JP2019028053A - New measurement method for sflt-1 (soluble type vascular endothelial growth factor-1) - Google Patents

Abstract

To provide a measuring method of a novel, simple and inexpensive, and practical sFlt-1 assay for sFlt-1 protein.SOLUTION: It is a method for measuring sFlt-1 in a sample by forming a complex between heparin and an anti sFlt-1 antibody with sFlt-1 sandwiched therebetween, and the method includes contacting the sample with a heparin or anti sFlt-1 antibody immobilized on a carrier, forming a complex between heparin or anti sFlt-1 antibody and sFlt-1 in the sample on the carrier, and detecting the presence of the complex using a labeled anti sFlt-1 antibody or anti sFlt-1 antibody and a labeled anti immunoglobulin antibody or a labeled heparin.SELECTED DRAWING: None

Description

  The present invention relates to sFlt-1 (soluble Fms-like tyrosine kinase-1) protein (soluble vascular endothelial growth factor receptor-1, sVEGFR-1 (soluble vascular endothelial growth factor receptor), which is a major cause of gestational hypertension syndrome. -1)), which is a technique that is simple and technically simple in a short time and inexpensive to manufacture by using one kind of specific antibody and heparin.

  In recent years, against the backdrop of late marriage and difficulties in child-rearing environments, the number of births worldwide has decreased, that is, the number of children has decreased, which has become a major social problem. Under such circumstances, it is an urgent issue to overcome diseases that damage the fetus and mother, and to ensure healthy birth for both mother and child. Pregnancy-induced hypertension syndrome (formerly called pregnancy toxemia) that occurs in 5-7% of pregnancy among obstetrical diseases has fatal consequences in both fetus and mother in severe cases Therefore, its early detection and early treatment are extremely important medical issues.

  It is becoming clear that the control mechanism of angiogenesis plays an important role in the development and maturation of the placenta on the maternal side, in addition to playing an important role in fetal development, individual maturation, wound healing, and the like. Many factors such as VEGF (vascular endothelial growth factor), FGF (fibroblast growth factor), and Angiopoietin are involved in angiogenesis. Among them, VEGF and its receptor VEGFR are central. It is becoming clear that these roles play a role (Non-Patent Documents 1 and 2). In 1990, the present inventors first isolated and reported a novel tyrosine kinase receptor gene Flt-1 (Fms-like tyrosine kinase-1) (Non-patent Document 3). Since Flt-1 binds to VEGF and acts as a receptor, it is also called VEGFR-1. The Flt-1 gene expresses a short mRNA for producing a protein only in the extracellular region of Flt-1 together with a mature mRNA that expresses a receptor (Non-patent Document 4). Mainly produces two types of short-chain Flt-1 mRNA and its products, e15a sFlt-1 and i13 sFlt-1 protein, in a larger amount than during normal pregnancy, reaching 5 to 10 times the serum level of normal pregnant women (Non-Patent Documents 5, 6 and 7). These abnormally produced sFlt-1 binds to VEGF and PlGF (placental growth factor) and suppresses their action, and also causes hypertension and proteinuria in the mother's body. The amount of sFlt-1 in the body, for example, the amount of sFlt-1 in the blood of pregnant women is now an important biological indicator / biomarker (Non-patent Document 7).

  Furthermore, last year (2016), blood levels of sFlt-1 were reported to be higher than those in light cases in acute pancreatitis as well as in pregnancy-induced hypertension syndrome in obstetrics (non-incidence) Patent Document 8). In other diseases, decreased sFlt-1 expression in retinal tissues in age-related macular degeneration, and decreased sFlt-1 expression in kidney and podocytes and onset of nephrotic-like chronic proteinuria in experimental animals (non-patented) Literature 9, 10). In the future, there is a high possibility that diseases in which sFlt-1 in blood or body fluid is used as a biomarker will increase further.

  Until now, the basic method for measuring sFlt-1 was the sandwich ELISA method using two specific antibodies against sFlt-1 (Non-patent Documents 5-7). However, it takes time and money to develop two types of specific antibodies and establish a measurement method. As a result, sandwich ELISA using two types of specific antibodies is expensive, and the measurement method is also technical. Since time is required at multiple stages, it has been difficult to use it widely in obstetrics from the early stages of pregnancy as a biomarker test method. The present invention overcomes the difficulties of the sFlt-1 measurement method and aims to develop a simple measurement method that can be widely used.

  So far, it has been known that sFlt-1 protein binds to heparin due to its sugar chain modification. Due to this property, heparin columns have been used to purify sFlt-1 obtained from tissues and cultured cells (Non-patent Document 11). As a method of trying to treat gestational hypertension syndrome, an attempt to reduce sFlt-1 by refluxing the blood of a pregnant woman who has developed this disease through a heparin column is also used in non-patent literature 12). However, it was unclear whether sFlt-1 bound to heparin could simultaneously bind to a specific antibody stably and be used as a simple measurement method.

Endocr. Rev. 25, 581-611, 2004. review Angiogenesis, 9: 225-230, 2006. review Oncogene 5, 519-524, 1990. Proc. Natl. Acad. Sci. USA 90, 10705-10709, 1993. J. Clin. Invest. 111, 649-658, 2003. J. Clin. Endocrinol. Metab. 88, 2348-2351, 2003. N. Engl. J. Med. 350, 672-683, 2004. Int J Mol Sci. 2016 Dec 6; 17 (12) .pii: E2038. Elife, 2013 Jun 18; 2: e00324.doi: 10.7554 / eLife.00324. Cell, 151: 384-399, 2012. Jpn. J. Cancer Res., 88, 867-876, 1997. Circulation 124, 940-950, 2011.

  An object of the present invention is to use an immunoassay based on a sandwich method in which sFlt-1 protein is bound to heparin, sandwiching sFlt-1 with a specific antibody against heparin and sFlt-1, The object is to provide a new, inexpensive and practical sFlt-1 measurement method.

  The present inventor has (1) a bead having heparin bound (already commercially available), (2) an anti-human sFlt-1-specific mouse antibody (hereinafter referred to as a primary antibody), and an sFlt-1 sample, and (3) A simple sandwich of immobilized heparin and specific antibody by mixing the anti-mouse immunoglobulin / goat antibody (hereinafter referred to as secondary antibody) labeled with alkaline phosphatase or HRP (horseradish peroxidase). The inventors have found that an sFlt-1 measurement method can be established, and have completed the present invention. That is, in the present invention, by utilizing the heparin-binding property of sFlt-1, the number of necessary anti-sFlt-1-specific antibodies is reduced from two to one, and the novel sFlt-1 that is inexpensive and shortens the reaction time A measurement method can be provided.

The present invention is as follows.
[1] A method for measuring sFlt-1 in a specimen by forming a sandwich complex sandwiching sFlt-1 with heparin and an anti-sFlt-1 antibody, comprising heparin or anti-sFlt-1 immobilized on a carrier A specimen is contacted with an antibody, a complex of heparin or anti-sFlt-1 antibody and sFlt-1 in the specimen is formed on the carrier, and the presence of the complex is labeled with the anti-sFlt-1 antibody or anti-sFlt- A method for measuring sFlt-1 in a sample, comprising detecting using 1 antibody and a labeled anti-immunoglobulin antibody that binds to the antibody, or labeled heparin.
[2] The sFlt- of [1], wherein the carrier for immobilizing heparin or anti-sFlt-1 antibody is a film-form product or a three-dimensional product formed by synthetic resin, synthetic fiber, or fiber derived from a natural product How to measure 1
[3] A method for measuring sFlt-1 of [1] or [2], which is an ELISA method or an immunochromatography method.
[4] A method for measuring sFlt-1 of any one of [1] to [3], using beads bound with heparin.
[5] Instead of heparin, a substance having structural similarity to heparin and binding to sFlt-1 of mammals, birds and amphibians including humans is used. How to measure.
[6] When sFlt-1 in a sample collected from a subject is measured by any of the methods of [1] to [5] and the sFlt-1 concentration in the sample is higher than that in a healthy subject, A method for detecting pregnancy-induced hypertension syndrome, wherein the subject is evaluated as suffering from pregnancy-induced hypertension syndrome.
[7] A kit for measuring sFlt-1 in a specimen, comprising at least a carrier on which heparin or anti-sFlt-1 antibody is immobilized, and a labeled anti-sFlt-1 antibody or heparin.
[8] A kit for measuring sFlt-1 in a sample, comprising at least a carrier on which heparin is immobilized, an anti-sFlt-1 antibody, and a labeled secondary antibody that binds to the anti-sFlt-1 antibody.
[9] The carrier on which the heparin or anti-sFlt-1 antibody is immobilized is a film-form product or a three-dimensional product formed by a synthetic resin, a synthetic fiber, or a fiber derived from a natural product, [7] or [8 A kit for measuring sFlt-1 in the specimen.
[10] A kit for measuring sFlt-1 of any one of [7] to [9], which is an ELISA method kit or an immunochromatography method kit.
[11] A kit for measuring sFlt-1 according to any one of [7] to [10], wherein heparin-bound beads are used.
[12] Instead of heparin, a substance having structural similarity to heparin and binding to sFlt-1 of mammals, birds and amphibians including humans is used. Kit for measuring.
[13] A kit for measuring sFlt-1 of any one of [7] to [12] for detecting pregnancy-induced hypertension syndrome.

  ADVANTAGE OF THE INVENTION According to this invention, the sFlt-1 measuring method with respect to sFlt-1 protein can be provided which is cheap, is easy also in a technique, and can shorten time. That is, in the present invention, the number of necessary anti-sFlt-1-specific antibodies is reduced to one by using the sFlt-1 binding property of heparin, and the cost is low by the sandwich method of solid-phased heparin and one specific antibody. Thus, a novel sFlt-1 measurement method with a shortened reaction time can be provided.

It is a figure which shows the outline of the novel sandwich sFlt-1 ELISA method by a heparin bead and an anti- sFlt-1 specific antibody. This figure shows an example in which an ordinary monoclonal antibody that is not labeled with an enzyme or the like is used as an anti-sFlt-1 specific antibody (primary antibody), and a labeled secondary antibody that binds to the primary antibody is added. FIG. 2 is a diagram showing an outline of a novel sandwich sFlt-1 ELISA method using heparin beads and an anti-sFlt-1 specific antibody, using a labeled primary antibody. In this case, since the labeled secondary antibody is not necessary, it is further simplified as compared with the case shown in FIG. It is a photograph which shows the result of sFlt-1 ELISA assay using a heparin bead and a specific antibody. It is a figure which shows the measured value of sFlt-1 ELISA assay using a heparin bead and a specific antibody. It is a graph which shows the result of sFlt-1 ELISA assay using a heparin bead and a specific antibody. It is a photograph which shows the result of sFlt-1 ELISA assay using a heparin bead and a commercially available specific antibody. It is a figure which shows the measured value of sFlt-1 ELISA assay using a heparin bead and a commercially available specific antibody. It is a photograph which shows the result of sFlt-1 ELISA assay using HRP (horseradish peroxidase) as an enzyme. It is a figure which shows the measured value of sFlt-1 ELISA assay using HRP (horseradish peroxidase) as an enzyme. It is a graph which shows the result of sFlt-1 ELISA assay using HRP (horseradish peroxidase) as an enzyme. It is a figure which shows the result of the example of measurement-1 (reaction temperature of 4 degreeC, reaction time of 3 hours) of serum sFlt-1 of a gestational hypertension syndrome patient. It is a figure which shows the result of the example of the measurement-2 (reaction temperature room temperature (about 25 degreeC), reaction time, about 30 minutes) of serum of the pregnancy hypertensive syndrome patient.

Hereinafter, the present invention will be described in detail.
The present invention is a method for measuring sFlt-1 in blood by an immunoassay based on the sandwich method. Here, the immunoassay based on the sandwich method of the present invention is to capture the target substance sFlt-1 by sandwiching it with heparin and anti-sFlt-1 antibody that bind to sFlt-1. , Refers to the method of measuring sFlt-1, also simply referred to as the sandwich method. A complex formed by sandwiching sFlt-1 with heparin and anti-sFlt-1 antibody is called a sandwich complex. Examples of the immunoassay based on the sandwich method include an immunochromatography method, an enzyme immunoassay adsorption method (ELISA (Enzyme-linked immune-sorbent assay) method), a Western blot method, and the like.
In the present invention, the term “measurement” includes any of quantitative, semi-quantitative, and detection.

  As an example of the method of the present invention, heparin is solid-phased on a solid-phased carrier, a specimen that may contain sFlt-1 is added to the solid-phased carrier and brought into contact, and heparin and sFlt-1 complex is formed. Furthermore, a specific antibody against human sFlt-1 (anti-sFlt-1 antibody) is added and contacted, and the anti-sFlt-1 antibody is bound to the complex of heparin and sFlt-1 on the immobilized carrier, The sandwich complex of heparin, sFlt-1 and anti-sFlt-1 antibody may be detected.

  For detection, anti-sFlt-1 antibody is enzyme such as alkaline phosphatase (ALP) or horseradish peroxidase (HRP), radioisotope, fluorescent substance, luminescent substance, colored particles such as colored polystyrene particles, colloidal particles such as gold colloid, etc. This can be performed by measuring the signal emitted from the labeling substance. When an enzyme is used, horseradish peroxidase (HRP) that enables measurement with high sensitivity is preferably used. In the measurement, when the label is an enzyme, a substrate for each enzyme is added to cause an enzyme reaction, and the color development may be measured by measuring the absorbance. When the label is a fluorescent substance, the fluorescence may be measured using a fluorescence detector. Labeling of the antibody can be performed by a known method.

  The anti-sFlt-1 antibody is not limited to a mouse monoclonal antibody, and an antibody produced in any animal such as a rat, rabbit, or goat can be used, and may be a polyclonal antibody.

  Further, instead of labeling the anti-sFlt-1 antibody, a labeled secondary antibody that binds to the anti-sFlt-1 antibody may be used. In this case, a sandwich complex of heparin, sFlt-1, anti-sFlt-1 antibody and secondary antibody is formed on the solid phase carrier, and the signal emitted from the labeling substance of the secondary antibody may be measured. As the secondary antibody, an antibody that specifically binds to the anti-sFlt-1 antibody can be used, and an antibody against the animal immunoglobulin used to produce the sFlt-1 antibody such as an anti-mouse immunoglobulin / goat antibody is used. That's fine.

  A schematic diagram of the ELISA method when a secondary antibody is used is shown in FIG. 1, and a schematic diagram of the ELISA method when a secondary antibody is not used is shown in FIG.

The antibody may be an immunoglobulin fragment such as Fab or F (ab ′) ₂ , or a recombinant antibody such as scFv, dsFv, diabody, minibody expressed as a recombinant. In the present invention, the term “antibody” also includes these fragments. These fragments can be prepared by a known method.

  When forming a sandwich complex of heparin, sFlt-1 and anti-sFlt-1 antibody or heparin, sFlt-1, anti-sFlt-1 antibody (primary antibody) and secondary antibody on the carrier, A two-step method in which a complex of heparin and sFlt-1 is formed, washed and then anti-sFlt-1 antibody is added to form a sandwich complex of heparin, sFlt-1 and anti-sFlt-1 antibody, or first To form a heparin-sFlt-1 complex, washed, and then contacted with an anti-sFlt-1 antibody to form a heparin-sFlt-1 and anti-sFlt-1 antibody sandwich complex, washed, and then secondary The antibody may be added to form a sandwich complex of heparin, sFlt-1, anti-sFlt-1 antibody (primary antibody) and secondary antibody. Alternatively, each reagent may be added simultaneously and sandwiched. One step to perform the complex formation reaction simultaneously It may be carried out in.

  The binding reaction between heparin and sFlt-1 and the antigen-antibody reaction can be performed at 4 ° C to 45 ° C, preferably 20 ° C to 40 ° C, more preferably 25 ° C to 38 ° C. The reaction time in one step is 10 minutes to 18 hours, more preferably 10 minutes to 3 hours, and even more preferably about 30 minutes to 2 hours.

  The above method is a method in which heparin is immobilized on a carrier, but in the immunoassay based on the sandwich method, it is possible to reverse the position of heparin and a specific antibody. . In other words, an anti-sFlt-1-specific antibody is immobilized on a carrier and bound to sFlt-1 in the sample, and then heparin of an appropriate size and labeled are bound to form a sandwich complex. It is also possible to measure.

  That is, the present invention is a method for measuring sFlt-1 in a specimen by forming a sandwich complex sandwiching sFlt-1 with heparin and an anti-sFlt-1 antibody, which comprises heparin or anti-antigen immobilized on a carrier. a specimen is contacted with the sFlt-1 antibody, a complex of heparin or anti-sFlt-1 antibody and sFlt-1 in the specimen is formed on the carrier, and the presence of the complex is labeled with the anti-sFlt-1 antibody or It is a method for measuring sFlt-1 in a sample, comprising detecting using an anti-sFlt-1 antibody and a labeled anti-immunoglobulin antibody that binds to the antibody, or labeled heparin.

  Examples of the carrier for immobilizing heparin or anti-sFlt-1 antibody include a film-form product or a three-dimensional product formed from a synthetic resin, a synthetic fiber, or a fiber derived from a natural product, specifically for ELISA. Various materials such as a microtiter plate, a slide glass, a nitrocellulose membrane, a cellulose membrane, a sepharose membrane, resin beads such as cellulose beads and sepharose beads, and magnetized beads can be used. In addition, commercially available resin beads to which heparin is bound can be used. For example, commercially available heparin beads in which heparin is bound to sepharose beads (Heparin Sepharose 6 Fast Flow, 17-0998-01, GE Healthcare) can be used. it can. Alternatively, a gel or fiber membrane immobilized so as not to elute heparin may be used. In the present invention, a method in which heparin or anti-sFlt-1 antibody-immobilized resin beads are used as a carrier and the resin beads are added to the wells of an ELISA microtiter plate and measured is also referred to as ELISA. Heparin or anti-sFlt-1 antibody can be bound to a carrier by a known method such as physical adsorption or covalent bonding using a functional group. The amount of the solid phase is not particularly limited, but when the carrier is a 96-well microtiter plate, it is preferably several ng to several tens of μg per well. The immobilization can be performed by bringing a solution of heparin or anti-sFlt-1 antibody to be immobilized into contact with a carrier. For example, when a microtiter plate is used, the solution can be immobilized by dispensing a solution of heparin or anti-sFlt-1 antibody into the well of the microtiter plate and placing it for a certain period of time. After immobilization of heparin or anti-sFlt-1 antibody, blocking is performed using a blocking solution containing bovine serum albumin, human serum albumin, rabbit serum albumin, ovalbumin, etc. to prevent non-specific binding during the assay. It is preferred to do so.

  Heparin is a kind of glycosaminoglycan chemically, and it is a high-molecular substance in which D-glucuronic acid or L-iduronic acid and glucosamine are polymerized by 1,4 bonds to form disaccharide repeating units. . Not only heparin but all compounds having a structure similar to heparin and binding to sFlt-1 can be used. As a compound having a structure similar to heparin and binding to sFlt-1, for example, a polysaccharide sulfated ester of mucopolysaccharide is exemplified, and a representative substance is heparan sulfate. It has been shown that sFlt-1 is expressed not only in humans but also in mice, i13 type sFlt-1 is expressed from vascular endothelial cells, placental trophoblast cells, etc. and binds to heparin. Therefore, the present invention can be applied to a mouse sFlt-1 measurement system. It can also be applied to the measurement of sFlt-1 in mammals, birds, and amphibians, including humans and mice, by using heparin that binds to sFlt-1 in mammals, birds and amphibians, or a substance having a similar structure to heparin. it can. The origin of a substance having a structure similar to heparin or heparin is not limited, and for example, a substance derived from bovine or porcine intestinal mucosa can be used.

  In the placenta of gestational hypertension syndrome, e15a sFlt-1 protein and i13 sFlt-1 protein, which are short isoforms of sFlt-1, are produced. It is contained at a concentration of 3 times or more, for example, 2 to 5 times, 3 to 5 times, or 5 to 10 times. In the method of the present invention, it is not necessary to distinguish between the e15a sFlt-1 protein and the i13 sFlt-1 protein, and either may be measured. Therefore, an anti-sFlt-1 antibody that binds to both the e15a sFlt-1 protein and the i13 sFlt-1 protein may be used. In the present invention, the term sFlt-1 includes e15a sFlt-1 protein and i13 sFlt-1 protein.

  The sFlt-1 measured in the present invention is not limited to human sFlt-1, and includes sFlt-1 of mammals, birds and amphibians including humans.

  Since sFlt-1 is expressed in many tissues of the body, in the present invention, specimens include not only serum, plasma, whole blood, but also almost all body fluids such as pleural effusion, ascites, cerebrospinal fluid, saliva, urine, etc. including. However, plasma prepared using heparin as a blood anticoagulant can be used after heparin removal.

  The amount of the sample and each reagent to be added is several μL to several hundred μL. The specimen may be used after being diluted several times to a dozen times.

  By measuring sFlt-1 in a sample collected from a subject, it can be evaluated whether or not the subject suffers from gestational hypertension syndrome, or auxiliary data for evaluation can be obtained. That is, when the sFlt-1 concentration in a sample collected from a subject is higher than that of a healthy person, the subject can be evaluated as suffering from a gestational hypertension syndrome.

  Alternatively, sFlt-1 in a sample of a healthy person may be measured in advance, and a cut-off value (threshold value) may be determined for the concentration measurement value of sFlt-1 based on the measurement value. Based on the cut-off value, when the sFlt-1 concentration in the sample collected from the subject is equal to or higher than the cut-off value or exceeds the cut-off value, the subject is suffering from pregnancy-induced hypertension syndrome. Can be evaluated.

  When measured by the method of measuring sFlt-1 in a specimen by forming a sandwich complex sandwiching sFlt-1 with the heparin of the present invention and anti-sFlt-1 antibody, the concentration of sFlt-1 in serum in normal delivery cases Is about 12 ng / ml (+/- about 7 ng / ml), the average is about 56 ng / ml (+/- about 33 ng / ml) in gestational hypertension syndrome cases, about 4.7 times that of normal delivery cases . Furthermore, in the first-onset cases (so-called severe cases) that delivered before 37 weeks and 6 days of pregnancy, the average value is about 70 ng / ml, which is about 5.8 times that of normal deliveries. Furthermore, in the late onset cases that gave birth after 38 weeks 0 days of pregnancy, the average value of sFlt-1 was about 24 ng / ml, which is about twice that of normal delivery cases.

  Therefore, for example, the value of 12 ng / ml × 2 to 5 can be set as the cut-off value of the sFlt-1 concentration in serum. This cut-off value is preferably a value obtained by measuring sFlt-1 in a specimen by forming a sandwich complex sandwiching sFlt-1 with the heparin of the present invention and an anti-sFlt-1 antibody. .

  The present invention also includes a method for obtaining auxiliary data for detecting pregnancy-induced hypertension syndrome.

  The present invention includes a kit for measuring sFlt-1 in a sample, the kit comprising at least heparin or a carrier on which a substance having structural similarity to heparin is immobilized, and further labeled anti-sFlt- 1 antibody or an anti-sFlt-1 antibody and a labeled secondary antibody that binds to the anti-sFlt-1 antibody. Alternatively, the kit includes a carrier on which an anti-sFlt-1 antibody is immobilized, and further includes labeled heparin or a substance having a structural similarity to heparin.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited to these examples.

<Example 1>
[SFlt-1 ELISA assay using heparin beads and specific antibodies]
In order to confirm whether the novel ELISA assay method in the present invention is effective, the following assay was attempted using an alkaline phosphatase-labeled secondary antibody.

  Culture of human sFlt-1 producing human 293 cell line by sandwich ELISA using heparin beads (GE Healthcare) and anti-sFlt-1 specific antibody (KM1750, Kanno S. et al. Oncogene 19, 2138-46, 2000) The supernatant was measured. Quantified human 7N-Flt-1 (obtained from the culture of insect cell lines using gene expression vectors, 7 of the extracellular region of the human Flt-1 receptor protein from 1 to 7 A peptide containing the immunoglobulin domain of. Cell, 151: 384-399, 2012.) was used as a control protein.

(1) Preparation of reaction solution
In 1 ml of solution, (a) 0, 20 or 50 ng of control 7N-Flt-1 protein (100 μl in saline) or sFlt-1 sample (human e15a sFlt-1 producing 293 cell line culture supernatant (CM) 100 μl), (b) fetal bovine serum 100 μl, (c) heparin beads 25 μl, (d) anti-human sFlt-1 mouse monoclonal specific antibody KM1750 about 75 ng, (e) alkaline phosphatase labeled anti-mouse immunoglobulin A reaction solution containing about 0.3 μl of goat antibody (KURABO ZT6550), (f) about 100 μg of skim milk (Snowmark-Hokkaido), and (g) 775 μl of physiological saline supplemented with 0.1% Tween-20 was prepared. For (a), the culture supernatant of the 293 cell parent strain, the medium alone, or the sample containing both the control 7N-Flt-1 protein and the sFlt-1 sample were also measured.

(2) Stirring of reaction solution and washing by centrifugation The reaction solution was placed in a 1.5 ml Eppendorf tube, the lid was closed, and the mixture was gently rotated at 4 ° C. for 3 to 4 hours and stirred. After stirring, the heparin bead conjugate was precipitated by centrifugation at 300 rpm for 7 to 8 seconds at low speed for a short time (twice). After sufficiently removing the supernatant, 1 ml of physiological saline containing 0.1% Tween-20 was added for the purpose of washing, the lid was closed, gently rotated at 4 ° C., and stirred for 5 minutes. After stirring, the heparin bead conjugate was precipitated by low-speed, short-time centrifugation (twice) at 300 rpm for 7 to 8 seconds, and the supernatant was sufficiently removed.

(3) Measurement with alkaline phosphatase reaction solution Measurement of alkaline phosphatase on washed heparin bead conjugate (sFlt-1 protein, primary antibody, secondary antibody, those expected to be bound by these three, approximately 15 μl) 0.2 ml of an alkaline solution (pH 8.8) containing a reagent (BCIP-NBT, Nacalai tesque) was added, stirred, and then transferred to a 96-well plate to observe the reaction. After about 20 to 30 minutes, it was confirmed that the reaction solution containing the control 7N Flt-1 protein turned blue, and measurement was performed at 595 nm using a plate reader.

(4) Results The results are shown as photographs in FIG. All of the same sample twice (this time 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18 have the same contents) Sample) was measured. The control reaction solution (No. 1, 2) to which 7N Flt-1 was not added was yellow without color formation due to alkaline phosphatase reaction, but 20N (No. 3, 4), 50 ng (No. .5, 6) In addition, it turns blue due to alkaline phosphatase reaction. The culture supernatant (293 CM) (Nos. 7 and 8) of the 293 cell parent strain is almost yellow, and no significant change is observed, but the culture supernatant of the 293 cell line expressing e15a sFlt-1 cDNA ( e15a CM) (No. 11, 12) shows a change to blue.

  In addition, in the sample (Nos. 9 and 10) obtained by adding 20 ng of control 7N Flt-1 protein to the culture supernatant of the 293 cell parent strain, a blue reaction close to No. 3 and 4 was obtained. It can be understood that no substance that suppresses the reaction nonspecifically is contained.

FIG. 4 shows measured values in each measurement.
In FIG. 4, [Measured Value] shows the value measured with a plate reader. [Average value] shows average values of 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, and 15 and 16. Furthermore, [average value−208] is a value obtained by subtracting the average value 208 of the background No. 1 and No. 2 to which sFlt-1 is not added. As shown in FIG. 5, the amount of sFlt-1 in the reaction solution is expected to be better quantitative when it is 20 ng or less.

<Example 2>
[SFlt-1 ELISA assay using heparin beads and commercially available specific antibody]
In order to confirm whether or not the novel ELISA assay method in the present invention exerts an effect by a commercially available anti-sFlt-1 antibody, the following assay was attempted.

  The method is basically the same as in Example 1, except that the anti-sFlt-1 antibody that is the primary antibody is the anti-human sFlt-1 mouse monoclonal antibody KM1750 used in Example 1, and a commercially available alternative. Both anti-human sFlt-1 mouse monoclonal antibodies (PK-AB704-64426, PromoKine) were compared. It was considered from the preliminary experiment that the antibody titer of the commercially available antibody was about one-half that of KM1750, so the amount of the antibody was doubled. The results are shown in FIG. As a control, 20 ng of 7N Flt-1 and 100 μl of culture supernatant of an e15a-expressing 293 cell line as a sample were measured.

  In FIG. 6, the results are shown as photographs. [A] from 1 to 8 using the same anti-human sFlt-1 mouse monoclonal antibody KM1750 as in Example 1, and [B] from 9 to 16 using a commercially available anti-human sFlt-1 mouse monoclonal antibody. It was measured. As the control 7N Flt-1, No. 1, 2, 3, 9, 10, and 11 used an amount of 0 ng, and No. 4, 5, 6, 12, 13, and 14 used an amount of 20 ng. In Nos. 7, 8, 15, and 16, an amount of 100 μl of the culture supernatant of the e15a-expressing 293 cell line was measured. Although there is a difference in the level of blue, a weak blue reaction was observed with 20 ng of 7N Flt-1 even when two types of anti-sFlt-1 antibodies were used (No. 4, 5, 6, 12, 13, 14). On the other hand, a strong blue reaction was observed in the culture supernatant of the e15a-expressing 293 cell line (No. 7, 8, 15, 16).

  FIG. 7 shows the measured values. Numerical values measured with a plate reader are shown in [Measured values] in FIG. [Average value] showed average values of 1 to 3, 4 to 6, 7 and 8, 9 to 11, 12 to 14, and 15 and 16. In addition, [average value -228] 0, 170, 435 in the upper half is the average value 228 of the background No. 1 to 3 to which sFlt-1 was not added, from [average value] 228, 398, and 663. Subtracted value. [Average value-210] 0, 78, 364 in the lower half subtracted the average value 210 of No. 9-11 in the background without addition of sFlt-1 from [Average value] 210, 288, and 574 Value. The amount of sFlt-1 in 100 μl of the culture supernatant of the e15a-expressing 293 cell line was [435/170 x 20] = 51 ng and [364/78 x 20] = 93 ng between the two antibodies. Yes. The average is 72ng / 0.1ml (720ng / ml). From the above, it was revealed that a commercially available primary antibody (anti-sFlt-1 antibody) can be used sufficiently.

<Example 3>
(1) Reaction method In order to increase measurement sensitivity, measurement was performed using an HRP (horseradish peroxidase) labeled secondary antibody (ab6789, Abcam) instead of the alkaline phosphatase labeled secondary antibody used in Examples 1 and 2. Went. As the amount of sFlt-1, 0, 0.1, 0.3, and 1 ng were used per reaction tube, and three reactions were performed for each. The volume of the reaction solution was the same as in Examples 1 and 2, but 150 ng / tube of the primary antibody KM1750 and about 200 ng / tube of the HRP-labeled secondary antibody were used. After the same reaction and washing as in Examples 1 and 2, 200 μl of HRP measurement solution (A + B solution, R & D Systems) was added, and after stirring, transferred to a 96-well plate. After reaction for about 20 minutes, measured at 595 nm did.

(2) Results FIG. 8 shows the results with photographs. Samples containing the same amount of sFlt-1 were measured 3 times (1-3, 0 ng; 4-6, 0.1 ng; 7-9, 0.3 ng; 10-12, 1 ng). The reaction liquid without addition of 7N Flt-1 (Nos. 1, 2, and 3) is weak blue, but becomes stronger blue as the amount of 7N Flt-1 increases.

(3) Measurement Value FIG. 9 shows measurement values in each measurement, and FIG. 10 shows a graph. The average of the measured values for 7N Flt-1 of 0, 0.1, 0.3 and 1 ng is 303, 386, 552 and 807, respectively, and the value obtained by subtracting the average of 0 ng is 83 for 0.1 ng and 0.3 ng for It was 249, and it became 504 at 1 ng, and increased to some extent quantitatively. By using this HRP-labeled secondary antibody, the sensitivity is increased about 100 times compared to the alkaline phosphatase-labeled secondary antibody method (7N Flt-1 10-50ng measured) used in Examples 1 and 2. Was made.

<Example 4>
[Measurement of clinical samples]
(1) Method It was examined whether this measurement method could actually measure sFlt-1 in clinical patient serum. Serum was measured from 5 cases of normal delivery and 13 cases of pregnancy-induced hypertension syndrome, which were provided by the Department of Obstetrics and Gynecology at Kashiwa University Hospital under the approval of the joint research agreement and ethics committee. For one measurement, 20 μl, 50 μl, or 100 μl of serum was used. The total amount of human serum and bovine serum in 1 ml of the reaction solution was adjusted to 100 μl. Specifically, bovine serum was 80 μl for human serum 20 μl, bovine serum 50 μl for human serum 50 μl, and bovine serum 0 μl for human serum 100 μl. The reaction time was measured at 4 ° C. for 3 hours (measurement-1) or at room temperature (about 25 ° C.) and 30 minutes (measurement-2).

(2) Results FIG. 11 is a photograph showing the results of an example of measurement-1 (reaction temperature 4 ° C., reaction time 3 hours) of serum sFlt-1 in patients with gestational hypertension syndrome. In the left lane, standard sFlt-1 (0 ng, 0.3 ng, 1 ng, 3 ng) was measured. In the right lane, sFlt-1 was measured before and after 5 days of delivery of gestational hypertension syndrome case a, and before and 5 days after delivery of gestational hypertension syndrome case b. 20 μl of serum was used. In both cases, the serum sFlt-1 level was as high as about 100 ng / ml before parturition, whereas the serum concentration of sFlt-1 was rapidly reduced 5 days after parturition.

  FIG. 12 is a photograph showing the results of an example of measurement of serum sFlt-1 in a gestational hypertension syndrome patient-2 (reaction temperature room temperature (about 25 ° C.), reaction time, about 30 minutes). In the left lane, standard sFlt-1 (0 ng, 0.3 ng, 1 ng, 3 ng) was measured. In the right lane, sera of gestational hypertension syndrome cases c (2 days before and immediately after delivery) and d (immediately after and before delivery) were measured. The blue reaction of standard sFlt-1 is slightly lower than that of the example with the reaction temperature of 4 ° C and the reaction time of 3 hours (Fig. 11), but the measurement method is a relative comparison of the reaction between the standard and the sample. There was no hindrance.

  As a result of the measurement of serum in 5 cases of normal delivery and 13 cases of gestational hypertension syndrome, sFlt-1 concentration (ng / ml serum) averaged around 12 (+/- about 7) in cases of normal delivery, gestational hypertension syndrome The average value in cases was around 56 (+/- about 33), and the value of this disease was about 4.7 times higher than that in normal delivery cases. Furthermore, in the first-onset cases (9 cases of so-called severe cases) that delivered before 37 weeks and 6 days of gestation, the average value was about 70, which was about 5.8 times that of normal delivery cases. On the other hand, in the late onset cases (4 cases) that gave birth after 38 weeks 0 days of pregnancy, the mean sFlt-1 was about 24, which was about twice that of normal delivery cases. The results obtained from this measurement, that is, that the average of serum sFlt-1 in pregnant hypertensive syndrome patients is about 2 to 5 times that of normal delivery cases (J. Clin. Invest. 111 , 649-658, 2003 .; J. Clin. Endocrinol. Metab. 88, 2348-2351, 2003 .; and N. Engl. J. Med. 350, 672-683, 2004.) It shows that the new sFlt-1 measurement method can be fully used for clinical tests.

  The sFlt-1 measurement method of the present invention can provide a clinically easy-to-use sFlt-1 screening method that is inexpensive, easy to perform by technique, and can be shortened in time.

Claims (13)

  A method for measuring sFlt-1 in a specimen by forming a sandwich complex sandwiching sFlt-1 with heparin and an anti-sFlt-1 antibody, the specimen being applied to heparin or anti-sFlt-1 antibody immobilized on a carrier An anti-sFlt-1 antibody or anti-sFlt-1 antibody labeled with the presence of the complex, and a complex of heparin or anti-sFlt-1 antibody and sFlt-1 in the sample is formed on the carrier. A method for measuring sFlt-1 in a sample, which comprises detecting using a labeled anti-immunoglobulin antibody or labeled heparin that binds to the antibody.   The sFlt-1 according to claim 1, wherein the carrier for immobilizing the heparin or the anti-sFlt-1 antibody is a film-form product or a three-dimensional product formed by a synthetic resin, a synthetic fiber, or a fiber derived from a natural product. How to measure.   The method for measuring sFlt-1 according to claim 1 or 2, which is an ELISA method or an immunochromatography method.   The method for measuring sFlt-1 according to any one of claims 1 to 3, wherein heparin-bound beads are used.   The sFlt-1 according to any one of claims 1 to 4, wherein a substance having structural similarity to heparin and binding to sFlt-1 of mammals, birds and amphibians including humans is used instead of heparin. How to measure.   If sFlt-1 in a sample collected from a subject is measured by the method according to any one of claims 1 to 5 and the sFlt-1 concentration in the sample is higher than that of a healthy subject, the test A method for detecting pregnancy-induced hypertension syndrome, in which the body is assessed as suffering from pregnancy-induced hypertension syndrome.   A kit for measuring sFlt-1 in a sample, comprising at least a carrier on which heparin or anti-sFlt-1 antibody is immobilized, and a labeled anti-sFlt-1 antibody or heparin.   A kit for measuring sFlt-1 in a sample, comprising at least a carrier on which heparin is immobilized, an anti-sFlt-1 antibody, and a labeled secondary antibody that binds to the anti-sFlt-1 antibody.   The specimen according to claim 7 or 8, wherein the carrier on which the heparin or anti-sFlt-1 antibody is immobilized is a film-form product or a three-dimensional product formed from a synthetic resin, a synthetic fiber, or a fiber derived from a natural product. Kit for measuring sFlt-1 in the inside.   The kit for measuring sFlt-1 according to any one of claims 7 to 9, which is an ELISA method kit or an immunochromatography method kit.   The kit for measuring sFlt-1 according to any one of claims 7 to 10, wherein heparin-bound beads are used.   The sFlt-1 according to any one of claims 7 to 11, wherein a substance having structural similarity to heparin and binding to sFlt-1 of mammals, birds and amphibians including humans is used instead of heparin. Kit for measuring.   The kit for measuring sFlt-1 according to any one of claims 7 to 12, for detecting pregnancy-induced hypertension syndrome.

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