JPH03220457A - Standard hemoglobin reagent - Google Patents

Abstract

PURPOSE:To obtain an Hb standard reagent with a high value of a stable HbAIC usable as control in the measurement of HbAIC utilizing a difference of an electric load by incorporating acetylated hemoglobin (Hb). CONSTITUTION:This reagent contains acetylated Hb to be used for the measurement of succharified hemoglobin. An acetylating reagent used for the preparation of the acetylated Hb is a compound which preferably has a skelton of acetanilide or acetylphenol in a molecule to isolate acetyl groups easily by a hydraulic decomposition. An acetylating reagent of such a type is preferably a compound which is soluble in water to a certain extent and the compound completely insoluble in water is not desirable because the acetylating reaction of the Hb is made in an aqueous solution. The acetylated Hb is formed by bringing the acelylating reagent into contact with a sample containing containing at least a red blood cell and Hb in the solution and the method is not limited specifically.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to glycated hemoglobin (hemoglobin A) in blood.
, ), particularly relates to a hemoglobin standard reagent used for measuring hemoglobin AtC.

(Prior Art) Glycated hemoglobin (hemoglobin AI), particularly hemoglobin Ale, in the blood is known as one of the indicators of diabetes, and its measurement is being carried out. (below.

Hemoglobin is designated as )Ib).

HbA,c is β of Hb as shown in the formula below
It is a complex in which one molecule of gluten is bound non-enzymatically to the amino group of valine present at the N-terminus of the chain. When gluten binds to the above amino group of Hb, first.

Unstable HbA, which is a ship base as shown in the following formula,
c (L-HbAtc) (1) is formed: In the above formula (1), β-A-NH2 represents Hb, in which NH2 connects the amino group of valine at the N-terminus of the β chain of Hb. show.

This L-HbA, c (+) production reaction is a reversible reaction, and the equilibrium changes depending on the glucose concentration.
c tilts toward the production direction or dissociation direction of (1). (1)
further undergoes Amatoli rearrangement to irreversibly stabilize Hb.
A. (5-HbA+c) (II). This 5
-HbA, c is blood glucose (i.e. blood sugar) M
It is an excellent indicator of diabetes because its amount does not change drastically and significantly, and it correlates well with the patient's blood sugar level about two months ago.

HbA,. Two different methods are known for measuring the value: mini-column, high-performance liquid chromatography (H
Methods applying various chromatography techniques such as PLC) are widely used. In these methods, HbA, for better quality control. Standard reagents of known concentration are required. By measuring the standard reagent in the same way as the measurement sample each time, it is possible to correct errors caused by differences in various conditions between measurements (differences in reagent lots, measurement equipment conditions, etc.), and to perform calibration. I can do it. In recent years, with the automation of measurement systems, the demand for standard reagents for calibration has been increasing. As an Hb standard reagent, 1 is usually used as HbA1C.
An Hb standard reagent whose value is in the normal range (approximately 4 to 6% of the total b amount) is used. However, 2 For example, diabetic patients' blood sugar is much higher than this, so Hb
In addition to the Hb standard reagent whose Atc value is in the normal range, it also has an abnormally high value range (relative to total Hblt).

It is considered that calipresicon can be performed more accurately by using in combination one or more of the Hb standard reagents (usually 6% or more). moreover.

When using the Hb standard reagent as an internal standard.

Especially 5-HbA among HbA, c. By using an Hb standard reagent with a high value, it is possible to obtain a more accurate measurement value regardless of the glucose concentration in the blood sample.

Therefore, HbA and c values are particularly high.
b Standard reagents are desired.

11b standard reagent, JP-A-58-3
No. 7561 "Standard or reference material for measuring glycosylated or total hemoglobin"; and JP-A-59-18
No. 3370 "Method for producing hemoglobin standard reagent" is known. In all of these documents, stable Hbll
Reagents are listed. j! to HbAl in the Hbm quasi-reagents prepared in these documents! , especially 5-HbA,
The amount of c depends on the 5-HbA and cl of the raw blood. Therefore, in order to produce an Hb standard reagent whose 1bA and c values are in the abnormally high range, it is necessary to use 5-Hb standard reagents such as the blood of diabetic patients as raw materials.
The only option is to use blood that has a high value of A, c (it. However, it is difficult to actually use the blood of a diabetic patient as a raw material for an Hb standard reagent, and it is difficult to use a Hb standard reagent that has a high 5-HbAIC value. is impossible to obtain stably in large quantities.

Another method for obtaining a Hb standard reagent with high values of 5-HbA and C is to artificially glycate [(b) in vitro.
-HbA,. Methods for synthesizing these are also being considered. but.

Christopher et al.
Chell, 260. 21. ppH611~
11618. (1985) in vitro converted Hb to D-
When converted to HbA by reacting with a reaction solution containing glucose, L-HbA I (bA, o)
Although C increases rapidly, L-) 1bA, 5-HbA, produced by Amatoli rearrangement of c. reported that the production rate was extremely slow.

Since L-HbA,o reversibly dissociates into glucose and Hb depending on the glucose concentration, it is unstable and is particularly unsuitable as a standard substance used as an internal standard.

Others, old rice (diabetes 23.679-684゜198
0), when Hb is reacted with acetaldehyde, H
It is reported that the peak of bA, , fraction (I (containing the acetaldehyde derivative of bA,C) increases).

However, when acetaldehyde is used, HbA
,. Not only the fraction peaks, but rather mainly HbA.

And the peak of HbA+b fraction increases, and H
b The fractionation pattern changes significantly. in this way.

5-FIbA,. To date, no Hb standard reagent has been obtained in which the value of the fraction is artificially increased.

(Problem to be solved by the invention) The present invention solves the above-mentioned conventional problems.

The purpose is to use HbAI using the difference in charge.
It can be used as a control in the measurement of C. 5-HbA
The purpose of the present invention is to provide an Hb standard reagent with a high lc value.

Another object of the present invention is to artificially 5-)1bA, without using blood with high HbA,c. The purpose of the present invention is to provide an Hb standard reagent with a high value fraction.

(Means for Solving the Problems) The inventors have proposed the following S-1 (
bA,. We considered using a substance that exhibits the same behavior as 5-HbA,c, rather than the component itself, and conducted various studies on such substances. As a result, the inventors discovered that acetylated Hb exhibits the same behavior as 5-HbA+c in HPLC, leading to the completion of the present invention.

The Hb standard reagent of the present invention is an Hb standard reagent used for glycated Hb measurement.
bml reagent containing acetylated hemoglobin, thereby achieving the above objectives.

The acetylation reagent used in the preparation of the above acetylation) 1b preferably has an acetanilide or acetylphenol skeleton in nine molecules.

A compound whose acetyl group can be easily released by hydrolysis is used. Such acetylation reagents.

This is because the acetylation reaction of Hb is carried out in an aqueous solution.

Compounds that have some degree of solubility in water are preferred, and compounds that are completely insoluble in water are not preferred.

Such compounds include, for example, ortho- or para-acetylsalicylic acid and N-7 cetyl aminobenzoic acid, which have some degree of solubility in water.

Examples include N-acetylaminobenzenesulfone and monoacetylcatechol. Salts, derivatives of the above compounds, and compounds having further substituents can also be used as acetylation reagents, as long as they have a certain degree of solubility in water and easily release an acetyl group by hydrolysis. . especially.

Acetylsalicylic acid is suitable because it is easily available, inexpensive, and has a strong acetylating effect.

Such an acetylation reagent 9 is usually dissolved in a suitable aqueous solvent such as a buffer and used as a medium for the acetylation reaction.

Acetylation) 1b is produced by bringing an acetylation reagent into contact with a sample containing at least red blood cells and/or Hb in blood in a solution, and the method is not particularly limited. For example, red blood cells washed with physiological saline or the like containing the acetylation reagent described above.

Incubating the Hb in an acetylation reaction solution isotonic with the red blood cells; dialyzing the Hb against an acetylation reaction medium containing the acetylation reagent;
is easily and quickly acetylated to form acetylated) 1b. The above-mentioned acetylated Hb is formed by acetylating the amine groups (N-terminal amine 7 group and lysine amino residue) of non-glycated 1b (HbAa) in red blood cells with an acetylation reagent. The rate of acetylation of I(b) by the acetylating reagent depends on the type of acetylating reagent.

Concentration 2 Varies depending on conditions such as reaction temperature and pH. These conditions will be explained below.

The amount of acetylation reagent used varies depending on the type of reagent,9 but usually 1 volume of acetylation reaction medium is used per 1 volume of red blood cells.
When using ~100 volumes, add 0.0
Preparation is carried out so that the content is 01 to 5 (v/v)%. For example, when acetylsalicylic acid is used as the acetylation reagent, the buffer solution contains acetylsalicylic acid at a ratio of 0.01 to 0.5%. A predetermined amount of acetylated Hb is formed by lengthening the reaction time when the acetylation reaction solution is used at a low concentration, and by shortening the reaction time when it is used at a high concentration.

During the reaction, the rate of acetylation generally increases as the reaction temperature increases. However, if the temperature is too high, Hb will be denatured, which is not preferable. Usually 4-5
Tolerance conditions of 0°C, preferably 25 to 40°C are adopted.

The rate of acetylation reaction becomes faster as the reaction system becomes more alkaline, and becomes slower as the pH becomes more acidic. For example, if 1 volume of red blood cells and 100 volumes of acetylation reaction medium (containing 0.3% acetylsalicylic acid) at pH 8.5 are mixed and incubated at 40°C for 6 hours, The bA and c values apparently increase approximately twice. However, even if the acetylation reaction was performed under the same conditions as above except that the pH was changed to 7.5,
HbA,. The value increases by only about 50%. in this way,
It is advantageous to carry out the acetylation reaction on the alkaline side.

However, raising the pH too high is not preferable because it causes denaturation of Hb. Usually, the pt+ of a mixed solution containing a sample and an acetylation reagent is 6.5 to 9. O9 preferably 7.
It is adjusted so that it becomes 5 to 8.5. Therefore, as a solvent for the acetylation reaction solution, a buffer solution having a buffering capacity at pH 6.s to 90 is preferable, for example.

Phosphate buffers are preferably used.

The reaction time for acetylation varies depending on the type and concentration of the acetylation reagent, pH conditions, temperature conditions, etc.

By lengthening one reaction time as described above, the amount of acetylated) 1b produced can be increased.

For example, when performing acetylation using an acetylation reaction solution with a pH of 8.5 containing 0.3% acetylsalicylic acid, 1 volume of red blood cells is mixed with 100 volumes of the acetylation reaction solution. Then, at 24℃ for 6 hours or 4
IbA (by incubating for 2 hours at 0°C)
,. The value increases by approximately 20-30%. moreover.

By incubating at 24°C for 18 hours or at 40°C for 6 hours, the HbA,c value appears to be approximately 2.
increase twice. However, if the reaction time is too long, H
This is not preferable because b may be denatured or acetylation may proceed too much, resulting in abnormal peaks.

After acetylating Hb under the above-mentioned suitable conditions, the reaction solution is processed using an HPLC apparatus (Kyoto Dai-ichi Co., Ltd.) using an ion exchange resin such as a cation exchange resin as a filler.
When fractionated using Kagaku's Auto AICHA-8110), acetylated) 1b is separated into the same fractions as S-1 (bA, c).
A, acetylated Hb obtained by acetylating the amine group of (non-[hemoglobin]) is 9 original HbA due to the introduced acetyl group. This is because it has a weaker affinity for the filler than HbA, and is eluted at the same retention time as HbA,c. Therefore, the peaks of HbA and C apparently increase. The fractionation pattern of Hb (Figure 3) is.

Actually HbA. There is almost no difference in the fractionation pattern from the blood samples with high values. In FIG. 3, Pl and P2 are.

The peak due to both HbA, and HbAl b + P5 is the peak due to 1bA0, and P6
shows peaks due to both HbA,c and acetylated) 1b. For comparison, the same H
The elution pattern when each component 1b of the blood sample before the acetylation reaction is separated using a PLC device is shown. In FIG. 1, P and P2 are peaks caused by lbAlm and HbA, and P3 is L-HbA.

r P4 is 5-HbA. , and P5 indicates a peak due to HbAo. HbA,. Since HbAlc has a weaker affinity for the filler than HbAo, HbAlc is eluted earlier than HbAo. In this way, acetylation) 1b is each 1 (saccharification using the difference in charge of component b)
In the method of measuring 1b, since it is eluted in the same fraction as 5-HbA,c, 1, for example, mini column method, H
It can be used as a standard material in PLC method, electrophoresis method, etc. Moreover, acetylated) 1b is chemically sufficiently stable.

For example, even when dialyzed against physiological saline, it is hardly hydrolyzed. Thus, acetylated Hb is suitable for use as a standard reagent. However, acetylated Hb is not HbA,c itself.

It cannot be used as a standard reagent in a measurement method using an affinity column that utilizes the bond between 1-2 cis diol of sugar and boric acid.

Next, two methods for producing the Hb standard reagent of the present invention will be described. First, a sample containing at least red blood cells and/or Hb is prepared. This sample may be whole blood, but the acetylation reaction may also occur with proteins other than Hb, which may reduce the production rate of acetylated Hb.

Therefore, it is preferable to use red blood cells or their hemolysates that have been sufficiently washed with physiological saline, or purified Hb.

The prepared sample is then acetylated as described above to obtain acetylated) 1b. After the acetylation reaction.

If red blood cells are used as a sample, perform hemolysis.

Hemolysis is performed using distilled water or surfactants. The surfactant is a higher aliphatic alcohol.

Alkylaryl polyether alcohol, polyoxyethylene ether of sulfonate compounds.

Examples include polyoxyethylene ether of sulfate compounds and polyoxyethylene adducts of sorbitol fatty acid esters. The amount of surfactant used varies depending on the type of surfactant, but it is normal.

It is 10-2000 mg per ml of blood. for example,
Hemolysis can be carried out by adding a hemolysis treatment solution containing a surfactant at a ratio of 0.01 to 2% (W/V) at a rate of 2 to 400 ml per 1 ml of blood and incubating. Excess surfactant makes it difficult to separate Hb by chromatography.

Next, it is washed with a water-insoluble organic solvent to remove lipids and centrifuged at 3,000-10,000 x g to obtain a supernatant. Benzene is the organic solvent.

Examples include toluene, xylene, hexafluoride, heptane, petroleum ether, etc. the resulting supernatant.

For example, it is used after being diluted to an arbitrary Hb concentration with a diluent such as physiological saline, a treatment solution for hemolysis, or a solution M used in chromatography. For example, two samples obtained in this way have an apparently high value of 5-HbA,c.

When stored at 4°C, there is no change over time even after 10 days, and it can be used directly as an Ib standard reagent. but.

Since it is not suitable for long-term storage, if it is not used immediately, it is preferable to freeze the supernatant or store it as a lyophilized product. Cryopreservation and preparation of lyophilized products can be carried out by conventional methods known to those skilled in the art. At this time, add a frost protection agent if necessary. As a frost protection agent.

Generally, polyhydroxy compounds are preferably used.

For example, inositol, sorbitol, sucrose,
In addition to glycerol, alkylene glycol, etc., various monosaccharides and polysaccharides can be used. The Ib standard reagent prepared in this manner is stable in a frozen or lyophilized state like ordinary proteins, and can be stored for a long period of time.

) It is convenient to dispense the Ib standard reagent into small bottles of 1 to 20 ml and store it frozen to avoid waste.

(Example) The invention is illustrated by the following examples.

In the Examples below, HbA+c was measured using Auto AH8HA-8110 manufactured by Kyoto Dai-Kagaku Co., Ltd. under appropriate conditions. This Auto AIC
) IA-8110 is) HbA by IPLc. This is a dedicated measurement device, and each Hb acid component 1 is measured by cation exchange.
Separate and elute in 3 minutes.

A dedicated eluent (phosphate buffer) is used as the elution buffer, and a dedicated hemolysis treatment solution (containing a hemolytic agent) is used for hemolysis. The elution pattern of Hb using this device is generally shown in FIG. In Figure 1, P and P2 are HbA, peaks due to a and Hb, b + P3 are L-HbA, c and p are 5-HbA,
C, and P are peaks caused by other Hb (HbAo).

Here, HbA,. (Total of S type and L type) is calculated by the following formula.

In this example, blood from the same person (healthy person) was used,
Immediately after blood collection, heparin was added and the blood was used as fresh blood. The acetylation reaction solution was prepared by adding a predetermined amount of acetylation reagent to 200mM phosphate buffer, and then adding 0. IN
The pH was adjusted with a Na0)l solution, and NaCl was added to make it isotonic with physiological saline using an osmometer.

Reference: 1.20ml of fresh surface from a healthy person. Centrifuge at soo'x g.

Only red blood cells were collected. 10ml of the obtained red blood cells
was dispersed in the same amount of physiological saline and centrifuged again at 1,500×g to collect red blood cells. This washing operation was repeated two more times to thoroughly wash the red blood cells.

Take 5 μl of this washed red blood cell and add 1.5 ml of hemolysis treatment solution.
After hemolyzing by adding Auto AICHA-
HbA by 8110. When measured, it was 3.9%. The fractionation pattern at this time is shown in FIG. 1st
In the figure, + h and P2 are peaks due to 1 (bA4. and Hb, , + p3 is L-HbA
,. , P4 indicates a peak attributable to 5-HbA, C, and P5 indicates a peak attributable to HbAo.

Three types of phosphate buffers containing 0.3% acetylsalicylic acid as an acetylation reagent and isotonic with physiological saline, pH 8, 5, 7, 5, and 6.5, were prepared. The above-mentioned washed red blood cells (obtained in the same manner as in the section for measurement of sofa rinse value) were added to 100 ml of these three types of acetylation reaction medium.
were dispersed in 1 ml each, and acetylation of Inkinive 1-L and Hb was performed at 40° C. while stirring slowly. The reaction solution was centrifuged 2 hours, 4 hours, and 6 hours after the start of the incubation, and the obtained red blood cells were collected in 5 μm portions.

1. Perform hemolysis by adding 5 ml of hemolysis treatment solution, A
HbA and c were measured using auto AICHA-8110. HbA over time. Fraction measurements (H
bA,. FIG. 2 shows the changes in the total amount of acetylated 11b and acetylated 11b. In Figure 2, the white circle mark (○) is pl+!
1.5, open square El] (b) indicates the result when an acetylation reaction medium of pH 7.5 was used, and open square mark (△) indicates the result when an acetylation reaction medium of pH 6.5 was used. From Figure 2, ink chamber/
The longer you wait, the better.

and p) The higher the 1 condition, the HbA,. It can be seen that the measured value of the fraction is high. In Figure 3, pH8
The fractionation pattern obtained when the acetylation reaction solution of ., 5 was used and incubated at 40'C for 6 hours is shown. )I
bA,. The fraction measured 84% HbA, before acetylation. The value was 4.5% higher than the actual value.

The red blood cells after the acetylation reaction in which the 1 (bA,. fraction increased to 8.4%) were collected and dispersed again in physiological saline.
Red blood cells were washed by incubation at 40° C. for 6 hours with slow agitation. After that, HbA, in the same way.

When the fraction was measured, the value was 8.1%, and there was no significant change in the measured value. Furthermore, 1 ml of the red blood cells after this acetylation reaction was stored refrigerated at 4°C for 10 days, and then the value of the HbA fraction was measured, and the value was 8.
．． 2%, and there was no significant change in the measured value.

Also, no major changes were observed in the measurement pattern.

Next, red blood cells (HbA) after the acetylation reaction.

1 ml of fraction 84%) was hemolyzed using the same amount of distilled water, and 2 ml of benzene was added thereto to defatte. 15.
The solids were removed by centrifugation at 00QXg for about 1 hour.

Approximately 3% inositol was added to the resulting supernatant as a cryoprotectant.
(W/V) and stored frozen in a deep freezer at -80'C. When I thawed this after 6 months and measured )lbA,c, the value was 8.3.
%, which was almost the same as the measured value before freezing.

Also, no major changes were observed in the measurement pattern. Thus, it was found that acetylated Hb has sufficient storage stability by freezing.

According to Example 1, 0.3% of acetylsalicylic acid was contained as an acetylation reagent. 2 of pH 8,5 and 6,5
Various acetylation reaction media were prepared. Using this acetylation reaction medium, temperature conditions were set at 25°C)
Acetylation of Ib was performed. HbA, respectively after incubation for a predetermined time. The results of fraction measurement are shown in 7.
It is shown in Figure 4. In Fig. 4.

The black circle (・) indicates pH 8.5, and the black triangle (mu) indicates p.
The results are shown when using an acetylation reaction medium for H6,5. From FIG. 4, it can be seen that as the reaction temperature is lowered, the acetylation rate of Hb becomes slower, and that acetylation hardly progresses under the conditions of 25° C. and pH 6.5.

In this way, by arbitrarily setting the pH and/or reaction temperature conditions, desired HbA+c'a degree Hb can be obtained.
Standard reagents may be prepared.

According to Example 1, 1.0% of para-N-acetylaminobenzoic acid was used instead of acetylsalicylic acid as the acetylation reagent.
Contains %. An acetylation reaction solution having a pH of 8.5 was prepared. Using this acetylation reaction solution, washed red blood cells were acetylated at 40°C for 6 hours, and then HbA. The fraction was measured and found to be 8.2%. This result is almost the same as the result of Example 1, and it was found that rose N-acetylaminobenzoic acid can also be used as an acetylation reagent like acetylsalicylic acid.

Ratio yヱ1゜A reaction solution containing 1% gluten in place of the acetylsalicylic acid of Example 1 and having a pH of 8.5 was prepared. When this reaction solution and washed red blood cells were combined and incubated at 40°C for 6 hours, the HbA and 0 fractions were measured, and a fractionation pattern as shown in FIG. 5 was obtained.

In Fig. 5, the peaks of P and ~P5 are W similar to Fig. 1.
The peak of b is shown. HbA at this time. The value is 7.0%
It was increasing. However, as is clear from Figure 5.

Although the L-HbAIC peak (P3) increased, 5-
Almost no increase in HbA1o peak (P4) was observed. Therefore, when the red blood cells after the above incubation were dispersed again in 100 ml of physiological saline and incubated at 40°C for 6 hours with slow stirring to wash the red blood cells, HbA. The value has returned to 3.9%.

From this, L-HbA. It can be seen that the increase in peaks is due to the reversible reaction between glucose and llb. Thus, it is not possible to maintain HbAlc at a stable high level using glucose.

A reaction solution of p) 18.5 containing 3% acetaldehyde instead of the acetylsalicylic acid of Example 1 was prepared.

This reaction solution and washed red blood cells were combined and incubated at 40°C for 6 hours, followed by HbA. When the fractions were measured, a fractionation pattern as shown in FIG. 6 was obtained. As is clear from Figure 6, HbA, and HbA
Only the peaks caused by , , increased, and the HbA,o peaks were almost no longer observed. Thus, HbA, using acetaldehyde. cannot be made too expensive.

(Effects of the Invention) According to the present invention, an Hb standard reagent having an apparently high HbAlc value is provided by acetylating Hb in blood. The Hb standard reagent of the present invention can use the blood of a healthy person or purified Hb as a raw material, and there is no need to use the blood of a diabetic patient with a high HbAlc level as in the past. Therefore.

It is possible to stably supply a sufficient amount.

Furthermore, the Hb standard reagent of the present invention is stable and can withstand long-term storage by freezing. Therefore, the same Hb standard reagent can be used for a long period of time,
HbA,. Calibration and accuracy control between measurements can be performed more effectively.

In addition, the desired AIC value can be controlled by arbitrarily setting the pH, reaction temperature, reaction time, and concentration of the acetylating reagent. This allows for more reliable HbA, c measurement, which can be used as an indicator of diabetes.

[Brief explanation of drawings]

Figure 1 shows HbA in the blood of a healthy person. AUTOA,
Chart showing the elution pattern when measured using cHA-8110; Figure 2 shows 1 reaction time and HbA when red blood cells were acetylated under different pH conditions at 40°C. Graph showing the relationship between the measured values of the fractions; Figure 3 shows that red blood cells were acetylated at pH 8, 5, and 40°C for 6 hours; ) A chart showing the elution pattern when measured using IA-8110;
Figure 4 is a graph showing the relationship between one reaction time and the measured value of HbA, 0 fraction when red blood cells are acetylated under different pH conditions at 25°C; Figure 5 is a graph showing the relationship between red blood cells and the measured value of the 0 fraction. 4.
After reacting for 6 hours at 0'C, pl+8.5, A
UTOA, c) A chart showing the elution pattern when measured using IA-8110; and Figure 6 shows the reaction of red blood cells and a reaction solution containing acetaldehyde at 40°C and pH 8.5 for 6 hours. After letting. At1TOA,. It is a chart figure showing the elution pattern when measured using HA-8110. that's all

Claims (1)

[Scope of Claims] 1. A hemoglobin standard reagent used for measuring glycated hemoglobin, which contains acetylated hemoglobin. 2. The acetylated hemoglobin is obtained by reacting a sample containing at least red blood cells and/or hemoglobin in blood with an acetylation reagent to acetylate non-glycated hemoglobin in the sample. Hemoglobin standard reagent as described. 3. The hemoglobin standard reagent according to claim 1, wherein the acetylating reagent is a compound that has an acetanilide or acetylphenol skeleton in its molecule and can easily release an acetyl group by hydrolysis. 4. The compound is orthoacetylsalicylic acid, paraacetylsalicylic acid, N-acetylaminobenzoic acid, N-
The hemoglobin standard reagent according to claim 3, which is acetylaminobenzenesulfonic acid or monoacetylcatechol.