JPH10227793A - Measuring method for carbamic hemoglobin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure CHb accurately and easily, by reacting an examined object containing carbamic hemoglobin with solid phase, and measuring anti-CHb antibody which is connected to solid phase due to reaction. SOLUTION: Solid phase, without being specified, is for example plate or polymeric particle, and the plate is for example micro titre plate made of plastic. The anti-CHb antibody is mono or poly chronal antibody, but mono chronal is preferable. An examined CHb containing object is material which is produced by hemolyzing and diluting all blood with hemolytic agent. As an example of measuring method, material made by first fixing anti-hemoglobin antibody on the plate, and performing blocking treatment of them is prepared as a solid phase. The solid and the examined object are mixed, and the anti-hemoglobin antibody on the solid phase and CHb in the examined object are reacted. After finish of reaction, the examined object is cleaned, the examined object which fails to react is removed, the anti-CHb antibody is reacted, and anti-CHb antibody without reacting is removed, and the anti-CHb antibody connected with the solid phase is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring carbamylated hemoglobin.

[0002]

2. Description of the Related Art Glycated hemoglobin is formed by combining sugar in blood with red blood cells in proportion to its concentration and then binding to hemoglobin, and its concentration is the average in blood for the past one to two months. It is said to reflect typical sugar concentration.
Since the blood sugar level and the urinary sugar level are less susceptible to physiological factors, the concentration of glycated hemoglobin in the blood is used as an optimal index for diagnosis of diabetes or follow-up of diabetic patients. For this reason, measurement of glycated hemoglobin by liquid chromatography is widely performed in the field of clinical testing.

[0003] Carbamylated hemoglobin (hereinafter referred to as CHb)
Is produced by the reaction of isocyanic acid generated from urea-derived cyanate ion in plasma with the N-terminal of hemoglobin. CHb in the blood is known to increase in patients with renal failure.

[0004] As a method for measuring CHb, Clinical Che
In Mistry, Vol. 36, No. 4, 607-610 (1990), N-terminal valine (carbamylated valine) of carbamylated hemoglobin was separated by acid hydrolysis, and the generated valine hydantoin was measured by high performance liquid chromatography. A method for doing so is disclosed. Also, Clinical Chemistry, Vol. 3
9, No. 12,2514-2518 (1993), CHb was converted to glycated hemoglobin A1 by cation exchange liquid chromatography.
c (hereinafter referred to as HbA1c).

[0005]

In the former method, the recovery after the hydrolysis treatment is as low as 70% or less, and there remains a problem in specificity. In the latter method, since the CHb peak does not overlap with the HbA1c peak, improvement in the quantification of HbA1c is described, but quantification of CHb itself is not described. Also, according to the chromatogram described, the CHb peak was found to be hemoglobin A0 (HbA
0) It is predicted that the accuracy is insufficient for CHb quantification because it overlaps the peak. Therefore, in the prior art, a method for measuring CHb with high accuracy and simpleness has not been established.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a method for measuring carbamylated hemoglobin with high accuracy and convenience.

[0007]

According to the method for measuring carbamylated hemoglobin of the present invention, a sample containing carbamylated hemoglobin in a solid phase is reacted, and then an anti-carbamylated hemoglobin antibody is reacted. Measuring the anti-carbamylated hemoglobin antibody bound to the solid phase.

[0008] As the solid phase used in the present invention,
There is no particular limitation on the solid phase that has been used in the field of immunoassay, and any of them can be used. Specifically, for example, a plate, polymer fine particles, and the like can be used. Examples of the plate include a plastic microtiter plate. Examples of the polymer fine particles include synthetic polymer fine particles such as polystyrene and polyacrylate; and natural polymer fine particles such as polyamino acids and high molecular weight polysaccharides. As the polymer fine particles,
In particular, latex particles are preferred. The particle size of the fine particles is preferably 0.01 to 10 μm.

In the solid phase, an anti-hemoglobin antibody may or may not be immobilized on its surface in advance.

When an anti-hemoglobin antibody is immobilized on the solid phase in advance, the anti-hemoglobin antibody is not particularly limited as long as it is a known anti-hemoglobin antibody, and may be a monoclonal antibody or a polyclonal antibody. As the anti-hemoglobin antibody, an anti-human hemoglobin antibody is particularly preferred. The anti-hemoglobin antibody can be immobilized on the solid phase by a known method. For example, the anti-hemoglobin antibody can be immobilized by adding the solid phase to an antibody solution having a concentration of about 1 μg / ml and leaving it at 4 ° C. overnight. Usually, after the above-mentioned immobilization treatment, a protein solution such as bovine serum albumin is brought into contact with the solid phase, for example, in order to cause the protein to be adsorbed to the protein adsorption site remaining without the antibody on the solid phase surface remaining adsorbed. (A so-called blocking treatment performed to prevent non-specific adsorption of proteins and the like when using a solid phase).

The anti-CHb antibody used in the present invention may be a monoclonal antibody or a polyclonal antibody.
Monoclonal antibodies are preferred for improving measurement accuracy. An anti-CHb monoclonal antibody can be obtained using a known cell fusion technique. Specifically, for example,
It can be obtained by culturing a hybridoma obtained by cell fusion between myeloma cells and spleen lymphocytes of a mouse immunized with CHb.

The sample containing CHb used in the present invention is hemolyzed blood, for example, whole blood collected from a blood collection tube containing a blood anticoagulant (eg, sodium fluoride). , Hemolytic agent (Triton X-1)
00, a buffer solution containing a surfactant such as Tween 20, etc.) and hemolyzed and diluted about 10 to 300 times.

As an example of a specific measurement method of the present invention, a method using a microtiter plate as a solid phase will be described below.

(1) First, an anti-hemoglobin antibody is immobilized on a microtiter plate by the above-mentioned method, and then a blocking solution is prepared as a solid phase. (2) An anti-hemoglobin antibody on the solid phase is reacted with CHb in the sample by mixing the solid phase and the sample.
The reaction conditions are preferably 10 to 40 ° C. for 0.5 to 5 hours, for example, about 2 hours at room temperature. (3) After the completion of the reaction, washing is performed to remove unreacted specimens and the like.

(4) React with an anti-CHb antibody. The reaction conditions are the same as the reaction conditions described in the above (2). (5) After completion of the reaction, washing is performed to remove unreacted anti-CHb antibody. (6) Quantification of anti-CHb antibody: Anti-CHb antibody bound to the solid phase via CHb is quantified. This quantification method includes:
A known method conventionally used in the field of immunoassay is used. For example, the anti-CHb antibody used in the above (4) is labeled in advance with an enzyme (usually called an ELISA method), a fluorescent dye or a radioactive substance,
A method for measuring the label; anti-C used in the above (4)
A method in which biotin is bound to an Hb antibody in advance, and the biotin is reacted with labeled avidin to measure the label; a labeled antibody that specifically reacts with the anti-CHb antibody is further reacted, and the label is measured. And the like.

(7) A calibration curve is prepared in advance between the amount of CHb in the sample and the activity of the label reflecting the amount of reacted anti-CHb antibody, and the calibration curve is applied to the calibration curve. C
The amount of Hb is measured.

As another specific example of the measuring method of the present invention,
A method using latex particles as a solid phase will be described below.

(1) First, CHb in the sample is adsorbed on the latex particles by mixing the latex particles with the sample. (2) After the completion of the reaction, washing is performed to remove unreacted specimens and the like.

(3) React with an anti-CHb antibody. As a result, the CHb adsorbed on the latex particles and the anti-CHb antibody bind. The reaction condition is 0.5 to 5 at 10 to 40 ° C.
Time, for example, about 2 hours at room temperature is preferable. (4) After completion of the reaction, washing is performed to remove unreacted anti-CHb antibody. (5) Quantification of anti-CHb antibody: Anti-CHb antibody bound to the solid phase via CHb is quantified. This quantification method includes:
A method of adding an antibody specifically reacting to a site other than the site reacting with CHb in the anti-CHb antibody, for example, an anti-IgG antibody is used. In this case, the anti-IgG antibody
By binding to the anti-CHb antibody bound to the solid phase via CHb, the solid phases are aggregated. Therefore, the amount of anti-CHb antibody can be measured by measuring the degree of aggregation by, for example, increasing the absorbance, and thereby the amount of CHb bound to the solid phase can be measured. (6) A calibration curve is prepared in advance between the amount of CHb in the sample and the degree of aggregation, and the amount of CHb in the sample is measured by applying the calibration curve.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to examples of the present invention, but the present invention is not limited thereto.

Reference Example 1 Preparation of Anti-CHb Monoclonal Antibody (1) Human CHb is sufficiently dispersed in Freund's complete adjuvant. (2) 100 μl of the dispersion obtained in the above (1) was added to B
Alb / c mice were immunized intraperitoneally with four injections every two weeks. (3) the immunized mice were sacrificed to obtain lymphocytes ¹⁰⁶ and removal of the spleen. (4) The lymphocytes and mouse myeloma cells were fused and cultured in the presence of polyethylene glycol. (5) The supernatant of the proliferating cells was collected.

(6) The supernatant was examined for the presence of anti-human CHb antibody by ELISA. (7) The cells that have become positive in (6) above are diluted and cultured by the limiting dilution method, and the anti-human CH in the culture solution is diluted.
b) The presence of the antibody was confirmed by ELISA, and anti-human C
Cells producing Hb antibodies were isolated. (8) The obtained anti-human CHb mouse monoclonal antibody-producing cells (hybridoma cells) were cultured in large quantities. (9) The obtained cultured cells were inoculated into the peritoneal cavity of mice. (10) Ascites was collected every three days from two weeks after inoculation to obtain an anti-human CHb mouse monoclonal antibody. This antibody was used in Example 1 below.

Example 1 Human CHb by ELISA
(1) 1.0 μg / ml of an anti-human Hb rabbit antibody at a concentration of 1.0 μg / ml was dispensed into wells of a 96-well microtiter plate, 50 μl each. (2) It was left at 4 ° C. for 12 hours. (3) After washing the wells 5 times with a phosphate buffered saline containing bovine serum albumin at a concentration of 2% by weight and Tween 20 at a concentration of 0.02% by weight,
00 μl was added to the wells and left at 4 ° C. for 12 hours.

(4) The supernatant in the well is removed, and CHb is reduced to 0, 1, 3, 6, 9, 12, 15, or 18% in total hemoglobin (CHb + glycated hemoglobin + non-glycated hemoglobin). [This sample was obtained as follows. That is, fresh blood 500 of a healthy person
To 500 μl, 500 μl of a physiological saline solution of cyanate (a 0.9% by weight aqueous solution of sodium chloride dissolved so that the concentration of sodium cyanate becomes 0.2 mg / dl) was added, and the temperature was kept at 40 ° C. Heated in a water tank. Sampling is performed at regular intervals, and a hemolytic agent (50 mM phosphate buffer containing Tween 20 at a concentration of 0.05% by weight, pH
After hemolysis and dilution by 20 times in 7.4), CHb, glycated hemoglobin and non-glycated hemoglobin were separated by the liquid chromatography method described in Japanese Patent Application No. 8-161860 previously filed by the present inventors. Then, the CHb concentration was determined from the peak area. The sample having a CHb concentration of 0% was prepared in the same manner as described above using only a physiological saline solution instead of the cyanate physiological saline solution. The apparatus and measurement method of the liquid chromatography method described in Japanese Patent Application No. 8-161860 are as follows. Liquid sending pump: Shimadzu Corporation, LC-9A, sample introduction device: Sekisui Chemical Co., Ltd.
ASU-420, detector: UV-visible absorptiometer, manufactured by Shimadzu Corporation, SPD-6AV, detection is at a wavelength of 415 nm
Was measured for absorbance. As eluents, phosphate buffer (A) (0.1 M, pH 6) and phosphate buffer (B)
(0.3M, pH 7.2), phosphate buffer (A)
And then the phosphate buffer (B) was sent. As a separation column, a column packed with a cation exchanger, manufactured by Sekisui Chemical Co., Ltd., trade name “Micronex A”
1c HS-II "was used. ] 150 μl is dropped,
It was left at room temperature for 2 hours. (5) After completion of the reaction, the resultant was washed four times with a physiological saline solution containing Tween 20 at a concentration of 0.02% by weight. (6) Next, peroxidase (POD) -labeled anti-human C
Hb mouse monoclonal antibody (concentration 500 ng / m
l) was added and left at room temperature for 2 hours.

(7) After the reaction is completed, Tween 20 is added to 0.
Washing was carried out four times with physiological saline containing a concentration of 02% by weight. (8) Next, at a concentration of 0.1% by weight, ABTS [2, 2 '
-Azino-bis- (3-ethylbenzot
hiazoline-6-sulfonic aci
d)] containing a 0.005% by weight H ₂ O ₂ solution
μl was added and left at room temperature for 30 minutes. (9) After completion of the reaction, color development was measured by colorimetry. The measurement is 41
The measurement is performed by measuring the absorbance at 5 nm and 492 nm, and the absorbance ratio (415 nm / 492n) is measured.
m) was calculated and the results are shown in Table 1.

[0026]

[Table 1]

[0027]

The structure of the method for measuring carbamylated hemoglobin of the present invention is as described above.
Carbamylated hemoglobin can be measured with high accuracy and in a simple manner. In addition, a large amount of sample can be measured at a time.

Claims (1)

[Claims] 1. After reacting a sample containing carbamylated hemoglobin on a solid phase, reacting with an anti-carbamylated hemoglobin antibody, and measuring the anti-carbamylated hemoglobin antibody bound to the solid phase via the carbamylated hemoglobin. A method for measuring carbamylated hemoglobin, which comprises: