JP4511847B2 - Method for measuring hemoglobin A1c - Google Patents

Description

  The present invention relates to a packing material for liquid chromatography, and more particularly to a packing material for ion exchange liquid chromatography having improved hydrophilicity without causing a decrease in strength.

  The ion exchange liquid chromatography method is an extremely effective method for the separation and analysis of various biological substances including the analysis of glycated hemoglobins. Up to now, the filler used in the ion exchange liquid chromatography method is the one in which an ion exchange group is introduced into a base composed of a silica compound, or the reaction of an ion exchange group-containing compound with crosslinkable particles composed of an organic synthetic polymer And the like obtained by reacting a crosslinkable monomer with an ion exchange group-containing compound (Patent Literature 2, Patent Literature 3, etc.) are known.

  In the above filler, the base portion other than the ion exchange group is desirably stronger and cross-linked particles in order to avoid swelling and shrinkage. However, when the strength is increased, there is a problem that the hydrophilicity is lowered, causing nonspecific adsorption of the hydrophobic substance, and the measurement accuracy is lowered.

  On the other hand, in order to suppress non-specific adsorption of hydrophobic substances, increasing the hydrophilicity by including a large amount of hydrophilic monomers tends to cause swelling and shrinkage as described above, resulting in a decrease in strength. To do. Therefore, there is a problem that analysis at a high flow rate cannot be performed, and equilibration is delayed when a plurality of eluents are used, resulting in measurement delay.

  On the other hand, as a method for solving the above problems, Patent Document 4 below discloses a method of adsorbing a compound having a hydrophilic group on the surface of a filler having an ion exchange group.

However, in the method described in Patent Document 4, if the adsorbing force of the adsorbed compound on the surface of the filler is weak, even if sufficient performance can be maintained in the initial state, There was a problem that the compound was detached from the surface and the retention time and measured value fluctuated.
JP-A-1-262468 Japanese Examined Patent Publication No. 63-59463 Japanese Patent Publication No. 8-7197 JP 2001-91505 A

  In view of the above-mentioned problems of the prior art, the object of the present invention is to increase the hydrophilicity without reducing the strength, and to suppress nonspecific adsorption of proteins and the like. An object of the present invention is to provide a packing material for ion exchange liquid chromatography that can improve accuracy and maintain performance for a long period of time.

The present invention is a packing material for ion exchange liquid chromatography, wherein a protein having an isoelectric point of 7.8 or more is adsorbed on the surface of the packing material having an ion exchange group.

  In the filler for ion exchange liquid chromatography according to the present invention, preferably, the ion exchange group of the filler is a sulfonic acid group.

In the packing material for ion exchange liquid chromatography according to the present invention, a protein having an isoelectric point of 7.8 or more is adsorbed on the surface of the packing material, so that the hydrophilicity is improved without reducing the packing material strength. Can do. Further, nonspecific adsorption of a measurement object, particularly a protein or a contaminant in a sample, can be suppressed, and the performance can be maintained for a long time.

  When the ion exchange group of the filler is a sulfonic acid group, the performance can be maintained for a longer period of time.

  In the present invention, the filler is not particularly limited as long as it is a filler used for ion-exchange liquid chromatography, and may have a cation exchange group or an anion exchange group. Good. Examples of the cation exchange group include a carboxyl group, a phosphate group, and a sulfonate group. Examples of the anion exchange group include a tertiary or quaternary amino group. The said filler should just have an at least 1 type of ion exchange group, and can use the filler which has a well-known ion exchange group in this invention. However, particularly preferably, fillers having sulfonic acid groups are used, whereby the performance can be maintained for a longer period.

  The method for preparing the filler having an ion exchange group is not particularly limited. For example, a method for introducing an ion exchange group into particles serving as a filler, or a monomer having an ion exchange group is polymerized to obtain a filler. A method of forming particles can be used.

  Examples of the fine particles include inorganic particles such as silica and zirconia, and organic particles such as polymer particles. Examples of the polymer fine particles include natural polymer particles such as cellulose, polyamino acid, and chitosan, polystyrene, Examples thereof include synthetic polymer particles such as polyacrylic acid esters. In particular, when synthetic polymer particles are used, it is desirable to use synthetic polymer particles having a high degree of crosslinking in order to enhance pressure resistance and swelling resistance.

  The method for introducing an ion exchange group into the polymer particles is not particularly limited, and a known method can be used. In the case of polymer particles, for example, after preparing polymer fine particles having a functional group, a method of introducing a compound having an ion exchange group into the functional group by a chemical reaction, or a monomer having an ion exchange group A method of mixing particles with a crosslinkable monomer and polymerizing in the presence of a polymerization initiator to form particles can be used. In addition, as described in JP-B-8-7197, after preparing crosslinkable polymer particles, a monomer having an ion exchange group is added, and a single unit having an ion exchange group near the surface of the polymer particles is added. A method of polymerizing a monomer may be used. In addition, a polymerizable ester compound such as methyl (meth) acrylate or ethyl (meth) acrylate is mixed with a crosslinkable monomer and polymerized in the presence of a polymerization initiator, and then the resulting particles are hydrolyzed. The ester compound may be converted to a cation exchange group by treatment.

The filler used in the present invention is in the form of particles, and the average particle diameter (diameter) is preferably 0.1 to 20 μm. Further, the particle size distribution is preferably 40% or less, more preferably 15% or less in terms of CV value (standard deviation ÷ average particle size × 100). If the average particle size is less than 0.1 μm, the inside of the column becomes too high and may cause separation failure. If it exceeds 20 μm, the dead volume in the column becomes too large and may cause separation failure. Even if the CV value exceeds 40%, the dead volume in the column becomes too large, and separation failure may occur.

In the present invention, the isoelectric point is a pH at which the charge of the whole molecule becomes ± 0 in an amphoteric electrolyte such as a protein. Examples of proteins having an isoelectric point of 7.8 or higher include lactoferrin (isoelectric point is 7.8), histone (isoelectric point is 10.5), avidin (isoelectric point is 9.5) and the like.

In the present invention, “adsorbing a protein having an isoelectric point of 7.8 or more on the surface of the filler” means the following methods (1) to (4) for the filler having an ion exchange group: It means applying at least one method. That is, two or more processes (1) to (4) may be combined as appropriate.

(1) Hydrophilization treatment at the time of slurry preparation A filler having an ion exchange group contains 0.0001 to 10% by weight, preferably 0.001 to 5% by weight of a protein having an isoelectric point of 7.8 or more. And the mixture is stirred for 0.5 hour or longer, preferably 1 hour or longer.

(2) Hydrophilization treatment at the time of packing 0.0001 to 10% by weight, preferably 0.001 to 5% by weight of a protein having an isoelectric point of 7.8 or more is dissolved in the packing liquid to fill the column body. Do. After filling, it is allowed to stand for 0.5 hour or longer, preferably 1 hour or longer.

(3) Hydrophilization treatment by passing a solution of a compound containing a hydrophilic group through the column after packing 0.0001 to 10% by weight, preferably 0.001 in a column packed with a packing material having an ion exchange group A solution in which a protein having an isoelectric point of ˜5 wt% is dissolved is 7.8 or more is allowed to flow for 1 minute or more at a flow rate of 0.01 to 10 mL / min, preferably 0.1 to 5 mL / min. Let stand for 0.5 hour or longer, preferably 1 hour or longer.

(4) Hydrophilization treatment by injecting a solution of protein having an isoelectric point of 7.8 or more into the column after packing 0.01 to 10% by weight, preferably in a column packed with a packing having an ion exchange group A solution in which 0.1 to 5% by weight of a protein having an isoelectric point of 7.8 or more is dissolved is injected in an amount of 1 to 1000 μL, preferably 10 to 500 μL, and a flow rate of 0.01 to 10 mL / min. Inject at 1-5 mL / min. After the injection, it is allowed to stand for 0.5 hour or longer, preferably 1 hour or longer.

  In the treatments (1) to (4), the treatment temperature is preferably 15 to 90 ° C., more preferably 40 to 90 ° C., or it is desirable to leave it.

  Prior to use, the packing material for ion exchange liquid chromatography obtained by the present invention is passed through the eluent having the strongest dissolution power used for measurement at a flow rate of 0.01 to 10 mL / min for 1 to 300 minutes. It is desirable.

  In the case of the treatment (1) above, the eluent having the strongest elution power used for the measurement is used for ion exchange liquid chromatography after the treatment (1) and before the column is packed. It is desirable to add 0.5 to 100 times the filler weight and stir for 1 to 300 minutes.

In the present invention, the protein having an isoelectric point of 7.8 or more is adsorbed on the surface of the filler, so that the protein is physically or chemically adsorbed on the hydrophobic portion of the filler surface, and the hydrophilicity is increased. Enhanced. In addition, non-specific adsorption between the hydrophobic portion of the surface of the packing material and the components to be measured, particularly proteins or contaminants in the measurement sample, is suppressed, and the performance as a packing material for ion-exchange liquid chromatography is extended over a long period of time. It is thought that it will be maintained.

  The packing material for ion exchange liquid chromatography according to the present invention is packed in a column main body made of stainless steel or the like to constitute a liquid chromatography column. The filling method is not particularly limited, and a known method can be used. For example, when a wet method (slurry method) is used, packing is performed by dispersing a packing material for ion exchange liquid chromatography in a dispersion medium such as a solvent used as an eluent and press-fitting the column body through a packer. Can do.

  In the analysis by liquid chromatography using the packing material for ion exchange liquid chromatography of the present invention, the eluent to be used is not particularly limited, and a known eluent can be used. For example, various buffer solutions containing inorganic acids such as phosphoric acid, nitric acid, hydrochloric acid, perchloric acid and salts thereof, organic acids such as acetic acid, malic acid, tartaric acid, succinic acid, citric acid and salts thereof, and the like can be used. . In addition, a salt such as sodium hydroxide or potassium hydroxide, an organic solvent such as acetone, acetonitrile, dioxane, methanol or ethanol may be added to the eluent.

  Next, the present invention will be described more specifically by giving specific examples and comparative examples. In addition, this invention is not limited to a following example.

[Example 1]
(Preparation of filler)
200 g of 2-acrylamido-2-methylpropanesulfonic acid (monomer A: manufactured by Tokyo Chemical Industry Co., Ltd.), 400 g of diethylene glycol dimethacrylate (crosslinkable monomer B: manufactured by Shin-Nakamura Chemical Co., Ltd.) and benzoyl peroxide (polymerization initiator) : Wako Pure Chemical Industries, Ltd.) 1.5 g was mixed and dispersed in 2.5 L of 4 wt% polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd.) aqueous solution. This was heated with stirring in a nitrogen atmosphere and polymerized at 80 ° C. for 8 hours. After polymerization, washing and classification were performed to obtain a filler having an average particle size of 8 μm.

(Hydrophilic treatment)
0.7 g of the above filler was dispersed in 10 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.

Next, 20 mL of 170 mmol / L phosphate buffer solution (pH = 5.7) containing 0.2 wt% lactoferrin (manufactured by Wako Pure Chemical Industries, Ltd., isoelectric point is 7.8 ) was added, and in a constant temperature water bath at 80 ° C. Then, the mixture was gently stirred for 24 hours and then taken out of the constant temperature water bath and left to reach room temperature. Then, after centrifuging at 3000 rpm × 10 minutes, the supernatant was removed, and then 5 mL of 300 mmol / L phosphate buffer (pH 8.5) was added. After centrifugation again at 3000 rpm × 10 minutes, the supernatant was removed. Next, 30 mL of 170 mmol / L phosphate buffer solution (pH 5.7) was added and gently stirred to obtain a hydrophilic filler-treated filler dispersion. The whole amount was injected into a packer (made by Umeda Seiki Co., Ltd.) connected to a stainless steel empty column (diameter: 4.6 mm × length: 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer, and the filler was filled at a constant pressure with a pressure of 200 kg / cm ² .

[Example 2]
(Preparation of filler)
The following hydrophilization treatment was performed on the same filler as in Example 1.

(Hydrophilic treatment)
0.7 g of the above filler was dispersed in 10 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.

Next, 20 mL of 170 mmol / L phosphate buffer solution (pH 5.7) containing 0.2 wt% histone (manufactured by Wako Pure Chemical Industries, Ltd., isoelectric point is 10.5) is added, and 24 hours in a constant temperature water bath at 80 ° C. After stirring gently for a period of time, it was taken out from the constant temperature water bath and left to reach room temperature. Then, after centrifuging at 3000 rpm × 10 minutes, the supernatant was removed. Next, 5 mL of 300 mmol / L phosphate buffer (pH 8.5) was added thereto, and centrifuged again at 3000 rpm × 10 minutes, and then the supernatant was removed. Next, 30 mL of 170 mmol / L phosphate buffer (pH 5.7) was added and gently stirred to obtain a hydrophilic hydrophilic filler dispersion. The whole amount was injected into a packer (made by Umeda Seiki Co., Ltd.) connected to a stainless steel empty column (diameter 4.6 mm × length 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer, and the filler was filled at a constant pressure with a pressure of 200 kg / cm ² .

Example 3
(Preparation of filler)
The following hydrophilization treatment was performed on the same filler as in Example 1.

(Hydrophilic treatment)
0.7 g of the above filler was dispersed in 30 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.

Next, the entire amount was injected into a packer (made by Umeda Seiki Co., Ltd.) connected with a stainless steel empty column (diameter: 4.6 mm × length: 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer and filled at a constant pressure of 200 kg / cm ² . Next, a 170 mmol / L phosphate buffer solution (pH 5.7) containing 0.1 wt% lactoferrin (manufactured by Wako Pure Chemical Industries, Ltd., isoelectric point is 7.8 ) in the above column at room temperature, LC-9A (manufactured by Shimadzu Corporation) was passed for 30 minutes at a flow rate of 2 mL / min.

  Then, it was left to stand in a constant temperature water bath at 60 ° C. for 168 hours, taken out from the constant temperature water bath and left to reach room temperature. Thereafter, 300 mmol / L phosphate buffer (pH 8.5) was passed through the column at room temperature for 30 minutes at a flow rate of 2 mL / min with a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation). Subsequently, 170 mmol / L phosphate buffer (pH 5.7) was passed through the column at room temperature for 30 minutes at a flow rate of 2 mL / min using a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation).

Example 4
(Preparation of filler)
The following hydrophilization treatment was performed on the same filler as in Example 1.

(Hydrophilic treatment)
0.7 g of the above filler was dispersed in 30 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.

Next, the entire amount was injected into a packer (made by Umeda Seiki Co., Ltd.) connected to an empty stainless steel column (diameter 4.6 mm × length 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer and filled at a constant pressure of 200 kg / cm ² . Next, a 170 mmol / L phosphate buffer solution (pH 5.7) containing 0.1 wt% histone (manufactured by Wako Pure Chemical Industries, Ltd., isoelectric point 10.5) in the above-mentioned column was fed at room temperature to a liquid feed pump ( LC-9A (manufactured by Shimadzu Corporation) was passed for 30 minutes at a flow rate of 2 mL / min.

  Then, it was left to stand in a constant temperature water bath at 60 ° C. for 168 hours, taken out from the constant temperature water bath and left to reach room temperature. Thereafter, 300 mmol / L phosphate buffer (pH 8.5) was passed through the column at room temperature for 30 minutes at a flow rate of 2 mL / min with a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation). Subsequently, 170 mmol / L phosphate buffer (pH 5.7) was passed through the column at room temperature for 30 minutes at a flow rate of 2 mL / min using a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation).

[Comparative Example 1]
(Preparation of filler)
Filling was performed as follows using the same filler as in Example 1, but no hydrophilization treatment was performed.

  0.7 g of the above filler was dispersed in 30 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.

Next, the entire amount was injected into a packer (made by Umeda Seiki Co., Ltd.) connected to an empty stainless steel column (diameter 4.6 mm × length 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer and filled at a constant pressure of 200 kg / cm ² .

[Comparative Example 2]
(Preparation of filler)
The following hydrophilization treatment was performed on the same filler as in Example 1.

(Hydrophilic treatment)
0.7 g of the above filler was dispersed in 10 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.

Next, 20 mL of 170 mmol / L phosphate buffer (pH 5.7) containing 0.2 wt% casein (manufactured by Wako Pure Chemical Industries, Ltd., isoelectric point is 4.6) is added, and in a constant temperature water bath at 80 ° C. After gently stirring for 24 hours, it was taken out from the constant temperature water bath and left to reach room temperature. Thereafter, the supernatant was removed by centrifugation at 3000 rpm × 10 minutes, 5 mL of 300 mmol / L phosphate buffer (pH 8.5) was added thereto, and the supernatant was removed again by centrifugation at 3000 rpm × 10 minutes. After adding 30 mL of 170 mmol / L phosphate buffer (pH 5.7) and stirring gently, a packer (Umeya Seiki) was formed by connecting a stainless steel empty column (diameter: 4.6 mm × length: 35 mm). Injected into the company). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer and filled at a constant pressure of 200 kg / cm ² .

[Comparative Example 3]
(Preparation of filler)
The following hydrophilization treatment was performed on the same filler as in Example 1.

(Hydrophilic treatment)
0.7 g of the above filler was dispersed in 30 mL of 170 mmol / L phosphate buffer (pH 5.7), subjected to ultrasonic treatment for 5 minutes, and then well stirred.

Next, the entire amount was injected into a packer (made by Umeda Seiki Co., Ltd.) connected with a stainless steel empty column (diameter: 4.6 mm × length: 35 mm). A liquid feed pump (manufactured by Sanuki Kogyo Co., Ltd.) was connected to the packer and filled at a constant pressure of 200 kg / cm ² . Next, a 170 mmol / L phosphate buffer solution (pH 5.7) containing 0.1 wt% phytene (manufactured by Wako Pure Chemical Industries, Ltd., isoelectric point is 3.5) in the above-mentioned column is fed at room temperature to a liquid feed pump ( LC-9A (manufactured by Shimadzu Corporation) was passed for 30 minutes at a flow rate of 2 mL / min.

  Then, it was left to stand in a constant temperature water bath at 60 ° C. for 168 hours, taken out from the constant temperature water bath and left to reach room temperature. Thereafter, 300 mmol / L phosphate buffer (pH 8.5) was passed through the column at room temperature for 30 minutes at a flow rate of 2 mL / min with a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation). Subsequently, 170 mmol / L phosphate buffer (pH 5.7) was passed through the column at room temperature for 30 minutes at a flow rate of 2 mL / min using a liquid feed pump (LC-9A, manufactured by Shimadzu Corporation).

(Evaluation of simultaneous reproducibility immediately after use)
Glyco-Hb (hemoglobin) control level 2 (manufactured by Kokusai Reagent Co., Ltd.) was dissolved in 200 μL of water for injection, and then diluted with a phosphate buffer (pH 7.0) containing 0.1 wt% Triton X-100) Using the sample diluted 100 times as the measurement sample, hemoglobin A1c was measured 10 times continuously under the following conditions, and the simultaneous reproducibility was compared. The reproducibility was evaluated by measuring the A1c value and the retention time of the A1c component. The results are shown in Tables 1 and 2 below.

Measurement conditions System: Liquid feed pump: LC-9A (manufactured by Shimadzu Corporation)
Autosampler: ASU-420 (manufactured by Sekisui Chemical Co., Ltd.)
Detector: SPD-6AV (manufactured by Shimadzu Corporation)
Eluent: First liquid: 170 mmol / L phosphate buffer (pH 5.7)
Second solution: 300 mmol / L phosphate buffer (pH 8.5)
Elution method: 1st liquid for 0 to 3 minutes, 2nd liquid for 3 to 3.2 minutes, 1st liquid for 3.2 to 4 minutes
The liquid was poured.

Flow rate: 1.0 mL / min Detection wavelength: 415 nm
Sample injection volume: 10 μL

（実検体の負荷におけるカラム劣化による測定値の変動の評価（耐久性試験））
健常人血をＮａＦ採血し、溶血希釈液（０．１％トリトンＸ−１００を含有するリン酸緩衝液（ｐＨ７．０））で溶血し、１５０倍に希釈したものを負荷検体とし、１日に３００検体を測定した。測定の前後でグリコＨｂコントロールレベル２（国際試薬社製）を２００μＬの注射用水で溶解した後、希釈液（０．１％トリトンＸ−１００を含有するリン酸緩衝液（ｐＨ７．０））で１００倍に希釈したものを測定試料として、上述した測定条件に従ってヘモグロビンＡ１ｃの測定を１０回連続で行い、その平均値を測定値とした。
本試験の評価は、Ａ１ｃ値（％）と、Ａ１ｃ成分の保持時間（秒）により行った。結果を下記の表３及び表４に示す。

(Evaluation of changes in measured values due to column deterioration under actual sample load (durability test))
Healthy human blood was collected from NaF, hemolyzed with a hemolysis dilution (phosphate buffer (pH 7.0) containing 0.1% Triton X-100), and diluted 150 times as a load sample. 300 samples were measured. Before and after the measurement, Glyco Hb Control Level 2 (made by Kokusai Reagent Co., Ltd.) was dissolved in 200 μL of water for injection, and then diluted with a diluent (phosphate buffer (pH 7.0) containing 0.1% Triton X-100). Using a sample diluted 100 times as a measurement sample, hemoglobin A1c was measured 10 times continuously according to the measurement conditions described above, and the average value was taken as the measurement value.
This test was evaluated based on the A1c value (%) and the retention time (seconds) of the A1c component. The results are shown in Tables 3 and 4 below.

Claims (2)

Measurement of hemoglobin A1c is performed by ion exchange liquid chromatography using a filler for ion exchange liquid chromatography in which a protein having an isoelectric point of 7.8 or more is adsorbed on the surface of the filler having ion exchange groups. Method.   The method for measuring hemoglobin A1c according to claim 1, wherein the ion exchange group of the filler is a sulfonic acid group.