JP2004317502A - Reagent for measuring anti-phosphatide antibody, and reagent kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple measurement method for quantitatively and decisively diagnosing an anti-phosphatide antibody that is a substance for causing antiphospholipid antibody syndrome, more specifically a loops anticoagulant(LA). <P>SOLUTION: A coagulation time measurement reagent measures coagulation time or performs immunological measurement, by using a coagulation reagent containing the antibody, serum, plasma, and immunological globulin of a vertebrate which exclude specimens containing LA that is a substance for causing the antiphospholipid antibody syndrome, and humans, or by using an immunological measurement reagent. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a blood coagulation reagent used for diagnosis of antiphospholipid antibody syndrome in fields such as clinical chemistry and pharmaceutical research, and a reagent kit using the reagent, and more specifically, a lupus anticoagulant positive in in vitro blood test. The present invention relates to a reagent, a reagent kit, and a method for detecting an antiphospholipid antibody, which can detect a disease with high accuracy and quantifiable lupus anticoagulant.

  Anti-phospholipid antibody is a general term for autoantibodies to negatively charged lipids such as cardiolipin, phosphatidylserine, and phosphatidic acid, and a typical example is lupus anticoagulant (hereinafter also referred to as `` LA ''). ). The antiphospholipid syndrome (hereinafter, also referred to as “APS”) is a general term for diseases in which the above-mentioned antiphospholipid antibody is positive and shows unique clinical symptoms such as thrombosis, premature birth, and thrombocytopenia.

  LA, a causative substance of the antiphospholipid antibody syndrome, is a heterogeneous antibody whose antigens are antibodies against a negatively charged phospholipid (phosphatidylserine) and a complex of β2-GPI with a cofactor or prothrombin. In recent years it has become clear. It is believed that the anticoagulant effect of LA occurs because LA inhibits prothrombin from converting to thrombin by binding to this complex. It is also known that LA is an immunoglobulin that inhibits the phospholipid-dependent coagulation reaction without suppressing the activity of individual coagulation factors (Procedure of Clinical Testing Methods, Kanehara Publishing, Revised 31st Edition, 442 page).

  As a reagent for detecting LA having such characteristics, a coagulation reagent containing a certain amount of phospholipid, for example, cephalin derived from rabbit brain, cephalin derived from bovine brain, soybean lecithin, snake venom, and the like have been conventionally used. If LA is present in the sample blood, LA will neutralize the phospholipids in the reagent, which will result in a shortage of phospholipids needed to initiate the cascade reaction that coagulates the sample. The clotting time of each test is prolonged in proportion to the amount of the phospholipid performed, and it can be diagnosed as LA-positive.

  Further, since the detection sensitivity of LA increases as the amount of the phospholipid in the coagulation reagent decreases, the reagent is diluted or a combination of reagents having different phospholipid concentrations (reagent kit) is used.

For example, an activated partial thromboplastin time measurement (hereinafter abbreviated as APTT) reagent in which two reagents having different phospholipid concentrations are combined is used, and the clotting time of each reagent is used to determine the Rosner Index and the Lupus ratio. (Lupus Ratio: LR) is calculated, and a method of determining the LA positive rate is known.
Commercially available coagulation measurement reagents include a test reagent by the Russell snake venom method called Gladipore LA (Gradipore; Australia) and a Staclot LA method (Roche) using hexagonal phosphifidylethanolamine.

  Furthermore, according to existing research reports (V. Chantarangkul, et.al., Thromb. Res. 1992, 67: 355-365 (Non-Patent Document 1); E. Rosner, et.al., Thromb. Haemost. 1987, 57: 144-149 (Non-Patent Document 2)), LA is arithmetically detected by utilizing the difference in phospholipid concentration between rabbit brain-derived phospholipids and the like.

  Each of these methods is a screening or qualitative test method, and the detection reagent reacts not only with the antiphospholipid antibody or lupus anticoagulant but also with other components. , Depending on the setting of the cut-off value, etc., it may be judged as positive even if it is not positive for LA, or conversely, negative for patients with low sensitivity to the detection reagent despite LA positive May be determined.

  In order to improve diagnostic accuracy, reagents and detection methods capable of quantitatively or specifically detecting immunoglobulins derived from antiphospholipid antibodies or lupus anticoagulants are desired.

  As a method for quantitatively measuring anti-phospholipid antibodies, a β2GPI-dependent anti-cardiolipin antibody measurement method, which is an immunoassay, has been developed, but this method is not a method for directly measuring anti-cardiolipin antibodies.

  Under such circumstances, at the International Society for Thrombosis and Hemostasis (ISTH), Brandt et al. Prepared guidelines for lupus anticoagulant (Thromb Haemost 1995; 74 (4): 1185-90 (Non-patent Document 3)) Sapporo Criteria (Arthritis Rheum 1999; 42 (7): 1309-11) for the diagnosis of antibody syndrome (APS) has been established. According to these, it is recommended that diagnosis of APS or LA be performed not by a method based on one measurement principle but by a combination of test methods using two or three different measurement principles.

  On the other hand, as a method for detecting with high accuracy using specificity, a method for measuring a phosphatidylserine-dependent anti-prothrombin antibody (Arthritis Rheum 2000; 43 (9): 1982-93 (Non-Patent Document 5)) However, since this method is an immunological measurement method, it cannot be easily measured as in a coagulation test method.

Thromb Res 1992; 67: 355-65 Thromb. Haemost. 1987, 57: 144-149 Thromb Haemost 1995; 74 (4): 1185-90 Arthritis Rheum 1999; 42 (7): 1309-11 Arthritis Rheum 2000; 43 (9): 1982-93

An object of the present invention is a coagulation test method that can be measured relatively easily, an anti-phospholipid antibody that is a causative substance of the anti-phospholipid antibody syndrome, specifically, LA, a coagulation time measurement reagent that can detect with high accuracy, and It is to provide a reagent kit using the same.
The present invention also provides a method for simply and accurately detecting an antiphospholipid antibody using the above-mentioned reagent kit.
Further, the present invention provides a method for specifically measuring an anti-phospholipid antibody in a sample so that it can be quantified.

  The present inventors, as a result of various studies, anti-phospholipid antibodies that are the causative agent of anti-phospholipid antibody syndrome, especially LA, mouse IgG, horse IgG, HBR, NMS, MAK33, mouse serum, horse serum, and the like, The present inventors have found that they specifically bind to antibodies of vertebrates other than humans, etc., and prevent the binding between anti-phospholipid antibodies and phospholipids contained in the specimen, thereby completing the present invention.

  That is, the reagent for measuring the coagulation time of the present invention comprises a coagulation composition; and at least one selected from the group consisting of antibodies, sera, plasma, and immunoglobulins of vertebrates other than humans.

  The anti-phospholipid antibody detection reagent kit of the present invention comprises the above-described clotting time measuring reagent (first clotting time measuring reagent) of the present invention and a clotting composition, but contains antibodies, serum, and plasma of vertebrates other than humans And a second clotting time measuring reagent which does not contain any of immunoglobulins.

  The method for detecting an antiphospholipid antibody of the present invention comprises the steps of contacting a sample with each of a first clotting time measuring reagent and a second clotting time measuring reagent; and a clotting time (first (Coagulation time) and a coagulation time (second coagulation time) when mixed with the second coagulation time measuring reagent.

  In addition, the method for measuring antiphospholipid antibody of the present invention comprises: at least one selected from the group consisting of a labeled vertebrate antibody other than human, serum, plasma, and immunoglobulin (labeled non-human-derived antibody and the like); A step of contacting the sample with a test subject; and a step of measuring a conjugate of the test subject with the labeled non-human-derived antibody or the like by measuring the label.

(Coagulation time measurement reagent)
The reagent for measuring coagulation time of the present invention comprises a coagulation composition; and at least one selected from the group consisting of antibodies, serum, plasma, and immunoglobulins of vertebrates other than humans.

  The coagulation composition refers to a component necessary for causing blood coagulation in vitro, and includes calcium, phospholipid, and at least one selected from the group consisting of an activator, snake venom, and tissue factor. .

  Examples of the phospholipid include phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and the like, and one or a combination of two or more thereof can be included.

  As the activator, it is preferable to use at least one selected from the group consisting of kaolin, celite, silica and ellagic acid. As the snake venom, it is preferable to use one or more selected from the group consisting of Russell snake venom, textalin snake venom, and ecarin snake venom. As tissue factor, it is preferable to use rabbit brain, human recombinant, and the like.

  Which of the activator, snake venom, and tissue factor to use is appropriately selected depending on the type of the coagulation time measuring reagent. For example, when it contains calcium, phospholipid and activator, it corresponds to an activated partial thromboplastin time measurement (APTT) reagent. If it contains calcium, phospholipids and snake venom, it falls under the Russell snake venom measurement time (RVVT) reagent. When calcium, phospholipid and tissue factor are contained, they correspond to prothrombin time measurement (PT) reagents.

  The content of the coagulation composition in the coagulation time measurement reagent of the present invention may be any amount that can initiate coagulation of blood or plasma as a test subject, and is appropriately selected according to the mixing ratio with the test subject. You.

  At least one selected from the group consisting of vertebrate antibodies other than humans, serum, plasma and immunoglobulin is contained in the clotting time measuring reagent of the present invention as a capture agent for capturing an antiphospholipid antibody to be detected. You. That is, the antiphospholipid antibody to be detected in the present invention, specifically, human lupus anticoagulant (human LA) binds to vertebrate antibodies other than humans, serum, plasma, and immunoglobulin to form a coagulant. The effect of anti-phospholipid antibodies on the clotting time initiated by the system composition can be ruled out. As a result, it is possible to prevent the coagulation time from being prolonged by the antiphospholipid antibody.

  Vertebrate antibodies, serum, plasma or immunoglobulin other than humans, which are contained to capture anti-phospholipid antibodies (hereinafter referred to as "non-human-derived antibodies etc." Preferably, mammalian antibodies other than human and pig, serum or immunoglobulin is used, specifically, mouse IgG, horse IgG, bovine IgG, mouse serum, horse serum, bovine serum, mouse, horse or cow Α-globulin and the like can be used. Commercially available products include HBR (Heterophile Blocking Reagent) manufactured by Scantibodies, MAK33 (manufactured by Roche), and NM8. HBR is a reagent added to the test system for the purpose of suppressing interference by heterophilic antibodies in the immunoassay method, the present inventors, anti-phospholipid antibody causing anti-phospholipid antibody syndrome, In particular, it was found that it specifically binds to LA and does not bind to components related to blood clotting time other than the antiphospholipid antibodies contained in the blood sample. Use as a lipid antibody capture agent became possible.

  As the non-human-derived antibody or the like contained for capturing the antiphospholipid antibody, an antibody or the like derived from only one kind of animal may be used, or an antibody or the like derived from two or more different kinds of animals, such as mouse IgG And horse IgG may be mixed and used.

  The content of the non-human-derived antibody or the like in the coagulation time measuring reagent of the present invention is preferably 0.1 to 50 mg, preferably 0.2 to 10 mg, more preferably 0.5 to 6 mg per mL of the subject. At 0.1 mg or less per 1 mL of the measurement sample, in the case of a sample of an anti-phospholipid antibody syndrome patient, the capture of the anti-phospholipid antibody becomes insufficient, and the prolonged coagulation time is recognized by the anti-phospholipid antibody not captured. It is.

  The coagulation time measuring reagent of the present invention may contain a buffer such as HEPES and Tris buffer, if necessary, in addition to the above-mentioned components (coagulation system composition, non-human antibody, etc.). The concentration of the buffer may be a concentration generally used in the field of clinical chemistry, and can be determined by a simple repeated experiment. The reagent for measuring a clotting time of the present invention may further contain a protein such as α-globulin.

  The coagulation time measuring reagent having the above composition may be contained in one container as a mixture of the above components, or may be a combination of the first partial reagent and the second partial reagent contained in separate containers. There may be. In this case, the first partial reagent includes at least one selected from the group consisting of vertebrate antibodies excluding non-human-derived antibodies, serum, plasma, and immunoglobulin; phospholipids; and activators, snake venom, and tissue factor. At least one selected from the group is included, and the second partial reagent includes calcium ions.

[Detection reagent kit]
The detection reagent kit of the present invention is a reagent kit for accurately detecting the presence or absence of an antiphospholipid antibody, particularly human lupus anticoagulant (human LA), in a blood or plasma sample. The target human LA may be any of the IgG, IgM, and IgA classes.

  The detection reagent kit of the present invention contains the above-described clotting time measuring reagent (first clotting time measuring reagent) of the present invention and a coagulation system composition, and includes any of vertebrate antibodies, serum, plasma, and immunoglobulin other than human. And a second clotting time measuring reagent not containing That is, the detection reagent kit of the present invention is a combination of two kinds of coagulation time measuring reagents which differ only in the presence or absence of a non-human-derived antibody or the like as an anti-phospholipid antibody capturing agent.

  The coagulation composition contained in the first coagulation time measurement reagent and the coagulation composition contained in the second coagulation time measurement reagent must be the same. That is, when the coagulation composition contained in the first coagulation time measurement reagent is the APTT reagent, the coagulation composition contained in the second coagulation time measurement reagent is also the APTT reagent, and the composition is the same. Is preferred.

  When the first clotting time measuring reagent is composed of a combination of the first partial reagent and the second partial reagent, it is preferable that the second clotting time measuring reagent is also a combination of the partial reagents. In this case, the partial reagent (third partial reagent) corresponding to the first partial reagent contains at least one selected from the group consisting of a phospholipid; and an activator, snake venom and tissue factor, (A fourth partial reagent) contains calcium ions.

  Since the second clotting time measurement reagent does not contain a non-human-derived antibody or the like serving as an anti-phospholipid antibody capturing agent, an anti-phospholipid antibody syndrome (APS) positive Prolongs the clotting time. On the other hand, when the sample is brought into contact with the first clotting time measuring reagent, the anti-phospholipid antibody is captured by the non-human-derived antibody or the like contained in the first clotting time measuring reagent. Even for an APS) -positive sample, the prolongation of clotting time due to the presence of an anti-phospholipid antibody (eg, LA) is prevented. Therefore, there is a significant difference between the clotting time when using the first clotting time measuring reagent (first clotting time) and the clotting time when using the second clotting time measuring reagent (second clotting time). If the result is positive, anti-phospholipid antibody is positive. If no significant difference is observed, it can be determined that anti-phospholipid antibody is negative.

(Detection method)
The method for detecting an antiphospholipid antibody of the present invention uses the above-described detection reagent kit of the present invention. That is, a step of bringing the sample into contact with each of the first clotting time measuring reagent and the second clotting time measuring reagent; a clotting time (first clotting time) when the sample is brought into contact with the first clotting time measuring reagent; And a step of comparing a coagulation time (second coagulation time) at the time of contact with.
If there is a difference between the first clotting time and the second clotting time, it is determined that the sample contains an antiphospholipid antibody, particularly LA.

  Further, from the measured first clotting time and the second clotting time, the inhibition ratio of the clotting time by the anti-phospholipid antibody may be calculated by the following formula, and the presence or absence of the anti-phospholipid antibody may be determined based on this. .

    Inhibition ratio = 1− (second clotting time−first clotting time) / first clotting time

  The inhibition ratio calculated by the above formula is almost 1 in the case of a normal plasma, as well as a sample whose clotting time has been prolonged due to a cause other than the antiphospholipid antibody (for example, a sample containing heparin which is an anticoagulant reagent). In contrast, a sample containing an antiphospholipid antibody generally shows a value of 1.2 or more, depending on the content.

  The subject to be measured may be blood or plasma collected from a patient, or a mixture with normal plasma. A blood-derived substance prepared by a general blood coagulation measurement system can be measured as a subject. Therefore, a blood sample obtained by collecting blood from a subject, or a blood sample obtained by adding an anticoagulant such as heparin, and a plasma or serum component obtained by separating components based on a conventional method such as centrifugation can be applied. is there. This is because a non-human-derived antibody or the like does not show an affinity for an anticoagulant, and thus it is considered that the anticoagulant hardly affects the first clotting time. In addition, in order to suppress non-specific adsorption due to the measurement method and to obtain a more accurate measurement, the plasma and serum components may be further separated by ultracentrifugation to obtain a specific fraction. . It is preferable to adjust the pH to 6 to 8 and the salt concentration to 1 to 500 mM in a state where the test substance and the coagulation time measuring reagent are mixed.

  In the contacting step between the coagulation time measuring reagent and the sample, it is preferable that both are contacted for a time sufficient for the non-human-derived antibody or the like contained in the first coagulation time to react and bind with the antiphospholipid antibody. Specifically, it is about 1 to 30 minutes, preferably about 2 to 10 minutes, and more preferably about 5 minutes. The contacting step is preferably performed at 30 to 40 ° C.

(Application of immunoassay method)
By using a non-human-derived antibody or the like that captures the anti-phospholipid antibody, the amount of the anti-phospholipid antibody in the sample can be determined.
That is, the method for measuring an anti-phospholipid antibody of the present invention involves contacting a sample with a solid phase on which a non-human-derived antibody or the like is immobilized, and measuring the amount of a labeled non-human-derived antibody or the like bound to the anti-phospholipid antibody. This is a method for measuring the amount of anti-phospholipid antibody (LA).

Specific methods for measuring antiphospholipid antibodies include a one-step method and a two-step method, and any of them may be used.
In the one-step method, a sample and a labeled non-human-derived antibody or the like (labeled secondary antibody) are added to a solid phase on which a non-human-derived antibody or the like (primary antibody) is immobilized, and incubated (for example, at 4 to 40 ° C.). (1 minute to 1 day) to form a complex of primary antibody-analyte (anti-phospholipid antibody) -labeled secondary antibody. After removing the labeled non-human-derived antibody and the like that did not constitute the complex, the amount of the anti-phospholipid antibody is quantified by measuring the amount of the labeled non-human-derived antibody and the like in the complex.

  In the two-step method, a sample is added to a solid phase on which a non-human-derived antibody or the like (primary antibody) is immobilized, incubated (for example, at 4 to 40 ° C. for 1 minute to 2 hours), and then washed. Next, a labeled non-human-derived antibody or the like (labeled secondary antibody) is added and incubated (for example, at 4 to 40 ° C. for 1 minute to 2 hours) to react with a sample bound to the primary antibody. A complex of primary antibody-analyte (anti-phospholipid antibody) -labeled secondary antibody is generated. After washing and removing excess labeled secondary antibody that has not bound to the sample, the amount of the antiphospholipid antibody is quantified by measuring the amount of the labeled non-human-derived antibody or the like of the complex.

  As a solid phase, a microtiter plate, polystyrene beads, or the like can be used. And leave it at 4 ° C overnight or more. Since it is not always the case that antibodies and the like are uniformly coated on the entire surface of the solid phase, it is preferable to dissolve bovine serum albumin in Tris buffer, contact this with the solid phase, and coat in the same manner.

  Instead of using a labeled secondary antibody, a complex of a primary antibody-analyte (anti-phospholipid antibody) -unlabeled secondary antibody is first formed using an unlabeled secondary antibody, and then a complex of the complex is formed. A label may be bound to the unlabeled secondary antibody. As a method for attaching the label, there is a method utilizing an avidin-biotin complex formation reaction.

  As the label, an enzyme such as alkaline phosphatase or peroxidase, a fluorescent coloring substance, a chemiluminescent substance, an isotope, or the like can be used.

  As a method for quantifying a label or the like, a method known in the art can be used depending on the type of the label. For example, when labeled with an enzyme, the enzyme activity can be measured, when labeled with a fluorescent coloring substance, it can be measured with a fluorometer, and when labeled with a chemiluminescent substance, chemical analysis using an enzyme can be performed. It can be measured by luminescence, and when labeled with a radioisotope, it can be measured by using a scintillation counter. Examples of the enzymatic reaction using a chromogenic substrate include, for example, p-nitrophenyl phosphate when labeled with alkaline phosphatase, and 2′-azino-bi- (3′-ethylbenzyl) when labeled with peroxidase. Thiazoline sulfonic acid) may be used as a substrate, and a product produced by reacting for a certain period of time may be colorimetrically measured with a spectrophotometer.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to the following Examples.

(Example 1) Preparation method of reagents Ellagic acid solution (0.1 mM) was mixed with 50 mM HEPES and 25 mM Tris buffer (pH 7.35), and phosphatidylserine (PS), phosphatidylethanolamine (PE) and Phosphatidylcholine (PC) was mixed to prepare a first partial reagent of the second clotting time measuring reagent of PS: 5 μg / mL, PE: 20 μg / mL, PC: 70 μg / mL.

To the second clotting time measuring reagent prepared above, HBR commercially available from Scantibodies as a heterophilic inhibitor was added to a concentration of 400 μg / mL, and the first clotting time measuring reagent was added to the first clotting time measuring reagent. A partial reagent was prepared. HBR is mainly composed of mouse-derived IgG.
(Measuring method)

  As the measurement sample, normal plasma (Coagtrol N manufactured by Sysmex Corporation), LA-positive patient plasma, warfarin-administered patient plasma, heparin-administered patient plasma, and factor VIII-deficient patient plasma were used. The patient sample used was a commercially available product from NIBSC, George King Biomedical, Gladipore, ADI, or Sanfuco.

表１から、LA含有血漿は、HBRを含有する第１凝固時間測定試薬に対する凝固時間（第１凝固時間）が、HBRを含有しない第２凝固時間測定試薬に対する凝固時間（第２凝固時間）と比べて、有意に凝固時間が短縮した。従って、HBRはヒトLAと選択的に結合して、凝固系組成物による凝固時間の延長を阻止したと考えられる。 Mix each of the above measurement samples (50 μL) with the first partial reagent of the first clotting time measuring reagent or the first partial reagent of the second clotting time measuring reagent prepared above and mix with 50 μL each, and heat at 37 ° C. for 5 minutes. After that, as a second partial reagent and a fourth partial reagent, a 25 mM / L calcium chloride solution was added so as to be 50 μL to initiate coagulation. The coagulation time was measured using a fully automatic blood coagulation analyzer Coagrex 800 (manufactured by Shimadzu Corporation). The measurement was performed twice each. Table 1 shows the results.

From Table 1, the LA-containing plasma shows that the coagulation time (first coagulation time) for the first coagulation time measurement reagent containing HBR is the same as the coagulation time (second coagulation time) for the second coagulation time measurement reagent not containing HBR. In comparison, the clotting time was significantly reduced. Therefore, it is considered that HBR selectively bound to human LA and prevented the coagulation composition from prolonging the coagulation time.

  On the other hand, for normal plasma, heparin plasma, warfarin plasma, factor VIII deficient plasma and factor IX deficient plasma, no significant difference was observed between the first clotting time and the second clotting time. It can be confirmed that HBR does not affect the coagulation system of normal plasma. In addition, anticoagulant samples (heparin plasma, warfarin plasma) and coagulation factor deficiency (factor VIII deficient plasma and factor IX deficient plasma) prolong the clotting time, It can be seen that no significant difference was observed between the first clotting time and the second clotting time when the extension was not due to lipid antibody dependence.

表２及び図１から、ヒトLA含有血漿では、HBRの濃度依存的に凝固時間が短縮すること、LAと非ヒト由来抗体(HBR)との反応性が定量的に行われることがわかる。一方、正常血漿、ヘパリン血漿、ワーファリン血漿、第VIII因子欠損血漿及び第IX因子欠損血漿については、HBR添加濃度の違いによる凝固時間の有意な違いは認められなかった。また、図１及び表３から、LA陽性検体50μLサンプルに対し、HBR5μg以上（サンプルに対するHBR濃度0.1mg/mL）の場合に阻害比率が1.2以上を示すことがわかる。 (Example 2)
The first clotting time having a final HBR concentration as shown in Table 2 was obtained by using thrombocheck APTT-SLA (manufactured by Sysmex Corporation), which is a commercially available APTT reagent having high sensitivity to LA, and adding HBR thereto. A measurement reagent was prepared. The HBR of 0 μg corresponds to the second clotting time measuring reagent containing no HBR.
As a measurement sample, 50 μL of a 1: 1 mixture of a specimen and normal plasma was used. Coagulation time measuring reagents having different HBR-containing concentrations were added to the measurement samples, and the coagulation times of the respective coagulation time measuring reagents were determined.
The measurement was performed twice using a fully automatic blood coagulation analyzer Coagrex 800 (manufactured by Shimadzu Corporation). The results are shown in Table 2 and FIG. For the obtained clotting time, the inhibition ratio of the clotting time (corresponding to the first clotting time) at each HBR concentration to the clotting time (corresponding to 2 clotting times) when the HBR concentration is 0 μg is represented by the following formula. Table 3 shows the inhibition ratio calculated based on the above.
Inhibition ratio = 1− (second clotting time−first clotting time) / first clotting time

From Table 2 and FIG. 1, it can be seen that in the human LA-containing plasma, the coagulation time is reduced in a HBR concentration-dependent manner, and the reactivity between LA and the non-human-derived antibody (HBR) is quantitatively performed. On the other hand, for normal plasma, heparin plasma, warfarin plasma, factor VIII-deficient plasma, and factor IX-deficient plasma, no significant difference in coagulation time was observed due to the difference in the concentration of HBR added. From FIG. 1 and Table 3, it can be seen that the inhibition ratio is 1.2 or more when the HBR is 5 μg or more (0.1 mg / mL of HBR to the sample) with respect to the 50 μL sample of the LA-positive sample.

表４、５より、非ヒトほ乳類由来血清については、LA陽性患者検体Ｎｏ．１、２のいずれも１以上の阻害比率を示したが、トリ由来血清では感度の悪い検体Ｎｏ．１に対して阻害比率が１以下であった。 (Example 3)
The relationship between the type of vertebrate from which it was derived and its affinity for human LA was examined.
In Example 2, a first clotting time measuring reagent containing 2 mg / mL of various vertebrate sera shown in Table 4 in place of HBR was prepared, and the first clotting time measuring reagent and the 5-fold diluted serum were used. The mixture was mixed at a ratio of 9: 1. As samples, plasma of two different LA-positive patients was used. GIBCO 38N2351 was used as bovine serum, GIBCO 35K3466 column purified product as bird-derived serum, GIBCO 1910DM as mouse-derived serum, and GIBCO 9M0644 as horse serum.
In the same manner as in Example 2, the coagulation time was measured using a fully automatic blood coagulation analyzer Coagrex 800 (manufactured by Shimadzu Corporation). Table 4 shows the results. As a control, the second clotting time when a second clotting time measuring reagent not containing vertebrate serum is used is shown. Table 5 shows the inhibition ratio calculated based on the above formula when various antibodies were used.

From Tables 4 and 5, it can be seen that for non-human mammal-derived serum, LA-positive patient sample No. 1 and 2 all showed an inhibition ratio of 1 or more, but in the case of avian-derived serum, sample No. The inhibition ratio was 1 or less with respect to 1.

ヒトLA含有血漿では、吸光度は1.308〜3.452であったが、正常血漿、ヘパリン血漿、ワーファリン血漿、第VIII因子欠損血漿及び第IX因子欠損血漿については、吸光度は0.084〜0.403の範囲であった。このことより、HBRが抗リン脂質抗体、特にLAに特異的に結合していることが確認できる。従って、吸光度とLA量の関係を利用することにより、LA量を定量することが可能となる。 (Example 4) Immunological measuring method using non-human derived antibody (HBR) (measuring method of LA by ELISA system)
Diluted with HBR (Scantibodies Inc.) 0.1% NaN ₃ · 0.1 M phosphate buffer (pH 7.5), was prepared immunoassay reagent for HBR concentration 20 [mu] g / mL. 100 μL / well of this reagent was added to each well of a 96-well microplate, and sensitized overnight in a refrigerator. Thereafter, the plate was washed with a washing solution (0.05% Tween 20 · PBS (pH 7.0)), and 50 mM PBS (pH 7.4) containing 1% bovine serum albumin (BSA) was added. 100 μL of antiphospholipid antibody syndrome (APS) patient or LA-positive patient plasma diluted 20-fold with 50 mM PBS (pH 7.4) containing 1% BSA was added to the HBR-immobilized plate, and the mixture was added at room temperature for 30 minutes. After reacting for 1 minute, the specimen was bound to HBR. After washing, HBR conjugated with biotin (0.1 μg / mL) was added, and further reacted at room temperature for 30 minutes to generate an HBR-analyte-biotin-conjugated HBR complex. After removing the biotin-bound HBR that did not form a complex and washing, 100 μL of streptavidin-labeled peroxidase (10 mU / mL) was added and reacted at room temperature for 30 minutes to label the biotin-bound HBR with an enzyme. After removing peroxidase that did not bind to biotin HBR, washing was performed, and an O-phenylenediamine (OPD) substrate solution was added. After reaction at room temperature for 15 minutes, 100 μL of a reaction stop solution (2 N sulfuric acid) was added. Colorimetry was performed with a spectrophotometer (main wavelength: 492 nm, sub-wavelength: 690 nm). Table 6 shows the results.

The absorbance of human LA-containing plasma was 1.308 to 3.452, while the absorbance of normal plasma, heparin plasma, warfarin plasma, factor VIII-deficient plasma, and factor IX-deficient plasma was in the range of 0.084 to 0.403. This confirms that HBR specifically binds to anti-phospholipid antibodies, particularly LA. Therefore, the amount of LA can be quantified by utilizing the relationship between the absorbance and the amount of LA.

  Anti-phospholipid antibody, especially LA, which is the causative agent of anti-phospholipid antibody syndrome, specifically binds to non-human-derived antibodies, etc., and blocks the binding between the anti-phospholipid antibody and the phospholipid contained in the specimen It is possible to quantify anti-phospholipid antibodies in anticophospholipid antibodies, specifically, clotting time measurement reagents, reagent kits, and samples that can detect LA with high accuracy. Thus, it has become possible to provide a method for performing specific measurement.

9 is a graph showing the measurement results of Example 2 (the relationship between the HBR concentration of each sample and the coagulation time).

Claims (14)

A coagulation composition; and at least one selected from the group consisting of antibodies, serum, plasma, and immunoglobulins of vertebrates other than humans;
A clotting time measuring reagent comprising: The reagent according to claim 1, wherein the vertebrate is a mammal excluding humans and pigs. The coagulation composition,
Calcium ions;
A phospholipid; and at least one selected from the group consisting of an activator, snake venom, and tissue factor;
The reagent for measuring a coagulation time according to claim 1, comprising: The reagent according to claim 1, wherein the activator is one selected from the group consisting of kaolin, celite, silica, and ellagic acid. The coagulation time measurement reagent comprises a first partial reagent and a second partial reagent,
The first partial reagent includes:
At least one selected from the group consisting of antibodies, serum, plasma and immunoglobulins of vertebrates other than humans;
A phospholipid; and at least one selected from the group consisting of an activator, a snake venom, and a tissue factor;
The reagent for measuring a coagulation time according to claim 3, which contains calcium ions. A coagulation system composition; and a first coagulation time measuring reagent comprising at least one selected from the group consisting of vertebrate antibodies other than human, serum, plasma, and immunoglobulin;
A reagent kit for detecting an anti-phospholipid antibody comprising a second coagulation time measuring reagent containing a coagulation composition but not containing any of vertebrate antibodies other than human, serum, plasma and immunoglobulin. The detection reagent kit according to claim 6, wherein the composition of the coagulation composition contained in the first coagulation time measurement reagent and the composition of the coagulation composition contained in the second coagulation time measurement reagent are the same. The detection reagent kit according to claim 6, wherein the antiphospholipid antibody is human lupus anticoagulant. The first clotting time measurement reagent comprises a first partial reagent and a second partial reagent,
The first partial reagent includes:
At least one selected from the group consisting of antibodies, serum, plasma and immunoglobulins of vertebrates other than humans;
A phospholipid; and at least one selected from the group consisting of an activator, a snake venom, and a tissue factor;
Contains calcium ions,
The second clotting time measuring reagent comprises a third partial reagent and a fourth partial reagent,
In the third partial reagent,
A phospholipid; and at least one selected from the group consisting of an activator, snake venom, and tissue factor;
The detection reagent kit according to claim 6, which contains calcium ions. Contacting the sample with each of a first clotting time measuring reagent and a second clotting time measuring reagent; and a clotting time (first clotting time) when mixed with the first clotting time measuring reagent; A method for detecting an anti-phospholipid antibody, comprising a step of comparing a clotting time (second clotting time) when mixed. 11. The method according to claim 10, further comprising the step of determining that the anti-phospholipid antibody is contained in the test substance if there is a significant difference between the first clotting time and the second clotting time as a result of the comparison. The detection method described in 1. When the value of the inhibition ratio calculated from the measured first clotting time and the second clotting time based on the following equation is 1.2 or more, it is considered that the anti-phospholipid antibody is contained in the test sample. The detection method according to claim 10, further comprising a determining step.
Inhibition ratio = 1− (second clotting time−first clotting time) / first clotting time The method according to claim 10, wherein the first clotting time measurement reagent uses at least a total amount of antibodies, serum, plasma, and immunoglobulins of vertebrates other than humans of 0.1 mg to 50 mg per 1 mL of a test substance. Detection method. Contacting a labeled vertebrate antibody, serum, plasma or immunoglobulin (such as a labeled non-human-derived antibody) with a test subject, and a non-human vertebrate antibody; A method for measuring an anti-phospholipid antibody, comprising a step of measuring a conjugate with the above by measuring the label.

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