JP2002328131A - Standards for fibrinogen measurement and quality control substances - Google Patents

Abstract

(57) [Problem] To provide a standard or quality control substance for fibrinogen measurement having good stability and improved measurement error. [MEANS FOR SOLVING PROBLEMS] By using a purified fibrinogen composition in which the activity of plasmin or plasminogen is substantially suppressed as a standard or a quality control substance for fibrinogen measurement, a highly reproducible and stable measured value can be obtained. Can be Substances that further inhibit the polymerization of fibrin, or F
By adding the DP component, the problem of the difference between the measured values by the Jacobson method and the Claus method is solved.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a fibrinogen composition. More specifically, the present invention relates to a fibrinogen composition used as a standard or a quality control substance when fibrinogen is measured mainly in a field such as a clinical test.

[0002]

2. Description of the Related Art Fibrinogen is contained in blood in healthy adults.
It is a protein containing 200-400 mg / dL, and is a major protein in blood clots and a plasma precursor of fibrin. Fibrinogen is a glycoprotein containing three pairs of polypeptides (Aα, Bβ, and γ) linked by disulfides. When the Aα and Bβ chains are degraded by thrombin, fibrinogen is converted to fibrin and blood clotting begins. Due to such an important role, measurement of fibrinogen in a clinical test is important for diagnosis of abnormal fibrinogenosis in a liver disease patient, a risk factor for acute myocardial infarction, and the like. Clinical tests for fibrinogen include the Claus method (thrombin addition method), PT
There are a derived method (clotting time method), a TIA (immune turbidity) method, a latex method, and the like, and a thrombin addition method is generally performed.

In the measurement of fibrinogen described above,
Standard products and quality control substances are required. There is a WHO standard as a standard for measuring fibrinogen, and the fibrinogen in the standard is determined by forming a coagulating protein using thrombin, dissolving the coagulating protein in an alkaline urea solution, and measuring the protein amount by absorbance. Method (Jaccobsson K; Scand.
J. Clin. Lab. Invest, 7, p. 1-54 (1955)) and the Claus method for measuring coagulation time by adding thrombin (Clauss
VA; Acta. Haemat, 17, p. 237-246 (1957)) and the like.

[0004] However, the values of the standard products deviate by the Jacobson method, the Claus method, etc., and as a result, there is a problem in the reliability of the measured values (Furlan M. et. Al .; Thrombosis Research, 5).
6, p.583 (1989), Hirai N. et.al .; Osaka City Medica
l Journal, 44, p.55 (1998), Jensen T. et.al .; Thrombo
sis Research, 100, p. 397 (2000)). Furthermore, when human plasma was used as a standard product, there were problems such as lack of stability and reproducibility, and errors tended to occur between facilities and between models. Therefore, it has good stability and reproducibility,
A standard or quality control substance for measuring fibrinogen with improved measurement error has been desired.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a standard or quality control substance for fibrinogen measurement which has high stability and reproducibility and has improved measurement errors.

[0006]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that the stability and reproducibility of the standard for fibrinogen measurement are poor due to plasmin or its precursor contaminating the standard. It was noted that certain plasminogens caused fibrinolysis of the formed fibrin and turbidity due to lipoproteins. Therefore, by using a purified fibrinogen composition in which the activity of plasmin or plasminogen has been substantially suppressed as a standard or a quality control substance for fibrinogen measurement, it is possible to obtain a highly reproducible and stable measured value. I found it. further,
For example, it has been found that the addition of a substance involved in the polymerization of fibrin such as a peptide having a specific sequence, or an FDP component eliminates the problem of the discrepancy in the measured values by the Jacobson method and the Claus method, and completed the present invention. did.

That is, the present invention provides: 1. A fibrinogen measurement standard, comprising a fibrinogen composition in which the activity of plasmin is substantially suppressed. 2. the standard product according to the above item 1, wherein the activity of plasminogen is substantially suppressed; 3. The fibrinogen measurement standard according to the above item 1 or 2, wherein a substance that inhibits fibrin polymerization is added. 4. The standard product according to item 3, wherein the substance that inhibits polymerization is a peptide; 5. The standard according to item 4, wherein the peptide comprises a sequence consisting of Gly-Pro-Arg; 6. a fibrinogen measurement standard, comprising a fibrinogen composition, to which an FDP component is added; 6. The preceding item 6, wherein the FDP component is an E fraction.
Standard products described in. 8. 8. A quality control substance for fibrinogen measurement, comprising the fibrinogen composition according to any one of items 1 to 7 above; 9. a fibrinogen measurement method, wherein the measurement is performed using the standard product or the quality control substance described in any one of the above items 1 to 8. A fibrinogen measurement reagent kit, comprising the standard product or the quality control substance according to any one of the above items 1 to 8.

[0008]

The object of the present invention is to provide a fibrinogen measurement standard which solves the problems of stability and reproducibility and eliminates the problem of deviation of measured values by the Jacobson method and the Claus method. In order to provide such a standard, a composition capable of approximating the value measured by the Jacobson method and the Claus method while removing a substance that inhibits the stability of fibrin or suppressing the activity of the inhibitor is provided. It is necessary to.

The fibrinogen standard of the present invention is characterized in that it is a fibrinogen composition in which the activity of plasmin or plasminogen is substantially suppressed. The fibrinogen composition in which plasmin or plasminogen is substantially suppressed means that plasmin or plasminogen is substantially not contaminated, or even if contaminated, their activity is substantially suppressed and fibrin Which do not affect the stability of the polymer. Such residual activity of plasmin or plasminogen contaminating the fibrinogen composition is 5% or less, preferably 2% or less, more preferably 0.
Those having 5% or less, more preferably 0.1% or less are suitable.

(Preparation of Fibrinogen Composition) A fibrinogen composition used as a standard for measuring fibrinogen is usually obtained from human or animal plasma. It is desirable to use fibrinogen obtained from human plasma as a raw material when performing a human blood test, and fibrinogen obtained from animal plasma when performing a blood test for 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 obtained.

For example, Fraction-I paste obtained from corn by the ethanol fractionation method from normal human plasma 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, added with glycine, and stirred. Further, plasmin or plasminogen can be substantially removed by adsorbing plasmin or plasminogen with lysine sepharose.

As another purification method, chromatographic isolation methods use gel filtration or ion exchange resins to produce intermediate-purity concentrates (Furlan M., Curr. Prob.
l. Clin. Biochem, 14,133-145,1984)) and ultrafiltration (Ko
pf and Morese, US Patent 5,354,682 (1993)). In order to purify fibrinogen from plasma with high purity, an affinity method can also be employed.

In order to more effectively suppress the activity of plasmin and the like, fibrinolytic inhibitors such as ε-aminocaproic acid and aprotinin may be added, or two or more thereof may be used in combination. The amount of fibrinolysis inhibitor is fibrinogen.
It is desirable to use 50 to 5000 units, preferably 500 to 3000 units for 80 mg.

(Pricing of fibrinogen standard) As described in the prior art, the standard for fibrinogen measurement is:
It is mainly performed by the Jacobson method or the Claus method. The Jacobson method is a method for forming a coagulable protein using thrombin for a sufficient time, dissolving the coagulable protein in an alkaline urea solution, and measuring the protein amount by absorbance at 280 nm. And 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 measured value measured by the Jacobson method.

Means for solving such a problem include adding a substance that inhibits the polymerization of fibrin.

When thrombin acts on fibrinogen, it first hydrolyzes the C-terminal of the α-chain of the fibrinogen molecule, cuts off fibrinopeptide A, and gives (α, β, γ)
Form a fibrin monomer consisting of _two . At this time, a Gly-Pro-Arg sequence appears at the C-terminal of the α chain. This sequence has the property of polymerizing, forming fibrin aggregates, and further forming fibrin polymers. this,
It was thought that by adjusting the formation of the fibrin polymer, it was possible to make the measured value by the Claus method 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 became possible to approximate the measured values of the Jacobson method and the Claus method.

More specifically, the composition to which the polymerization inhibitor is added at different concentrations is measured by the Jacobson method and the Claus method, respectively. The point where the values measured by both the measurement methods coincide with each other is judged to be the optimum amount of the polymerization inhibitor. As the fibrin polymerization inhibitor, a fibrin polymerization inhibitor peptide is known (Laudano, AP and Doolit).
tle, RF: Proc.Nati.Sci.USA, 75, 3085 (1978)).
Preferably, a peptide that prevents polymerization of the Gly-Pro-Arg sequence at the C-terminus of the α chain is suitable,
More preferably a peptide comprising the sequence Gly-Pro-Arg,
It is more preferable to use a peptide having the sequence of Gly-Pro-Arg-Pro. The peptide having such a sequence is hereinafter referred to as “GPRP”. The effective addition amount of GPRP for a fibrinogen-containing composition at 200 mg / dL is 10 to 500 μmol / L, preferably 20 to 200 μmol / L, and more preferably 50 to 150 μmol / L.

Another method for eliminating the difference between the measured values obtained by the Jacobson method and the Claus method is to add an FDP component. Here, FDP refers to a degradation product of fibrin by activated plasmin,
DD / E, X, Y, D, E fractions and the like. When the purified fibrin composition was used as a standard, the measured values of the Jacobson method and the Claus method tended to be more divergent because the composition was different from the plasma composition conventionally used. Natural fibrinogen is a high molecular weight (HM) that is an intact Aα chain.
W) Fibrinogen (molecular weight 340 kD), low molecular weight (LMW) fibrinogen lacking the C-terminus (molecular weight 300 kD)
And LMW 'fibrinogen (molecular weight 280 kD) are constituents (Torstein Jansen; Thrombosis Res. 10
0, 397-403 (2000)), the purification treatment tends to increase the proportion of HMW fibrinogen. Then, F
It was thought that when the composition of the fibrinogen was brought close to the natural fibrinogen composition by adding the DP component, the measured values of both the measurement methods were similar. Among the FDP components, the FDPE 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 5 μg / mL for a 200 mg / dL fibrinogen-containing composition.
00 μg / mL is preferred.

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, sugars or sugar alcohols, amino acids, and the like can be added (Japanese Patent Application Laid-Open No. 08-151330).

The fibrinogen composition of the present invention can be used as a standard product or a quality control substance. The present invention further extends to a method for measuring fibrinogen using the fibrinogen composition of the present invention as a standard or a quality control substance, and a measurement reagent kit.

[0021]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

[0022]

Example 1 (Fibrinogen composition) The fibrinogen composition of the present invention was prepared by the following method. Fraction-I obtained from normal human plasma by the corn ethanol fractionation method
The paste was dissolved with a citrate buffer. 0.3% bird (n
The virus was inactivated by treatment with a solution containing -butyl) phosphoric acid and 1% Tween 80 at 30 ° C. for 6 hours, and then a fibrinogen original fraction was obtained by ethanol precipitation. This fraction
6 volumes of buffer (0.15% trisodium citrate,
300 ml of 0.25% NaCl, pH 7.0 ± 0.1) and add 1 at 37 ° C.
The mixture was stirred and dissolved for an hour. The residue was removed by centrifugation (3000 rpm, 10 minutes) and adjusted to a 2.5 w / v% solution.

Glycine is added to 10 mL of the above fibrinogen solution and stirred, and 2% w / v of fibrinogen and 0.5% of glycine are added.
A solution containing w / v% was obtained, and after sterile filtration, the volume was 10 ml.
4 ml was dispensed into the vial. This is quickly frozen at -80 ° C, freeze-dried, and after completion of reduced pressure, left at 30 ° C overnight,
A freeze-dried fibrinogen was obtained.

[0024]

Example 2 (Confirmation of Stability) The composition of Example 1 and commercially available freeze-dried products (Coagtrol IX (manufactured by International Reagents) and Coagtrol IIX (manufactured by International Reagents)) were checked for stability. The composition of Example 1 was prepared using purified water at 250 mg /
Prepared to be dL and 125 mg / dL, and 1
It was dissolved in mL of purified water to obtain each measurement sample. Each test sample is a thermostat at 18 ° C, and is 0, 1, 2, 4, 8, 12, 24 and 4
Stored each hour for 8 hours. The fibrinogen concentration of the sample stored at each time is determined by Thrombocheck Fib (manufactured by International Reagents).
Was used as a reagent, and the measurement was performed using an analyzer Coagrex 700 (manufactured by Shimadzu Corporation). As a result, as shown in FIG.
Coagtrol IX (conventional product 1) and Coagtrol II
For X (conventional product 2), a decrease in the measured value was observed over time, but for the composition of Example 1 (products 1 and 2 of the present invention), no decrease in the measured value was observed and the composition was stable.

[0025]

Example 3 (Confirmation of Reproducibility) Confirmation of measurement reproducibility of the composition of the present invention (product of the present invention) and the conventional products Coagtrol N (manufactured by International Reagents) and Coagtrol IX (manufactured by International Reagents) Was done. The measurement was performed 10 times for each sample, and the variation was calculated as a CV value. as a result,
As shown in FIG. 2, it was confirmed that the present invention had a lower CV value and less variation as compared with the conventional product.

[0026]

Example 4 (Confirmation of Measurement Values by Jacobson Method and Claus Method (1)) GPRP was added to the composition of Example 1 at different concentrations, and the composition was examined. As a result, as shown in FIG. 3, the measured value by the Jacobson method was 180 m.
In the case of the fibrinogen-containing composition prepared to be g / dL, the measured value of the Claus method was similar when 100 μmol / L of GPRP was added.

[0027]

Example 5 (Confirmation of Measurement Values by Jacobson Method and Claus Method (2)) A composition obtained by adding FDP-E to the composition of Example 1 at different concentrations was examined. As a result, as shown in FIG. 4, the composition containing 210 mg / dL (measured by the Claus method) of fibrinogen contained 400 μg / m 2.
When the L FDP component E fraction was added, the value measured by the Jacobson method showed the closest value.

[0028]

As described above, the fibrinogen composition of the present invention has high stability, improved measurement error,
It was confirmed to be useful as a standard product or a quality control substance for fibrinogen measurement.

[Brief description of the drawings]

FIG. 1 is a diagram showing 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 graph showing the effect of adding GPRP. (Example 4)

FIG. 4 is a graph showing the effect of adding FDP-E. (Example 5)

Continuation of the front page (72) Inventor Yahiro Uemura 1-2-1 Muroya, Nishi-ku, Kobe-shi, Hyogo F-term in the International Reagents Research and Development Center (reference) 2G045 AA10 AA13 AA20 BB37 CA25 DA20 DA36 JA02 JA05 JA06

Claims (10)

[Claims] 1. A fibrinogen measurement standard product comprising a fibrinogen composition in which plasmin activity is substantially suppressed. 2. The standard product according to claim 1, wherein the activity of plasminogen is substantially suppressed. 3. The standard product for measuring fibrinogen according to claim 1, wherein a substance that inhibits the polymerization of fibrin is added. 4. The standard product according to claim 3, wherein the substance that inhibits polymerization is a peptide. 5. The standard product according to claim 4, wherein the peptide comprises a sequence consisting of Gly-Pro-Arg. 6. The fibrinogen measurement standard according to claim 1, wherein an FDP component is added. 7. The standard product according to claim 6, wherein the FDP component is an E fraction. 8. A fibrinogen measurement quality control substance comprising the fibrinogen composition according to claim 1. 9. A method for measuring fibrinogen, which is carried out by using the standard product or the quality control substance according to any one of claims 1 to 8. 10. A reagent kit for measuring fibrinogen, comprising the standard product or the quality control substance according to any one of claims 1 to 8.