JPH11201969A - Simple free hemoglobin measuring method and kit for measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for easily, quickly and accurately measuring free hemoglobin present in a liquid sample and a kit for measurement. SOLUTION: By bringing the liquid sample containing the free hemoglobin, a hemoglobin-haptoglobin complex and free haptoglobin into contact with one end of a water absorptive carrier containing an anti-haptoglobin antibody directly or through a hemocyte separation film, the hemoglobin-haptoglobin complex and the free haptoglobin in the liquid sample are captured. Then, by combining the free hemoglobin developed on the water absorptive carrier without being captured and a first anti-hemoglobin antibody marked by a colored particle and contained in the water absorptive carrier and then capturing a colored combined material developed on the water absorptive carrier by a second anti- hemoglobin antibody made into a solid phase at the water absorptive carrier, the free hemoglobin in the liquid sample is visibly detected and measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring free hemoglobin and a kit for measuring free hemoglobin.
Specifically, the present invention relates to a simple free hemoglobin measurement method and a measurement kit, which can be used to measure free hemoglobin simply and quickly by an immunological technique, which is used in the field of clinical testing and the like.

[0002]

2. Description of the Related Art Extracorporeal circulation, burns, enterohemorrhagic Escherichia coli infection, incompatible blood transfusion, hemolysis after blood transfusion, hemolytic disease, etc. may cause a large amount of hemolysis, Often accompanied by complications such as disability. Usually, hemoglobin (hereinafter H) in plasma or serum
b) exists as a hemoglobin-haptoglobin complex (hereinafter abbreviated as Hb-Hp) and free Hb (hereinafter abbreviated as F-Hb), and the cause of this hemolytic renal disorder is F-Hb It is said that there is.

More specifically, Hb-Hp in blood
Is rapidly taken into the liver and metabolized to bilirubin via the normal pathway, but F-Hb passes through the renal glomeruli and is excreted in urine. This is taken up by tubular epithelial cells and causes hemolytic kidney injury. Therefore, F-Hb in blood
Can be measured separately from Hp-bound Hb, or F-
Measuring Hb has clinical significance.

[0004] In the measurement of F-Hb, it is necessary to separate it from Hb-Hp.
F-Hb adsorption method (column method) and the like are known. The simple differential quantification method is based on the binding ratio between Hb and Hp based on the quantitative value of haptoglobin (hereinafter abbreviated as Hp) in serum by the one-way radial immunodiffusion method and the quantitative value of Hb by the cyanmethemoglobin method, based on the binding ratio between Hb and Hp. It is to be calculated. However, in the simple fractionation and quantification method, F-
Since this method is for obtaining the amount of Hb, there is a problem that F-Hb below the quantification limit of Hb and Hp cannot be measured.

In the F-Hb adsorption method, after Hp is immobilized on a carrier, F-Hb is adsorbed on the immobilized Hp, and the amount of Hb bound to Hp, that is, the amount of F-Hb is measured based on the color tone of Hb. is there. More specifically, Hp is immobilized on activated Sepharose, which is uniformly packed in a cylindrical column. 0.1 ml of the test serum is injected into a column which is set up vertically, and washed sufficiently with a 3 to 10-fold amount of physiological saline.
The measurement is performed at room temperature. Measure the length of the colored portion of Hb,
The amount of F-Hb is read from a calibration curve prepared in advance with a known concentration of F-Hb solution. (Takeshi Oshiro: Clinical Haptoglobin, Nagai Shoten, 1987)

However, this method has a problem of lack of reproducibility and quantitativeness because the length of the colored layer varies depending on the uniformity of the Hp-immobilized Sepharose, the method of injecting the sample, the column diameter, and the like. Further, after inserting a high molecular compound column immobilized with an anti-Hb-Hp antibody into a centrifuge tube and adding serum as disclosed in JP-A-5-322888, F-Hb in the serum is separated by centrifugation. Methods for quantitative determination of the fraction have also been reported. In this method, after Hb-Hp is removed by a column,
-Quantitation of Hb is inferior in measurement speed and convenience.

Further, a method of quantifying F-Hb using an Hp-immobilized plate based on an enzyme immunoassay or a method of treating Hb-Hp or free Hp (hereinafter referred to as FH) in serum by anti-Hp antibody treatment.
(hereinafter abbreviated as p) is inactivated, and the method of measuring F-Hb is disclosed in JP-A-3-272698 and JP-A-7-103.
978. These are excellent in quantification and reproducibility, but lack in quickness and simplicity of measurement due to use of enzyme immunoassay.

Regarding the rapidity of measurement, when measuring components in serum or plasma such as blood F-Hb, the collected blood is allowed to coagulate. Since a complicated operation such as separation from a blood cell portion is required, a considerable time is required after blood collection. Further, there is a problem that the amount of blood required for these operations increases, and the burden on the patient is large. In addition, physical stimulation of red blood cells during these operations, or leaving whole blood unattended, causes hemolysis, which is a major cause of inability to obtain accurate F-Hb data.

[0009]

Means for Solving the Problems The present inventors have made intensive studies for many years and have found that Hb-Hp and F-H in blood by anti-Hp antibody treatment by the enzyme immunoassay described above.
Based on the method of measuring F-Hb after inactivating p, serum and plasma based on immunochromatography, which has been applied to commercial kits such as pregnancy diagnostics in recent years, for further simplification. In addition, they have found a method for accurately, quickly and easily measuring F-Hb present in liquid samples such as whole blood and urine, and have completed a simple method for measuring free hemoglobin and a measurement kit.

That is, in the present invention, a liquid sample containing F-Hb, Hb-Hp and F-Hp is brought into contact with one end of a water-absorbing carrier containing an anti-Hp antibody directly or via a blood cell separation membrane. Hb-H in the liquid sample
p and F-Hp were captured, and then F-Hb developed on the water-absorbing carrier without being captured and the first anti-Hb antibody labeled with colored particles and contained in the water-absorbing carrier were bound. Subsequently, the colored conjugate developed on the water-absorbing carrier is captured by a second anti-Hb antibody immobilized on the water-absorbing carrier, whereby F-Hb in the liquid sample is visually detected and measured. The present invention provides a simple immunological measurement method characterized in that:

The present invention further provides a simple F-Hb assay kit comprising a water-absorbing carrier containing an anti-Hp antibody, a first anti-Hb antibody labeled with colored particles, and a second anti-Hb antibody. I do. Anti-H that can be used in the present invention
The p antibody and the anti-Hb antibody include polyclonal antibodies obtained by immunizing heterologous animals such as rabbits and goats with Hp and Hb, respectively, or cell fusion of spleen cells and myeloma cells of mice immunized with Hp and Hb, respectively. After that, a monoclonal antibody obtained by culturing the fused cells or inoculating the mouse into a mouse can be used. These antibodies may be prepared in-house,
It is available from Nippon Biotest Co., Ltd., Wako Pure Chemical Co., Ltd., Institute for Medical Biology, Daiichi Kagaku, and so on, so it can be easily obtained.

As the colored particles which can be used in the present invention, colloidal metal particles or colloidal nonmetallic particles, synthetic polymer latex and the like are well known. However, stable particle diameter, variety of color tones, immunochromatographic development, etc. Considering later clarity, a synthetic polymer latex is preferred.
Commercially available synthetic high-polymer latex can be selected so that this chromatography can be performed under optimum conditions. Polymer Laboratories, Sekisui Chemical Co., Ltd.
It can be easily obtained from Rhone Poulin. The particle size is commercially available in the range of 0.05 to 5 μm, and it is important to appropriately select the sensitivity and accuracy depending on the antibody to be labeled, but preferably 0.4 to 1.0 μm.
μm.

The water-absorbing carrier which can be used in the present invention has a capillarity, and a conjugate of an antibody labeled with a solute or a synthetic polymer latex particle in a liquid sample and a substance to be detected is used as a developing agent. For example, any carrier can be used as long as it can be rapidly diffused and moved with water or a buffer solution, and a nitrocellulose sheet, glass fiber filter paper, filter paper, nonwoven fabric, nylon sheet and the like can be used. However, water absorption for performing the main reaction of immunochromatography (reaction of F-Hb with the first anti-Hb antibody labeled with colored particles, development of the colored conjugate, and reaction with the second anti-Hb antibody) A nitrocellulose sheet is most suitable as a carrier (hereinafter, a support for immunochromatography). Nitrocellulose sheets are commercially available from Schleicher-Schuell, Millipore, Germanic Science and the like and are readily available. In recent years, a nitrocellulose sheet of Mylar backing having excellent strength as a sheet exclusively for immunochromatography has been commercially available from each manufacturer.

Next, a method for labeling an antibody with synthetic polymer latex particles will be described. The labeling method is not particularly limited as long as it is a commonly used method. In general, it can be achieved by mixing latex particles with an anti-Hp antibody or an anti-Hb antibody at room temperature for 1 hour or more. In the case of the present invention, white latex particles are desirable for labeling the anti-Hp antibody. For the labeling of the anti-Hb antibody, colored latex particles such as blue and red are used for easy determination. The latex particle-labeled antibody is collected by a method such as centrifugation, washed well with a commonly used buffer solution, for example, a phosphate buffer solution, and then unblocked with a blocking agent. Bovine serum albumin (BSA), milk protein, and the like are often used as the blocking agent.

Next, a method for mounting the labeled antibody will be described. The anti-human Hp antibody labeled with white latex may be diluted to an optimum concentration, impregnated in a water-absorbing carrier such as a nonwoven fabric, dried by air drying or freeze drying, and then mounted on a support for immunochromatography. It may be applied directly onto a support for immunochromatography, dried and mounted. The anti-human Hb antibody labeled with a colored latex may be diluted to an optimal concentration, impregnated in a water-absorbing carrier such as a nonwoven fabric, dried by air drying or freeze drying, and then attached to a support for immunochromatography. It may be applied directly onto a support for immunochromatography, dried and mounted.

Next, Hb-Hp by anti-Hp antibody treatment
-A method for capturing and removing F-Hp will be described. The Hb-Hp.F-Hp in the liquid sample is desirably removed before the F-Hb contacts the first anti-Hb antibody labeled with the colored particles. As a removal method, a liquid sample is reacted with an anti-Hp antibody in a test tube, and Hb-Hp and an anti-Hp antibody, FH
Hb-Hp.F-Hp is captured and removed by first contacting a liquid sample with a water-absorbing carrier containing an anti-Hp antibody to form and inactivate an immune complex of p and anti-Hp antibody. Although the latter method is mentioned, the latter method is desirable from the viewpoint of simplicity. Therefore, the water-absorbing carrier containing the anti-Hp antibody is optimally placed as a pretreatment site in the liquid sample dropping (immersion) portion, which is the immunochromatography development start site. In the present invention, a free antibody may be used as the anti-Hp antibody to be contained in the water-absorbing carrier for the pretreatment. However, as shown in Example 2 below,
To enhance p-capturing ability, it is desirable to label with white latex.

Since a large number of inventions have already been disclosed with respect to a kit for measurement relating to immunochromatography, it is not necessary to specifically refer to the present invention. However, examples are given below in which the method of the present invention can be reliably and easily implemented. A support for immunochromatography, such as a nitrocellulose sheet, may be formed into a rectangle (5 to 10 mm in width, 20 to 1 in length) of any size in consideration of ease of use.
00 mm is preferably used). On the upstream side in the developing direction of the support, a pretreatment site (a water-absorbing carrier containing a latex-labeled anti-human Hp antibody), a liquid sample absorbing site (a water-absorbing carrier for accurately and rapidly developing an immunochromatography) ), A colored particle-labeled antibody holding site (a water-absorbing carrier containing a colored latex-labeled anti-human Hb primary antibody) is provided.

The pretreatment site, the liquid sample absorption site, and the colored particle-labeled antibody holding site may be prepared in separate water-absorbing carriers (FIG. 1). Alternatively, the respective portions may be provided on the same water-absorbing carrier to be integrated (FIG. 2).
Alternatively, the pretreatment site and the liquid sample absorption site may be provided on the same water-absorbing carrier, and the colored particle-labeled antibody holding site may be provided on the immunochromatography support (FIG. 3).

Furthermore, a blood cell separation site (blood cell separation membrane) is placed on the pretreatment site in the sample dropping (immersion) portion so that the blood can be directly measured as a sample, and the collected blood is directly dropped (immersed). ), A method of adding a developing solution, or a method of diluting blood with a developing solution and then dropping (immersing) the blood, the measurement can be performed without performing a serum / plasma separation operation (FIGS. 4 to 6). . Regarding the blood cell separation membrane, JP-A-1-302161 and JP-T-63-501594, W
O9603194, etc., and are publicly known. Particularly preferred are cytosep, hemasep, hemadine, etc., manufactured by Germanic Science, Inc., which can be appropriately selected.

H is used as a support for immunochromatography.
b Detection site (zone where anti-human Hb second antibody is immobilized)
Is provided. In addition, a control site (a zone in which an antibody against the IgG of the animal used to prepare the first antibody, for example, an anti-mouse IgG antibody immobilized in a mouse, is immobilized) is provided further downstream on the same support. If this is done, it can be a method for ending and confirming the measurement. On the downstream side of the support for immunochromatography, an excess liquid sample absorption site (a water-absorbing carrier for accurately and rapidly developing immunochromatography) is provided. Filter paper is suitable as the water-absorbing carrier.

[0021] The above-mentioned pretreatment site, (blood cell separation site),
The liquid sample absorption site, the colored particle-labeled antibody holding site, the immunochromatographic support, and the excess liquid sample absorption site are arranged in the developing direction, and the whole is fixed with an adhesive tape to obtain a test paper for measurement. The test paper for measurement may be used as it is as a kit for measurement, or may be placed in a plastic case provided with a sample dropping port and a judgment window and used as a kit for measurement. Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0022]

[Example 1] Preparation of F-Hb measurement kit 1) Preparation of immunochromatography support on which anti-human Hb secondary antibody is immobilized Nitrocellulose sheet (BA
S-85, manufactured by Schleicher-Schell)
Cut to 30 mm, 0.5 mg / ml of anti-human Hb monoclonal antibody solution (manufactured by Nippon Biotest) at a position 10 mm from the end, and 0.5 mg / ml of anti-mouse IgG antibody solution (manufactured by Kappel) at a position of 20 mm ) Were applied linearly using an airbrush (manufactured by Olympus Corporation) to prepare lines for anti-human Hb antibody and anti-mouse IgG antibody. After drying at room temperature for 2 hours, the plate was immersed in phosphate saline (PBS) containing 1% skim milk (manufactured by Difco) and 0.1% Tween 20 at 37 ° C. for 2 hours to perform blocking, dried sufficiently, and then subjected to immunochromatography. A photographic support was prepared.

2) Preparation of Latex Particle Labeled Antibody a. Colored latex particle labeled anti-human Hb primary antibody Red latex particle dispersion (PL-Latex, 10
%, Particle diameter 450 nm, manufactured by Polymer Laboratories)
1.2 ml of PBS was added to 300 μl, and 13,000
Centrifugation was performed at rpm for 5 minutes. 1 ml of an anti-human Hb monoclonal antibody solution (0.5 mg / ml) (manufactured by Nippon Biotest) was added, mixed well, and reacted at room temperature for 1 hour. To remove unreacted anti-human Hb monoclonal antibody, centrifugation was performed at 13,000 rpm for 5 minutes, and the precipitate was suspended in 1.5 ml of PBS and centrifuged again. The precipitate was suspended in 1.5 ml of PBS containing 1% skim milk-0.01% sodium azide and stored refrigerated.

B. White latex particle-labeled anti-human Hp antibody White latex particle dispersion (PL-Latex, 10
%, Particle diameter 450 nm, manufactured by Polymer Laboratories)
And an anti-Hp antibody (manufactured by Medical Biology Laboratory) by the same operation as above. c. Carrier holding colored latex particle-labeled anti-human Hb first antibody 10 μl of 5 mm × 5 mm non-woven Bemliese nonwoven fabric (manufactured by Asahi Kasei Corporation) was impregnated with colored latex particle-labeled anti-human Hb first antibody and air-dried. d. Carrier holding white latex particle-labeled anti-human Hp antibody A white latex particle-labeled anti-human Hp antibody was impregnated with 20 μl of 5 mm × 5 mm glass fiber filter paper, blocked with PBS containing 0.1% skim milk, and air-dried.

3) Preparation of Measurement Kit The immunochromatographic support on which the anti-human Hb second antibody has been immobilized, a colored latex particle-labeled anti-human Hb first antibody holding carrier is placed at a position 2.5 mm from one end. Stacked. Further, a carrier for absorbing a liquid sample (filter paper No. 526) was placed on the carrier holding the colored latex particle-labeled anti-human Hb primary antibody.
(Advantech Toyo) 5 × 15 mm was overlapped to a position 5 mm from the end. In addition, a carrier containing 5 × 10 mm of a white latex particle-labeled anti-human Hp antibody holding carrier was overlaid so as to cover it. In addition, the water-absorbing carrier 5 mm × 20 m from the other end of the support for immunochromatography to a position 5 mm from the other end.
m (filter paper No. 526 made by Advantech Toyo). Finally, paste a transparent tape on the back side to fix the whole,
A measurement kit was placed in a plastic case provided with a liquid sample dropping port and a judgment window. In the case of whole blood measurement, a blood cell separation membrane was further superimposed on the carrier holding the white latex particle-labeled anti-human Hp antibody, and placed in a plastic case provided with a liquid sample dropping port and a judgment window to prepare a measurement kit.

Example 2 Comparison of Hp-Capturing Ability of Water-Absorbent Carrier Containing Anti-Hp Antibody and White Latex Particle-Labeled Anti-Hp Antibody For comparison with a water-absorbing carrier containing an antibody, the amount of anti-Hp antibody per kit was 0.1, 1.
A measurement kit in which an anti-Hp antibody solution is directly applied to a support for immunochromatography so as to be 0 or 10 μg, and a measurement kit in which a glass filter impregnated with a white latex-labeled anti-Hp antibody solution is attached to the support for immunochromatography. Was prepared according to the method of Example 1.
To each kit, serum S1 containing no free Hb (Hb-Hp only as a type of Hb in the serum) was dropped, and the Hp capturing ability was examined. As a result, in the case of the anti-Hp antibody direct application kit, H
b-Hp was not captured completely and was Hb-positive. A sufficient amount of anti-Hp antibody was applied at 10 μg to completely capture Hb-Hp in serum, which was a negative result. White latex labeled anti-H
When a kit is prepared by impregnation with p antibody, the antibody amount is 0.1
μg can completely capture Hb-Hp,
It was possible for p antibody to act efficiently (Table 1).

[0027]

[Table 1]

[Example 3] Performance test of kit for measuring F-Hb in serum / plasma 1) Reactivity test using standard solution Human Hb (manufactured by Sigma) was 1, 0.5, 0.1 mg / d
1 was dissolved in PBS containing 0.1% BSA to obtain a standard solution. 200 μl of each standard solution was added to the liquid sample dropping port of the measurement kit, developed, and visually judged after 10 minutes. When PBS containing 0.1% BSA was developed, the result was negative (Table 2).

[0029]

[Table 2]

2) Reactivity test using standard serum Human pooled human serum was added with 1 m of human Hb (manufactured by Sigma).
g / ml and used as F-Hb standard serum. F
As the -Hb negative serum, a healthy human pooled serum was used as it was. Each serum was diluted with PBS so that it became 5000 times, and 200 μl was added to the liquid sample dropping port of the measurement kit, developed, and visually judged 10 minutes later. The results are shown in Table 3. The results were positive for the serum containing Hb and negative for the serum of a healthy individual.

[0031]

[Table 3]

3) Examination of serum dilution ratio 0.1% BSA using standard serum and healthy subject serum as standard
-The measurement was performed on the sample diluted with PBS. Table 4
As shown in the figure, the magnification at which the healthy serum was negative and the standard serum was positive was 5 × 10 ³ times.

[0033]

[Table 4]

4) Examination of judgment time The reaction time until the visual judgment was 5, 10, 20, 30, 60
After examining the minutes, as shown in Table 5,
Good results were obtained in 20 minutes. The judgment time was set to 10 minutes in consideration of the quick measurement time.

[0035]

[Table 5]

5) Examination of Sensitivity Standard human Hb was added to diluted serum to examine the measurement sensitivity. As shown in Table 6, the original serum concentration was 0.1 mg / d
1 could be determined.

[0037]

[Table 6]

6) Reproducibility test Two samples of normal human serum and standard serum having different Hb added concentrations (S
1, S2), a simultaneous reproducibility test (n = 5) was performed. All sera from healthy subjects gave negative results, and two standard sera gave positive results (Table 7).

[0039]

[Table 7]

7) Cross-reactivity When the reactivity of other animals to Hb was confirmed, as shown in Table 8, no cross-reactivity to goat, cow, and pig Hb (5 mg / dl) was observed.

[0041]

[Table 8]

8) Correlation with enzyme immunoassay Assay was performed on 62 human sera by enzyme immunoassay (ELIS).
The correlation with the F-Hb measurement in serum in A) was examined. In ELISA, 2.2 mg / dl or more was regarded as positive. The agreement rate was 94%, the sensitivity was 100%, and the specificity was 90%, and almost good results were obtained (Table 9).

[0043]

[Table 9]

[Example 3] Performance test of kit for measuring F-Hb in whole blood 1) Test for confirming performance of blood cell separation membrane The collected blood was immediately mixed with an anticoagulant commonly used. Saline or P to prevent hemolysis
A sample diluted 5000 times with BS was used as a sample. As a positive sample, the whole blood after blood collection was vigorously shaken to physically lyse the blood. Table 10 shows the comparison results with and without the blood cell separation membrane. The use of a blood cell separation membrane enabled measurement of F-Hb in whole blood.

[0045]

[Table 10]

2) Examination of Sensitivity Standard human Hb was added to non-hemolyzed whole blood diluted 5000-fold to examine the measurement sensitivity. As shown in Table 11, determination was possible up to 0.1 mg / dl in the stock solution concentration.

[0047]

[Table 11]

3) Reproducibility test For each of the five human blood samples, physically hemolyzed and non-hemolyzed blood were prepared and diluted 5000-fold, and the measurement was performed. As a negative result, positive results were obtained in all cases of physical hemolysis.

[0049]

[Table 12]

[0050]

As is clear from the above examples, according to the present invention, F-Hb, which is an indicator of hemolysis in a living body, can be quickly, simply and accurately determined from serum, plasma, urine or whole blood immediately after blood collection. Provided are an immunological measurement method and a measurement kit. Rapid confirmation is required for in-vivo hemolysis by extracorporeal circulation, burns, enterohemorrhagic Escherichia coli infection, blood transfusion, and the like, which cause hemolytic nephropathy. Therefore, the present invention is useful for avoiding and preventing hemolytic kidney injury caused by delay in confirmation of hemolysis.

[Brief description of the drawings]

FIG. 1 shows a simple embodiment of the present invention, and is a top view and a cross-sectional view of the measurement kit. (For serum and plasma measurement)

FIG. 2 shows a simple embodiment of the present invention, and is a top view and a sectional view of the measurement kit. (Integrated serum and plasma measurement 1)

FIG. 3 shows a simple embodiment of the present invention, and is a top view and a cross-sectional view of the measurement kit. (Integrated serum and plasma measurement 2)

FIG. 4 shows a simple embodiment of the present invention, and is a top view and a cross-sectional view of the measurement kit. (For whole blood measurement)

FIG. 5 shows a simple embodiment of the present invention, and is a top view and a cross-sectional view of the measurement kit. (Integrated whole blood measurement 1)

FIG. 6 shows a simple embodiment of the present invention, and is a top view and a cross-sectional view of the measurement kit. (Integrated whole blood measurement 2)

[Description of Signs] a Pretreatment site (water-absorbent carrier holding latex-labeled anti-haptoglobin antibody) b Liquid sample absorption site c Color-particle-labeled antibody holding site (water-absorbent carrier holding latex-labeled anti-human hemoglobin primary antibody) d Hemoglobin detection Site (anti-human hemoglobin second antibody immobilized zone) e Control site (anti-mouse IgG antibody immobilized zone) f Support for immunochromatography g Excess liquid sample absorption site h Blood cell separation site (blood cell separation membrane)

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Eriko Matsuyama 1-5-3 Nihonbashi Muromachi, Chuo-ku, Tokyo Wakamoto Pharmaceutical Co., Ltd. (72) Inventor Haruhisa 1-5-3 Nihonbashi Muromachi, Chuo-ku, Tokyo Wakamoto Pharmaceutical Co., Ltd.

Claims (4)

[Claims] 1. A liquid sample containing free hemoglobin, a hemoglobin-haptoglobin complex and free haptoglobin is brought into contact with one end of a water-absorbing carrier containing an anti-haptoglobin antibody directly or through a blood cell separation membrane. The hemoglobin-haptoglobin complex and free haptoglobin in the liquid sample were captured, and then free hemoglobin developed on the water-absorbing carrier without being captured, and the first particle was labeled with colored particles and contained in the water-absorbing carrier. Free hemoglobin in the liquid sample is visible by binding the anti-hemoglobin antibody and then capturing the colored conjugate developed on the water-absorbing carrier with the second anti-hemoglobin antibody immobilized on the water-absorbing carrier. A simple immunological measurement method for free hemoglobin, characterized in that it is detected and measured. 2. The method according to claim 1, further comprising an anti-haptoglobin antibody labeled with white particles instead of the anti-haptoglobin antibody.
The simple immunological measurement method described in the above. 3. A simple free hemoglobin measurement kit comprising an anti-haptoglobin antibody, a first anti-hemoglobin antibody labeled with colored particles, and a water-absorbing carrier containing a second anti-hemoglobin antibody. 4. The kit for measuring free hemoglobin according to claim 3, wherein the kit contains an antibody labeled with white particles instead of the anti-haptoglobin antibody.