JPH02231565A - Reagent kit for measuring free hemoglobin and method for measuring free hemoglobin by using this kit - Google Patents

Abstract

PURPOSE:To directly and quantitatively measure free hemoglobin with a high sensitivity by using the solid phase human haptoglobin formed by conjugating human haptoglobin with a solid phase and the oxygen labeled antibody formed by conjugating oxygen with an anti-human haptoglobin antibody. CONSTITUTION:A free hemoglobin-contg. specimen which is adjusted in concn. with a suitable buffer soln. at need is brought into reaction with the solid phase human haptoglobin. The supernatant is thereafter removed, by which the free hemoglobin conjugated specifically with the solid phase human haptoglobin is made to remain on the solid phase. The oxygen labeled antibody is then added thereto, by which the oxygen labeled antibody is conjugated with the free hemoglobin remaining on the solid phase. After the excess oxygen labeled antibody failing to be conjugated is removed, the oxygen activity is measured. On the other hand, the relation between the free hemoglobin and the oxygen activity is formed by making the similar operation on the sample solution of the standard free hemoglobin diluted to 3-5 stages. The free hemoglobin quantity in the specimen is determined by contrasting the resulted oxygen activity and a calibration curve.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a reagent kit for measuring free hemoglobin used in the field of clinical testing and a method for measuring free hemoglobin using the same. More specifically, a reagent kit for measuring free hemoglobin comprising immobilized human haptoglobin and an enzyme-labeled anti-human hemoglobin antibody, an enzyme immunoassay method using the same, and a free hemoglobin measurement comprising human haptoglobin-sensitized particles and an anti-human hemoglobin antibody. The present invention relates to a reagent kit for human haptoglobin and a human haptoglobin-sensitized particle aggregation method using the same.

[Prior Art] Massive blood transfusion, extracorporeal circulation, hemodialysis, organ perfusion, etc. often cause severe hemolysis, which raises concerns about various complications including hemolytic kidney damage. The hemoglobin that causes the disease is called free hemoglobin, which is not bound to habtoglobin, and clinically, it is necessary to quantify hemoglobin in plasma and serum separately into habtoglobin-bound hemoglobin and free hemoglobin. There is.

As described above, the measurement of free hemoglobin contained in plasma, serum, etc. has important clinical significance, and its measurement methods include cellulose acetate membrane electrophoresis,
A simple fractional quantitative method and a free hemoglobin adsorption quantitative method (column method) are conventionally known.

To measure free hemoglobin using the cellulose acetate membrane electrophoresis method described above, first, the total amount of hemoglobin in a sample (plasma and serum) is quantified using the cyanmethemoglobin method. Next, the same sample was separated into free hemo-1 globin, habtoglobin-hemoglobin complex, and methemoalbumin (albumin-bound hemoglobin) fractions by cellulose acetate membrane electrophoresis, and the hemoglobin was stained by peroxidase reaction. The degree of staining in each segment is scanned with a densitometer, and the ratio of the amount of free hemoglobin to the amount of total hemoglobin (free hemoglobin + habutogubin-hemoglobin complex + methemoalbumin) is determined. The amount of free hemoglobin in the specimen is calculated by multiplying the calculated ratio by the total amount of hemoglobin, which has been determined in advance by the cyanmethemoglobin method.

To measure free hemoglobin using the simple differential quantitative method, total hemoglobin is determined using the cyanmethemoglobin method and total habutoglobin is determined using the one-way immunodiffusion method (Manc1n1 method), and the binding ratio between habtoglobin and hemoglobin is used. This is to calculate the amount of free hemoglobin. In addition, when quantifying total habutoglobin by the one-way immunodiffusion method, the serotype of habutoglobin is identified in advance by starch gel electrophoresis, immunoelectrophoresis, or acrylamide gel electrophoresis, and the serotype is prepared for each serotype. If a standard curve prepared using pooled serum is used, or a standard curve prepared using pooled serum is used, the measured value obtained from the standard curve must be multiplied by the coefficient for each serotype of the Habtog mouth bottle to obtain the quantitative value.

In addition, to measure free hemoglobin using the free hemoglobin adsorption quantitative method, a fixed amount of sample (plasma or serum) is injected into a cylindrical ram uniformly filled with Sepharose immobilized in a Habtog bottle. After washing with physiological saline, the amount of free hemoglobin is determined by measuring the length of the colored part due to free hemoglobin adsorbed on the buttoglobin-immobilized Sepharose.

[Problems to be Solved by the Invention] In the above-mentioned conventional technology, the method using cellulose acetate membrane electrophoresis is a method of indirectly determining the amount of free hemoglobin by multiplying the total amount of hemoglobin by the fractionation ratio. There are problems in that the operation is complicated and takes a long time, and free hemoglobin below the quantification limit of total hemoglobin and the detection limit of cellulose acetate membrane electrophoresis cannot be measured.

In addition, in the measurement method using the simple fractional quantitative method, since the amount of free hemoglobin is indirectly determined from the total amount of hemoglobin and the total amount of habutogbin, free hemoglobin below the quantification limit of total hemoglobin cannot be measured.

Furthermore, there is a problem in that the hemoglobin bound to the amount of habtoglobin below the quantification limit of the total habtog bottle is ultimately calculated as free hemoglobin.

On the other hand, in the free hemoglobin adsorption quantitative method, a fixed amount of sample (serum or plasma) is injected into a column filled with Habtog bottle-immobilized Sepharose, and the sample is colored by the free hemoglobin adsorbed to the Habtog bottle-immobilized Sepharose. Since this method determines the amount of free hemoglobin in a sample by measuring the length of the colored layer of the column, the length of the colored layer depends on the uniformity of the habtoglobin-immobilized Sepharose in the column, the injection method of the sample, the washing method, etc. In addition, there are problems in that the end point of the colored layer is unclear, and quantitativeness and reproducibility are lacking.

The present invention has been made to solve the above-mentioned drawbacks of the prior art, and is a free hemoglobin that can be used to specifically and directly quantify free hemoglobin in a specimen (e.g., serum, plasma, urine, feces, etc.) with high sensitivity. The purpose of the present invention is to provide a reagent kit for hemoglobin measurement and a measurement method using the same.

[Means and effects for solving the problem] The reagent kit for measuring free hemoglobin of the present invention consists of: (1) immobilized human haptoglobin in which human haptoglobin is bound to a solid phase, and an enzyme bound to an anti-human hemoglobin antibody; A reagent kit for measuring free hemoglobin, characterized by comprising an enzyme-labeled antibody; and 1. Free hemoglobin measurement, characterized by comprising human haptoglobin-sensitized particles in which human haptoglobin is bound to a particle carrier, and an anti-human hemoglobin antibody. It is a reagent kit for

In addition, the method for measuring free hemoglobin of the present invention includes (1) contacting a sample containing free hemoglobin with immobilized human haptoglobin in which human haptoglobin is bound to a solid phase, and removing free hemoglobin in the sample from the human haptoglobin on the solid phase. After binding to haptoglobin, an enzyme-labeled antibody in which an enzyme is bound to an anti-human hemoglobin antibody is further applied to immobilize the enzyme-labeled antibody on a solid phase via free hemoglobin, and then the enzyme activity is measured. A method for measuring free hemoglobin characterized by: (11) contacting a sample containing free hemoglobin with human haptoglobin-sensitized particles in which human haptoglobin is bound to a particle carrier, and converting free hemoglobin in the sample into human haptoglobin on the particles; After the binding, an anti-human hemoglobin antibody is further applied, and the free hemoglobin in the sample is measured based on the degree of aggregation between the particles caused by the binding of the free hemoglobin on the particles and the anti-human hemoglobin antibody. This is a method for measuring hemoglobin.

The present invention has the above-mentioned configuration, and in the case of the free hemoglobin measurement method shown in (1) above, the enzyme-labeled antibody, which is an enzyme-conjugated antibody to immobilized human haptoglobin and anti-human hemoglobin shown in (2) above, is used. The enzyme-labeled antibody is bound to the solid phase via the free hemoglobin in the sample (
The enzyme activity of the enzyme-labeled antibody is determined by the amount of bound enzyme-labeled antibody, and the amount of enzyme-labeled antibody is determined by the amount of free hemoglobin bound to immobilized human haptoglobin. . Furthermore, the amount of free hemoglobin that binds to immobilized human haptoglobin is determined by the amount of free hemoglobin in the specimen. Therefore, since the enzyme activity is correlated with the amount of free hemoglobin in the sample, the amount of free hemoglobin in the sample can be determined by measuring the enzyme activity.

In the case of the free hemoglobin measurement method shown in (11) above, human haptoglobin-sensitized particles in which human habtobin is bound to the particle carrier shown in (1) above and an anti-human hemoglobin antibody are used, and the analyte is transferred onto the sensitized particles. After binding the free hemoglobin in the sample, anti-human hemoglobin antibody is applied to measure the amount of free hemoglobin in the sample based on the degree of aggregation of the sensitized particles. The amount of free hemoglobin bound to the sensitized particles is determined by the amount of free hemoglobin bound to the sensitized particles, and the amount of free hemoglobin bound to the sensitized particles is determined by the amount of free hemoglobin in the specimen.

Therefore, since the degree of aggregation of sensitized particles correlates with the amount of free hemoglobin in the specimen, the amount of free hemoglobin in the specimen can be determined by measuring the degree of aggregation of sensitized particles.

The first reagent kit for measuring free hemoglobin of the present invention includes:
This is a reagent kit consisting of immobilized human haptoglobin and an enzyme-labeled antibody made by binding an enzyme to anti-human hemoglobin.

As the immobilized human haptoglobin used here, human haptoglobin prepared from human plasma by a known method (Clinical Habtoglobin, written by Meng Oshiro, published by Nagai Shoten, see p. 223) is used, and the adsorption method, Promsian method, etc. As a solid phase material for immobilizing human haptoglobin, which can be prepared by a known method (enzyme immunoassay, 3rd edition: published by Igaku Shoin, see page 395), there are solid phase materials conventionally used in enzyme immunoassay, etc. Any of the solid phase materials known in the art can be used, and examples thereof include polysaccharides such as agarose and sepharose, glass, ceramics, and plastics. Although the shapes of these materials are not particularly limited, microplates, plastic tubes, or plastic balls are more preferable from the viewpoint of convenience in measurement operations. The amount of human haptoglobin immobilized on the carrier is not particularly limited, and can be adjusted as appropriate depending on the desired sensitivity, etc., and this adjustment can be performed by adjusting the Promcyan group introduced into the carrier, the concentration of human habtoglobin, etc. , the human haptoglobin concentration of the human haptoglobin solution used for immobilization was 5 μt.
It is preferably about r/m to 5/I.

On the other hand, enzyme-labeled anti-human hemoglobin antibodies are produced using known methods such as the glutaraldehyde method, maleimide method, and periodate oxidation method (enzyme-linked immunosorbent assay, 3rd edition) using antibodies against human hemoglobin (polyclonal antibodies and monoclonal antibodies). (Published by Igaku Shoin, see p. 109). Any antibody against human hemoglobin (polyclonal antibody or monoclonal antibody) can be used as the enzyme-labeled anti-human hemoglobin antibody or the anti-human hemoglobin antibody used for preparation. It can be obtained from serum by conventional methods. More preferably purified human hemoglobin antibodies are used, for example, as described by George Hz et al.
at. Ils. J. CIin. Pathol. V
ol. 69, 342-346. (1978), and can also be prepared from human hemoglobin antibodies (polyclonal antibodies and monoclonal antibodies) by affinity stomatography in which purified human hemoglobin is bound. Furthermore, as the enzyme used here, any of the enzymes conventionally used in enzyme immunoassay can be used, for example,
Examples include peroxidase, alkaline phosphatase, glucosidase, β-D-galactosidase, and glucose oxidase.

The second reagent kit for measuring free hemoglobin of the present invention is a reagent kit comprising human haptoglobin-sensitized particles in which human habtobin is bound to a particle carrier and an anti-human hemoglobin antibody.

The human haptoglobin sensitizing particles used here are:
Human haptoglobin was prepared from human plasma by a known method (Clinical Habtoglobin, written by Meng Oshiro, published by Nagai Shoten, see p. 223), and hemoglobin-habtoglobin was determined by affinity stomatography coupled with an anti-human hemoglobin antibody. Using human haptoglobin (unadsorbed fraction) excluding the bottle complex (adsorbed fraction), adsorption to particle carriers, hydrophobic binding method, coupling agent (e.g.
Particle carriers for immobilizing human haptoglobin, which can be prepared by known means such as methods using tannic acid, glutaraldehyde, bisdiazobenzene, tolylene diisocyanate, carbodiimide, etc., include formalin treatment, glutaraldehyde treatment, etc. Any of the carriers conventionally used in agglutination reaction tests (particle immunoassay method) using artificial particles such as red blood cells and boristyrene latex can be used.In addition to the red blood cells and latex mentioned above, gelatin particles and bentonite Examples include particles, plastic particles, kaolin particles, ceramic particles, and polysaccharide particles such as agarose and sepharose.
From the viewpoint of convenience in measurement operations, etc., the shape is more preferably spherical. In addition, the particle size of the particle carrier is 0.1 to 2.0μ.
About m is used. The amount of human haptoglobin on the particle carrier is not particularly limited, and can be adjusted as appropriate depending on the desired sensitivity, etc., and this adjustment can be performed by a method such as adjusting the human haptoglobin concentration of the human haptoglobin solution that is brought into contact with the particle carrier. The concentration of human haptoglobin in the human haptoglobin solution used for adsorption is preferably about 0.005 to 0.05%.

On the other hand, as the anti-human hemoglobin antibody, any antibody (polyclonal antibody or monoclonal antibody) against human hemoglobin can be used, and these can be obtained by conventional methods from the serum of an animal immunized with human hemoglobin. Can be done. More preferably, a purified human hemoglobin antibody is used, and the purified human hemoglobin antibody can be prepared from anti-human hemoglobin antibodies (polyclonal antibodies and monoclonal antibodies) by affinity stomatography to which purified human hemoglobin is bound.

Among the reagent kits of the present invention, the reagent kit shown in item (1) above is composed of at least immobilized human haptoglobin in which human haptoglobin is bound to a solid phase, and an enzyme-labeled antibody in which an enzyme is bound to an anti-human hemoglobin antibody. The immobilized human haptoglobin is usually kept in a dry state using a freeze-drying method in order to improve its storage life and stability. It may be in a liquid form dissolved in an appropriate buffer solution such as a buffer solution. Furthermore, it may additionally contain an enzyme substrate reagent for measuring enzyme activity, a free hemoglobin standard used for creating a calibration curve, and a habtoglobin reagent containing a certain amount of habtog vial used as a confirmation test reagent. good.

The above-mentioned enzyme substrate reagent may be the enzyme substrate used in the enzyme-labeled antibody itself or a solution thereof dissolved in an appropriate buffer solution to an appropriate concentration. Further, the free hemoglobin standard product can be prepared, for example, by the method described in Example 5 below, and the Habtog bottle reagent can be prepared by the method described in Example 8 below.

In addition, the reagent kit shown in (1) above comprises at least human haptoglobin-sensitized particles in which human haptoglobin is bound to the particle carrier and an anti-human hemoglobin antibody,
The above-mentioned sensitized particles are usually made into a lyophilized preparation or a suspension using an appropriate buffer solution, and the anti-human hemoglobin antibody can be prepared in a powdered form such as a lyophilized preparation or a suitable solution such as a phosphate buffer solution. It may also be in liquid form dissolved in a buffer solution. Additionally, a free hemoglobin positive control containing a fixed amount of free hemoglobin is used as a comparative control for the degree of aggregation, a free hemoglobin negative control containing substantially no free hemoglobin, and a fixed amount of haptoglobin is used as a confirmation test reagent. It may also contain a quantity of Habtog bottle reagent. These free hemoglobin positive controls, free hemoglobin negative controls, and habutoglobin reagents are as described in Examples 6, 7, and 8 below.
It can be prepared by the means described in .

Next, the method for measuring free hemoglobin using the reagent kit of the present invention will be explained in more detail. After reacting a sample containing free hemoglobin (serum, plasma, urine, fecal extract, etc.) with immobilized human haptoglobin, the free hemoglobin that has specifically bound to immobilized human haptoglobin is removed by removing the supernatant. remains on the solid phase. Next, by adding an enzyme-labeled antibody, the enzyme-labeled antibody binds to the free hemoglobin remaining on the solid phase. After removing unbound excess enzyme-labeled antibody, enzyme activity is measured. On the other hand, by performing the same operation as above on sample solutions prepared by diluting the free hemoglobin standard product to 3 to 5 levels of concentration, a relational expression (calibration curve) between free hemoglobin concentration and enzyme activity was created, and the equation obtained above was By comparing the enzyme activity obtained with the calibration curve, the amount of free hemoglobin in the sample can be determined.

In the above method, various methods are used to measure the enzyme activity depending on the type of enzyme, but methods include adding a substrate for the enzyme of the enzyme-labeled antibody and measuring the amount of change in the substrate (for example, a colorimetric method). , fluorescence method, chemiluminescence method, bioluminescence method, etc.) are preferred because they are excellent in operability and quantitative performance. In particular, it is easy to measure the change in substrate by measuring absorbance. Examples of such enzymes and their substrates include combinations of peroxidase, hydrogen peroxide, and 0-phenylenediamine, and β-D-galactosidase. Examples include a combination of and 0-nitrophenyl-β-D-galactoside, a combination of alkaline phosphatase and p-nitrophosphate, and the like.

On the other hand, to explain in more detail the method for measuring free hemoglobin using the reagent kit for measuring free hemoglobin shown in (1) above, for example, a sample (serum, plasma, urine, fecal extract, etc.) diluted with an appropriate buffer solution can be used. by reacting with human haptoglobin-sensitized particles, free hemoglobin in the specimen is bound to human haptoglobin on the particles. Furthermore, by adding an anti-human hemoglobin antibody, the anti-human hemoglobin antibody specifically binds to the free hemoglobin bound on the particles, but the anti-human hemoglobin antibody
Because it has two antigen-binding sites, aggregation occurs between particles bound to free hemoglobin. Using a sample solution in which the specimen is diluted several times with an appropriate buffer solution, the highest dilution factor that causes agglutination is determined, and on the other hand, a sample solution in which a standard solution containing a known concentration of free hemoglobin is diluted several times (positive control) is used.
The amount of free hemoglobin in the sample can be measured semi-quantitatively by determining the highest dilution factor that causes agglutination using the method and comparing the two. The degree of agglutination can be measured in the same manner as the procedure commonly used in red blood cell agglutination tests using microplates, latex agglutination tests using slide glasses (observation plates), and the like.

Furthermore, the method for measuring free hemoglobin of the present invention is not limited to the above-mentioned method, and includes, for example, a method of optically counting the number of unagglomerated human haptoglobin-sensitized particles other than the formed aggregates (particle force counting immunoassay method). ), a method of measuring the turbidity of aggregates using near-infrared rays (0.8-2.4 μm) (near-infrared turbidimetry), etc. to quantitatively measure free hemoglobin. You can also do it.

[Effects of the Invention] According to the reagent kit for measuring aM hemoglobin of the present invention and the method for measuring free hemoglobin using the same, an enzyme immunoassay method and a human haptoglobin-sensitized particle agglutination method are used. Free hemoglobin contained in plasma, urine, feces, etc.) can be measured specifically, highly sensitively, and directly quantitatively (or semi-quantitatively), and the measurement operation is simple and can be performed in a short time. It has the effect of ending.

[Examples] Hereinafter, the present invention will be explained in more detail based on Examples and Experimental Examples, but the present invention is not limited to these Examples.

Example 1 Preparation of immobilized human haptoglobin Purified human haptoglobin (■Midori Juji Co., Ltd.) prepared from hemoglobin-free normal human pool plasma was added to 0.05
Dissolve habtoglobin in M sodium carbonate buffer (pH 9.6) to a concentration of 1 mg/xl.
．． 2xl each was added to the wells of an EIA microplate and left standing at 4°C for 15 hours. After the contents of the wells were removed by suction, 0.31 f of phosphate buffered saline (PBS) containing 0.02% Tveen-20 was added, and the contents were removed by suction again.

After repeating the addition of Tveen-20-containing PBS and suction removal three times in total, PI3s (0.3xl) containing 0.5% bovine serum albumin was added, left at room temperature for 3 hours, and suction removed. Through the above treatment, a microplate with immobilized Habtog mouth bottle was prepared.

Example 2 Preparation of enzyme-labeled antibodies George H. et at.
．． J. CIin. Pathol. Vol. 69
．． 342-346. Purified anti-human hemoglobin goat IgG prepared by the method of 1978) was digested with Bevcin in a conventional manner to obtain F (ab) 2, which was further reduced with 2-mercabutethylamine to prepare Fab''. , horseradish peroxidase (hereinafter referred to as HR
0.3 mg of 4-g was dissolved in 0.1 M sodium phosphate buffer (pH 7.0).
(N-maleimidomethyl)cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester with N,N-
A solution (crosslinking reagent) dissolved in 30 μg of dimethylformamide was added, and the mixture was reacted at 30° C. for 60 minutes. Next, the precipitate was removed by centrifugation, gel filtrated with a Sephadex G-50 column (eluent: 0.IM sodium phosphate buffer, pH 6.0), and both fractions showing absorption of 403n* were fractionated and concentrated. , maleimidated HRpo was prepared.

Maleimidized HRPOI obtained above. 8 ■ and Fab-
Mix 2mg, 0.1 containing 2.5s MEDTA
Add M sodium phosphate buffer (pH 6.0) and dilute the total volume to 1'I! The mixture was reacted at 30°C for 60 minutes. after that,
After stopping the reaction by adding 10 μg of 50 nM N-ethylmaleimide,
The complex fraction was collected using a (LKB) column, and the amount of Fab per 11 was 0.025 to 0.05! The antibody was diluted with PBS containing 0.25% bovine serum albumin to give a peroxidase-labeled anti-human hemoglobin antibody solution.

Example 3 Preparation of Habtog mouth bottle sensitized particles Hemoglobin-Habtog mouth bottle complex was obtained from the purified human haptoglobin (v4 Midori Juji) described in Example 1 by affinity stomatography coupled with an anti-human hemoglobin antibody. Highly purified human haptoglobin (unadsorbed fraction) from which the body (adsorbed fraction) was removed was dissolved in PBS (1)H.
7.2) The habtoglobin solution dissolved so that the habtoglobin concentration was 0.01% and the particle size of 1.8 to 26
0 μm artificial particles (manufactured by Japan Synthetic Rubber, IMMUTEX)
A suspension of 2 (V/V)% of 2 (V/V)% of 1 product name) was mixed with the same amount, and the mixture was left standing at 37° C. for 1 hour.

After removing the supernatant by suction, add the same PBS in an amount 100 to 200 times the volume of the particles, shake gently to suspend, centrifuge, and remove the supernatant by suction. Repeat the centrifugal washing operation 5 times in total. Uta. Next, PBS containing 0.25% bovine serum albumin was added in an amount approximately 100 times the volume of the particles, and the particles were suspended by shaking gently, and then allowed to stand at 4° C. for 15 hours. After removing the supernatant by suction, centrifugal washing was repeated 5 times in total using the method described above, and then PBS was added so that the particle amount was 0.2% by volume.
was added to obtain a Habtog bottle sensitized particle suspension. Through the above treatment, a habtoglobin-sensitized particle suspension was prepared.

Example 4 Preparation of anti-human hemoglobin antibodies George H. et al.
．． J. CIin. Pathol. Vol. 69.
842-346. Purified anti-human hemoglobin goat IgG prepared by the method of (1978) was added to the above PBS.
It was dissolved so that the absorbance at 280 ns was 0.28. An anti-human hemoglobin antibody solution was prepared by the above treatment.

Example 5 Preparation of free hemoglobin standard solution Williams fllllllaws. Anal. B1
oehes. Vol, 54. 137-145. 1
From human hemoglobin prepared by the method of 973), the unadsorbed fraction was collected by affinity chromatography using an anti-human haptoglobin antibody, and the adsorbed fraction (hemoglobin-habtoglobin complex) was removed. The amount of hemoglobin in the separated unadsorbed fraction was measured by the cyanmethemoglobin method and used as a free hemoglobin standard solution.

Example 6 Preparation of free hemoglobin positive control The free hemoglobin standard solution obtained in Example 5 was mixed with PBS until the final amount of free hemoglobin was 20 μg/1! The diluted solution was used as a free hemoglobin positive control solution.

Example 7 Preparation of free hemoglobin negative control Unadsorbed fractions were collected from pooled normal human plasma that does not contain hemoglobin by affinity chromatography coupled with anti-human hemoglobin antibodies, and adsorbed fractions (free hemoglobin and Hemoglobin-haptoglobin complex) was removed. The separated unadsorbed fraction was used as a free hemoglobin negative control solution.

Example 8 A purified Habtog bottle (manufactured by II Midori Juji) prepared from pooled normal human plasma that does not contain hemoglobin was diluted with PBS until the habtoglobin concentration was 0. It was diluted to a concentration of 1 (W/V)%, and the resulting diluted solution was used as a nobutoglobin reagent.

[Experimental example] Human haptoglobin-immobilized microplate obtained in the example, peroxidase-labeled anti-human hemoglobin antibody, free hemoglobin standard solution, human haptoglobin-sensitized particles,
Free hemoglobin was measured using an anti-human hemoglobin antibody solution, a free hemoglobin positive control solution, a free hemoglobin negative control solution, and a habutoglobin reagent as follows.

Experimental Example 1 Creating a calibration curve using free hemoglobin standard The free hemoglobin standard solution obtained in Example 5 was used as 11! The concentration was adjusted with PBS containing 0.5% bovine serum albumin so that the amount of free hemoglobin in the sample was 1.000 ng, and then diluted stepwise with the same PBS. The diluted solution was used as the measurement sample solution.

Next, in the wells of the human haptoglobin-fixed microplate obtained in Example 1, add the above PBS and 1x to 1.024
The diluted solution was added in 0.1 volt increments and allowed to stand at 37°C for 1 hour. After the contents of the wells were removed by suction, the addition of Tveen-20-containing PBS (0.311>) and removal by suction were repeated three times in total. Then, the peroxidase-labeled anti-human hemoglobin antibody solution obtained in Example 2 was .1d was added to each well and left to stand at 37°C for 1 hour.The contents of the wells were removed by suction again, and PBS containing Tveen-20 was added.
(0.311>) and suction removal were repeated 3 times. Then, 0.1M phosphate monocitrate buffer (pH
5.0) with hydrogen peroxide (0.015% v/v), O-
0.1 d of a substrate solution in which phenylenediamine (1.5 μ/If) was dissolved was added to the well, reacted for 30 minutes at room temperature, and then 2.5 M sulfuric acid (0.0517) was added to stop the reaction. . Using a well to which PBS was added instead of the measurement sample solution as a control, the absorbance (492rv) of the well to which the measurement sample was added was measured using a microplate meter. The results are shown in Figure 1.

Experimental Example 2 Specificity Test of Enzyme Immunoassay Method Plasma containing about 1 to 20 mg/dl of free hemoglobin was aliquoted into two test tubes, each containing 0.5111 ml of plasma, and the habtoglobin obtained in Example 8 was placed in one of the tubes. 0.5w of reagent
1 and left at 37°C for 1 hour (referred to as post-inhibition sample); and 0.5xl of PBS instead of Habtog bottle reagent and left at 37°C for 1 hour (referred to as post-inhibition sample).
(referred to as the pre-blocking sample) was prepared. Each was diluted 1.000 times with PBS containing 0.5% bovine serum albumin, and then treated according to the method shown in Experimental Example 1.
The absorbance at ns was measured. The inhibition rate (%) by the Habtog bottle reagent was determined by subtracting the absorbance of the sample after inhibition from the absorbance of the sample before inhibition and dividing the value by the absorbance of the sample before inhibition.

Table 1 shows the test results conducted using the plasma of three people (sample numbers 1 to 2). As shown in Table 1, the addition of Habtog bottle reagent significantly inhibited the reaction, confirming that the enzyme immunoassay of the present invention is a highly specific test method for free hemoglobin.

Table 1 Experimental Example 3 Dilution test example 5 using Habtog bottle sensitized particle aggregation method
After adjusting the concentration of the free hemoglobin standard solution obtained in PBS so that the free hemoglobin concentration was 100 μg/III, dilution was performed step by step with the same PBS, and 1.
(100 μg/wl) to 512 times (0.20
Ten types of diluted solutions up to μg/xi) were used as measurement samples.

Microplate [vertical 8 wells (A-H rows), horizontal 12 wells (1-12)] (see Figure 2) H row (1-12)
50 μg each of the free hemoglobin positive control solution obtained in Example 6, the free hemoglobin negative control solution obtained in Example 7, and the above 10 types of measurement samples were added to the wells of ~12 wells)
After adding 25 μg of PBS to each column, dilutions were performed from column H to column A using an autodiluter (for 25 μg) according to a conventional method.

Next, 25 μg of the Habtog mouth bottle sensitized particle suspension obtained in Example 3 was added to all wells, and after shaking lightly,
It was left at ℃ for 30 minutes. Furthermore, after adding 25 μg of the anti-human hemoglobin antibody solution obtained in Example 4 and shaking gently,
After standing at room temperature for about 2 hours, the degree of aggregation of the habtoglobin-sensitized particles in each well was observed.

A schematic diagram of the results is shown in FIG. In the figure, - indicates non-aggregation, 2 indicates aggregation intermediate between aggregation and non-aggregation, and 2 indicates aggregation. As is clear from Figure 2, the positive control (2
0 μg; / If ) and each sample solution (100.0 to 0
．． 2μg/1! (10 types) both 03 39 to 0.2
μg/1! When diluted to a concentration of

Experimental Example 4 Specificity Test of Habtog Bottle Sensitized Particle Aggregation Method The post-inhibition sample and the pre-inhibition sample prepared in Experimental Example 2 were used as measurement samples. Each post-blocking sample and pre-blocking sample was diluted 16 times with PBS, and the diluted solution was added to the H of the microplate (see Figure 3).
50μ each in wells 1 and 2, 3 and 4, and 5 and 6 of rows.
After adding 25 μp of PBS to other wells (wells 1 to 6 in row G-A), use an autodiluter (for 25 μg) in the usual manner to add PBS from row H to A. Multiple dilutions were made in columns.

Next, 25 μg of the habutoglobin-sensitized particle suspension obtained in Example 3 was added to all wells, and after shaking gently,
It was left at ℃ for 30 minutes. Furthermore, after adding 25 μg of the anti-human hemoglobin antibody solution obtained in Example 4 and shaking gently,
After standing at room temperature for about 2 hours, the degree of aggregation of the habtoglobin-sensitized particles in each well was observed, and the aggregation values before and after inhibition were compared.

A schematic diagram of the results is shown in FIG. In the figure, - indicates non-aggregation and + indicates aggregation. As shown in Figure 3, aggregation was significantly inhibited by the addition of Habtog bottle reagent, confirming that the habtoglobin-sensitized particle agglutination method of the present invention is a highly specific test method for free hemoglobin. It was done.

Experimental Example 5 Comparison test with conventional method Hemolyzed patient plasma was diluted stepwise with physiological saline (1 to 3
The immobilized human haptoglobin of the present invention and the enzyme-labeled anti-human hemoglobin antibody were measured using the diluted solution (2-fold dilution) and the free hemoglobin positive control solution and free hemoglobin negative control solution obtained in Examples 6 and 7, respectively, as measurement samples. Free hemoglobin was measured by the enzyme immunoassay method and human haptoglobin-sensitized particle agglutination method used, as well as the conventionally used simple fractional quantitative method and adsorption quantitative method (column method) using a habtoglobin-immobilized Sepharose column.

Measurement by enzyme immunoassay was performed using the method shown in Experimental Example 1, and the amount of free hemoglobin in the sample (1 g
/old) was sought. Measurement using the human haptoglobin-sensitized particle agglutination method was performed using the method shown in Experimental Example 3, and the highest dilution factor (agglutination value) that showed agglutination was determined, and the amount of free hemoglobin in the sample was determined using the free hemoglobin positive control solution as a reference. (converted value, ag/old) was calculated.

Measurement by simple fractional quantitative method and adsorption quantitative method (column method)
Each measurement was carried out according to Oshiro's method (Clinical Habtogu mouth bottle, written by Meng Oshiro, published by Nagai Shoten, see page 33).

The results are shown in Table 2. As shown in Table 2,
Although it is not possible to measure low concentrations of free hemoglobin using conventional methods such as the simple fractional quantitative method and the adsorption (column) method, the present invention can quantify (
or semi-quantitative).

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a standard curve for measuring free hemoglobin by enzyme immunoassay using immobilized human haptoglobin and an enzyme-labeled human hemoglobin antibody of the present invention, and FIG. FIG. 2 is a schematic diagram showing the degree of aggregation of human haptoglobin-sensitized particles relative to free hemoglobin concentration in measurement of free hemoglobin by a sensitized particle aggregation method. In the figure, 1 indicates non-aggregation, 2 indicates aggregation intermediate between aggregation and non-aggregation, and + indicates aggregation. FIG. 3 is a schematic diagram showing that human haptoglobin-sensitized particles 1 specifically aggregate with free hemoglobin in a specimen in the measurement of free hemoglobin by the human haptoglobin-sensitized particle agglutination method of the present invention. In the figure, 1 indicates non-aggregation and + indicates agglutination. Free hemoglobin concentration (ng/11)

Claims (1)

[Scope of Claims] 1. A reagent kit for measuring free hemoglobin, comprising solid-phase human haptoglobin, which is human haptoglobin bound to a solid phase, and an enzyme-labeled antibody, which is an anti-human hemoglobin antibody bound to an enzyme. 2. The reagent kit for measuring free hemoglobin according to claim 1, wherein the solid phase of immobilized human haptoglobin is a microplate, a plastic tube, or a plastic ball. 3. The enzyme of the enzyme-labeled antibody is peroxidase, β-D-
The reagent kit for measuring free hemoglobin according to claim 1 or 2, which is galactosidase or alkaline phosphatase. 4. The method for measuring free hemoglobin according to any one of claims 1 to 3, further comprising a free hemoglobin standard product containing a certain amount of free hemoglobin, a haptoglobin reagent containing a certain amount of human haptoglobin, and an enzyme substrate reagent. Reagent kit. 5. A reagent kit for measuring free hemoglobin comprising human haptoglobin-sensitized particles in which human haptoglobin is bound to a particle carrier and an anti-human hemoglobin antibody. 6. The reagent kit for measuring free hemoglobin according to claim 5, wherein the particle carrier is fixed red blood cells or latex particles. 7. Claim 5 or 6 additionally comprising a free hemoglobin positive control reagent containing a certain amount of free hemoglobin, a free hemoglobin negative control reagent substantially free of free hemoglobin, and a haptoglobin reagent containing a certain amount of human haptoglobin. The reagent kit for measuring free hemoglobin described above. 8. A specimen containing free hemoglobin is brought into contact with immobilized human haptoglobin, in which human haptoglobin is bound to a solid phase, to cause the free hemoglobin in the specimen to bind to human haptoglobin on the solid phase, and then further coated with an anti-human hemoglobin antibody. 1. A method for measuring free hemoglobin, which comprises applying an enzyme-labeled antibody to which an enzyme is bound, immobilizing the enzyme-labeled antibody on a solid phase via free hemoglobin, and then measuring the enzyme activity. 9. After contacting a specimen containing free hemoglobin with human haptoglobin-sensitized particles in which human haptoglobin is bound to a particle carrier to bind the free hemoglobin in the specimen to the human haptoglobin on the particles, an anti-human hemoglobin antibody is further applied. 1. A method for measuring free hemoglobin in a specimen, which comprises: measuring free hemoglobin in a specimen based on the degree of aggregation between particles caused by binding of free hemoglobin on the particles with an anti-human hemoglobin antibody.