JP4527901B2 - Standards for fibrinogen measurement and quality control substances - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to fibrinogen compositions. More specifically, the present invention relates to a fibrinogen composition that is used as a standard product or a quality control substance when measuring fibrinogen mainly in the field of clinical examination and the like.
[0002]
[Prior art]
Fibrinogen is a protein that is contained in blood at 200 to 400 mg / dL in healthy adults, and is a plasma precursor of fibrin, the main protein in clots. Fibrinogen is a glycoprotein containing three pairs of polypeptides (Aα, Bβ, and γ) joined by disulfides. When the Aα and Bβ chains are degraded by thrombin, fibrinogen is converted to fibrin and blood clotting begins. Because of having such an important role, measurement of fibrinogen in clinical examination is important in diagnosing abnormal fibrinogenosis in liver disease patients, risk factors of acute myocardial infarction, and the like. The clinical tests for fibrinogen include the Claus method (thrombin addition method), PT derived method (coagulation time method), TIA (immunoturbidimetric method), latex method, etc. The thrombin addition method is generally used Yes.
[0003]
When measuring the above fibrinogen, reference standard products and quality control substances are required. There is a WHO standard product as a standard product for fibrinogen measurement. The fibrinogen price of the standard product is as follows: (1) a thrombin is used to form a coagulable protein, and the coagulable protein is dissolved in an alkaline urea solution. Jacobson method (Jaccobsson K; Scand. J. Clin. Lab. Invest, 7, p.1-54 (1955)), and (2) Claus method to measure clotting time by adding thrombin (Clauss VA; Acta. Haemat, 17, p.237-246 (1957)).
[0004]
However, the values of the standard products differed due to the Jacobson method, the Claus method, etc., and as a result, there was a problem in the reliability of the measured values (Furlan M. et.al .; Thrombosis Research, 56, p.583 (1989), Hirai N. et.al .; Osaka City Medical Journal, 44, p.55 (1998), Jensen T. et.al .; Thrombosis Research, 100, p.397 (2000)). Furthermore, when human plasma is used as a standard product, there are problems such as lack of stability and reproducibility, and errors are likely to occur between facilities and models. Therefore, a standard product or quality control material for fibrinogen measurement that has good stability, reproducibility, and improved measurement error has been desired.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a standard product or a quality control substance for fibrinogen measurement having high stability and reproducibility and improved measurement error.
[0006]
[Means for Solving the Problems]
As a result of extensive research, the present inventors have found that fibrinogen measurement standard products have problems with stability and reproducibility because plasmin that is contaminated with the standard products or plasminogen, which is a precursor thereof, has formed fibrin. We paid attention to fibrinolysis and turbidity caused by lipoproteins. Therefore, by using a fibrinogen composition that has been purified to substantially suppress the activity of plasmin or plasminogen as a standard product for fibrinogen measurement or a quality control substance, a highly reproducible and stable measurement value can be obtained. I found it. Further, for example, it has been found that by adding a substance involved in fibrin polymerization, such as a peptide having a specific sequence, or an FDP component, the problem of deviation in measured values by the Jacobson method and the Claus method is solved. Was completed.
[0007]
That is, the present invention
1. A fibrinogen measurement standard characterized by comprising a fibrinogen composition in which the activity of plasmin or plasminogen is substantially suppressed , characterized in that a substance that inhibits fibrin polymerization or an FDP component is added ;
2 . The standard product according to item 1 above, wherein the substance that inhibits fibrin polymerization is a peptide,
3 . The standard product according to item 2 above, wherein the peptide comprises a sequence consisting of Gly-Pro-Arg,
4. The standard product according to item 1 above, wherein the FDP component is the E fraction,
5 . A quality control substance for measuring fibrinogen, comprising the fibrinogen composition according to any one of items 1 to 4 ,
6 . Fibrinogen measurement method, characterized by measuring using the standard product or quality control substance according to any one of items 1 to 5 above,
7 . A reagent kit for fibrinogen measurement, comprising the standard product or the quality control substance according to any one of items 1 to 5 ,
Consists of.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
It is an object of the present invention to provide a fibrinogen measurement standard product in which the problems of stability and reproducibility are solved, and the problem of measurement value deviation by the Jacobson method and the Claus method is solved. In order to provide such a standard product, a composition capable of approximating the measured value by the Jacobson method and the Claus method while removing the substance that inhibits the stability of fibrin or suppressing the activity of the inhibitor It is necessary to.
[0009]
The fibrinogen standard of the present invention is a fibrinogen composition in which the activity of plasmin or plasminogen is substantially suppressed. The fibrinogen composition in which the plasmin or plasminogen is substantially suppressed means that the activity is substantially suppressed even if the plasmin or plasminogen is not substantially contaminated, or fibrin Those that do not affect the stability of the. Such residual activity of plasmin or plasminogen contaminating the fibrinogen composition is 5% or less, preferably 2% or less, more preferably 0.5% or less, more preferably compared to plasmin or plasminogen contained in normal plasma. A content of 0.1% or less is suitable.
[0010]
(Preparation of fibrinogen composition)
The fibrinogen composition used as a fibrinogen standard is usually obtained from human or animal plasma. It is desirable to use fibrinogen obtained from human plasma when a human blood test is performed, and fibrinogen obtained from the animal plasma as a raw material when performing a blood test of an animal. The purification method and additives of fibrinogen are not particularly limited as long as plasmin or a composition in which plasminogen activity is substantially suppressed can be used.
[0011]
For example, a fraction-I paste obtained from normal human plasma by corn ethanol fractionation can be used as a starting material (Cohn. EJ et. Al .; J. Am. Chem, 68, p. 459 ( 1946)). The fraction-I paste can be dissolved in a citrate buffer, glycine can be added and stirred. Furthermore, these can be substantially removed by adsorbing plasmin or plasminogen by lysine sepharose.
[0012]
As an alternative purification method, chromatographic isolation uses gel filtration or ion exchange resins to produce intermediate purity concentrates (Furlan M., Curr. Probl. Clin. Biochem, 14, 133-145, 1984). )) And ultrafiltration (Kopf and Morese, US Patent 5,354,682 (1993)) method and the like can be employed. In order to purify fibrinogen from plasma with high purity, an affinity method can also be employed.
[0013]
In order to more effectively suppress the activity of plasmin or the like, a fibrinolysis inhibitor such as ε-aminocaproic acid or aprotinin may be added, or two or more of these may be used in combination. The blending amount of the fibrinolysis inhibitor is 50 to 5000 units, preferably 500 to 3000 units, with respect to 80 mg of fibrinogen.
[0014]
(Price for fibrinogen standard product)
As described in the prior art, fibrinogen measurement standard products are mainly performed by the Jacobson method or the Claus method. The Jacobson method uses thrombin to form a coagulable protein for a sufficient period of time, dissolves the coagulable protein in an alkaline urea solution, and measures the amount of protein by absorbance at 280 nm. In addition, there is a disadvantage that the measurement procedure is complicated. On the other hand, the Claus method measures the coagulation time by adding thrombin. However, although it can be measured relatively easily, there is a problem that there is a difference from the measurement value measured by the Jacobson method.
[0015]
As a means for solving such a problem, a substance that inhibits fibrin polymerization may be added.
[0016]
When thrombin acts on fibrinogen, first, the C-terminus of the α chain of the fibrinogen molecule is hydrolyzed to cleave fibrinopeptide A to form a fibrin monomer composed of (α, β, γ) ₂ . At this time, a Gly-Pro-Arg sequence appears at the C-terminus of the α chain. This arrangement has the property of polymerizing, forming fibrin aggregates and further forming fibrin polymers. It was considered that by adjusting the formation of the fibrin polymer, the measured value by the Claus method can be brought close to the measured value by the Jacobson method. That is, by adding a substance that inhibits the polymerization of fibrin as a compounding agent, it was possible to approximate the measured values of the Jacobson method and the Claus method.
[0017]
Specifically, the measurement is performed by the Jacobson method and the Claus method, respectively, for the composition to which the polymerization inhibitor is added at different concentrations. The place where the measured values by both measurement methods agree is judged as the optimum addition amount of the polymerization inhibitor. Fibrin polymerization-inhibiting peptides are known as the above-mentioned fibrin polymerization-inhibiting substances (Laudano, AP and Doolittle, RF: Proc. Nati. Sci. USA, 75, 3085 (1978)). Preferably, it is suitable to use a peptide that blocks the polymerization of the Gly-Pro-Arg sequence at the C-terminus of the α chain, more preferably a peptide containing the sequence Gly-Pro-Arg, more preferably Gly It is preferable to use a peptide having the sequence -Pro-Arg-Pro. The peptide having such a sequence is hereinafter referred to as “GPRP”.
The effective addition amount of GPRP is 10 to 500 μmol / L, preferably 20 to 200 μmol / L, more preferably 50 to 150 μmol / L for a 200 mg / dL fibrinogen-containing composition.
[0018]
As another method for eliminating the difference between the measured values by the Jacobson method and the Claus method, there is a method of adding an FDP component. Here, FDP refers to the degradation product of fibrin by activated plasmin, and includes DD / E, X, Y, D, E fractions, and the like. When the purified fibrin composition was used as a standard product, the measured values of the Jacobson method and the Claus method tended to be more dissimilar because they differed from the plasma composition conventionally used. Natural fibrinogen is composed of high molecular weight (HMW) fibrinogen (molecular weight 340 kD), low molecular weight (LMW) fibrinogen (molecular weight 300 kD) and LMW 'fibrinogen (molecular weight 280 kD) lacking the C-terminal. (Torstein Jansen; Thrombosis Res. 100, 397-403 (2000)), the proportion of HMW fibrinogen tends to increase as a result of purification treatment. Therefore, it was considered that when the FDP component was added, the measured values of both methods approximated the natural fibrinogen composition. Of the FDP components, the FDP E fraction is preferably used. The effective addition amount of the E fraction is 50 to 1000 μg / mL, preferably 100 to 750 μg / mL, and more preferably 250 to 500 μg / mL for a 200 mg / dL fibrinogen-containing composition.
[0019]
In addition, the fibrinogen composition of the present invention can be in a lyophilized state and can be stably stored for a long period of time. In addition, additives generally used as stabilizers and solubilizers, for example, surfactants, human serum albumin, sugar or sugar alcohol, amino acids and the like can be added (Japanese Patent Laid-Open No. 08-151330).
[0020]
The fibrinogen composition of the present invention can be used as a standard product or quality control substance. Furthermore, the present invention extends to a fibrinogen measurement method and measurement reagent kit using the fibrinogen composition of the present invention as a standard product or quality control substance.
[0021]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, it is not limited to a present Example.
[0022]
[Example 1]
(Fibrinogen composition)
The fibrinogen composition of the present invention was prepared by the following method.
Fraction-I paste obtained from normal human plasma by corn ethanol fractionation was dissolved in citrate buffer. After inactivating the virus by treatment with a solution containing 0.3% tri (n-butyl) phosphate and 1% Tween 80 at 30 ° C. for 6 hours, an original fraction of fibrinogen was obtained by ethanol precipitation. To 50 g of this fraction, 300 ml of a 6-fold amount of buffer solution (0.15% trisodium citrate, 0.25% NaCl, pH 7.0 ± 0.1) was added and dissolved by stirring at 37 ° C. for 1 hour. The residue was removed by centrifugation (3000 rpm, 10 minutes) and adjusted to a 2.5 w / v% solution.
[0023]
Glycine was added to 10 mL of the fibrinogen solution and stirred to obtain a solution containing 2 w / v% fibrinogen and 0.5 w / v% glycine. After sterilization and filtration, 4 ml was dispensed into a 10 ml vial. This was rapidly frozen at -80 ° C. and then lyophilized. After completion of the decompression, it was left at 30 ° C. overnight to obtain a freeze-dried fibrinogen product.
[0024]
[Example 2]
(Confirmation of stability)
The stability of the composition of Example 1 and commercially available lyophilized products (Coagtrol IX (made by Kokusai Reagent) and Coagtrol IIX (made by Kokusai Reagent)) were confirmed.
The composition of Example 1 was prepared to be 250 mg / dL and 125 mg / dL using purified water, and a commercially frozen product was dissolved in 1 mL of purified water, and used as each measurement sample. Each measurement specimen was stored at 18 ° C. for 0 hours, 1, 2, 4, 8, 12, 24 and 48 hours.
The fibrinogen concentration of the specimens stored for each time was measured using an analyzer Coagrex 700 (manufactured by Shimadzu Corporation) using thrombocheck Fib (manufactured by International Reagents) as a reagent.
As a result, as shown in FIG. 1, the measured values of Coagtrol IX (Conventional Product 1) and Coagtrol IIX (Conventional Product 2) were found to decrease over time. Regarding 1 and 2), no decrease in the measured value was observed, and the results were stable.
[0025]
[Example 3]
(Reproducibility check)
The reproducibility of the measurement was confirmed for the composition of Example 1 (product of the present invention) and the conventional products Coagtrol N (made by Kokusai Reagent) and Coagtrol IX (made by Kokusai Reagent). The measurement was performed 10 times for each specimen, and the variation was calculated as a CV value. As a result, as shown in FIG. 2, it was confirmed that the present invention has a lower CV value and less variation than the conventional product.
[0026]
[Example 4]
(Confirmation of measured values by Jacobson method and Claus method (1))
The composition of Example 1 was examined by adding and adding GPRP at different concentrations. As a result, as shown in FIG. 3, in the case of a fibrinogen-containing composition prepared so that the measured value by the Jacobson method is 180 mg / dL, the measured value by the Claus method is approximated when 100 μmol / L GPRP is added. The value to show.
[0027]
[Example 5]
(Confirmation of measured values by Jacobson method and Claus method (2))
The composition obtained in Example 1 with FDP-E added at different concentrations was examined. As a result, as shown in FIG. 4, for the fibrinogen-containing composition of 210 mg / dL (measured by the Claus method), the value closest to the measured value of the Jacobson method when the FDP component E fraction of 400 μg / mL was added. showed that.
[0028]
【The invention's effect】
As described above, it was confirmed that the fibrinogen composition of the present invention has high stability, improved measurement error, and is useful as a standard product or a quality control substance for fibrinogen measurement.
[Brief description of the drawings]
FIG. 1 shows the stability of a fibrinogen composition. (Example 2)
FIG. 2 is a diagram showing the reproducibility of fibrinogen measurement values of a fibrinogen composition. (Example 3)
FIG. 3 is a diagram showing the effect of adding GPRP. Example 4
FIG. 4 is a diagram showing the effect of adding FDP-E. (Example 5)

Claims (7)

A fibrinogen measurement standard product comprising a fibrinogen composition in which the activity of plasmin or plasminogen is substantially suppressed, wherein a fibrin polymerization inhibiting substance or an FDP component is added . 2. The standard product according to claim 1 , wherein the substance that inhibits the polymerization of fibrin is a peptide. The standard product according to claim 2 , wherein the peptide contains a sequence consisting of Gly-Pro-Arg. The standard product according to claim 1, wherein the FDP component is an E fraction. A quality control substance for fibrinogen measurement, comprising the fibrinogen composition according to any one of claims 1 to 4 . 6. A fibrinogen measurement method, wherein the measurement is performed using the standard product or the quality control substance according to any one of claims 1 to 5 . A reagent kit for fibrinogen measurement, comprising the standard product or quality control substance according to any one of claims 1 to 5 .

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