JPH02208568A - Haptoglobin-determining-reagent kit and haptoglobin-determining method using said kit - Google Patents

Abstract

PURPOSE:To disregard the genotype of haptoglobin and to make it possible to determines the quantities of haptoglobin for many specimens quickly and accurately at a low cost by picking up large amounts of the specimens which do not incorporate hemoglobin, picking up small amounts of the specimens which incorporate the hemoglobin, and applying a color determining method. CONSTITUTION:An aqueous solution (reagent 1) incorporates tetramethyl bezene (TMB). Another aqueous solution (reagent 2) incorporates strontium peroxide or hydrogen peroxide. At least one of either of standard solution, i.e. human hemoglobin (Hb) or human haptoglobin (Hp), and a standard solution, which does not incorporate said material, is added into said aqueous solutions (reagents 1 and 2). Thus, an Hp determining reagent kit is provided. The reagents 1 and 2 are mixed. A specimen incorporating Hp is added into the mixture, and coloring reaction is made to occur. Thus, colorimetry is performed. In this way, a trace quantity of Hp incorporated in plasma and serum can be determined, and the determination can be performed with an automatic analyzer.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for quantifying /% butoglobin in the field of clinical testing and a quantitative reagent kit used therefor.
This invention relates to the determination of lX butoglobin by the so-called color method using tetramethylbenzidine as an oxidative coloring agent.

[Conventional technology]

Normally, about 20 to about 200 mg/dN of haptoglobin (hereinafter abbreviated as Hp) exists in the blood of healthy people, and it is turned over with a blood half-life of 2 to 4 days.

Hp specifically binds to hemoglobin (hereinafter abbreviated as Hb) to form an Hp-Hb complex. Hb produced by hemolysis forms a complex with Hp in the blood and is transported to the liver where it is metabolized. However, when Hb and Q exceed the amount of Hp bound, excess Hb is released into the blood in a free state. exists in
Causes Hb leukemia and Hburia. For Hb leukemia and Hburia, treatment methods include massive blood transfusion and administration of diuretics.
There is a treatment method that administers haptoglobin (haptoglobin therapy). Haptoglobin therapy does not cause free fiHb to be excreted from the kidneys. The aim is to combine 1tHb with Hp and metabolize it in the liver.

In recent years, extracorporeal circulation using a heart-lung machine has rapidly become popular during surgical operations, and 71 butoglobin therapy is being adopted to prevent postoperative renal failure that develops as a result of hemolysis caused by extracorporeal circulation. In haptoglobin therapy, H in the patient's blood
It is extremely important to know the p and Hb concentrations. Currently, the blood concentration of Hp is determined by a) cellulose acetate membrane electrophoresis, b) - optical radial immunodiffusion method (Mancini method), C) laser nephelometer method, d) rapid spectral quantitative method, and e) latex agglutination method. JPJ can be determined by methods such as (Meng Oshiro, "Clinical/X Butoglobin", Nagai Shoten, 1987).

However, these methods other than method C) are unsuitable for processing a large number of samples quickly and accurately in a short period of time, and C) requires the purchase of expensive equipment, making it difficult to widely disseminate it. seems to be difficult. In addition, the Muncie method (b) is an accurate quantitative method, but in addition to the above-mentioned inconvenience, it is inconvenient to multiply the measured value by the respective type coefficient to obtain the quantitative value, considering the three genetic types specific to Hp. There is.

[Means to solve the problem]

The present inventors, in collaboration with others, have exploited the peroxidase-like action of hemoglobin to transform the originally colorless 3.3', 5'
．． He devised a method for colorimetric determination of Hb in which 5'-tetramethylbenzidine (hereinafter abbreviated as TMB) is oxidized via strontium peroxide to develop a color, and filed a patent application (Japanese Patent Application No. 63-276387, hereinafter referred to as (simply referred to as "first application").

This method is used to quantify Hb in plasma, serum, urine, etc., and the quantitative reagent kit consists of (a) an aqueous solution in which poorly soluble TMB is solubilized by adding citric acid, and (b) strontium peroxide. It consists of an aqueous solution containing For quantitative determination, mix the aqueous solutions of (a) and (b) before use, add a small amount of sample to the mixture, and incubate at a specific temperature (10 to 37°C) for a specific time (1 to 60 minutes) to develop a reaction color. . Color development is 365-375nm or 645-655r+
The absorbance of n+ at a specific wavelength is measured by a conventional method using an autologous blank as a control. If necessary, hydrogen peroxide at a concentration of about 1% can be added to the reaction system to promote color development. In the same way, instead of the sample, a standard test solution prepared by adding Hb to a series of concentrations to a self-contained blank containing no Hb was used to quantify Hb and create a calibration curve (a straight line). Find the Hba degree. An automatic analyzer can be used for colorimetric determination, and a large amount of specimen can be easily processed in a short period of time.

The present inventor has discovered that the mixture of aqueous solutions (a) and (b) used in the so-called color method described above, which is a method for quantifying Hb,
Both exhibit weak reactivity, and the color development is similar to Hb.
It was found that it has specific absorption at 5-375 rv or 645-655 rv. That is, it has been experimentally revealed that Hp has peroxidase-like activity, and its activity is 1/45 to 1152.5 times that of Hb in the range of 50 to 200 μg.

This was demonstrated through experiments when Hp was added to Hp- and Hb-free plasma at various final concentrations, and the peroxidase-like activity was expressed as a 0D37° value using the color method described above, as shown in Table 1.

Table 2 shows the peroxidase-like activity of Ib.

Table 2 Color development reaction time: 10 minutes The OD values in Tables 1 and 2 are linearly related to Hp and )lbb addition, indicating quantification.

The peroxidase-like activities of Hp and Hb can be compared from the values of OD/Hp (μg)/action time (minutes) in Tables 1 and 2. For example, Hp: Hb-1,56/'
12m1n:84/10iIn =1/45, similarly Hp:Hb-1,925/12:58.6/10-
Using this peroxidase-like activity of Hp, it is possible to quantify Hp contained in Hb-free, unhemolyzed plasma or serum using a color method that conforms to the Hb determination method described above. It is.

On the other hand, for hemolyzed plasma containing Hb, the peroxidase-like activity of Hp is much weaker than that of Hb, as mentioned above, so the presence of Hp in the sample does not seem to have a major effect on the quantification of Hb. However, it was found that it actually has a large effect.

That is, when a fixed amount of Hb and Hp is added to plasma that does not contain Hb and Hp and the OD is measured by the color method, an irrationally excessive result is given.

This relationship is shown in Table 3 and FIG.

Table 3 Table 4 shows the OD per 1 μg of Hb from Table 3.

From Table 4, Table 3.4 and Figure 1, the coexistence of Hb gives an activity that far exceeds the peroxidase-like activity of Hp itself.
Moreover, it can be seen that the linearity of the OD value is not disturbed for each addition of Hp. Based on this unexpected result, H
It will be understood that by coexisting Hp in the specimen, it becomes possible to quantify a trace amount of Hp in the specimen using the amplified OD, C, 1L, and Yamabetsu 1U values.

Thus, the method of the previous patent's empty constant amount of Hb method is similar to that in the Hb quantitative method in the case of a sample that does not contain Hb by collecting a relatively large amount during the color reaction, and in the case of a sample that contains Hb. It has been found that by collecting a relatively small amount, it can be applied as is.

Based on the above findings, the present invention provides a quantitative reagent kit that enables micro-quantification of haptoglobin contained in plasma, serum, etc. and is suitable for use with an automatic analyzer, and a method for quantifying haptoglobin using the same. According to the present invention, Hp
Haptoglobin can be quantified quickly, inexpensively, and accurately in a large number of samples without regard to genetic type.

Although the present invention has been described above with reference to the prior application, the gist of the present invention can also be satisfied by using hydrogen peroxide in place of strontium peroxide. That is, the aqueous hydrogen peroxide solution can be used not only as an oxidizing agent but also as the main oxidizing agent. This is clear from the fact that a method for quantifying Hb using the same reaction mechanism using hydrogen peroxide solution and TMB aqueous solution as reagents is already known (Medical Te
Chnology.

Volume 13, No. 12, Pages 1264-1265, 198
5, Teruo Yamada, Futoshi Chuo, “Plasma hemoglobin”).

The reagent kit for Hb quantification of the present invention essentially consists of (a) Reagent 1: an aqueous solution containing TMB or a water-solubilized derivative thereof, and (b) Reagent ■: an aqueous solution containing strontium peroxide or hydrogen peroxide. . The kit may further include at least one of (c) a human hemoglobin standard solution, (d) a human haptoglobin standard solution, and (e) a standard solution free of human hemoglobin and human λ-ptoglobin.

In reagent I, TMB is sparingly soluble in water, but the amount of TMB required in the Hp amount method of the present invention is 0, which corresponds to a trace amount of peroxidase-like active substance containing actual Hp.
Since it is 2 to 0.3 rmg/m, it is below the solubility of TMB. However, depending on the storage temperature, precipitation may occur. Therefore, it is preferable to make TMB water-soluble. In the present invention, water-soluble TMB dihydrochloride is used in an approximately equivalent amount of 0.25 to 0.5 mg/mN as TMB.

In addition to using the above-mentioned hydrochloride, any method for water solubilization may be used as long as it does not inhibit the coloring reaction based on the peroxidase-like activity of Hp, which is the basis of the present invention. Focusing on being soluble in citric acid aqueous solution, TMB 20-3 Clng/100
It has been recognized that an aqueous solution containing mII citric acid at a ratio of 150 to 250 mg/100 rrl is suitable for the above-mentioned method, is relatively stable, is safe, and does not cause corrosion of quantitative equipment. In this case, TMB is likely to exist as a salt of citric acid;
B is also defined as a water-solubilized TMB derivative.

All of the above aqueous solutions are preferably prepared by dissolving a predetermined amount of the solution in a predetermined amount of purified water and then filtering the solution with a filter (preferably 0.45 μm pore size).

The aqueous solution of Reagent H contains sufficient strontium peroxide to oxidize TMB, e.g.
It is obtained by dissolving 90 mg in 100 mj7 of water (preferably purified water) and then filtering with a filter (preferably 0.45 μm pore size).

Hydrogen peroxide solution that can be used for reagent H has a concentration of 1
~4% or is a relatively stable, highly concentrated solution that can be diluted to this concentration before use.

The human Hb standard solution (c) has a constant concentration accurately determined by the Cyanmeth international standard method, and is used for preparing Hb-containing specimens and Hb-containing standard test solutions.

The Hp standard solution in (d) has a constant concentration accurately determined by the Muncie II method, and is added to the Hp- and Hb-free standard solution in (e) and the Hb-containing standard test solution to obtain a series of Hp concentrations. Prepare a standard test solution and use it to create a calibration curve.

The Hp- and Hb-free standard solution (e) can be prepared by treating unhemolyzed healthy human plasma with affinity chromatography coupled with an anti-haptoglobin antibody. In addition, when human urine is used as a sample, the urine of a healthy person can be used directly as a standard solution. Hp is hardly detected in the urine of healthy people.

Using the kit of the present invention, H in a specimen such as plasma or serum can be
To quantify p, mix reagent 1 and reagent 2, and add H to the mixture.
The method consists of adding a sample containing p, causing a color reaction, and performing colorimetric determination.

For convenience, the method will be explained using a standard test solution for drawing a calibration curve. Reagents (1) and (2) are mixed in approximately equal amounts (0.5 to 2 mN). The Hb-free standard test solution added to the mixture is Hp
from about 1000mg/dΩ to about 300mg/dΩ
Adjust to a series of concentrations of 50 to 1000 μl each.
A minimum of 20 μg and a maximum of 1000 μg of Hp are reacted by adding g to the reagent mixture.

On the other hand, in the Hb-containing standard test solution, the Hb concentration may be adjusted to any desired concentration, but it is preferably 100 to 600 ■/dR, and the Hpa level is adjusted to a series of concentrations of about 40,011 g/dJl at the maximum. Then, 5 to 10 μg of each is added to the reagent mixture, and 1 μg or more of Hp is reacted.

The reaction was carried out under common conditions for each standard test solution.
The reaction temperature is selected to be constant from 10 to 37°C, and the reaction time is selected from 1 to 601n. In order to promote the reaction, 1 mg or less of a 1-4% hydrogen peroxide solution can be added to the reaction system.

After the reaction is complete, change the color to 365-375ni or 645-6
The absorbance at a specific wavelength selected from 55 nm is measured by a conventional method using an autologous blank as a control. Create a calibration curve from the measured values.

Samples such as plasma from healthy people who have not had hemolysis, have similar reactions.
A sample to which fb (the same amount as the Hb standard test solution) was added,
For samples such as hemolyzed plasma with the same amount of Hb as the Hb standard test solution, color development is measured and up is quantified based on the respective calibration curves.

In addition, due to the linearity of the calibration curve, the wavelength used is 365 to 375 nm when quantifying a sample that does not contain hemoglobin, such as the plasma of a healthy person without hemolysis, and 365 to 375 nm when quantifying in the presence of a predetermined amount of Hb. , 645 to 655 ni+.

In addition, in amplifying the quantitative nature of Hp due to the coexistence of Hb,
In the case of a specimen that already contains Hb, such as hemolyzed plasma, it is desirable to set the predetermined amount of Hb in the Hb standard test solution to a higher concentration than the level of Hb contained in the specimen. The present method can be conveniently carried out by quantifying the existing Hb in the specimen by the color method or the cyanmeth international standard method, and using the specimen to which the missing amount has been added.

The present invention will be explained in more detail with reference to Reference Examples and Examples below. The reagents used in these examples are as follows.

Reagent 1: In reference examples and Examples 1 to 5, citric acid 192 mg/100mj7 S7M826 mg/I
Q Q Aqueous solution contained in a concentration of ml.

In Example 6, TMB/dihydrochloric acid Aqueous solution containing salt at a concentration of 40 mg/100 mg.

Reagent ■: Aqueous solution containing strontium peroxide at a concentration of 77 og/100 mg.

Reference Examples The data shown in Tables 2 and 3 (therefore, FIG. 1) were obtained as follows.

1) Hp-0μg Hb and Hp-free plasma are added with Hb accurately determined by the cyanmeth international standard method, and diluted to 0.20 μg.
, 40, 60. Hb at a concentration of 80, 100a+g/dp
Standard plasma was prepared.

Coloring reagent 2 prepared by thoroughly mixing reagent I and reagent II
rrl was added with 5 μg of Hb gold-containing serum of each concentration and mixed, and incubated at 37° C. for exactly 10 minutes.The absorbance at 370 tv was measured using a spectrophotometer using a self-blank as a control, and a calibration curve was created.

The results are shown in Table 2 and are also incorporated in Table 3, Figure 1.

2) Hp-5μg Hb standard plasma and Hp 100mg/dj? 1) above was repeated except that it further included.

The results are included in Table 3 and Figure 1.

3) Hp - 10 μg Same as above 2 except that Hp was 200 IIg/'d 1)
) was repeated.

The results are included in Table 3 and Figure 1.

4) Hp-15 μg The above 2) was repeated except that Hp was 300 mg/dN.

The results are included in Table 3 and Figure 1.

5) Hp-20μg The above 2) was repeated except that Hp was changed to 400ff+g/dff.

The results are included in Table 3 and Figure 1.

6) Hp-25 μg The above 2) was repeated except that Hp was changed to 500 mg/djJ.

The results are included in Table 3 and Figure 1.

Example I Hp that was accurately determined by the Munchii method was added to Hb- and Hp-free plasma, and this was diluted to 0°50.100.
, 150, 200, 250, 300 mg/d, 17 concentrations of Hp standard plasma were prepared. Add each concentration of Hp to 2mM of coloring reagent prepared by mixing equal amounts of Reagent I and Reagent II.
Add and mix 100 μg of plasma containing plasma and incubate at 37°C for exactly 12 hours.
After incubating for a minute, the absorbance of 370 r+a+ was measured using a spectrophotometer using a blank as a control, and a calibration curve was created.

A similar operation was performed using 100 μg of a non-hemolyzed specimen to determine the absorbance at 370 nm, and the Hpa degree in the specimen was determined from the calibration curve.

The results for the trademarked plasma are shown in Table 1, and the calibration curve is shown in Table 2.
As shown in the figure.

Example 2 Example 1 was repeated. However, the Hp concentration of Hp standard plasma is 0.200, 400, 600, 800゜1000mg/
dII and the incubation time was exactly 6 minutes.

The calibration curve is shown in Figure 3.

Example 3 Hb accurately quantified by the cyanmeth international standard method was added to Hb- and Hp-free plasma to bring the final Hba degree to 20.
0 mg/dΩ, then add Hp accurately quantified by the Mancini method to give a final Hp/a degree of 0 and 50 degrees, respectively.
,100,150゜200.250,300 g/df
A standard plasma of I was prepared.

Add and mix 5 μg of standard plasma of each concentration to 2 mfl of the coloring reagent prepared by mixing equal amounts of Reagent I and Reagent II,
After incubating at 37° C. for exactly 8 minutes, the absorbance at 650 r+m was measured using a spectrophotometer using an autologous blank as a control, and a calibration curve was created.

Quantitate Hb in the hemolyzed plasma using the constant Kara method or the Cyanmeth standard international method, add the missing amount of Hb to make the concentration 200 mg / d, the same as the concentration in standard plasma °, and use 5 μg of this, The absorbance was determined to 71-1 by subjecting it to a similar coloring reaction, and the Hpa degree was determined from the calibration curve.

The calibration curve is shown in Figure 4.

Example 4 Example 3 was repeated. However, the final concentration of Hb in the standard plasma was set to 400,111 g/djJ.

The calibration curve is shown in Figure 4.

Example 5 Example 3 was repeated except that the final Hb concentration in the standard plasma was 500ff+g/dΩ.

The calibration curve is shown in Figure 4.

Example 6 Hp that was accurately determined by the Munchii method was added to Hb- and Hp-free plasma, and this was diluted to 0.100, 2.
Hp standard plasma was prepared at a concentration of 00,400,600,800°1000 mg/mΩ.

Coloring reagent 2 prepared by mixing equal amounts of reagent 1 and reagent 2
After adding 50 μg of each concentration of I (p-containing plasma) to the
The absorbance of 370no+ was measured using a spectrophotometer using the autologous blank as a control, and a detection ffi line was created.

A similar operation was performed using 50 μg of a non-hemolyzed specimen, the absorbance at 370 nm was determined as atj, and the Hp concentration in the specimen was determined from the calibration curve.

The calibration curve is shown in FIG.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the influence of absorbance of haptoglobin depending on the hemoglobin concentration in plasma in Reference Example and Example 1, and FIGS. 2 and 3 are diagrams showing haptoglobin in hemoglobin-free plasma of Examples 1 and 2, respectively. Figure 4 is a diagram showing the calibration curve of haptoglobin in the hemoglobin-containing plasma of Examples 3, 4, and 5, respectively, and Figure 5 is a diagram showing the calibration curve of haptoglobin in the hemoglobin-free plasma of Example 6. FIG. Patent applicant Takashi Terashima, patent attorney representing Midori Juji Co., Ltd.

Claims (1)

[Claims] 1. Consisting of (a) an aqueous solution containing 3,3',5,5'-tetramethylbenzidine or its water-solubilized derivative, and (b) an aqueous solution containing strontium peroxide or hydrogen peroxide. Haptoglobin quantitative reagent kit. 2. The kit according to claim 1, further comprising at least one of (c) human hemoglobin standard solution, (d) human haptoglobin standard solution, and (e) haptoglobin and hemoglobin-free standard solution. 3. The kit according to claim 1, wherein the aqueous solution of (a) contains 3,3',5,5'-tetramethylbenzidine hydrochloride at a ratio of 25 to 50 mg/100 ml. 4. Claim 1, wherein the aqueous solution of (a) contains 20 to 30 mg/100 ml of 3,3',5,5'-tetramethylbenzidine and 150 to 250 mg/100 ml of citric acid.
Kit as described. 5. The aqueous solution of (b) contains strontium peroxide from 60 to 9
The kit according to claim 1, containing the kit at a ratio of 0 mg/100 ml. 6. (a) Mixing an aqueous solution containing 3,3',5,5'-tetramethylbenzidine and or its water-solubilized derivative and (b) an aqueous solution containing strontium peroxide or hydrogen peroxide before use. Add a sample containing haptoglobin to the solution,
A method for quantifying haptoglobin consisting of reaction color development and colorimetric determination. 7. The method according to claim 6, wherein the specimen contains hemoglobin. 8. The method according to claim 6, wherein the specimen is human plasma or serum. 9. The method according to claim 6, wherein the colorimetric determination is based on absorbance at a wavelength of 645-655 nm or 365-375 nm. 10. The method according to claim 7, wherein the colorimetric determination is performed based on a calibration curve obtained by measuring a standard test solution having a hemoglobin concentration that is the same as the hemoglobin concentration of the specimen and a series of haptoglobin concentrations. 11. Claim 7 or 1, wherein the colorimetric determination is performed based on absorbance at a wavelength of 645 to 655 nm.
The method described in 0. 12. The method according to claim 6, comprising carrying out the colorimetric determination by using an automatic analyzer.