Reagent composition, method for producing same, and inspection method

Abstract

Provided are: a reagent composition for measuring activated partial thromboplastin time, the reagent composition containing an antibody that binds to a blood coagulation factor as an antigen or an antigen-binding fragment thereof; and an inspection method including a step for mixing the reagent composition and a plasma specimen to be inspected at a prescribed ratio and measuring if the blood coagulation time thereof is T, and a step for determining that the plasma specimen is abnormal when the blood coagulation time T is longer than a designated reference.

Description

Reagent composition, its manufacturing method, and testing method

[0001] The present invention relates to a reagent composition, a method for producing the same, and a testing method. This application claims priority based on Japanese Patent Application No. 2024-044197, filed on March 19, 2024, the contents of which are incorporated herein by reference.

[0002] There are two mechanisms by which blood clots: the intrinsic coagulation pathway and the extrinsic coagulation pathway. Abnormalities in blood coagulation factors increase the time required for blood clotting during bleeding. A known test method for detecting abnormalities in the intrinsic coagulation pathway is to measure activated partial thromboplastin time (APTT). Another known method for detecting abnormalities in the extrinsic coagulation pathway is to measure prothrombin time (PT). In these test methods, a test reagent is mixed with plasma derived from the blood of the test subject, and the blood clotting time (APTT or PT) required for clotting is measured in seconds. Plasma with abnormalities in the intrinsic or extrinsic coagulation pathway has a blood clotting time several seconds to several tens of seconds longer than healthy plasma. Samples with long blood clotting times may be abnormal, so retesting or detailed testing is performed. An example of a conventional APTT measurement reagent is that disclosed in Patent Document 1.

[0003] Japanese Unexamined Patent Publication No. 59-91899

[0004] In blood coagulation tests, when a plasma sample is mixed with a test reagent and the difference in blood clotting time between a normal sample and an abnormal sample is small, the abnormal sample may be overlooked due to the variability in the measured values ​​of the normal sample. For example, even in an abnormal sample in which factor IX (FIX) activity is reduced to approximately 30-45% of normal activity, conventional test reagents have low sensitivity to FIX, making it difficult to detect the abnormal sample because the clotting time is not sufficiently long. In this situation, there is a need for a test reagent that can sufficiently extend the blood clotting time of an abnormal sample compared to the blood clotting time of a normal sample in order to reliably detect the abnormal sample.

[0005] The present invention provides a reagent composition having improved sensitivity for detecting abnormal specimens compared to conventional methods, a method for producing the same, and a testing method using the reagent composition.

[0006] As a result of extensive investigations, the present inventors have found that the detection sensitivity of a conventional test reagent for a blood coagulation factor for which detection sensitivity is to be increased can be increased by adding to the test reagent an antibody or an antigen-binding fragment thereof that binds as an antigen to the blood coagulation factor for which detection sensitivity is to be increased, and have completed the present invention. That is, the present invention includes the following aspects.

[0007] [1] A reagent composition for measuring activated partial thromboplastin time, comprising an antibody or an antigen-binding fragment thereof that binds to a blood coagulation factor as an antigen. [2] The reagent composition according to [1], wherein the content of the antibody or the antigen-binding fragment thereof relative to the total mass of the reagent composition is such that the blood coagulation time ratio, expressed as {(T4 / T2) / (T3 / T1)}, is 1.2 or greater, when a control reagent obtained by omitting the antibody or the antigen-binding fragment thereof from the reagent composition is mixed with normal plasma derived from a healthy individual in a predetermined ratio, the blood coagulation time is T1 second when the reagent composition is mixed with the normal plasma in the predetermined ratio, the blood coagulation time is T2 second when the control reagent is mixed with abnormal plasma having a lower content of the blood coagulation factor than the normal plasma in the predetermined ratio, and the blood coagulation time is T4 second when the reagent composition is mixed with the abnormal plasma in the predetermined ratio. [3] The reagent composition according to [2], wherein the predetermined mixing ratio of the control reagent to the normal plasma is, in volume ratio, 8:2 to 2:8 (control reagent:normal plasma). [4] The reagent composition according to [2] or [3], wherein the blood coagulation factor is factor V, factor VIII, factor IX, factor X, factor XI, or factor XII. [5] The reagent composition according to any of [2] to [4], wherein the blood coagulation factor is factor IX, and the content of the blood coagulation factor in the normal plasma is 0.30 to 0.38 mg / 100 ml of plasma. [6] The reagent composition according to any of [2] to [5], wherein the blood coagulation factor is factor IX, and the content of the blood coagulation factor in the abnormal plasma is 0.05 to 0.12 mg / 100 ml of plasma. [7] The reagent composition according to any of [2] to [6], wherein the antibody is a polyclonal antibody or a monoclonal antibody. [8] The reagent composition according to any one of [2] to [7], comprising one or more selected from the group consisting of phospholipids, activators, and metal salts.[9] A testing method comprising the steps of: mixing the reagent composition according to any one of [1] to [8] with a plasma sample to be tested in a predetermined ratio, measuring that the blood clotting time is T6 seconds, and determining that the plasma sample is abnormal if the T6 seconds is longer than an arbitrary standard.

[10] A testing method comprising the steps of: mixing the reagent composition according to any one of [2] to [8] with a plasma sample to be tested in the predetermined ratio, measuring that the blood clotting time is T6 seconds, mixing the control reagent with the plasma sample in the predetermined ratio, measuring that the blood clotting time is T5 seconds, calculating a blood clotting time ratio expressed as {(T6 / T2) / (T5 / T1)}, and determining that the plasma sample is abnormal if the blood clotting time ratio is 1.2 or more.

[11] A method for producing a reagent composition according to any one of [2] to [8], comprising preparing an antibody or its antigen-binding fragment that binds to the blood coagulation factor as an antigen, and blending the antibody or its antigen-binding fragment with the total mass of the reagent composition so that the blood coagulation time ratio, {(T4 / T2) / (T3 / T1)}, is 1.2 or greater.

[12] A method for producing a reagent composition according to

[11] , wherein the blood coagulation factor is factor V, factor VIII, factor IX, factor X, factor XI, or factor XII.

[13] A method for producing a reagent composition according to

[11] or

[12] , comprising blending a polyclonal or monoclonal antibody that binds to the blood coagulation factor as an antigen.

[0008] According to the present invention, it is possible to provide a reagent composition having improved sensitivity for detecting abnormal specimens compared to conventional methods, a method for producing the same, and a testing method using the reagent composition.

[0009] FIG. 1 is a conceptual diagram showing an example of the relationship between the amount of antibody contained in the reagent composition of the first embodiment of the present invention and whether or not the blood coagulation time of a sample is prolonged.

[0010] <Test Reagent> A first aspect of the present invention is a reagent composition for measuring activated partial thromboplastin time (APTT), comprising an antibody or an antigen-binding fragment thereof that binds to a blood coagulation factor as an antigen. The reagent composition of this aspect is not limited to use in measuring APTT, but can also be used as a reagent composition for measuring prothrombin time (PT). While the following describes the case where APTT is measured, PT can also be measured in a similar manner.

[0011] The blood coagulation factor for measuring APTT may be one or more selected from the group consisting of Hagman factor (factor XII: FXII), PTA (factor XI: FXI), antihemophilic factor (factor VIII: FVIII), Christmas factor (factor IX: FIX), Stewart factor (factor X: FX), AC globulin (factor V: FV), and prothrombin (factor II: FII). On the other hand, the blood coagulation factor for measuring PT may be one or more selected from the group consisting of proconvertin (factor VII: FVII), FX, FV, and FII.

[0012] The antibody may be a polyclonal antibody or a monoclonal antibody. An antibody against a desired blood coagulation factor can be produced by a conventional method. The antibody may be any antibody, including IgG, IgA, IgM, IgD, or IgE, as long as it binds to a blood coagulation factor contained in a plasma sample to be mixed and reduces the activity of the blood coagulation factor, and the antibody may be derived from any animal species.

[0013] The antigen-binding fragment of the antibody is a portion of the antibody that binds to the blood coagulation factor. Examples of antigen-binding fragments include Fab, F(ab'), scFV, and other recombinant proteins or peptides (e.g., proteins or peptides that contain the amino acid sequence of the variable region of the antibody or a modified amino acid sequence close to the amino acid sequence and mimic the antigen-binding ability of the antibody).

[0014] The reagent composition of this embodiment may contain one or more types of antibodies or antigen-binding fragments thereof. In this specification, unless otherwise specified, antibodies and antigen-binding fragments thereof may be collectively referred to as "antibodies" without distinction.

[0015] The content of the antibody contained in the reagent composition of this embodiment is set arbitrarily depending on the type of antibody, its binding strength to the antigen, the type and amount of the blood coagulation factor that is the antigen, and the like.

[0016] The amounts of blood coagulation factors contained in the plasma of healthy individuals (normal plasma) generally fall within a certain range, which is generally known as the range described below.

[0017] As used herein, "abnormal plasma" refers to plasma in which the content of one or more of the blood coagulation factors of interest contained in normal plasma is lower than that in normal plasma. The amount of a blood coagulation factor contained in abnormal plasma may be 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or 0%, when the content in normal plasma is taken as 100%. In clinical tests, the abnormality to be detected may vary depending on the type of blood coagulation factor, the patient's condition, and the physician's judgment. For example, the detection target may be any of 0-70%, 10-60%, 20-50%, or 25-40%.

[0018] The content of the antibody contained in the reagent composition of this embodiment can be, for example, in a range of 1 to 1,000 parts by mass, 10 to 500 parts by mass, or 50 to 200 parts by mass relative to 100 parts by mass of an arbitrarily selected blood coagulation factor in normal plasma. However, if the content of the antibody is excessive relative to the amount of the blood coagulation factor, the difference in blood clotting time between normal plasma and abnormal plasma is unlikely to occur, and the blood clotting time of both normal plasma and abnormal plasma will be prolonged. On the other hand, if the content of the antibody is too low relative to the amount of the blood coagulation factor, the difference in blood clotting time between normal plasma and abnormal plasma is unlikely to occur, and the abnormal plasma may be overshadowed by the variability in the measured values ​​of the normal plasma. Therefore, a method for determining an appropriate antibody content will be described with reference to FIG. 1.

[0019] From the top row to the bottom row in Figure 1, the FIX activity in each plasma sample increases in 5% increments within the range of 0 to 100%. A sample with 100% activity represents the activity of normal plasma from a healthy individual. A sample with 40% activity is a plasma sample prepared by mixing abnormal plasma (0% activity) containing no FIX with normal plasma in a 6:4 ratio, resulting in an abnormal sample with an activity reduced to 40% of normal. Normal plasma with 100% activity and abnormal plasma with 0% activity, including various blood coagulation factors, can be purchased from blood product manufacturers.

[0020] From the leftmost column to the rightmost column in Figure 1, the concentration of the anti-FIX antibody in the test reagents in each column increases in increments of 0.02 mg / ml within the range of 0.00 to 0.26 mg / ml. For example, test reagents adjusted to each concentration can be prepared by adding a commercially available anti-FIX antibody to a conventional APTT assay reagent.

[0021] First, the blood clotting time (APTT) was measured when an APTT reagent containing no anti-FIX antibody was mixed with normal plasma (FIX activity 100%), and this was designated the basic APTT. In the corresponding square in Figure 1 (the square where 0.00 mg / ml intersects with 100% activity), the APTT was entered as "not prolonged." Here, let's assume that the basic APTT was measured for multiple normal plasmas, and the average value was 30 seconds with a standard deviation of 5 seconds.

[0022] Next, as listed in Figure 1, if the measured blood clotting time (APTT) when an APTT reagent containing anti-FIX antibody at each concentration is mixed with a plasma sample containing FIX at each concentration exceeds 35 seconds (basic APTT + standard deviation of normal plasma), write "APTT is prolonged" in the corresponding box in Figure 1; if the measured value is within 35 seconds, write "APTT is not prolonged" in the corresponding box in Figure 1.

[0023] By conducting such experiments and creating a table such as that shown in Figure 1, it is possible to determine the appropriate amount of antibody to be added to the test reagent. For example, when an APTT reagent containing 0.00 to 0.08 mg / ml of anti-FIX antibody is used, it is not possible to distinguish between abnormal plasma in which FIX activity has been reduced to 30% and normal plasma in which activity is 100% (both are "non-prolonged"). Therefore, it can be concluded that the above antibody concentration is insufficient for the purpose of detecting abnormal plasma in which FIX activity has been reduced to 30%.

[0024] On the other hand, an APTT reagent containing anti-FIX antibody at a concentration of 0.10 mg / ml can distinguish between abnormal plasma with a 30% reduction in FIX activity and normal plasma with a 100% activity (the former is "prolonged"). Therefore, the above antibody concentration can be considered appropriate for detecting abnormal plasma with a 30% reduction in FIX activity.

[0025] 1 , if the objective is to distinguish from normal plasma abnormal plasma in which FIX activity has been reduced to 30%, which has been difficult to detect with conventional APTT reagents, then the APTT reagent should contain at least 0.10 mg / ml of anti-FIX antibody. Furthermore, if the objective is to distinguish from normal plasma abnormal plasma in which FIX activity has been reduced to 30-45%, and not to determine abnormal plasma with an activity of 50% or more as abnormal, then the APTT reagent should contain 0.10-0.16 mg / ml of anti-FIX antibody.

[0026] In the example of Figure 1 described here, the case where the standard deviation of the basic APTT is exceeded is determined to be "prolonged," but the case where any threshold based on empirical rules is exceeded may also be determined to be "prolonged."

[0027] In this way, the content of the antibody contained in the reagent composition of this embodiment can be set to "an amount that allows for distinguishing between normal plasma and abnormal plasma" or "an amount that allows for a longer APTT than that of conventional APTT reagents that do not contain antibodies."

[0028] The content of the antibody contained in the reagent composition of this embodiment is preferably the amount explained below: First, the blood clotting time (APTT) represented by T1 to T4 (seconds) is defined.

[0029] The blood clotting time when a "control reagent" obtained by removing the antibody from the reagent composition of this embodiment is mixed with "normal plasma" derived from a healthy individual in an arbitrary predetermined ratio is defined as T1 second. The blood clotting time when the "reagent composition" of this embodiment is mixed with the "normal plasma" in the predetermined ratio is defined as T2 second. The blood clotting time when the "control reagent" is mixed with the "abnormal plasma" in the predetermined ratio is defined as T3 second. The blood clotting time when the "reagent composition" is mixed with the "abnormal plasma" in the predetermined ratio is defined as T4 second. Note that in this specification, the "reagent composition" may be referred to as the reagent, and the "control reagent" may be referred to as the control reagent composition. Here, the predetermined ratios when measuring each of the blood clotting times T1 to T4 seconds are the same.

[0030] The predetermined ratio of the control reagent to the normal plasma, expressed as a volume ratio, is preferably selected from the group consisting of (8:2) to (2:8), (7:3) to (3:7), (6:4) to (4:6), and 5:5. This predetermined ratio can improve the reliability of blood coagulation time measurements. The predetermined ratio of the reagent composition to the normal plasma determined when measuring T2 seconds, the predetermined ratio of the control reagent to the abnormal plasma determined when measuring T3 seconds, and the predetermined ratio of the reagent composition to the abnormal plasma determined when measuring T4 seconds are the same as the predetermined ratio of the control reagent to the normal plasma determined when measuring T1 seconds.

[0031] In the reagent composition of this embodiment, the content of the antibody relative to the total mass of the reagent composition is preferably an amount such that the blood coagulation time ratio, represented by (T2 / T1), is 1.0 to 1.2. This range can prevent erroneous determination of "normal plasma" as "abnormal plasma" (misjudgment) in a test in which the reagent composition of this embodiment is mixed with a plasma sample and the APTT measurement time is measured (hereinafter referred to as an APTT measurement test).

[0032] In the reagent composition of this embodiment, the content of the antibody relative to the total mass of the reagent composition is preferably an amount that results in a blood clotting time ratio expressed as {(T4 / T2) / (T3 / T1)} of 1.2 or more, more preferably 1.5 or more, even more preferably 1.8 or more, and particularly preferably 2.1 or more, 2.4 or more, 2.7 or more, or 3.0 or more. The higher this blood clotting time ratio, the higher the sensitivity for detecting "abnormal plasma."

[0033] The content of the antibody contained in the reagent composition of this embodiment may be determined by reference to the content of each blood coagulation factor contained in 100 mL of normal plasma. Examples of the content of each blood coagulation factor include the following ranges: Factor XII: 2.2 to 2.9 mg, preferably 2.4 to 2.6 mg Factor XI: 0.40 to 0.80 mg, preferably 0.45 to 0.60 mg Factor X: 0.65 to 0.95 mg, preferably 0.70 to 0.80 mg Factor IX: 0.28 to 0.42 mg, preferably 0.30 to 0.38 mg Factor VIII: 0.7 to 3.0 μg, preferably 0.8 to 1.5 μg Factor VII: 0.04 to 0.10 mg, preferably 0.045 to 0.07 mg Factor V: 2.1 to 2.9 mg, preferably 2.3 to 2.7 mg Factor II: 12 to 24 mg, preferably 15 to 20 mg Factor I: 200 to 450 mg, preferably 250 to 400 mg

[0034] When determining the content of the antibody contained in the reagent composition of this embodiment, the content of each blood coagulation factor contained in 100 mL of abnormal plasma can be set, for example, within the following ranges, which may make it difficult to distinguish abnormal plasma from normal plasma using conventional test reagents. Factor XII: 0.10 to 2.00 mg, preferably 0.25 to 1.50 mg Factor XI: 0.01 to 0.35 mg, preferably 0.05 to 0.30 mg Factor X: 0.05 to 0.50 mg, preferably 0.075 to 0.45 mg Factor IX: 0.03 to 0.25 mg, preferably 0.034 to 0.204 mg Factor VIII: 0.05 to 0.65 μg, preferably 0.10 to 0.60 μg Factor VII: 0.001 to 0.035 mg, preferably 0.005 to 0.030 mg Factor V: 0.10 to 2.00 mg, preferably 0.25 to 1.50 mg Factor II: 1.50 to 11.0 mg, preferably 1.75 to 10.5 mg Factor I: 20 to 190 mg, preferably 30 to 180 mg

[0035] In addition to the antibody, the reagent composition of this embodiment preferably contains the same components as conventional APTT or PT measurement reagents, for example, one or more selected from the group consisting of buffers, polysaccharides, inclusion compounds, amino acids, phospholipids, activators, and metal ion-forming compounds such as metal salts.

[0036] The buffer is a pH buffering agent, and is contained together with water to form a pH buffer solution. Examples of pH buffering agents include PIPES, ACES, HEPES, TAPSO, POPSO, EPPS, and CHES. The pH of the pH buffer solution is preferably 4 to 9, more preferably 6 to 8. When the reagent composition of this embodiment contains a buffer, the concentration of the buffer in the reagent composition is preferably 0.1 to 1000 mM, more preferably 0.5 to 500 mM, even more preferably 1 to 200 mM, and particularly preferably 10 to 100 mM.

[0037] The polysaccharide or inclusion compound may include, for example, one or more selected from the group consisting of dextran, cyclodextrin, sulfated polysaccharides, and salts thereof. Of these, sulfated polysaccharides and salts thereof are preferred. Examples of sulfated polysaccharides include dextran sulfate and cyclodextrin sulfate. Examples of sulfated polysaccharide salts include sodium dextran sulfate and sodium cyclodextrin sulfate. Examples of cyclodextrin sulfate include α-cyclodextrin sulfate, β-cyclodextrin sulfate, and γ-cyclodextrin sulfate. Examples of salts include sodium salts. When the reagent composition of this embodiment contains a polysaccharide or inclusion compound, the concentration of the polysaccharide or inclusion compound in the reagent composition is preferably 0.0001 to 0.15 mg / mL, more preferably 0.001 to 0.05 mg / mL, and even more preferably 0.001 to 0.01 mg / mL.

[0038] Examples of the amino acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, methionine, phenylalanine, proline, serine, threonine, valine, leucine, and isoleucine. These amino acids may be contained alone or in combination of two or more. Furthermore, dipeptides such as glycylglycine may be contained as components similar to the amino acids. When the reagent composition of this embodiment contains an amino acid, the concentration of the amino acid in the reagent composition is preferably 10 to 500 mM, more preferably 20 to 300 mM, even more preferably 25 to 140 mM, and particularly preferably 30 to 135 mM.

[0039] Examples of the phospholipid include natural and synthetic phospholipids. These phospholipids can be used alone or in combination of two or more. The phospholipid may be contained in the form of a liposome. When the reagent composition of this embodiment contains a phospholipid, the concentration of the phospholipid in the reagent composition is preferably 0.005 to 2 mM, more preferably 0.02 to 0.5 mM.

[0040] Examples of the activator include ellagic acid, kaolin, celite, colloidal silica, polyphenol compounds, and silicic anhydride. The activators exemplified here can be used alone or in combination of two or more. When the reagent composition of this embodiment contains an activator, the concentration of the activator in the reagent composition is preferably 0.001 to 2 mg / mL, more preferably 0.001 to 0.5 mg / mL, even more preferably 0.001 to 0.1 mg / mL, and particularly preferably 0.005 to 0.05 mg / mL.

[0041] The metal ion-forming compound is a compound that supplies metal ions. Examples of the metal ions include Zn. ２＋ , Mn ２＋ , Cu ２＋ , Fe ２＋ , Al ３＋ and the like. Examples of metal ion-forming compounds include salts containing these metal ions (i.e., metal salts). The metal ion-forming compounds are sometimes classified as activators. When the reagent composition of this embodiment contains a metal ion-forming compound (e.g., a metal salt), the concentration of the metal ion-forming compound in the reagent composition is preferably 0.001 to 2 mg / mL, more preferably 0.001 to 0.5 mg / mL, even more preferably 0.001 to 0.1 mg / mL, and particularly preferably 0.005 to 0.05 mg / mL.

[0042] The reagent composition of this embodiment is preferably a first reagent composition containing the antibody and one or more selected from water, a buffer, an activator, a metal salt, a phospholipid, an amino acid, a polysaccharide, and an inclusion compound.

[0043] When the reagent composition of this embodiment is a first reagent composition for use in measuring APTT, it is preferably used by mixing it in any ratio with a second reagent composition containing a component that initiates blood coagulation.

[0044] The component that initiates blood coagulation is preferably calcium ions. The second reagent composition may contain, for example, a water-soluble calcium salt such as calcium chloride, calcium lactate, calcium gluconate, calcium glucuronate, or calcium tartrate. One or more of these calcium salts may be included. The concentration of the calcium salt contained in the second reagent composition is, for example, preferably 5 mM to 100 mM, more preferably 10 mM to 50 mM.

[0045] The reagent composition of this embodiment can also be understood as a mixed reagent composition obtained by mixing the first and second reagent compositions at the time of or before use. The present invention may also be a reagent kit including the first and second reagent compositions.

[0046] The above-mentioned reagent compositions may further contain additives to improve storage stability or stability. Examples of additives include preservatives, antioxidants, dispersants, stabilizers, etc. Preservatives include ciprofloxacin, propionic acid, sodium benzoate, sodium azide, and a mixture of 2-methyl-1,2-thiazol-3(2H)-one and 5-chloro-2-methyl-1,2-thiazol-3(2H)-one (e.g., Proclin 300). Antioxidants include citric acid and butylhydroxyanisole. Dispersants include phenol, collagen, peptides, etc. Stabilizers include polyethylene glycol, polyvinylpyrrolidone, polymeric polysaccharides such as dextran and polysucrose (e.g., Ficoll), salts such as sodium chloride, amino acids, sugars, etc.

[0047] The reagent compositions may be in the form of liquid, frozen solid, or dried. In the case of a dried form, it is dissolved in water, a buffer solution, or the like at the time of use to prepare a liquid. The above-mentioned concentrations of the various components contained in the first and second reagent compositions represent the concentrations in the liquid reagent compositions.

[0048] <Method of Use> One example of a method of using the reagent composition of this embodiment is a method in which a specimen and the reagent composition are mixed to prepare a sample solution and the blood clotting time of the sample solution is measured. The specimen is preferably a subject's plasma. An anticoagulant commonly used during blood collection may be added to the specimen. For example, blood is collected using a blood collection tube containing sodium citrate, an anticoagulant, and then centrifuged to obtain plasma. The specimen is diluted with a diluent as needed. A first reagent composition is added to the prepared specimen, and after incubation for a predetermined period of time, a second reagent composition is added to initiate the clotting reaction. The time from the addition of the second reagent composition until the sample solution coagulates can be measured as the APTT.

[0049] The reagent composition of this embodiment contains an antibody that binds to a blood coagulation factor contained in a normal sample as an antigen, and therefore, in principle, has a longer blood clotting time than a control reagent composition that does not contain the antibody. For example, when an abnormal sample containing only about 50% of the normal amount of factor IX is mixed with a reagent composition containing an anti-factor IX antibody, the factor IX contained in the abnormal sample binds to the antibody and loses its function, resulting in a longer blood clotting time. Conventionally, there has been a problem in that the difference in blood clotting time between an abnormal sample containing only about 50% of the normal amount of factor IX and a normal sample is small. However, by mixing the reagent composition of this embodiment with an abnormal sample and adding an antibody in an amount that reduces the amount of factor IX by, for example, 30%, the amount of functioning factor IX in the sample solution can be reduced to about 20% of the normal amount, thereby significantly prolonging the blood clotting time and facilitating the identification of an abnormal sample. On the other hand, even if the reagent composition of this embodiment is mixed with a normal sample, the same amount of antibody is bound to factor IX, and the amount of functioning factor IX in the sample solution is reduced to about 70% of normal, the blood coagulation time is only slightly or not at all prolonged, and the sample can be determined to be normal.

[0050] <Testing Method> A second aspect of the present invention is a testing method comprising the steps of: mixing the reagent composition of the first aspect with a plasma sample to be tested in a predetermined ratio, measuring the blood clotting time (T6 seconds) of the mixture, and determining that the plasma sample is abnormal if the T6 seconds is longer than an arbitrary standard. Here, the "arbitrary standard" may be a blood clotting time generally considered normal in this technical field, a blood clotting time obtained using the "control reagent," or whether the blood clotting ratio (described below) is equal to or greater than a predetermined value.

[0051] The testing method of this embodiment can be carried out in the same manner as conventional methods, except for using the reagent composition of the first embodiment. For example, the first reagent composition is mixed with a sample diluted as necessary, and the resulting mixture is heated. Heating conditions include, for example, 30°C to 40°C, preferably 35°C to 39°C. The second reagent composition is then added to the mixture to initiate the coagulation reaction. The coagulation time (APTT) of the mixture (sample solution) after the addition of the second reagent composition is measured. Examples of measurement methods include optical methods that measure the amount of scattered light, transmittance, or absorbance of the sample solution, or mechanical methods that measure the viscosity of the sample solution. The temperature of the sample solution during measurement can be, for example, 30°C to 40°C, preferably 35°C to 39°C. The concentrations of each reagent composition in the sample solution are preferably such that the APTT of normal plasma falls within the range of, for example, 15 to 80 seconds, preferably 15 to 60 seconds, and more preferably 20 to 50 seconds.

[0052] The start of the coagulation reaction can typically be defined as the time when the first reagent composition is mixed with the sample and then the second reagent composition is mixed. Alternatively, the start of the reaction may be defined as the time when another operation is completed. The time for continuing measurement of the coagulation reaction can be, for example, several tens of seconds to approximately 7 minutes from the time when the sample and the second reagent composition are mixed. The measurement may be terminated at an arbitrarily determined fixed time, or may be continued until the completion of the coagulation reaction of the sample is detected. During the measurement period, it is preferable to repeatedly measure the progress of the coagulation reaction (e.g., by measuring the amount of scattered light) at predetermined intervals (e.g., 0.1 second intervals). The coagulation time (APTT) is calculated from the measured coagulation reaction according to a known method.

[0053] The series of operations in the testing method of this embodiment may be performed using an automatic analyzer. In this case, an operator may perform some of the operations manually. For example, the operator may prepare the sample, and the subsequent operations may be performed by the automatic analyzer.

[0054] Here, the definitions of T1 seconds to T6 seconds will be confirmed. The blood clotting time when a "control reagent" obtained by removing the antibody from the reagent composition of the first embodiment used in this embodiment is mixed with "normal plasma" derived from a healthy individual at the predetermined ratio is defined as T1 second. The blood clotting time when the "reagent composition" and the "normal plasma" are mixed at the predetermined ratio is defined as T2 second. The blood clotting time when the "control reagent" and the "abnormal plasma" are mixed at the predetermined ratio is defined as T3 second. The blood clotting time when the "reagent composition" and the "abnormal plasma" are mixed at the predetermined ratio is defined as T4 second. The blood clotting time when the "control reagent" and the "plasma sample" to be tested are mixed at the predetermined ratio is defined as T5 second. The blood clotting time when the "reagent composition" and the "plasma sample" are mixed at the predetermined ratio is defined as T6 second. Here, the predetermined ratios when measuring each of the blood clotting times T1 to T6 seconds are the same. However, when T6 seconds is measured and evaluated independently of T1 to T5 seconds, the predetermined ratio when measuring T6 seconds can be set independently of the predetermined ratio when measuring T1 to T5 seconds.

[0055] In the testing method of this embodiment, the reagent composition and the control reagent used preferably satisfy a relationship in which the blood coagulation time ratio, expressed as {(T4 / T2) / (T3 / T1)}, is 1.2 or more. This range facilitates the detection of abnormal samples.

[0056] In the testing method of this aspect, it is preferable to determine that the plasma sample is abnormal when the blood coagulation time ratio expressed as {(T6 / T2) / (T5 / T1)} is 1.2 or more. This method makes the determination easier.

[0057] An example of an embodiment of the testing method of this aspect includes the steps of mixing the control reagent and a plasma sample to be tested in the predetermined ratio and measuring that the blood clotting time is T5, mixing the reagent composition of the first aspect and a plasma sample to be tested in the predetermined ratio and measuring that the blood clotting time is T6, calculating a blood clotting time ratio expressed as {(T6 / T2) / (T5 / T1)}, and determining that the plasma sample is abnormal when the blood clotting time ratio is 1.2 or higher.

[0058] <Modifications> As a modification of the reagent composition of the first aspect or the testing method of the second aspect, it is possible to adopt the following T0 second instead of the T1 second. Here, T0 second is defined as a typical blood clotting time when the "control reagent" and the "normal plasma" are mixed in any predetermined ratio. An example of a typical blood clotting time is the median of the reference range of blood clotting time for "normal plasma" mixed with the "control reagent," which is described in the package insert of the "control reagent."

[0059] 1) Samples The following samples were used. 1-1) Pseudo-samples Pooled normal plasma (PNP) (pooled plasma from healthy individuals purchased from George King) 1% FVIII activity sample (prepared by mixing FVIII factor-deficient plasma purchased from George King with PNP) 30% FVIII activity sample (prepared by mixing FVIII factor-deficient plasma purchased from George King with PNP) 1% FIX activity sample (prepared by mixing FIX factor-deficient plasma purchased from George King with PNP) 30% FIX activity sample (prepared by mixing FIX factor-deficient plasma purchased from George King with PNP) 1% FXI activity sample (prepared by mixing FXI factor-deficient plasma purchased from George King with PNP) 30% FXI activity sample (prepared by mixing FXI factor-deficient plasma purchased from George King with PNP) 1% FXII activity sample (prepared by mixing FXI factor-deficient plasma purchased from George King with PNP) FXII activity 15% sample (prepared by mixing FXII factor deficient plasma purchased from George King with PNP)

[0060] One of the abnormal samples, the "FVIII activity 1% sample," had a factor VIII activity reduced to 1% of that of normal plasma (PNP) from a healthy individual. It was prepared by mixing PNP and FVIII-deficient plasma in a ratio of 1:99. Other abnormal samples were prepared in the same manner.

[0061] 1-2) Actual samples Normal plasma (sample from a healthy individual, N=1) FVIII-deficient sample (sample from a patient with FVIII deficiency, N=1) FIX-deficient sample (sample from a patient with FIX deficiency, N=3) FXI-deficient sample (sample from a patient with FXI deficiency, N=1) LA-positive sample (sample from a patient with LA-positive sample, N=1) Here, "N" indicates the number of samples. "LA" stands for lupus anticoagulant.

[0062] 2) APTT measurement reagent 2-1) Basic recipe

[0063]

[0064] 2-2) Test Reagents Test Reagents 1 to 3: Anti-Factor IX / PTC antibody (Abcam, ab128038), an antibody that binds to factor IX, was added to the basic formulation R1 under the conditions shown in the table below. The basic formulation reagent (reagent without antibody added) was used as a control.

[0065]

[0066] 2-3) Preparation of R1 and R2 The above components were mixed to the predetermined concentrations to prepare each test reagent and the control R1 and R2. R1 was mixed under ice cooling.

[0067] 3) Clotting Time Measurement After 50 μL of sample was heated in a cuvette at 37°C for 45 seconds, 50 μL of first reagent (R1) was added and the mixture was further heated for 171 seconds. 50 μL of second reagent (R2) was added to the heated mixture to initiate the clotting reaction. The reaction was carried out at 37°C. The sample (sample solution) to which R2 had been added was irradiated with light of 660 nm wavelength, and the optical change (change in scattered light intensity) due to the clotting reaction was measured. The clotting time was calculated from the measured clotting reaction. Clotting time measurements were performed using a CP3000 automatic blood coagulation analyzer (manufactured by Sekisui Medical Co., Ltd.).

[0068] For the pseudo-samples, the ratio of the clotting time of each sample to the clotting time of PNP (APTT) (APTT ratio) was calculated using the formula [APTT ratio = (APTT with test reagent) / (APTT with control)]. An APTT ratio greater than 1.00 indicates a prolonged clotting time compared to PNP. The results are shown in Table 3.

[0069] For the actual samples, the variability (%) of the clotting time (APTT) of the actual sample mixed with the test reagent relative to the clotting time (APTT) of the actual sample mixed with the control (basic formulation) was calculated using the formula [variability (%) = (APTT with test reagent - APTT with control) / APTT with control x 100]. A variability of more than 100% indicates that the clotting time was prolonged compared to the control. The results are shown in Table 4.

[0070] 3-1) Evaluation results of pseudo-samples When the test reagent containing antibody against factor IX was used, the clotting time of the low FIX activity sample was significantly prolonged compared to PNP, low FVIII activity sample, low FXI activity sample, and low FXII activity sample (Table 3).

[0071]

[0072] The test results using the above pseudo-samples showed that the concentration of added antibody was sufficient in Test Reagent 1. In the next test using actual samples, Test Reagent 2 was used, which contains a high concentration of antibody to allow for some leeway.

[0073] 3-2) Evaluation results of actual samples When the test reagent containing antibody against factor IX was used, the clotting time of the FIX-deficient sample was significantly prolonged compared to normal plasma, FVIII-deficient sample, FXI-deficient sample, and LA-positive sample (Table 4). In Table 4, the units of values ​​other than the rate of variation are "seconds."

[0074]

[0075] In the above test results, the antibody content in Test Reagent 2 is an amount such that the blood coagulation time ratio, expressed as (T4 / T2) / (T3 / T1), is (77.6 / 30.3) / (42.1 / 27.8) = 1.69 or more.

[0076] As can be seen from the above experimental results, test reagents 1 to 3 according to the present invention contain anti-factor IX antibodies, and by using these, factor IX-deficient specimens can be easily detected from various actual specimens.

Claims

1. A reagent composition for measuring activated partial thromboplastin time, comprising an antibody or an antigen-binding fragment thereof that binds to a blood coagulation factor as an antigen.

2. The reagent composition according to claim 1, wherein the content of the antibody or its antigen-binding fragment relative to the total mass of the reagent composition is such that the blood clotting time ratio, expressed as {(T4 / T2) / (T3 / T1)}, is 1.2 or greater, in the following cases: the blood clotting time when a control reagent, in which the antibody or its antigen-binding fragment is omitted from the reagent composition, and normal plasma derived from a healthy individual are mixed in a predetermined ratio is T1 second; the blood clotting time when the reagent composition and the normal plasma are mixed in the predetermined ratio is T2 second; the blood clotting time when the control reagent and abnormal plasma, which contains a lower amount of the blood clotting factor than the normal plasma, are mixed in the predetermined ratio is T3 second; and the blood clotting time when the reagent composition and the abnormal plasma are mixed in the predetermined ratio is T4 second.

3. The reagent composition according to claim 2, wherein the predetermined ratio of the control reagent to the normal plasma is, expressed as a volume ratio, control reagent:normal plasma = 8:2 to 2:

8.

4. The reagent composition according to claim 2, wherein the blood coagulation factor is factor V, factor VIII, factor IX, factor X, factor XI, or factor XII.

5. The reagent composition according to claim 2, wherein the blood coagulation factor is factor IX, and the content of the blood coagulation factor in the normal plasma is 0.30 to 0.38 mg / 100 ml of plasma.

6. The reagent composition according to claim 2, wherein the blood coagulation factor is factor IX, and the content of the blood coagulation factor contained in the abnormal plasma is 0.05 to 0.12 mg / 100 ml of plasma.

7. The reagent composition of claim 2, comprising said antibody, said antibody being a polyclonal antibody or a monoclonal antibody.

8. The reagent composition according to claim 2, comprising one or more members selected from the group consisting of phospholipids, activators, and metal salts.

9. A testing method comprising the steps of: mixing the reagent composition according to any one of claims 1 to 8 with a plasma sample to be tested in the specified ratio; and measuring that the blood clotting time is T6 seconds; and determining that the plasma sample is abnormal if the T6 seconds is longer than an arbitrary standard.

10. A testing method comprising the steps of: mixing the reagent composition according to any one of claims 2 to 8 with a plasma sample to be tested in the specified ratio and measuring that the blood clotting time is T6 seconds; mixing the control reagent with the plasma sample in the specified ratio and measuring that the blood clotting time is T5 seconds; calculating a blood clotting time ratio expressed as {(T6 / T2) / (T5 / T1)}; and determining that the plasma sample is abnormal when the blood clotting time ratio is 1.2 or greater.

11. A method for producing a reagent composition according to any one of claims 2 to 8, wherein when the antibody or its antigen-binding fragment that binds to the blood coagulation factor as an antigen is prepared and then blended to obtain the reagent composition, the amount of the antibody or its antigen-binding fragment blended relative to the total mass of the reagent composition is an amount such that the blood coagulation time ratio, expressed as {(T4 / T2) / (T3 / T1)}, is 1.2 or greater.

12. The method for producing a reagent composition according to claim 11, wherein the blood coagulation factor is factor V, factor VIII, factor IX, factor X, factor XI, or factor XII.

13. The method for producing a reagent composition according to claim 11, further comprising compounding a polyclonal or monoclonal antibody that binds to the blood coagulation factor as an antigen.

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