JPH07103978A - Measurement of free hemoglobin - Google Patents

Abstract

PURPOSE:To realize an easy, quick and highly accurate measurement of free Hb by measuring the free Hb after an anti-haptoglobin Hp antibody is added to a test sample to react it with a hemoglobin Hb-Hp complex therein. CONSTITUTION:After an anti-Hp antibody is added to a free Hp and an Hb-Hp complex in a test sample (for example, serum, plasma, urine, etc.) in order to form an immunological complex, it is allowed to react on an anti-Hb antibody fixing solid phase and only a free Hb is allowed to joint to the solid phase selectively. Further, after the solid phase is cleaned, the anti-Hb antibody labeled with oxigen is made to react and excessive oxigen-labeled anti-Hb antibody is cleaned and removed, then an enzyme substrate and a color coupler are allowed to react each other and a reaction stopping agent is added thereto to measure its cloring intensity with a colorimeter. Finally, the free Hb is quntitatively determined using the standard curve which is prepared based on the data of Hb standard liquid measured in advance as a control.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring free hemoglobin and a free hemoglobin measuring kit. More specifically, free hemoglobin used in the field of clinical examination can be simply, quickly and accurately measured by an immunological method. The present invention relates to a free hemoglobin measurement method and a free hemoglobin measurement kit.

[0002]

BACKGROUND OF THE INVENTION Problems to be Solved by the Invention: Extracorporeal circulation, burns, incompatible blood transfusion, post-transfusion hemolysis, hemolytic disease, etc. may cause a large amount of hemolysis, and complications such as hemolytic renal damage. Often accompanied by. The cause of this hemolytic nephropathy is haptoglobin (hereinafter abbreviated as Hp).
It is said that it is free hemoglobin that is not bound with, and therefore measuring free hemoglobin in blood has clinically important significance.

More specifically, the hemoglobin-haptoglobin complex in blood is promptly taken up by the liver,
It is metabolized to bilirubin through the normal pathway, while
Free hemoglobin flows in the blood and is taken up by the reticuloendothelial system,
While being partially metabolized by bilirubin, it partially passes through the renal glomerulus and is excreted in the urine, but at that time, it is taken up by renal tubular epithelial cells and causes renal damage.

Usually, hemoglobin (hereinafter abbreviated as Hb) in plasma or serum is a Hb-Hp complex and free Hb.
Therefore, when quantifying this substance, it is necessary to separate and quantify it. For the measurement of free Hb, a simple fractional quantification method, a free Hb adsorption quantification method (column method) and the like are known. The simple fractional quantification method is to calculate free Hb from the binding ratio of Hp-Hb from the quantified value of serum Hp by the one-way radial immunodiffusion method and the quantified value of total Hb by the cyanmethemoglobin method. However, since the simple fractional quantification method is a method of indirectly determining the free Hb amount from the total Hb amount and the total Hp amount, total Hb and total Hp
There is a problem that free Hb below the quantitation limit of 1 cannot be measured.

The free Hb adsorption quantitative method is a method in which Hp is immobilized on a carrier, free Hb is adsorbed on the immobilized Hp, and the amount of Hb-Hp complex, that is, the amount of free Hb is measured by the color tone of Hb. Is. More specifically, Hp is immobilized on activated sepharose, and this is uniformly packed in a cylindrical column. Various free Hb solutions having known concentrations are prepared in advance, and a calibration curve is prepared using a certain amount of this solution. Inject 1.0 ml of test serum into a vertically standing column and
Wash thoroughly with 0 times the amount of physiological saline. The measurement is performed at room temperature. Measure the colored portion of Hb in mm and use the calibration curve to find Hb
It is to read the quantity. (Mr. Oshiro: Clinical Haptoglobin, Nagai Shoten, 1987)

However, this method has a problem that reproducibility and quantitativeness are lacking because the length of the colored phase varies depending on the uniformity of the Hp-immobilized sepharose, the injection method of the sample, the length of the column aperture and the like. In addition, in recent years, EL using a Hp solid-phased plate
The ISA method has been reported. (Yuro Kanamori, Midori Nagatomo: Clinical Examination, 36 , 1350-1354, 1992) As a result of intensive studies over the years, the present inventors found that anti-Hp
Focusing on the fact that the antibody reacts with free Hp, Hb-Hp complex and not with Hb, the amount of free Hb present in plasma, serum, or urine can be accurately and simply
We found a method for rapid measurement, and completed the "free hemoglobin measurement kit".

[0007]

The method for measuring free Hb according to the present invention is applied to Hp and Hb in a sample (eg, serum, plasma, urine, etc.).
-Hp complex was reacted with anti-Hp antibody to form an immune complex, and then free Hb was reacted with anti-Hb antibody-immobilized solid phase E
It is characterized in that the measurement is performed by the LISA method. The free Hb measurement kit of the present invention is a measurement kit used in the measurement of the present method described above, and is an anti-H that reacts with the free Hp, Hb-Hp complex present in the sample.
It means a measurement kit comprising a reagent consisting of p antibody.

Specifically, free Hp and Hb-H in the sample
After the anti-Hp antibody was added to the p-complex to form an immune complex, it was reacted with the anti-Hb antibody-immobilized solid phase and free H
Only b can be selectively bound. Further, after washing the solid phase, an enzyme-labeled anti-Hb antibody is reacted and excess enzyme-labeled anti-Hb antibody is removed by washing, and then an enzyme substrate and a color developing agent are allowed to act. After adding the reaction terminator,
The color development intensity is measured with a colorimeter. As a control, Hb standard solution was similarly measured in advance, and free Hb was directly quantified from the prepared standard curve.

As the anti-Hp antibody which can be used in the present invention, a heterologous animal such as a rabbit or goat is immunized with Hp, and a splenic cell of a Hp-immunized mouse is fused with a myeloma cell. The monoclonal antibody obtained by the method is used, and several types of polyclonal antibodies are commercially available, such as Wako Pure Chemical Industries, Ltd., Medical and Biological Research Institute, and monoclonal antibodies are commercially available from Daiichi Pure Chemicals Co., Ltd. Because it is available, it is easily available. For the anti-Hb antibody, a polyclonal antibody or a monoclonal antibody can be used as in the above. Usually, commercially available products include polyclonal antibodies for medical and biological research, and monoclonal antibodies for MEDIX BIOTECH Co., Ltd., etc., which are easily available.

To further increase the antibody purity, anti-Hp
Affinity chromatography using Hp-Sepharose for the antibody and Hb-Sepharose for the anti-Hb antibody can be appropriately used. Next, the ELIS generally used for the measurement of free Hb in the present invention.
The measurement can be carried out by any of the A method such as the sandwich method and the competitive method, but the sandwich method is preferable for the measurement of this method. As the solid phase material for immobilizing the anti-Hb antibody, any of the solid phase materials conventionally used in enzyme immunoassays and the like can be used. The body bead method, the plastic bead method and the like are preferable.

There are various enzymes for the enzyme-labeled substance which can be used in the present invention. Among them, peroxidase, alkaline phosphatase, β-galactosidase and the like are particularly preferable. There are several methods for labeling an antibody with an enzyme, but the most important conditions imposed on each are: 1) The activity of the enzyme and the antibody is not reduced. 2) The bond is stable and can be stored for a long time.
3) The procedure is as simple as possible and the labeled antibody can be easily obtained. The enzyme labeling methods that have been developed so far to satisfy these conditions can be roughly classified into two types. One of them is a method of chemically linking an enzyme and an antibody, which is the glutaraldehyde method (S. Av.
rameas, Immunochemistry , 6 , 4
3, 1969) and the periodate oxidation method (P.K.Naka).
ne, A.N. Kawaoi, J .; Histochem . C
ytochem . , 22 , 1084, 1974) correspond to this. The other is a method of connecting an enzyme and a first antibody by using an antibody or the like as an intermediary. In this method, the avidin-biotin method (SM Hsu, L. Raine, H. Fan) is used.
ger, Am . J. Clin . Pathol . , 75 ,
734-738, 1981) is known. In the present invention, any labeling method can be used for measurement.

As one mode of producing the labeled antibody used in the present invention, for example, a method of obtaining an antibody labeled with peroxidase is known. Specifically, all amino groups of peroxidase are blocked with 1-fluoro-2,4-dinitrobenzene (FDNB), and then the adjacent hydroxyl group of the dinitrobenzenated sugar moiety of peroxidase is cleaved with periodate to remove the aldehyde group. Reborn.
Unreacted periodate is decomposed by adding ethylene glycol, and the reaction is stopped.

Next, 0.01M sodium carbonate buffer (p
After dialysis with H9.5), the newly formed aldehyde group is reacted with the amino group of the antibody to form a Schiff base. The Schiff base reaction product is then reduced with sodium borohydride to chemically stabilize it. After reduction, excess sodium borohydride is removed by dialysis to obtain the desired enzyme-labeled antibody. Further, it is preferable to purify the obtained labeled antibody by gel filtration before use.

As the color-developing substrate and color-developing agent, hydrogen peroxide and 3,3'-5,5 'are used in the case of peroxidase.
-Tetramethylbenzidine and 3,3'-diaminobenzidine are used, and in the case of alkaline phosphatase bromo-chloroindole phosphate nitro-
A blue tetrazolium drug substance is used. Further, the reaction conditions of the color former may be the same as those of the usual enzyme reaction, and the temperature is not particularly specified, but it is most preferably carried out at room temperature.

[0015]

EXAMPLES Examples will be shown below, but the present invention is not limited to these examples.

Example 1 Anti-human Hp antibody and human Hb-human Hp complex and release
Reactivity of human Hb [Preparation of human Hb- human Hp complex] Serum of healthy person 2 m
20 mg of purified human Hb was added to 1 and reacted at room temperature for 2 hours. After the reaction, perform a cooling centrifuge to remove insoluble matter.
It was performed at 000 rpm for 5 minutes. Sephacryl obtained by equilibrating 1 ml of the centrifugal supernatant with 0.1 M phosphate buffer (pH 7.2) containing 0.15 M sodium chloride in advance.
It was loaded on an S-200HR column (φ1.8 cm × 69 cm, manufactured by Pharmacia) and gel filtration was performed. The human Hb content of the eluted fraction was measured by an ELISA method using an anti-human Hb antibody. As shown in FIG. 1, a human Hb-human Hp complex fraction was obtained from the first peak, which was detected as two peaks, and a human Hb fraction was obtained from the second peak.

[Measurement of human Hb, human Hb-human Hp complex] An ELISA method (sandwich method) was used for measuring the human Hb-human Hp complex of peak 1 and the Hb of peak 2. That is, 200 μl of each diluted test serum was added to a 96-well microplate on which anti-human Hb antibody was immobilized in advance. Standard human Hb (0-160n
g / ml) was similarly added in 200 μl increments. After stirring for 1 minute with a micromixer, the mixture was reacted at 37 ° C. for 1 hour. After removing the liquid in each well, 250 μl of the washing liquid was added, and after stirring, the liquid was removed. This operation was repeated 3 times, 200 μl of the peroxidase-labeled anti-human albumin antibody solution was added, and the mixture was similarly reacted at 37 ° C. for 1 hour. After the reaction, the liquid was removed, and the same washing operation was performed using the above washing liquid. Next, a substrate solution (hydrogen peroxide + O -phenylenediamine) is added,
Reacted for 15 minutes at room temperature. Stop reaction by 4N H ₂ SO ₄
Perform with 50 μl, and use the colorimeter for microplate (492
nm / 630 nm). The human Hb value was calculated from a standard curve prepared from standard human Hb in advance.

[Reactivity with Anti-Human Hp Antibody] The human Hb-human Hp complex fraction (peak 1) and the human Hb fraction (peak 2) of the above-mentioned items were preliminarily added in amounts of 10 to 100 in each 0.5 ml.
0.5 m each of anti-human Hp antibody (manufactured by BML) diluted 0-fold
1 was added and reacted at 37 ° C. for 1 hour. Next is 10,000
Centrifugation was performed at 0 × g for 20 minutes, and the human Hb content was measured by an ELISA method using an anti-human Hb antibody. Reactivity was expressed as residual activity with 100% without addition of anti-human Hp antibody, and the results are shown in FIG. As shown in FIG. 2, the residual activity of the human Hb-human Hp complex was remarkably decreased as the concentration of the anti-human Hp antibody was increased. ) Is formed. On the other hand, anti-human H
It was also found that the p antibody did not react with human Hb at all.

Example 2 Reactivity of Immune Complex with Anti-Human Hb Antibody In Example 1, the immune complex (anti-human Hp antibody-human Hp-human Hb complex) was removed by centrifugation and then removed.
The supernatant was used to measure free human Hb. However, in consideration of easier operation, if the measurement can be performed in the presence of the immune complex, the simplification can be further achieved. Therefore, the case where the immune complex was treated by centrifugation was compared with the case where it was not treated. That is, human Hb-human Hp complex fraction (peak 1) and human Hb fraction (peak 2) 0.5 m each
Anti-human Hp preliminarily diluted 10 to 1,000 times in 1
0.5 ml of each (manufactured by BML) was added and reacted at 37 ° C. for 1 hour. Then, the human Hb content was measured by an ELISA method using an anti-human Hb antibody. As a control, centrifugation (10,000 × g, 20 minutes) was performed in the same manner.

[0020]

[Table 1]

Example 3 Addition recovery test using anti-human Hp antibody-treated serum [Preparation of anti-human Hp antibody-treated serum]
It was diluted 10 ⁴ times with phosphate saline containing 1% BSA. The diluted serum was diluted 500-fold with phosphate saline containing 0.1% BSA to prepare anti-human Hp.
An equal amount of antibody (MBL, IgG fraction) was mixed, and 37
The reaction was carried out at 1 ° C for 1 hour to obtain anti-human Hp antibody-treated serum.

[Additional Recovery Test] 500 μl of anti-human Hp antibody-treated serum was added with 500 μl of human Hb having a known concentration, and the mixture was stirred at room temperature for 10 minutes, and then free human Hb was measured and collected by an ELISA method (sandwich method). I asked for the rate. As shown in Table 2, human Hb addition amount 4 to 220 n
The recovery was 92 to 110% at g / ml, and good results were obtained.

[0023]

[Table 2]

Example 4 Construction of Free Human Hb Quantitative Kit One example of the assay of the quantitative assay kit is shown below, but the present invention is not limited to this example. A. The configuration of the kit comprises the components shown in (1) to (11) below. (1) Anti-human Hp antibody (goat anti-human Hp antibody, lyophilized product) (2) Anti-human Hb-immobilized plate (anti-human Hb antibody-binding microplate, sodium azide-containing phosphate buffered saline solution added) ( 3) Buffer solution (phosphate buffered saline containing 0.1% bovine serum albumin) (4) Enzyme-labeled antibody (peroxidase-labeled anti-human Hb
Antibody, lyophilized product) (5) Enzyme-labeled antibody solution (0.1% bovine serum albumin-containing phosphate buffered saline) (6) OPD (containing 16 mg of O -phenylenediamine,
(Phosphate buffer, lyophilized product) (7) OPD solution (purified water) (8) Standard human Hb (containing human Hb, phosphate buffered saline, lyophilized product) (9) Hydrogen peroxide solution (0 6% hydrogen peroxide solution) (10) Washing stock solution (5 times concentrated solution) (0.5% bovine serum albumin phosphate buffered saline) (11) Reaction stop solution (4N sulfuric acid)

B. The measuring method was carried out according to the following operating procedures (1) to (13). (1) The anti-human Hp antibody was dissolved in a buffer solution. (2) The test serum was diluted 10 ⁴ with a buffer solution. (3) An anti-human Hp antibody solution and 0.5 ml of diluted serum were mixed and reacted at 37 ° C. for 1 hour. (4) Standard human Hb is diluted with a buffer solution to give 0 to 200 ng.
/ Ml standard human Hb solution was prepared. (5) Anti-human Hb was added to each well of the anti-human Hb-immobilized plate.
200 μl of p-antibody-treated serum and standard human Hb solution were added. (6) The mixture was stirred for 1 minute with a microplate mixer, covered, and left at 37 ° C. for 1 hour. (7) After incubation, the plate was turned upside down, and the solution remaining in the well was discarded at once. About 250 μl of the washing solution was added to all wells, stirred for 30 seconds with a microplate mixer, and then discarded at once. Repeat this wash 3 times, and finally, flip the plate on a paper towel
The washing solution was completely removed from the wells. (8) Except for the blank wells, 200 μl of enzyme-labeled antibody solution was rapidly added to each well in the same order, and the mixture was stirred for 30 seconds with a microplate mixer and allowed to stand at 37 ° C. for 1 hour. (9) Washing was performed in the same manner as (7). (10) Add 200 μl of substrate solution (hydrogen peroxide + O -phenylenediamine) to all wells in the same order and at the same time intervals, stir for 30 seconds with a microplate mixer, and shield from light for 15 minutes at room temperature (15-25 ° C.). I put it. (11) 50 μl of the reaction stop solution was added to all wells in the same order and at the same time intervals. (12) A blank well was used as a control and measured with a microplate colorimeter (492 nm / 630 nm). (13) Standard curve from the absorption value of standard human Hb (Fig. 3)
Was prepared and free human Hb was calculated.

[Brief description of drawings]

FIG. 1 is a diagram showing an elution pattern of human hemoglobin-added serum by gel filtration.

FIG. 2 is a diagram showing the reactivity of anti-human haptoglobin antibody with respect to human hemoglobin-human haptoglobin complex.

FIG. 3 is a diagram showing a standard curve of human hemoglobin.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Ryomin Ohashi 5-3, Minamigaoka 44-3, Hadano-shi, Kanagawa (72) Inventor Makoto Maeda 3-4-3 Shin-Chiba, Chuo-ku, Chiba-shi, Chiba

Claims (2)

[Claims] 1. An anti-haptoglobin antibody is added to a free hemoglobin-containing sample to obtain hemoglobin-existing in the sample.
A method for measuring free hemoglobin, which comprises reacting the antibody with a haptoglobin complex and then measuring free hemoglobin by an enzyme immunoassay. 2. A test kit for measuring free hemoglobin using the assay method according to claim 1, comprising a reagent comprising an anti-haptoglobin antibody that reacts with a hemoglobin-haptoglobin complex present in a sample. Characteristic measurement kit.