JP2004132816A - Method for determining blood anticoagulation factor antibody by enzyme-linked immunosorbent assay - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining a blood anticoagulation factor antibody capable of highly sensitively determining the blood anticoagulation factor antibody, by enzyme-linked immunosorbent assay, from a specimen in which a blood coagulation factor and the blood anticoagulation factor antibody coexist. <P>SOLUTION: By adding an enzyme 4, which specifically breaks down the blood coagulation factor 1 in the process of blood coagulation reaction, to the specimen at least at either point in time before or after the treatment of bonding the blood anticoagulation factor antibody 2 to a primary antibody 5 by the enzyme-linked immunosorbent assay, the blood anticoagulation factor antibody 2 is bonded to a secondary antibody 7 and determined without the need for performing the treatment of deactivating a specific catabolic enzyme 4 or being affected by the steric hindrance of the blood coagulation factor 1 afterwards. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for quantifying an anticoagulation factor antibody by enzyme immunoassay, and in particular, under the coexistence of blood coagulation factor and anticoagulation factor antibody, for example, blood coagulation factor in immunoaffinity chromatography. Suitable for quantifying anti-coagulant factor antibodies from eluates in which anti-coagulant factor antibodies are slightly mixed in the elution step or blood samples of patients who have produced autoantibodies to the administered blood coagulation factors The present invention relates to a method for quantifying anti-coagulation factor antibodies by various enzyme immunoassays.
[0002]
[Prior art]
Conventionally, there is known a disease in which blood is difficult to coagulate due to a lack of blood coagulation factors and causes bleeding disorders. For example, hemophilia is congenitally blood coagulation factor VIII or blood coagulation factor. There are also known diseases in which factor IX is deficient, factor V deficiency in which blood coagulation factor V is deficient, factor XIII deficiency in which blood coagulation factor XIII is deficient, and the like. Treatment of these diseases requires supplementation with blood clotting factors, and fresh frozen plasma or blood clotting factor concentrates are administered according to the symptoms. Among patients with congenital bleeding, the most common in Japan is hemophilia A patients who are quantitatively or qualitatively deficient in blood coagulation factor VIII. In this hemophilia A patient, a blood coagulation factor VIII concentrated preparation in which only blood coagulation factor VIII is separated and purified has been developed and administered by self-injection therapy.
[0003]
The blood coagulation factor VIII concentrated preparation is usually produced through purification treatment using ion exchange chromatography, virus removal membrane or immunoaffinity chromatography using cryoprecipitate as a raw material. Immunoaffinity chromatography is a purification method that utilizes an antigen-antibody reaction, and has high specificity and can efficiently remove impurities. Therefore, immunoaffinity chromatography is widely used for high-purity protein purification. The main steps of this immunoaffinity chromatography will be described with reference to FIG. First, in order to purify blood coagulation factor VIII 1, anti-blood coagulation factor VIII mouse monoclonal antibody (2) (hereinafter referred to as “mouse IgG (2)”) that specifically reacts with this is shared by gel carrier 9 They are combined and packed in the column 10 (FIG. 9A).
[0004]
Subsequently, by putting the cryolysis solution 11 into the column 10, blood coagulation factor VIII 1 and mouse IgG (2) are specifically bound by antigen-antibody reaction (FIG. 9 (b)). Thereafter, the impurities 12 are removed with a washing solution (FIG. 9 (c)), and the eluate is poured to weaken the antigen-antibody reaction between mouse IgG (2) and blood coagulation factor VIII 1, and blood coagulation factor VIII 1 is removed. Elute (FIG. 9 (d)). Through these steps, blood coagulation factor VIII 1 is purified with high purity.
[0005]
However, in the immunoaffinity chromatography described above, as shown in FIG. 9 (d), mouse IgG (2) is immobilized on the gel carrier 9 by the strongest bond among chemical bonds called covalent bonds. In addition, when the blood coagulation factor VIII 1 is eluted, the mouse IgG (2) may be peeled off from the gel carrier 9 in a small amount and mixed into the eluate. Thus, depending on the amount of mouse IgG (2) mixed, there is a risk of affecting the quality of the blood coagulation factor VIII concentrated preparation, so it is necessary to accurately grasp the amount of contamination.
[0006]
Then, in order to measure how much mouse IgG (2) is mixed in the blood coagulation factor VIII concentrated preparation, for example, an enzyme-linked immunosorbent assay (also referred to as ELISA) can be mentioned. This enzyme immunoassay is a method of measuring the amount of antigen or antibody by developing a chromogenic substrate using a secondary antibody labeled with an enzyme, and it can be measured with a spectrophotometer and has excellent detection sensitivity. It is considered preferable.
[0007]
[Patent Document 1]
JP 2001-21559 A
[0008]
[Problems to be solved by the invention]
However, since blood coagulation factor VIII and mouse IgG (2) are in an antigen-antibody relationship, they both form an immune complex, and the molecular weight of blood coagulation factor VIII 1 is also high, resulting in enzyme immunization. In the measurement method, there is a problem that the binding of the mouse IgG (2) to the primary antibody or the binding of the secondary antibody is inhibited, and the mouse IgG (2) cannot be quantified with sufficient sensitivity.
[0009]
This will be described in more detail with reference to FIG. 10. In the enzyme immunoassay, first, as shown in FIG. 10 (a), an anti-mouse which is an antibody against mouse IgG (2) is placed on a plastic plate 6. IgG (5) is bound, and a solution in which mouse IgG (2) and blood coagulation factor VIII 1 coexist is added thereto. Then, as shown in FIG. 10 (b), mouse IgG (2) binds to anti-mouse IgG (5). After making this react well, enzyme-labeled anti-mouse IgG (7) as a secondary antibody labeled with an enzyme is added. Here, when an HRP-labeled anti-mouse IgG (7) labeled with an enzyme called horseradish peroxidase (HRP) is added, the HRP-labeled anti-mouse IgG (7) binds to mouse IgG (2) (FIG. 10 (c)). Then, it is washed well, a substrate is added, and color is developed with HRP (FIG. 10 (d)). As a result, color development of an amount corresponding to the concentration of mouse IgG (2) in the solution appears, enabling quantitative analysis.
[0010]
However, as shown in FIG. 10 (c), blood coagulation factor VIII and mouse IgG (2) in an antigen-antibody relationship form an immune complex, and the molecular weight of the blood coagulation factor VIII 1 is Since it is as large as 300 KDa, when binding of mouse IgG (2) to anti-mouse IgG (5) or secondary antibody HRP-labeled anti-mouse IgG (7) was added, HRP-labeled anti-mouse IgG (7) and mouse There is a problem in that binding to IgG (2) is three-dimensionally inhibited by blood coagulation factor VIII 1 and cannot be bound and washed away.
[0011]
As a method for solving such a problem, it is conceivable to use a plurality of monoclonal antibodies having different antigenic determinants (epitopes) as primary antibodies and secondary antibodies. For example, when a monoclonal antibody that reacts with both an antigen and an antibody is a primary antibody and a secondary antibody, the immune complex is captured by the primary antibody, and the secondary antibody is also a mixture of antibodies against both. Detection by enzyme immunoassay is possible without being obstructed by the three-dimensional structure of the body.
[0012]
However, such a method may be used for the purpose of detecting an immune complex, but is insufficient for quantifying only one of the immune complexes as in the problem of the present invention. In particular, it is not suitable for quantifying the minute amount of mouse IgG (2) as described above. Another possible solution is to perform an enzyme immunoassay under conditions that dissociate the binding of the immune complex.
[0013]
However, under conditions that weaken the binding power of the immune complex, the binding power between the primary antibodies of mouse IgG (2) and anti-mouse IgG (5), and in some cases mouse IgG (2) and HRP-labeled anti-mouse Since the binding between the secondary antibody and IgG (7) also becomes weak, the sensitivity of the entire measurement system is lowered.
[0014]
Furthermore, in JP-A-2001-21559, a protein-degrading protein is added to a protein-binding glycated protein to decompose non-specifically an antigenic protein to expose a protein glycation reaction product. An invention is described in which the proteolytic enzyme is inactivated by immersing it in boiling water at a temperature not lower than ° C.
[0015]
However, since such a method is a general protein denaturation method such as addition of a protein denaturant or heating, mouse IgG (2) to be quantified may be denatured and accurate quantification cannot be performed. there is a possibility.
[0016]
The present invention has been made in order to solve such problems, and the anticoagulation factor antibody is quantified by enzyme immunoassay for a sample in which a blood coagulation factor and an anticoagulation factor antibody coexist. In this case, only the blood coagulation factor that inhibits the binding of the anti-blood coagulation factor antibody to the primary antibody or the secondary antibody is specifically decomposed without affecting the properties of the anti-blood coagulation factor antibody. An object of the present invention is to provide a method for quantifying an anti-coagulation factor antibody by an enzyme immunoassay that can be quantified with high sensitivity.
[0017]
[Means for Solving the Problems]
The anti-blood coagulation factor antibody quantification method by the enzyme immunoassay method of the present invention is characterized in that the anti-coagulation factor antibody and the anti-blood coagulation factor antibody specifically reacting with the blood coagulation factor coexist with the anti-coagulation factor antibody by the enzyme immunoassay. A method for quantifying a blood coagulation factor antibody, wherein the anticoagulation factor antibody is bound to a primary antibody by the enzyme immunoassay before the treatment and at least one time point after the treatment. In the process, an enzyme that specifically degrades the blood coagulation factor is added to specifically degrade the blood coagulation factor in the specimen, and then the anti-degradation enzyme is deactivated without performing the treatment for inactivating the specific degradation enzyme. The blood coagulation factor antibody is quantified by binding to the secondary antibody.
[0018]
By adopting such a configuration, an enzyme that specifically decomposes by activating a blood coagulation factor in the blood coagulation reaction process specifically decomposes only the blood coagulation factor, and the target to be measured The anti-coagulation factor antibody is not affected by the properties of the anti-coagulation factor antibody, so that the degradation treatment is performed before or after the primary antibody binding and / or after the treatment. It has the effect of preventing the binding between the primary antibody and the secondary antibody from being inhibited by blood coagulation factors.
[0019]
In the present invention, the treatment for specifically degrading a blood coagulation factor in a specimen with a specific degrading enzyme is preferably performed under conditions of in-vivo temperature. As a result, the degradation process of the blood coagulation factor proceeds under the condition that the specific degrading enzyme exhibits the original function in the blood coagulation reaction, that is, the temperature in the living body. Play.
[0020]
Furthermore, in the present invention, when the blood coagulation factor is any one of blood coagulation factor VIII, blood coagulation factor V, and blood coagulation factor XIII, the enzyme for the specific degradation treatment uses thrombin. It is like that. This reliably degrades blood coagulation factor VIII, blood coagulation factor V, and blood coagulation factor XIII, which inhibit the quantification of anti-coagulation factor antibodies.
[0021]
In the present invention, it is preferable to add thrombin having a titer of 0.00125 U or more to a blood coagulation factor having a titer of 25 U. This determines the concentration and amount of thrombin appropriate for degrading the blood coagulation factors VIII, VIII, and XIII.
[0022]
Furthermore, in the present invention, it is preferable that the decomposition treatment performed by adding thrombin to the specimen is allowed to react for 20 minutes or longer. Thereby, it is possible to secure a time during which thrombin can sufficiently decompose each blood coagulation factor of blood coagulation factor VIII, blood coagulation factor V, and blood coagulation factor XIII.
[0023]
The present invention is also characterized in that the anti-blood coagulation factor VIII antibody is quantified by enzyme immunoassay from a sample in which blood coagulation factor VIII and anti-blood coagulation factor VIII antibody coexist. Before the treatment for binding the coagulation factor VIII antibody to the primary antibody by the enzyme immunoassay, thrombin with a titer of 0.00125 U or more against a blood coagulation factor VIII with a titer of 25 U is added to the specimen. The blood coagulation factor VIII is specifically decomposed by incubating for 20 minutes or more, and then the anti-coagulation factor VIII antibody is bound to the primary antibody and the secondary antibody without inactivating the thrombin. The point is to quantify.
[0024]
By adopting such a configuration, thrombin that activates blood coagulation factor VIII in the blood coagulation reaction process specifically decomposes only blood coagulation factor VIII, and the anticoagulation subject to be measured is measured. Since the properties of the factor VIII antibody are not affected, such a degradation treatment is performed before the primary antibody binding treatment, whereby the anti-blood coagulation factor VIII antibody can be bound to the primary antibody and the secondary antibody. There is an effect that accurate quantification can be performed without being inhibited by blood coagulation factor VIII.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, an example of an embodiment of a method for quantifying an anti-blood coagulation factor antibody by an enzyme immunoassay according to the present invention will be described with reference to the drawings.
[0026]
The anti-blood coagulation factor antibody quantification method according to the present embodiment uses the enzyme immunoassay method from among samples coexisting with a blood coagulation factor and an anti-blood coagulation factor antibody that specifically reacts with the antigen. This is a quantitative analysis of coagulation factor antibodies with high sensitivity. Specifically, an enzyme that specifically degrades the blood coagulation factor at least one time before and after the treatment of binding the anti-blood coagulation factor antibody to the primary antibody by enzyme immunoassay, in this embodiment, Without adding the enzyme that activates the blood coagulation factor in each step of the blood coagulation reaction (also called cascade), specifically decomposing the blood coagulation factor, and then inactivating the decomposing enzyme The anti-blood clotting factor antibody is quantified by binding to a secondary antibody.
[0027]
The blood coagulation factor decomposition treatment with a specific decomposing enzyme must be performed at least before binding the anti-blood coagulation factor antibody to the secondary antibody in order to remove the three-dimensional structural disorder. Before or after the treatment with which the antibody is bound can be selected, but in this embodiment, it is preferably performed before the binding with the primary antibody so that the antibody can be efficiently bound. Furthermore, in addition to the decomposition treatment before the binding with the primary antibody, the decomposition treatment is more preferably performed after the binding with the primary antibody for the purpose of decomposing the blood coagulation factor remaining without being decomposed.
[0028]
Here, blood coagulation factor VIII purified as the most typical blood coagulation factor VIII preparation among blood coagulation factor preparations will be described as a representative example of this embodiment with reference to FIGS. To do.
[0029]
The specimen used in the present embodiment is a blood coagulation factor VIII concentrated preparation. In the production process, blood coagulation factor VIII 1 is adsorbed to mouse IgG (2) by immunoaffinity chromatography, and impurities are washed away. Purified.
[0030]
Then, using thrombin, which is an enzyme that specifically degrades blood coagulation factor VIII, the quantification method of the present embodiment mainly includes the thrombin before quantification by enzyme immunoassay as shown in FIG. 4 is added to the first thrombin treatment (treatment S1) for decomposing blood coagulation factor VIII 1 and the thrombin-treated specimen is placed in a plate 6 to which anti-mouse IgG (5) is bound, and then mouse IgG (2 ) And anti-mouse IgG (5) are combined with a primary antibody (treatment S2), and after this primary antibody treatment, thrombin 4 is added again to decompose the remaining blood coagulation factor VIII 1 (treatment S3). ) And a secondary anti-antibody that binds to mouse IgG (2) by adding HRP-labeled anti-mouse IgG (7) labeled with an enzyme to the sample after this thrombin treatment Treatment and (process S4) are sequentially performed.
[0031]
Each of these processes will be described in more detail with reference to the schematic diagrams of FIGS.
[0032]
In the first thrombin treatment, 0.00125 U or more of thrombin 4 is added to 25 U of blood coagulation factor VIII 1. For example, in the case of a sample containing 0.5 ml of 50 U / ml blood coagulation factor VIII 1, it is necessary to add 0.025 ml or more of 0.05 U / ml thrombin 4.
[0033]
The temperature condition for the reaction is an enzyme that acts so that thrombin 4 activates blood coagulation factor VIII in vivo, and thus the in vivo temperature is preferably about 37 ° C. The time condition is about 10 to 30 minutes, preferably 20 minutes or more, and more preferably 30 minutes or more because blood coagulation factor VIII 1 can be more reliably decomposed. This reaction is carried out on a heat block or a water bath suitable for temperature adjustment.
[0034]
By such first thrombin treatment, thrombin 4 is an activating enzyme for blood coagulation factor VIII 1, and thus specifically degrades blood coagulation factor VIII 1 as shown in FIG.
[0035]
Next, as shown in FIGS. 3A to 3C, in the primary antibody treatment, the thrombin-treated specimen is placed in a 96-well plate 6 coated with anti-mouse IgG (5) for about 2 hours at room temperature. Incubate. At this time, since blood coagulation factor VIII 1 is finely decomposed, mouse IgG (2) is likely to bind to anti-mouse IgG (5). After incubating, wash well with washing solution.
[0036]
Subsequently, in the second thrombin treatment, the thrombin 4 is again added to the washed plate 6 to decompose the remaining blood coagulation factor VIII. This ensures and facilitates the binding of HRP-labeled anti-mouse IgG (7) in the subsequent secondary antibody treatment. In the second thrombin treatment, a thicker amount of thrombin 4 is added than in the first thrombin treatment.
[0037]
Next, in the secondary antibody treatment, the second thrombin treatment is followed by washing with a washing solution, and HRP-labeled anti-mouse IgG (7) is added and incubated at room temperature for about 2 hours. Thereby, as shown in FIG. 3D, the HRP-labeled anti-mouse IgG (7) is bound to mouse IgG (2) without being inhibited by blood coagulation factor VIII. And after washing | cleaning with a washing | cleaning liquid, a substrate is added, the substrate is made to color with HRP, and an absorbance etc. are measured.
[0038]
In place of the HRP-labeled anti-mouse IgG (7) in the secondary antibody treatment described above, as shown in FIG. 4, a biotinylated anti-mouse IgG (7a) is added to the specimen after the second thrombin treatment, and the secondary antibody. Processing may be performed, and further avidinized HRP8 may be added. Since four avidinylated HRP8s bind to one biotin, the degree of color development becomes high and detection is possible with high sensitivity even when mouse IgG (2) is at a low concentration.
[0039]
Next, in order to confirm the effect of the thrombin treatment in the present embodiment described above, the following experiment was conducted to measure the absorbance and the like.
[0040]
【Example】
First, as the first thrombin treatment, 0.5 μ / ml human thrombin 4 (manufactured by Mitsubishi Pharma) was added in an amount of 25 μl to 500 μl of the blood coagulation factor VIII concentrated preparation having a titer of 100 U / ml, and 37 ° C. Incubated for 30 minutes.
[0041]
Subsequently, 100 μl of each thrombin-treated specimen was placed in a 96-well plate 6 coated with goat anti-mouse IgG (5) and incubated at room temperature for 2 hours for primary antibody treatment.
[0042]
Next, after washing 5 times with a washing solution (0.05% Tween-PBS), 100 μl of 5 U / ml thrombin 4 was added for the purpose of decomposing the remaining blood coagulation factor VIII, and then at 37 ° C. for 30 minutes. The second thrombin treatment was performed by incubating.
[0043]
Then, after washing 5 times with a washing solution, 100 μl of biotinylated anti-mouse IgG (7a) (manufactured by Seikagaku Corporation) diluted to 1 / 60,000 was added and incubated at room temperature for 2 hours. And after washing | cleaning 5 times with a washing | cleaning liquid, 100 microliters of avidinized HRP (made by GIBCO) diluted 1/1000 was added at a time, and it incubated at room temperature for 1 hour. Finally, after washing 5 times with the washing solution, 100 μl of 3,3 ′, 5,5′-tetramethylbenzidine (TMB) (manufactured by BIO-RAD) was added 10 μl after 10 minutes, and the absorbance was measured at 450 nm. . For the quantification of mouse IgG (2), the same operation was first performed for mouse IgG (2) of known concentration, the absorbance for each concentration was determined to create a standard line, and the absorbance of mouse IgG (2) was determined from the absorbance of this standard line. The concentration was calculated. These operations were performed using an automatic ELISA apparatus (Behring ELISA Processor III).
[0044]
After performing the treatment in the present embodiment as described above, in order to confirm the effect of thrombin 4, a standard solution containing only a sample that has undergone thrombin treatment, an untreated sample, and mouse IgG (2) to which no sample is added. The absorbance with respect to the concentration of mouse IgG (2) was measured. In addition, in order to investigate the influence of thrombin 4 on blood coagulation factor VIII 1 and mouse IgG (2), the properties were confirmed by polyacrylamide gel electrophoresis (Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis: hereinafter referred to as SDS-PAGE). did.
[0045]
"Confirmation of thrombin treatment effect"
When measuring the absorbance with respect to the mouse IgG concentration, the sample was first diluted with a diluting solution so that the concentration of mouse IgG (2) was 0 to 0.5 ng / ml as a standard solution serving as a reference for comparison. In addition, as a sample to be treated with thrombin and a sample not to be treated with thrombin, mouse IgG (2) was added to a blood coagulation factor VIII concentrated preparation so that the concentration of mouse IgG (2) was 0 to 0.5 ng / ml. Added to. At this time, the amount of mouse IgG (2) mixed in the blood coagulation factor VIII concentrated preparation is not included in the calculation of the concentration.
[0046]
FIG. 5 and FIG. 6 show the results of measuring the absorbance by applying light having a wavelength of 450 nm to the standard solution of each concentration of mouse IgG (2) and the thrombin-treated and untreated specimens. FIG. 6 is a graph showing the numerical values of FIG. However, in these figures, 1 / 10,000 biotinylated anti-mouse IgG (7a) is used for color development. Absorbance A is log ₁₀ (I ₀ / I), I ₀ Is the intensity of the incident light, and I is the intensity of the transmitted light. According to these results, the sample not treated with thrombin showed a lower absorbance at a mouse IgG concentration of 0.05 ng / ml or higher than the standard straight line, and showed an absorbance of the mouse IgG concentration or lower added. It was. As pointed out in the conventional problem, mouse IgG (2) and blood coagulation factor VIII 1 form an immune complex, so that mouse IgG (2) and anti-mouse IgG (primary antibody) This is a result of inhibition of binding to 5) or binding to the secondary antibody, biotinylated anti-mouse IgG (7a).
[0047]
On the other hand, the absorbance of the sample treated with thrombin has the same slope as the standard straight line, indicating that mouse IgG (2) can be accurately quantified.
[0048]
"Influence of thrombin on blood coagulation factor VIII and mouse IgG (2)" Next, SDS-PAGE was performed to examine the effect of thrombin 4 on blood coagulation factor VIII 1 and mouse IgG (2). . The SDS-PAGE sample was prepared by mixing SDS sample buffer and protein solution 1: 1 and then heating at 100 ° C. for 2 minutes. For SDS-PAGE, a gel composed of a gel for electrophoresis and a gel for concentration and a migration buffer were used, and the electrophoresis was performed at a constant current of 10 mA. As the gel for electrophoresis, a gel containing 8.5% acrylamide was used in the case of blood coagulation factor VIII1, and a gel containing 10% acrylamide was used in the case of mouse IgG (2).
[0049]
Then, after completion of SDS-PAGE, the protein on the gel was transferred to a nitrocellulose membrane using a semi-dry blotting apparatus. Transfer is 1.5 mA / cm ² The nitrocellulose membrane was blocked with 3% bovine serum albumin and then reacted with anti-human factor VIII mouse monoclonal antibody as the primary antibody and biotinylated goat anti-mouse IgG as the secondary antibody. Then, after reacting with avidinized alkaline phosphatase (manufactured by Harlan), a chromogenic substrate was added to detect blood coagulation factor VIII. For detection of mouse IgG (2), biotinylated goat anti-mouse IgG, which is the secondary antibody used in the detection of blood coagulation factor VIII 1, was used as the primary antibody, and the subsequent steps were the same as for blood coagulation factor VIII 1. The procedure was performed.
[0050]
FIG. 7 shows the change over time when blood coagulation factor VIII 1 was treated with thrombin. As shown in FIG. 7, when the elapsed time after the introduction of thrombin 4 is 0 minutes, that is, blood coagulation factor VIII 1 when thrombin is not treated, the heavy chain is detected as a band having a molecular weight of 200 KDa or more. The light chain has been detected as a double chain having a molecular weight of 80 KDa. Thereafter, the heavy chain and the light chain are degraded with the passage of time after the thrombin treatment, and after 20 minutes, most of the molecules are detected as short bands of 45 KDa or less, and after 30 minutes, most of them are 45 KDa. It was detected as a band and fragmented. Although not shown in the figure, the molecules were not further decomposed even after 30 minutes or longer.
[0051]
Meanwhile, the effect of thrombin treatment on mouse IgG (2) was examined. In FIG. 8, non-reduced mouse IgG and reduced mouse IgG were prepared with and without thrombin treatment, and their molecular weights were detected by SDS-PAGE, respectively. The reduction treatment is a treatment for cleaving the disulfide bond (SS bond) between the light chain and heavy chain of mouse IgG.
[0052]
Thus, as shown in FIG. 8, both lane 1 and lane 2 are non-reduced mouse IgG (2), lane 1 is thrombin-treated mouse IgG (2), and lane 2 is unthrombin-treated. Mouse IgG (2). It can be seen that all samples have a molecular weight of 150 KDa and are not affected by the degradation by thrombin treatment.
[0053]
Similarly, lane 3 and lane 4 are both reduced mouse IgG (2), lane 3 is thrombin-treated mouse IgG (2), and lane 4 is thrombin-untreated mouse IgG (2). is there. In both cases, a 50 KDa heavy chain and a 25 KDa light chain appear and are not different.
[0054]
From these results, it was confirmed that there was no change in the secondary structure of mouse IgG (2) and that mouse IgG (2) was not degraded by thrombin 4.
[0055]
Next, the detection limit in the quantification method of the present embodiment was calculated according to the following formula 1.
Absorbance detection limit = μB + (3.3 × σB) Equation 1
μB = average response obtained from blank or test substance
σB = standard deviation of blank response
The average of the blank value in 20 tests performed for collecting validation data is 0.075, and the standard deviation is 0.017. Substituting these numbers into the above formula,
Absorbance detection limit = 0.075 + (3.3 × 0.017) = 0.131
[0056]
Therefore, in the quantification method of this embodiment, it is possible to measure a specimen having an absorbance of 0.131 or more. According to the data collected this time, when the mouse IgG concentration is 0.05 ng / ml or more, the absorbance is 0.131 or more. As a result, the quantification limit of the enzyme immunoassay with thrombin treatment was 0.05 ng / ml.
[0057]
As described above, according to the above-described embodiment, when a blood coagulation factor and an anti-blood coagulation factor antibody coexist, when the anti-blood coagulation factor antibody is quantified by an enzyme immunoassay, the binding to the secondary antibody is performed. By specifically decomposing only the blood coagulation factor that inhibits the above, it is possible to quantify with high sensitivity without affecting the properties of the anti-blood coagulation factor antibody.
[0058]
When protein purification using immunoaffinity chromatography is performed, the antibody may be detached from the gel carrier and become an impurity for the target protein. When measuring the antibody concentration, the target protein that serves as the antigen is present. Therefore, in the sandwich assay method such as enzyme immunoassay, the binding of the secondary antibody is inhibited if the target protein is large. , Measurement sensitivity decreases. In such a case, the sensitivity can be improved by treating the specimen with an enzyme that degrades only the target protein as in this embodiment.
[0059]
In addition, each structure of this embodiment of this invention is not restricted to what was mentioned above, It can change suitably.
[0060]
For example, in this embodiment, blood coagulation factor VIII 1 is shown as an example as a representative example of blood coagulation factor, but other blood coagulation factors such as blood coagulation factor V and blood coagulation factor XIII are shown. Furthermore, since the molecular weights are large, when quantifying the anti-blood coagulation factor V monoclonal antibody and anti-blood coagulation factor XIII monoclonal antibody, which are each antibody, in the coexistence, they are enzymes activated in the blood coagulation reaction process. By adding a specific degradation treatment with thrombin 4, it is possible to perform more accurate quantitative analysis. Similarly, the blood coagulation factor IX concentration preparation, which is a concentrate for hemophilia B patients, is also used in the blood coagulation reaction process in the blood coagulation reaction process when the mixed anti-coagulation factor IX monoclonal antibody is quantified. It is considered that more accurate quantitative analysis is possible by adding blood coagulation factor XI, which is an activator of the factor, and specifically decomposing it.
[0061]
Moreover, although mouse IgG (2) is used as an antibody for each blood coagulation factor, the present invention is not limited to this and can be applied to a polyclonal antibody.
[0062]
Furthermore, in the present embodiment, a blood coagulation concentrated preparation purified by immunoaffinity chromatography is exemplified, but the present invention is not limited to this. For example, a disease that produces an autoantibody against blood coagulation factor VIII 1 The antibody can be used for quantification of the antibody, or not only for the relationship between the antigen and antibody, but also for the quantification of one of them under conditions where two substances having affinity exist.
[0063]
【The invention's effect】
As described above, according to the present invention, when an anti-blood coagulation factor antibody is quantified by an enzyme immunoassay for a sample in which a blood coagulation factor and an anti-blood coagulation factor antibody coexist, The effect of specifically degrading only the blood coagulation factor that inhibits the binding to the primary antibody or the secondary antibody, and can be quantified with high sensitivity without affecting the properties of the anti-coagulation factor antibody Play.
[Brief description of the drawings]
FIG. 1 is a flow chart showing the main steps of a method for quantifying an anticoagulant factor antibody by enzyme immunoassay according to the present invention.
FIG. 2 is a schematic diagram showing a state of a specimen when first thrombin treatment is performed in the present embodiment.
FIG. 3 is a schematic diagram showing a method for quantifying an anti-blood coagulation factor antibody by an enzyme immunoassay using an HRP-labeled secondary antibody in the present embodiment, and (a) is a diagram in which anti-mouse IgG is bound to a plate. The figure which shows a state, (b) is a figure which shows the state which injected | thrown-in the sample after thrombin processing, (c) is a figure which performs a primary antibody binding process, and shows after washing | cleaning, (d) is an HRP labeled anti-antibody as a secondary antibody. It is a figure which shows the state couple | bonded with mouse | mouth IgG.
FIG. 4 is a schematic diagram showing a method for quantifying an anti-coagulation factor antibody by an enzyme immunoassay using a biotinylated secondary antibody in the present embodiment, wherein (a) is an anti-mouse IgG bound to a plate. The figure which shows a state, (b) is the figure which shows the state which supplied the test substance after thrombin processing, (c) is a figure which performs a primary antibody binding process and after washing | cleaning, (d) is a biotinylated anti-antibody as a secondary antibody. The figure which shows the state couple | bonded with mouse | mouth IgG, (e) is a figure which shows the state which couple | bonded the avidinized HRP with biotin.
FIG. 5 is a table showing the absorbance results for each concentration of mouse IgG in the present embodiment.
6 is a graph showing the numerical values in FIG.
FIG. 7 is a graph showing changes over time in blood coagulation factor VIII by thrombin treatment, and shows the results of blotting a sample after SDS-PAGE.
FIG. 8 is a diagram showing the influence of thrombin treatment on mouse IgG, showing the result of blotting after SDS-PAGE.
9A and 9B are diagrams showing immunoaffinity chromatography steps in the process of purifying blood coagulation factor VIII, wherein FIG. 9A shows a state in which a cryolysis solution is put into a column, and FIG. 9B shows a cryolysis solution. The figure which shows the state which passed through the column and made blood coagulation factor VIII adsorb | suck to mouse | mouth IgG, (c) is a figure which shows the state which flows a washing | cleaning liquid and removes impurities, (d) is the figure which shows blood coagulation factor VIII It is a figure which shows the state which elutes.
FIG. 10 is a schematic diagram showing a method for quantifying anti-blood coagulation factor VIII mouse monoclonal antibody by a conventional enzyme immunoassay method, wherein (a) shows a state in which anti-mouse IgG is bound to a plate; (b) is a diagram showing a state in which the eluate is put into the plate and the primary antibody binding between mouse IgG and anti-mouse IgG is performed; (c) is a diagram showing a state in which HRP-labeled anti-mouse IgG is introduced as a secondary antibody; (D) is a view showing a state in which HRP-labeled anti-mouse IgG is bound to mouse IgG.
[Explanation of symbols]
1 Blood coagulation factor VIII
2 Mouse IgG
4 Thrombin
5 Anti-mouse IgG
6 plates
7 HRP labeled anti-mouse IgG
7a Biotinylated anti-mouse IgG
8 Avidinized HRP
9 Gel carrier
10 columns
11 Clio solution
12 Impurities

Claims (6)

A method of quantifying the anti-coagulation factor antibody by an enzyme immunoassay from a sample in which a coagulation factor and an anti-coagulation factor antibody that specifically reacts with an antigen-antibody coexist with the anti-coagulation factor antibody, By adding an enzyme that specifically degrades the blood coagulation factor in the blood coagulation reaction process to the sample at least at one time point before and after the treatment to bind to the primary antibody by enzyme immunoassay. The blood coagulation factor is specifically decomposed, and then the anti-coagulation factor antibody is bound to a secondary antibody and quantified without performing the treatment of inactivating the specific degrading enzyme. A method for quantifying an anti-coagulation factor antibody by enzyme immunoassay. 2. The method for quantifying an anti-coagulant factor antibody by an enzyme immunoassay method according to claim 1, wherein the treatment for decomposing the blood coagulation factor with the specific degrading enzyme is performed under conditions of in-vivo temperature. . 3. The degradation for specific degradation treatment according to claim 1 or 2, wherein the blood coagulation factor is any one of blood coagulation factor VIII, blood coagulation factor V, and blood coagulation factor XIII. A method for quantifying an anti-coagulation factor antibody by an enzyme immunoassay characterized by using thrombin as an enzyme. 4. The method for quantifying anti-blood clotting factor antibodies by enzyme immunoassay according to claim 3, wherein thrombin having a titer of 0.00125 U or more is added to a blood clotting factor having a titer of 25 U. 5. The method for quantifying an anticoagulant factor antibody by an enzyme immunoassay method according to claim 3 or 4, wherein the degradation treatment performed by adding thrombin to the specimen is allowed to react for 20 minutes or more. A method for quantifying the anti-coagulation factor VIII antibody from a sample in which blood coagulation factor VIII and anti-coagulation factor VIII antibody coexist by enzyme immunoassay, wherein the anti-coagulation factor VIII antibody is converted into the enzyme Prior to treatment with the primary antibody by immunoassay, thrombin with a titer of 0.00125 U or more against blood coagulation factor VIII with a titer of 25 U is added to the specimen and incubated for 20 minutes or more. The blood coagulation factor VIII is specifically decomposed, and then the anti-coagulation factor VIII antibody is bound to the primary antibody and the secondary antibody and quantified without inactivating the thrombin. A method for quantifying an anti-coagulation factor antibody by enzyme immunoassay.

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