillin, monesicin, pyrrolenitrin, phosphonomycin, etc. However, for example, minocycline, tetracycline, hydroxytetracycline, chronocycline, teramycin, nitrocycline, amysai Phosphorus, anthracyclines, methacycline, deoxy anthracyclines, glycolate cyclin, tetracycline antibiotics such as en Hydro tetracycline, acetyl spiramycin,
It is preferable to use macrolide antibiotics such as erythromycin, picromycin, pimaricin, resensomycin, amphotericin B, candicin A and candicin B. The above-mentioned non-penicillin antibiotics to be added to hemoglobin are used alone or in combination of two or more, and are used in a weight ratio of 0.002 to 5,000,000 with respect to hemoglobin 1, but 0.004 to 5 with respect to hemoglobin 1. It is preferable to use it at a weight ratio of 2,000,000.

The hemoglobin-containing sample may be any sample as long as it contains hemoglobin, but feces, blood, urine, sputum, etc. are preferably used.

The test sample is usually dissolved in a buffer solution or the like to which a non-penicillin antibiotic is added, and then used for the measurement of hemoglobin. The amount of non-penicillin antibiotics added is
0.005-0.5% by weight in buffer, preferably 0.1.
It is added so as to have a concentration of 01 to 0.2% by weight. 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. A range of 8.5 is good. The buffer solution may contain salts such as sodium chloride, stabilizers such as albumin, antibacterial agents such as sodium azide, lactic acid and the like.

As albumin contained in the buffer solution,
Examples thereof include animal albumin such as human, rabbit, sheep, goat, horse, cow, pig, etc., and ovalbumin. In order to enhance the effects of non-penicillin antibiotics, albumin in buffer,
When sodium azide, lactic acid, etc. are added, albumin 0.05 to 5% by weight, preferably 0.1 to 2% by weight, sodium azide 0.05 to 0.5% by weight, preferably 0.1 to 0% It is preferable to add it at a concentration of 0.3% by weight, lactic acid 0.1 to 15% by weight, preferably 0.5 to 5.0% by weight. These additives were added to the sample hemoglobin in a weight ratio of 1 to hemoglobin 1 of albumin 0.0.
20-50 million, sodium azide 0.02-500
Lactic acid may be used in a ratio of 0.04 to 150 million.

The test liquid may be used as it is for the hemoglobin measurement method, but it may also be used for the hemoglobin measurement method after being stored at 4 to 50 ° C. for 1 to 30 days.

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.

As an immunological assay method 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 using enzyme-labeled anti-hemoglobin antibody, latex using latex particles sensitized with anti-hemoglobin antibody Examples thereof include an agglutination method, a latex agglutination inhibition method, and a gold colloid agglutination method using gold colloid particles sensitized with an anti-hemoglobin antibody.

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

Ordinary Polyclonal Antibody Preparation 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 bond method [protein chemistry 1 (amino acid, peptide), 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.

[0017]

【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
0.2 M Tris-HCl buffer (PH 8.2) in which 2 ml of Tris-HCl buffer (PH8.2) and 1% polystyrene latex [particle size 0.35 μm; manufactured by Kyowa Hakko Kogyo Co., Ltd.] were suspended.
8.2) 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-mentioned Tris-hydrochloric acid buffer solution using a centrifuge, and 0.2M Tris-hydrochloric acid buffer solution (PH) containing 0.5% bovine serum albumin (BSA) so that the latex content became 1%.
The suspension was suspended in 8.2) to prepare an anti-human hemoglobin antibody-sensitized latex reagent.

(2) Fecal dissolution buffer As a fecal dissolution buffer, sodium azide 0.1%,
The amounts of non-penicillin antibiotics shown in Table 1 were dissolved in 0.05M borate buffer (PH8.0) containing 0.2% BSA and 0.85% sodium chloride at different concentrations. .

(3) Comparative Buffer Solution As a comparative control of the fecal lysis buffer solution prepared in (2), 0.05M borate buffer solution (PH8.0) containing penicillin in the amount shown in Table 1 was used. It was made.

(4) Fecal lysis buffer for control 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. .05M borate buffer (PH8.
0) was prepared.

(5) Immediate detection operation The concentration of human hemoglobin in the fecal lysis buffer prepared in (2), the comparative buffer prepared in (3) or the control fecal lysis buffer prepared in (4). Was changed to dissolve the stool, and the feces of a healthy person 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]

According to Table 1, human hemoglobin up to 125 ng / ml can be measured by the method of the present invention, the comparative control method and the control method in the immediate detection operation.

(Detection after 7 days of storage) Human was added to the fecal lysis buffer prepared in (2), the comparative buffer prepared in (3), or the control fecal lysis buffer prepared in (4). Hemoglobin was dissolved at different concentrations, and the stools of healthy people were mixed with 6
It was dissolved at a concentration of mg / ml. The obtained solution was stored at 23 ° C. for 7 days, and then the latex reagent was added dropwise in the same manner as in (5) to observe an aggregation image. The results are shown in Table 2.

[0025]

[Table 2]

According to Table 2, hemoglobin could be detected at 2000 ng / ml or more of hemoglobin in the control without addition of antibiotics. With the hemoglobin of the comparative example, the detection sensitivity was slightly improved, and the hemoglobin was 1000 ng.
It was possible to detect by weak aggregation at / ml. In contrast, acetylspiramycin and minocycline
Both can be detected by weak aggregation of hemoglobin 125 ng / ml at a concentration of 0.2%, 0.01-0.05%
It was possible to detect by strong aggregation of hemoglobin of 250 ng / ml even at the concentration, and the detection sensitivity was remarkably improved as compared with the comparative example.

(6) Detection after high temperature storage Human hemoglobin was added to the fecal lysis buffer prepared in (2), the comparative buffer prepared in (3) or the control fecal lysis buffer prepared in (4). Was dissolved in various concentrations, and the feces of 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 (5) to observe an aggregation image. The results are shown in Table 3.

[0028]

[Table 3]

According to Table 3, hemoglobin was detectable at 4000 ng / ml in the control without addition of antibiotics. With the hemoglobin of the comparative example, the detection sensitivity was slightly improved, and with 2000 ng / ml of hemoglobin, detection by weak aggregation was possible. In contrast, acetylspiramycin and minocycline are associated with hemoglobin 250
The detection was possible by weak aggregation of ng / ml, and the detection sensitivity was significantly improved compared to the comparative example.

[0030]

EFFECTS OF THE INVENTION By applying the method for suppressing the degradation of hemoglobin in a stool sample of the present invention to a method for long-term storage of a hemoglobin-containing sample, it is possible to accurately measure hemoglobin in a clinical test method used for diagnosing colorectal cancer. become.

Claims (2)

[Claims] 1. A method for suppressing the degradation of hemoglobin, which comprises allowing a non-penicillin antibiotic to coexist in a hemoglobin-containing sample. 2. A method for measuring hemoglobin in a test sample, which comprises coexisting a non-penicillin antibiotic in the sample.