JP2003262633A - Evaluation method for contamination by endocrine- disrupting substance, antibody used for the same as well as reagent and kit for detection by using the antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for evaluating a state of a contamination by an endocrine-disrupting substance, by using an animal suitable as a monitoring object, when the contamination by the endocrine-disrupting substance is investigated. <P>SOLUTION: The evaluation method for the contamination by the endocrine- disrupting substance comprises an immunoreaction process, in which a vitellogenin antibody used to specifically recognize vitellogenin, derived from a freshwater turtle, is made to react with a body fluid of the freshwater turtle as a detection object of the vitellogenin, and an immunoreaction decision process used to decide the existence of an immunoreaction by the vitellogenin antibody. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for evaluating endocrine disrupting substance contamination using an antibody against vitellogenin in freshwater turtles, an antibody used therefor, a detection reagent and a detection kit using the antibody. Is.

[0002]

2. Description of the Related Art Vitellogenin is an egg yolk protein precursor, estrogen (estroge).
n) Produced by stimulation. In females, after sexual maturity, the estrogen, which is one of the female hormones, acts on liver cells to express the vitellogenin gene and synthesize vitellogenin protein. The produced vitellogenin reaches the ovaries through the bloodstream and matures into yolk proteins.

Even in males, the vitellogenin gene is retained, but vitellogenin is not normally synthesized. However, it is known that vitellogenin is also synthesized in males and appears in blood when exposed to a substance having an estrogenic activity from the outside (Environ Health Perspect 1995 May; 103 Suppl 4: 1.
9-25 Palmer BD et al.). Endocrine disruptor
Uptors) It is well known that so-called environmental hormones have estrogenic activity. Therefore, by monitoring the appearance of blood vitellogenin in males, it is possible to evaluate the state of environmental pollution due to endocrine disrupting substances in the region where the monitored organism inhabits.

As a method for evaluating the above-mentioned environmental pollution situation, for example, Japanese Patent Laid-Open No. 2000-125867 proposes a pollution evaluation method for a freshwater environment in which carp is to be monitored. The above-mentioned evaluation method, an antibody against carp vitellogenin and vitellogenin in the body fluid of male carp are reacted, and an increase in the concentration of vitellogenin measured by this reaction is used as an index, and the state of contamination by endocrine disruptors in the freshwater environment is evaluated. It is a method of evaluation. According to this, it is possible to evaluate the pollution situation of endocrine disrupting substances in the freshwater environment such as rivers and lakes where carp inhabits. The carp vitellogenin detection kit is already on the market.

In addition to carp, medaka has also been reported as a monitoring target in the case of evaluating the state of pollution by endocrine disrupting substances in a freshwater environment.

[0006]

However, when evaluating the state of contamination by endocrine disrupting substances in a freshwater environment, the above-mentioned conventional fish such as carp and medaka fish are not suitable for monitoring.

[0007] Since fish swim, there is a problem that it is uncertain to specify a growing point or a living water area.
Fish are highly likely to swim and move from the water bodies to be evaluated for the endocrine disruptor pollution status to other water bodies, and the probability of exposure to environmental hormones in the water bodies to be evaluated is expected to be low. . Since individuals of fish are fragile as organisms, they often die due to capture, blood collection, and retention for various treatments, and it is difficult to release them after the treatment for follow-up investigation. In addition, water contaminated with endocrine disruptors can also be contaminated with other toxins at the same time, and fish can die from other types of toxins before they are affected by endocrine disrupters. .

[0008] Further, since fishes are exposed to toxic substances through the body surface or when they ingest water or food, the presence state of environmental hormones at this time greatly affects the exposure.
However, since environmental hormones are poorly soluble in water,
It does not penetrate into the body through the body surface of fish. Due to its fat-soluble nature, environmental hormones are bound to particulate solids in water. Most of the particulate solids to which environmental hormones are bound are accumulated in the sedimented soil at the bottom of rivers and lakes. Fish wander in water where the proportion of endocrine disrupters is lower than in sedimented soil at the bottom of rivers and lakes. Therefore, unless fish actively take in the mud on the bottom of the water, the probability of exposure to environmental hormones is low.

[0009] That is, when the presence of environmental hormones is confirmed when the pollution situation of freshwater environment is evaluated using fish as a monitoring target, the contamination by environmental hormones has already occurred in the sedimentation soil at the water bottom of the water area being evaluated. But,
It is speculated that it is progressing to some extent. Therefore, when a fish is used as a monitoring target, it is difficult to determine the presence of the endocrine disrupter in the early stage of the process in which contamination with the endocrine disrupter progresses.

Furthermore, since fish are not located at higher levels in the food chain and follow-up surveys are difficult as described above, they are not suitable as targets when investigating bioconcentration of environmental hormones.

The present invention has been made in view of the above-mentioned conventional problems, and an object thereof is to make it possible to evaluate a body of water with severe contamination and investigate the bioconcentration of environmental hormones. It is an object of the present invention to provide a method for evaluating endocrine disrupting substance contamination in a freshwater environment, which can prolong the period for measuring the contamination by hormones and improve the accuracy of measurement data regarding the contamination by environmental hormones.

[0012]

[Means for Solving the Problems] The present inventors have made it possible to evaluate a body of water with severe contamination and investigate the bioconcentration of environmental hormones, and to extend the period for measuring the environmental hormone contamination, In addition, for the purpose of providing an evaluation method for endocrine disruptor contamination that can improve the accuracy of measurement data regarding contamination with environmental hormones, monoclonal and polyclonal antibodies against vitellogenin in freshwater turtles were prepared. As a result, freshwater turtles can be used as a monitoring target when evaluating the state of pollution by endocrine disrupting substances in the freshwater environment, thus completing the present invention.

First, the method for evaluating endocrine disrupter contamination according to the present invention comprises immunizing a vitellogenin antibody that specifically recognizes vitellogenin derived from freshwater turtles with a body fluid of freshwater turtles to be detected by the vitellogenin. It is characterized by including a reaction step and an immune reaction determination step of determining the presence or absence of an immune reaction by a vitellogenin antibody.

Further, the method for evaluating the endocrine disrupter contamination according to the present invention is the same as the method for evaluating the endocrine disrupter contamination, further comprising the step of determining the amount of vitellogenin produced in a freshwater turtle based on the immune reaction by the vitellogenin antibody. It is characterized by having a vitellogenin quantification step for quantification.

Furthermore, the method for evaluating endocrine disrupter contamination according to the present invention is the same as the above method for evaluating endocrine disrupter contamination, wherein the vitellogenin quantification step estimates the amount of vitellogenin based on the measured absorbance. It is characterized by quantification by using a calibration curve.

According to the above invention, when assessing the pollution situation of endocrine disrupting substances in the freshwater environment, so-called environmental hormones, freshwater turtles that can live even in heavily polluted waters are targeted for monitoring. It will be possible to obtain data on the pollution status of water areas that were difficult to achieve.

The above-mentioned highly polluted water area is, for example, a water area which is contaminated with environmental hormones and other types of poisons and which makes it difficult for fish to survive. Freshwater turtles have the ability to inhabit and reproduce in domestic wastewater and soil sedimentation tanks of rivers and lakes where environmental hormones are accumulated.

Further, freshwater turtles have the property of wandering in the mud on the bottom of the water, and are easily affected by the environmental hormones present in the soil sedimentation tanks on the bottom of the rivers and lakes where fish are less susceptible. Therefore, when assessing the state of pollution of the freshwater environment by environmental hormones, the presence of environmental hormones can be determined at an early stage in the process of progressing pollution by environmental hormones, by monitoring freshwater turtles for monitoring. It will be possible.

Furthermore, freshwater turtles have the property of sticking to specific water areas, have a life span of 10 years or more, can identify their age, are strong in blood collection and experimental treatment, and can be discharged after treatment. is there. As a result, if freshwater turtles are to be monitored for pollution assessment,
Because you can carry out regular surveys for each individual over a long period of time,
It becomes easier to measure the contamination situation for a long time, and more accurate measurement data can be obtained.

Further, freshwater turtles are omnivorous, are located higher in the food chain than fishes, and can carry out long-term regular surveys as described above. It becomes possible to investigate.

Therefore, in the case of investigating the polluted state by the endocrine disrupting substance, so-called environmental hormone, in the freshwater environment, it is possible to evaluate the water area where the severe pollution has progressed and to investigate the bioconcentration of the environmental hormone, and It is possible to provide an evaluation method for endocrine disrupting substance contamination that can prolong the period for measuring the contamination and improve the accuracy of measurement data regarding the contamination by environmental hormones.

The method for evaluating endocrine disrupting substance contamination according to the present invention may be further specified by the characteristics of the vitellogenin antibody described above in any of a) to d) below. a) Recognizing vitellogenin derived from the Japanese turtle as an antigen. b) A polyclonal antibody. c) It is a monoclonal antibody. d) A monoclonal antibody whose immunoglobulin class is IgG.

According to the above-mentioned a) of the present invention, when assessing the state of contamination of endocrine disrupting substances in a freshwater environment, the object to be monitored can be a turtle.

Further, the method for evaluating the endocrine disrupting substance contamination according to the present invention is the same as the method for evaluating the endocrine disrupting substance contamination described above, wherein in the immune reaction determination step, an ELISA method or an immunoprecipitation method is used as a method for determining the immune reaction. The method is characterized in that any one of the Western blotting method and the affinity chromatography method is used.

According to the above invention, the presence or absence of an immune reaction due to the vitellogenin antibody can be determined by any one of the above methods for determining an immune reaction.

Secondly, the monoclonal antibody according to the present invention is a monoclonal antibody used in the above-mentioned method for evaluating the endocrine disruptor contamination, and comprises mouse spleen lymphocytes immunized with freshwater turtle-derived vitellogenin and mouse myeloma cells. It is characterized in that it is produced by a hybridoma obtained by fusing the above, and specifically binds to vitellogenin derived from the freshwater turtle.

The polyclonal antibody according to the present invention is a polyclonal antibody used in the method for evaluating the endocrine disruptor contamination, which is purified from immune serum of a rabbit immunized with freshwater turtle-derived vitellogenin and is derived from the freshwater turtle. It is characterized by specifically binding to vitellogenin.

Furthermore, examples of the monoclonal antibody according to the present invention include a monoclonal antibody that specifically binds to vitellogenin derived from freshwater turtles, and a monoclonal antibody in which the vitellogenin is derived from turtles.

Further, examples of the polyclonal antibody according to the present invention include a polyclonal antibody that specifically binds to vitellogenin derived from freshwater turtles, and a polyclonal antibody in which the vitellogenin is vitellogenin derived from turtles.

Thirdly, the reagent for detecting vitellogenin according to the present invention is characterized by containing at least one of the above-mentioned monoclonal antibody and the above-mentioned polyclonal antibody.

Further, the kit for evaluating the endocrine disrupter contamination according to the present invention is characterized by containing the above-mentioned reagent for detecting vitellogenin.

[0032]

BEST MODE FOR CARRYING OUT THE INVENTION An embodiment of the present invention will be described below with reference to FIGS. The present invention is not limited to this.

The present inventor, in the case of investigating the pollution by endocrine disrupting substances, aims to establish a method for evaluating the state of pollution by endocrine disrupting substances by using an animal suitable as a monitoring target, An antibody against Vitellogenin (VTG) was prepared.

The method for evaluating the endocrine disruptor contamination of the present embodiment comprises an immunoreaction step of reacting a vitellogenin antibody that specifically recognizes vitellogenin derived from freshwater turtles with a body fluid of freshwater turtles to be detected by the vitellogenin. ,
An immune reaction determination step of determining the presence or absence of an immune reaction by the vitellogenin antibody.

Vitellogenin is an egg yolk protein precursor and has a molecular weight of about 214,000 as shown in FIG.
It is a phospholipoglycoprotein consisting of two (214 kDa) polypeptide chains. FIG. 1 shows the molecular weight of vitellogenin (VTG) derived from the reed turtle, which is a kind of freshwater turtle. MWM in FIG. 1 indicates a molecular weight marker.

Examples of freshwater turtles include Japanese turtles, Mississippia turtles, and reed turtles.
Reed turtles are more abundant than other types of freshwater turtles and therefore have a higher catch. In addition, since it can live in a clean environment from a highly polluted environment, it does not escape and escape to other basins even if pollution progresses, and it has strong resistance to poisonous substances,
It is suitable for monitoring when conducting pollution investigations.

In the present embodiment, the reed turtle is used, for example, but other freshwater turtles can be used. Hereinafter, each step will be described in detail.

First, in the immunoreaction step, a conventionally known ELISA (enzyme-linked immunosorbent a) method is used.
ssay, enzyme-linked immunosorbent assay) is preferably used to confirm whether or not vitellogenin is contained in the body fluid of the freshwater turtle. Specifically, a commercially available ELISA plate is coated with the vitellogenin antibody, washed, and blocked, blood is collected from a freshwater turtle, and the serum prepared by centrifugation is appropriately diluted, and each well of the ELISA plate is sampled. And react.

Next, in the immunological reaction determination step, a secondary antibody, such as HRP (horseradis), which is bound to the vitellogenin antibody and labeled so as to develop a color with a chromogenic substrate.
h peroxidase: Horseradish peroxidase)
The labeled antibody is used to determine the immune reaction step. Specifically, the ELISA plate is washed after the reaction in the immunoreaction step, and the HRP-labeled antibody is added to the ELISA plate and reacted. After the reaction, the ELISA plate is washed, and the ELISA plate is used as a color-developing substrate.
In this case, for example, hydrogen peroxide and o-phenylenediamine are added and reacted. Whether or not vitellogenin is contained in the body fluid of the freshwater turtle can be determined by the presence or absence of color development in the reaction. In other words, the immune reaction determination step is a color development step of the ELISA method.

After the color reaction in the immunological reaction determination step, the immunological reaction can be more accurately determined by measuring the color development intensity of each well using a commercially available plate reader.

Further, in the above immunoreaction step, it is possible to use two kinds of the above vitellogenin antibodies. For example, a monoclonal antibody (m) that specifically binds to vitellogenin derived from freshwater turtles.
Ab) and vitellogenin derived from freshwater turtle (polyclonal antibody: pAb)
And can be used in combination. Also, for example,
A monoclonal antibody that specifically binds to vitellogenin derived from a turtle and a polyclonal antibody that specifically binds to vitellogenin derived from a turtle can be used in combination.

When two kinds of the above-mentioned vitellogenin antibody are used in the above-mentioned immunoreaction step, the conventionally known Sandwich ELISA method can be preferably used. In the sandwich ELISA method, one of two types of primary antibodies used in an immunoassay system is immobilized on an ELISA plate. The primary antibody fixed to this ELISA plate is the immobilized antibody. The sandwich ELISA immunoassay system uses two types of primary antibodies: a solid-phased antibody that is immobilized on an ELISA plate and used, and a primary antibody that binds to the secondary antibody.

When a monoclonal antibody that specifically binds to vitellogenin derived from freshwater turtle and a polyclonal antibody that specifically binds to vitellogenin derived from freshwater turtle are used in combination in the above-mentioned immunoreaction step, which is used as the immobilized antibody I don't mind.

For example, in the immunoreaction step, the polyclonal antibody that specifically binds to the turtle-derived vitellogenin is used as a solid-phase antibody, and the monoclonal antibody that specifically binds to the turtle-turtle vitellogenin is used as the secondary antibody. The sandwich ELISA method used as the antibody on the binding side is performed as follows.

First, a commercially available ELISA plate is coated with a polyclonal antibody that specifically binds to vitellogenin derived from the above-mentioned turtle, which is used as the immobilized antibody, and is immobilized. After immobilization, the above ELISA plate is washed and blocked. Next, blood is collected from the reed turtle and the serum prepared by centrifugation is added to the above ELISA plate. As a result, the above-mentioned polyclonal antibody of the solid-phased antibody is reacted with vitellogenin derived from the horseshoe turtle. After the reaction, the above ELISA plate is washed, and a monoclonal antibody that specifically binds to the above-mentioned turtle-derived vitellogenin is added to the above ELISA plate.
As a result, vitellogenin derived from horseshoe turtle bound to the immobilized antibody is reacted with the above-mentioned monoclonal antibody that is the primary antibody on the side to which the secondary antibody binds (immunological reaction step). After the reaction, the above ELISA plate was washed, and then a commercially available HRP-labeled anti-mouse IgG was used as a chromogenic substrate.
An antibody (Anti-Mouse IgG-HRP) is added to the ELISA plate and reacted with the monoclonal antibody. After the reaction, the ELISA plate is washed, hydrogen peroxide and o-phenylenediamine are added to the ELISA plate, and the presence or absence of an immune reaction is determined (immune reaction determination step).

A system 1 (Sandwich ELISA System I) is used when a polyclonal antibody that specifically binds to vitellogenin derived from the above-mentioned turtle is used as a solid-phased antibody, as in the sandwich ELISA method (see FIG. 2).

Further, for example, in the immunoreaction step, the monoclonal antibody that specifically binds to the turtle-derived vitellogenin is used as a solid phase antibody, and the polyclonal antibody that specifically binds to the turtle-derived vitellogenin is secondary. The sandwich ELISA method used as the antibody on the side to which the antibody binds is performed as follows.

The above-mentioned turtle-derived vitellogenin-specific monoclonal antibody is used as an immobilized antibody, and the above-mentioned turtle-derived vitellogenin-specific polyclonal antibody is used as the antibody on the side to which the secondary antibody is bound. The immunoreaction step and the immunoreaction determination step are performed in the same manner as in System 1 above, except that a commercially available HRP-labeled anti-rabbit IgG antibody (Anti-Rabbit IgG-HRP) is used as a color-developing substrate.

System 2 (Sandwich ELISA System II) in the case where the polyclonal antibody that specifically binds to vitellogenin derived from the above-mentioned turtle is used as the antibody on the side to which the secondary antibody binds, as in the above sandwich ELISA method.
(See FIG. 3).

Before the above immunoreaction step, for example,
Blood was collected from freshwater turtles and centrifuged to prepare serum. Using this serum as a sample, SDS (sodium d
odecyl sulfate: sodium dodecyl sulfate
oresis: PAGE), and after the electrophoresis, an immunoreaction trial step of confirming the reactivity with the above vitellogenin antibody by Western blotting may be added. That is, after the electrophoresis, the protein on the gel is transferred to a nitrocellulose membrane, and the nitrocellulose membrane to which the protein is transferred and the vitellogenin antibody are allowed to act to confirm the presence or absence of the reaction.

To confirm the presence or absence of the above reaction, a secondary antibody which is bound to the above vitellogenin antibody and labeled so as to develop a color with a chromogenic substrate, for example, an HRP-labeled antibody is used. Whether or not vitellogenin is contained in the body fluid of a freshwater turtle can be confirmed in advance before the immunoreaction step by adding a color-developing substrate and developing color.

When there are a plurality of the vitellogenin antibodies, the immune reaction trial step may be used as an index when selecting which antibody to use.

The method for evaluating endocrine disrupting substance contamination of the present embodiment further includes a vitellogenin quantification step for quantifying the amount of vitellogenin produced in the freshwater turtle body based on the immune reaction by the vitellogenin antibody.

The vitellogenin quantification step is quantified by using a calibration curve for estimating the amount of vitellogenin based on the measured absorbance (Absorbance).

In the quantification step, in order to quantify the amount of vitellogenin produced in the freshwater turtle body, for example, a calibration curve capable of estimating the amount of vitellogenin from the absorbance is used. At the time of color development in the immunological reaction determination step, the absorbance is measured with a plate reader. By substituting this absorbance value into the equation of the above calibration curve, the amount of vitellogenin produced in the freshwater turtle body is calculated.

The above-mentioned calibration curve was obtained by performing the above-mentioned immunoreaction step and the above-mentioned immunoreaction determination step using a serially diluted vitellogenin derived from purified freshwater turtle and the above-mentioned vitellogenin antibody, and at the time of color development. The absorbance can be measured with a plate reader, and it can be produced based on the measured value.

The above calibration curve is, for example, as shown in FIG.
Alternatively, the calibration curve shown in FIG. 5 (see Examples below) may be used. When the calibration curves shown in FIGS. 4 and 5 are used, the absorbance is measured at 450 nm by a commercially available plate reader at the time of color development in the immune reaction determination step. 4 and 5
The vertical axis represents the value measured by the plate reader at 450 nm, and the horizontal axis represents the vitellogenin concentration.

When the calibration curve of FIG. 4 is used, the above system 1 is carried out. In this system 1, of the monoclonal antibodies that specifically bind to vitellogenin derived from the above-mentioned turtle, ACVTG-5 (see Examples described later) is used as the antibody on the side to which the secondary antibody binds.

When the calibration curve of FIG. 5 is used, the above system 2 is carried out. In this system 2, the ACVTG-5 is used as the immobilized antibody among the monoclonal antibodies that specifically bind to the turtle-derived vitellogenin.

The vitellogenin antibody used in the method for evaluating the endocrine disruptor contamination of the present embodiment is preferably an antibody that recognizes the turtle-derived vitellogenin as an antigen.

Furthermore, the vitellogenin antibody used in the method for evaluating the endocrine disruptor contamination according to the present embodiment is
It may be a polyclonal antibody.

The vitellogenin antibody used in the method for evaluating the contamination of endocrine disruptor according to the present embodiment may be a monoclonal antibody. Furthermore, the immunoglobulin class of the above vitellogenin antibody (immunoglobulin
It is preferred that class) is an IgG monoclonal antibody.

Further, in the above-mentioned immune reaction determination step of the method for evaluating endocrine disrupting substance contamination according to the present embodiment, any of an ELISA method, an immunoprecipitation method, a Western blotting method and an affinity chromatography method can be used as a method for determining an immune reaction. Is used.

When the ELISA method is used as a method for determining an immune reaction, as described above, whether or not vitellogenin is present in the body fluid of a freshwater turtle is determined by the immunoreaction using the above vitellogenin antibody and the above HRP-labeled antibody. Determine from the presence or absence of.

When the immunoprecipitation method is used as a method for determining the immune reaction, in the serum prepared from the blood of freshwater turtle,
The above-mentioned vitellogenin antibody is added, and further, anti-IgG antibody immobilized on agarose beads is added. After the reaction, the agarose beads are washed to extract vitellogenin contained in the serum. This extract is SDS-
As a sample for PAGE, SDS-PAGE may be carried out to determine whether or not the target vitellogenin is present depending on whether the molecular weights match. In addition, SDS-PAGE
After that, Western blotting may be performed, and it may be determined from the result of the antigen-antibody reaction whether or not the target vitellogenin derived from freshwater turtle.

When the affinity chromatography method is used as a method for determining an immune reaction, the above vitellogenin antibody is bound to an insoluble support and packed in a column to prepare a specific adsorbent, that is, an antibody column. By passing the serum prepared from the blood of freshwater turtle through the antibody column, the target vitellogenin derived from freshwater turtle is adsorbed to the antibody column. After that, all those that do not bind to the antibody column are washed from the column, and it is confirmed that there is no protein in the passing solution by measuring the protein concentration of the passing solution. Then, for example, the pH is adjusted to 2.5 to
0M 0.2M glycine buffer solution, or citrate buffer solution, or elution solution such as phosphate buffer solution (PBS) containing potassium thiocyanate or sodium thiocyanate was added to the antibody column to The vitellogenin derived from freshwater turtles bound to the column is released and eluted. A transient peak is obtained by measuring the protein concentration of this eluate. From this, it can be confirmed that the protein is present in the eluate.

The proteins in the above eluate were treated with SDS-PA.
As a sample for GE, SDS-PAGE may be performed to determine whether or not the target is vitellogenin by whether or not the molecular weights match. Furthermore, after SDS-PAGE,
Western blotting may be performed, and it may be determined from the result of the antigen-antibody reaction whether or not the target vitellogenin derived from freshwater turtle.

Furthermore, the monoclonal antibody of the present embodiment is a monoclonal antibody used in the above-mentioned method for evaluating the endocrine disruptor contamination, and comprises a mouse spleen lymphocyte immunized with fresh water turtle-derived vitellogenin and a mouse myeloma cell. It is produced by a hybridoma that is fused and specifically binds to the above-mentioned freshwater turtle-derived vitellogenin.

The polyclonal antibody of the present embodiment is a polyclonal antibody used in the method for evaluating the endocrine disruptor contamination, which is purified from immune serum of a rabbit immunized with vitellogenin derived from freshwater turtle, It specifically binds to the vitellogenin of origin.

Furthermore, the monoclonal antibody of the present embodiment is a monoclonal antibody that specifically binds to vitellogenin derived from freshwater turtles, and it is more preferable that the vitellogenin is vitellogenin derived from turtles.

Further, the polyclonal antibody of the present embodiment is a polyclonal antibody which specifically binds to vitellogenin derived from freshwater turtle,
More preferably, it is a vitellogenin derived from a turtle.

The monoclonal antibody and the polyclonal antibody of the present embodiment can be prepared by using a vitellogenin protein derived from freshwater turtle or reed turtle, fragments or other derivatives thereof, analogs thereof, or cells expressing them as an immunogen. Can be produced. These are normal immune operations,
Or hybridoma method (Kohler, G. and Milstein, C.,
Nature 256, 495-497 (1975)), trioma method, human B-cell hybridoma method (Kozbor, Immunology Today
4, 72 (1983)) and EBV-hybridoma method (Mo
noclonal Antibodies and Cancer Therapy, Alan R Lis
s, Inc., 77-96 (1985)).

Furthermore, the vitellogenin detection reagent of the present embodiment contains at least one of the above monoclonal antibody and the above polyclonal antibody.

The monoclonal and polyclonal antibodies used in the reagent for detecting vitellogenin of the present embodiment are 1M to 10M known buffer solutions (pH 7 to
8) Dilute with 0.001 μg / mL to 1 μg / mL
It can be used as a reagent for evaluation of endocrine disruptor contamination. Examples of the buffer solution include phosphate buffer solution (PBS), Tris buffer solution, glycine buffer solution and the like.

The buffer solution may contain, for example, commercially available glycerol as a component.

Further, the kit for evaluating the contamination of endocrine disrupting substances of the present embodiment contains the above-mentioned detection reagent.

The monoclonal antibody and the polyclonal antibody used in the above detection reagent can be used by immobilizing them on a carrier, for example. Carriers include commercially available gel filtration agents such as Sepharose. Further, for example, the monoclonal antibody or the polyclonal antibody used in the above detection reagent can be used by binding to a carrier, dispersing in a buffer solution, or packing in a column.

In addition, in the above-mentioned kit for evaluating endocrine disrupting agent contamination, for example, in the description, based on the measured absorbance when the monoclonal antibody and polyclonal antibody used in this evaluation kit are used, A calibration curve for estimating the amount of vitellogenin is shown.

Further, the kit for evaluating the endocrine disruptor contamination further includes, for example, commercially available glycerol, bovine serum albumin, gelatin, skim milk (manufactured by Difco),
Blocking reagents such as Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.) may be included.

[0080]

EXAMPLES Examples and FIGS. 1, 2, and 6 to 1 will be described below.
The present invention will be described more specifically on the basis of 0, but the present invention is not limited thereto. The vitellogenin used in the examples was purified as follows. Moreover, the polyclonal antibody and the monoclonal antibody used in the examples were prepared as follows. Furthermore, the antibody titer was evaluated as follows.

[Purification Example of Vitellogenin Derived from Tortoises] A commercially available 17β-estradiol having an estrogenic effect was diluted in corn oil (1 μg / g turtle body weight) into the abdominal cavity of female Tortoises, and 0.1 mL of this was added.
Injections were made once a day for 7 days, then every other day from the first injection day until the 21st day, injecting the same amount every other day. On the day following the last injection, that is, on day 22, whole blood was collected and plasma was collected. 20 mL of EDTA (ethylene
diaminetetraacetic acid: ethylenediaminetetraacetic acid)
20 mL of the solution was added and stirred. Then 0.5M Mg
1.6 ml of Cl ₂ was added and gently stirred, and then a precipitate was formed by centrifugation. The supernatant was discarded and the precipitate produced by centrifugation contained 1M NaCl 50
The precipitate was dissolved by adding 3 mL of mM Tris-HCl buffer solution, and then the precipitate was formed again by centrifugation.
The supernatant is discarded, and the precipitate generated by centrifugation is added to the above 1
The precipitate was redissolved by adding 3 mL of 50 mM Tris-HCl buffer solution containing M NaCl.

About this solution, commercially available Sepharose-6B was used.
It was possible to obtain the purified horseshoe vitellogenin by performing gel filtration chromatography using the above and collecting a single peak. Using a part of this purified product as a sample, SDS polyacrylamide gel electrophoresis was performed. As a result, vitellogenin derived from the Japanese turtle,
As shown in FIG. 1, it was confirmed that the protein had a molecular weight of about 214,000.

[Example of Preparation of Polyclonal Antibody] 1 mg / mL of the above-mentioned purified horseshoe vitellogenin used as an antigen and an equal amount of commercially available Freund's adjuvant were mixed to prepare an emulsion, and 1 mL / animal was prepared in the back skin of a rabbit. I was immunized. Booster immunization (1 mL / animal) was performed every 3 weeks. In the final third immunization, 0.5 ml of the purified horseshoe vitellogenin (1 mg / mL) alone was injected without mixing the adjuvant. One week after the final immunization, blood was collected and serum was collected. This serum was added to a commercially available affinity chromatography, protein A column for purification.

[Example of Preparation of Monoclonal Antibody] Commercially available mouse-derived myeloma cells (X63.Ag.653) and spleen cells of BALB / c mice immunized with turtle-derived vitellogenin were fused by the polyethylene glycol method. After that, HAT selection was performed. An antibody-producing positive cell group was selected by screening by the ELISA method, and the cell group was cloned to establish an antibody-producing hybridoma. The hybridoma was transplanted into the abdominal cavity of a mouse, and ascites was collected to obtain a purified monoclonal antibody using a commercially available affinity chromatography and protein G column. Four types of monoclonal antibodies that specifically bind to vitellogenin derived from the reed turtle have been established. They were named ACVTG-1, ACVTG-3, ACVTG-4, and ACVTG-5, respectively.

[Evaluation of antibody titer] The antibody titer of the antibody was evaluated by the ELISA method.

The method for evaluating the ELISA antibody titer of the polyclonal antibody that specifically binds to the vitellogenin derived from the above-mentioned turtle will be specifically described below.

100 μL / wel of a phosphate buffer solution (PBS) containing 10 μg / mL vitellogenin derived from turtles.
l Add to a commercially available ELISA plate and leave it at 4 ℃ overnight.
It was solid-phased. After removing the antibody solution, 0.05% Tween2
Wash twice with PBS containing 0%, then skim 10% (Di
PBS containing fco) was added at 200 μL / well, and the temperature was 37 ° C.
It was left for 1 hour for blocking. Then, as the primary antibody, the above polyclonal antibody serially diluted with PBS containing 0.05% Tween 20 was added to 100 μL / well of the above ELISA plate and reacted at 37 ° C. for 1 hour. After the same washing operation as above was repeated 5 times, 100 μL of HRP-labeled anti-rabbit IgG antibody (Bio-Rad) diluted 3000 times with PBS containing 0.05% Tween20 was further used as a secondary antibody. / Well In addition to the above ELISA plate,
The reaction was carried out at 37 ° C for 1 hour. Next, 0.4 mg / mL o-phenylenediamine (o-phenylenediamine) aqueous solution containing 0.006% hydrogen peroxide was added to the above ELISA plate in an amount of 150 μL / well, and a color reaction was carried out at 37 ° C. for 30 minutes, which was commercially available. Absorbance was measured at 450 nm with a plate reader of.

The results are shown in FIG. The ELISA antibody titer of the above polyclonal antibody at 100 ng / mL is
As a result of measuring the absorbance at 450 nm, it was 3.00. The vertical axis of FIG. 6 is 450 n according to the plate reader.
The measured value in m is shown, and the horizontal axis shows the concentration of the polyclonal antibody.

The method for evaluating the ELISA antibody titer of the monoclonal antibody that specifically binds to the vitellogenin derived from the above-mentioned turtle will be specifically described below.

As the primary antibody, the above monoclonal antibody ACVTG-1 serially diluted with PBS containing 0.05% Tween 20.
HRP-labeled anti-mouse I, which was diluted 3000-fold with PBS containing 0.05% Tween 20 as a secondary antibody
The ELISA antibody titer of the above-mentioned monoclonal antibody was measured in the same manner as the above-mentioned method for evaluating the ELISA antibody titer of the polyclonal antibody except that the gG antibody was used. Also, with respect to the above-mentioned monoclonal antibodies ACVTG-3, ACVTG-4, and ACVTG-5, the ELISA antibody titer was measured in the same manner as the above-mentioned evaluation method of the ELISA antibody titer of the polyclonal antibody.

The results are shown in FIG. The ELISA antibody titers of the monoclonal antibodies ACVTG-1, ACVTG-3, ACVTG-4, and ACVTG-5 at 100 ng / mL were measured by measuring the absorbance at 450 nm using a commercially available plate reader, and the values are shown in Table 1. Became. The vertical axis of FIG. 7 represents the value measured by the plate reader at 450 nm, and the horizontal axis represents the concentration of the monoclonal antibody.

In addition, the above-mentioned monoclonal antibody ACVTG-
1, ACVTG-3, ACVTG-4, and ACVTG-5 all had an immunoglobulin subclass of mouse IgG ₁ . Table 1 shows various properties of the monoclonal antibody that specifically binds to vitellogenin derived from the Japanese turtle.

[0093]

[Table 1]

[Confirmation of Reactivity between Monoclonal Antibody and Antigen] SDS polyacrylamide gel electrophoresis was carried out using purified turtle turtle-derived vitellogenin as a sample, and then Western blotting was used to detect the above-mentioned four types of the mouse turtle. The reactivity with vitellogenin was confirmed. As shown in FIG. 8, ACVTG-5
Showed a particularly strong reaction, and only ACVTG-4 showed no reaction.

[Example 1] Sandwich ELISA using vitellogenin derived from Triggered turtle as an antigen, using the above-mentioned polyclonal antibody as a solid-phased antibody, and using ACVTG-1 among the above-mentioned monoclonal antibodies as an antibody on the side to which a secondary antibody binds The method (system 1) was performed (see FIG. 2).

Specifically, one of the above polyclonal antibodies was used.
PBS containing 0 μg / mL was added to a 100 μL / well commercially available ELISA plate and coated overnight at 4 ° C. After removing the antibody solution, the plate was washed twice with PBS containing 0.05% Tween 20, and P containing 10% skim milk (manufactured by Difco)
Add 200 μL / well of BS to the above ELISA plate, and add 3
It was left standing at 7 ° C for 1 hour for blocking. 100% purified vitellogenin derived from horseshoe turtle diluted serially with PBS
μL / well was added to the above ELISA plate and reacted at 37 ° C. for 1 hour. After the same washing operation as above was repeated 5 times, 100 μL of PBS containing 1 μg / mL of ACVTG-1 was used.
/ Well was added to each well and reacted at 37 ° C. for 1 hour.

The same washing procedure as above was repeated 5 times, and then HRP-labeled anti-mouse IgG antibody (manufactured by Bio-Rad) diluted 3000 times with PBS containing 0.05% Tween 20 was added at 100 μL / well to the above-mentioned ELISA. The plate was added and reacted at 37 ° C. for 1 hour. Next, 0.4 mg / mL o-phenylenediamine (o-ph) containing 0.006% hydrogen peroxide was added.
enylenediamine) aqueous solution on the above ELISA plate 15
0 μL / well was added, color reaction was carried out at 37 ° C. for 30 minutes, and the absorbance was measured at 450 nm by a commercially available plate reader. The result is shown in FIG.

The vertical axis of FIG. 9 shows the value measured by the plate reader at 450 nm, and the horizontal axis shows the vitellogenin concentration.

[Example 2] ACVTG-3 among the above-mentioned monoclonal antibodies was used as the antibody to which the above-mentioned secondary antibody binds.
The evaluation method according to the present invention was carried out in the same manner as in Example 1 except that was used. The result is shown in FIG.

[Example 3] ACVTG-4 among the above-mentioned monoclonal antibodies was used as the antibody to which the above-mentioned secondary antibody binds.
The evaluation method according to the present invention was carried out in the same manner as in Example 1 except that was used. The result is shown in FIG.

Example 4 ACVTG-5 among the above-mentioned monoclonal antibodies was used as the antibody to which the above-mentioned secondary antibody binds.
The evaluation method according to the present invention was carried out in the same manner as in Example 1 except that was used. The result is shown in FIG.

[Example 5] ACVTG-among the above-mentioned monoclonal antibodies was prepared using vitellogenin derived from the Japanese turtle as an antigen.
The sandwich ELISA method (system 2) was performed using 1 as the immobilized antibody and the above polyclonal antibody as the antibody on the side to which the secondary antibody binds (see FIG. 3).

Specifically, the above ACVTG-1 was added at 10 μg /
PBS containing 100 mL / well of a commercially available ELISA plate was added and coated overnight at 4 ° C. After removing the antibody solution, wash twice with PBS containing 0.05% Tween 20,
20% PBS containing 10% skim milk (Difco)
Add 0 μL / well to the above ELISA plate and add 1 at 37 ℃.
It was left for a time and blocked. 100 μL / we of purified vitellogenin derived from horseshoe turtle diluted serially with PBS
ll It was added to the above ELISA plate and reacted at 37 ° C. for 1 hour. After the same washing procedure as above was repeated 5 times, 100 μl of PBS containing 1 μg / mL of the above polyclonal antibody was used.
L / well The mixture was added to the above ELISA plate and reacted at 37 ° C. for 1 hour.

After the same washing operation as above was repeated 5 times, HRP-labeled anti-rabbit IgG antibody (manufactured by Bio-Rad) diluted 3000 times with PBS containing 0.05% Tween20.
100 μL / well to the above ELISA plate, and add 37 ° C
And reacted for 1 hour. Next, 0.4 mg / mL o-phenylenediamine (o-containing 0.006% hydrogen peroxide)
phenylenediamine) aqueous solution to the above ELISA plate 1
50 μL / well was added, color reaction was carried out at 37 ° C. for 30 minutes, and the absorbance was measured at 450 nm by a commercially available plate reader. The result is shown in FIG.

The vertical axis of FIG. 10 shows the value measured by the plate reader at 450 nm, and the horizontal axis shows the vitellogenin concentration.

[Example 6] As the immobilized antibody, except that ACVTG-3 was used among the above monoclonal antibodies,
The evaluation method according to the present invention was carried out in the same manner as in Example 5. The result is shown in FIG.

[Example 7] [0107] As the immobilized antibody, ACVTG-4 was used among the above monoclonal antibodies,
The evaluation method according to the present invention was carried out in the same manner as in Example 5. The result is shown in FIG.

[Example 8] As a solid-phased antibody, ACVTG-5 among the above-mentioned monoclonal antibodies was used, except that
The evaluation method according to the present invention was carried out in the same manner as in Example 5. The result is shown in FIG.

From the above results, the above-mentioned monoclonal antibody and the above-mentioned polyclonal antibody prepared by using vitellogenin derived from horsetail turtle as an antigen can be used for determining the presence or absence of vitellogenin in the body fluid of turtles. Therefore, when assessing the pollution situation of endocrine disrupting substances in the freshwater environment, it is possible to monitor reed turtles.

Further, in the system 1, in the case of the above-mentioned Example 4 in which ACVTG-5 was used as the antibody on the side to which the secondary antibody binds, the small amount of vitellogenin was satisfactorily captured (see FIG. 9). When ACVTG-5 was used, the minimum detected vitellogenin concentration was 10 ng / mL and 100 ng
A calibration curve showing excellent linearity up to / mL was obtained (Fig. 4).
reference).

Furthermore, in System 2, in the case of Example 8 using ACVTG-5 as the immobilized antibody, a small amount of vitellogenin was captured best (see FIG. 10). When ACVTG-5 was used, the minimum detected vitellogenin concentration was 1 ng / mL, and a calibration curve showing excellent linearity up to 50 ng / mL was obtained (see FIG. 5).

[0112]

As described above, the method for assessing endocrine disrupting substance contamination according to the present invention comprises a vitellogenin antibody that specifically recognizes vitellogenin derived from freshwater turtles and a body fluid of freshwater turtles to be detected as vitellogenin. It includes an immune reaction step of reacting and an immune reaction determining step of determining the presence or absence of an immune reaction by the vitellogenin antibody. Preferably, it has a vitellogenin quantification step of quantifying the amount of vitellogenin produced in the freshwater turtle body based on the immune reaction by the vitellogenin antibody.

Further, the vitellogenin quantification step is as follows.
It is quantified by using a calibration curve for estimating the amount of vitellogenin based on the measured absorbance.

Further, in the method for evaluating the endocrine disrupting substance contamination according to the present invention, the above vitellogenin antibody recognizes vitellogenin derived from the herring turtle as an antigen.
In addition, the evaluation method of endocrine disruptor contamination according to the present invention,
The vitellogenin antibody is a polyclonal antibody.
Furthermore, in the method for evaluating endocrine disrupter contamination according to the present invention, the vitellogenin antibody is a monoclonal antibody, and preferably the immunoglobulin class is an IgG monoclonal antibody.

In the method for evaluating endocrine disrupting substance contamination according to the present invention, any one of the ELISA method, the immunoprecipitation method, the Western blotting method and the affinity chromatography method can be used as the method for determining the immune reaction in the above-mentioned immune reaction determining step. Is used.

The monoclonal antibody according to the present invention is a monoclonal antibody used in the above-described method for evaluating endocrine disruptor contamination, which comprises fusing mouse spleen lymphocytes immunized with freshwater turtle-derived vitellogenin with mouse myeloma cells. Is produced by the hybridoma and specifically binds to vitellogenin derived from the freshwater turtle.

Further, the polyclonal antibody according to the present invention is a polyclonal antibody used in the above-mentioned method for evaluating the endocrine disruptor contamination, which is purified from immune serum of a rabbit immunized with vitellogenin derived from freshwater turtle and derived from the freshwater turtle. Specifically binds to vitellogenin.

Furthermore, the monoclonal antibody according to the present invention is a monoclonal antibody that specifically binds to vitellogenin derived from freshwater turtle, and preferably the vitellogenin is a vitellogenin derived from turtle.

Further, the polyclonal antibody according to the present invention is a polyclonal antibody which specifically binds to vitellogenin derived from freshwater turtle, and preferably the vitellogenin is a vitellogenin derived from turtle.

Therefore, the method for evaluating the endocrine disrupting substance pollution according to the present invention is used to evaluate the water area where severe pollution has progressed, and to evaluate the endocrine disrupting substance in the freshwater environment, that is, environmental hormones. It is possible to investigate the bioaccumulation of the plant, to prolong the period for measuring the environmental hormone contamination, and to improve the accuracy of the measurement data regarding the environmental hormone contamination.

Furthermore, the reagent for detecting vitellogenin according to the present invention contains at least one of the above monoclonal antibody and the above polyclonal antibody.

The kit for evaluating the contamination of endocrine disrupting substances according to the present invention contains the above reagent for detecting vitellogenin.

Therefore, the reagent for detecting vitellogenin and the kit for evaluating the contamination of endocrine disrupting substances of the above-mentioned invention have the same effects as those described in the method for evaluating the contamination of endocrine disrupting substances.

[Brief description of drawings]

FIG. 1 is a drawing-substituting photograph showing the results of an experiment in which the molecular weight of Reeve's turtle vitellogenin was confirmed by SDS polyacrylamide gel electrophoresis-Western blotting.

FIG. 2 is a schematic diagram showing a system 1, which is a vitellogenin quantification method using the sandwich ELISA method according to the present invention.

FIG. 3 is a schematic diagram showing System 2, which is a method for quantifying vitellogenin using the sandwich ELISA method according to the present invention.

FIG. 4 is a graph showing a vitellogenin calibration curve when the monoclonal antibody ACVTG-5 that specifically binds to velvet genin derived from the turtle according to the present invention is used in the system 1.

FIG. 5 is a graph showing a vitellogenin calibration curve when the monoclonal antibody ACVTG-5 that specifically binds to velvet genin derived from the turtle according to the present invention is used in the system 2.

FIG. 6 is a graph showing the ELISA antibody titer of the polyclonal antibody that specifically binds to the produced turtle-derived vitellogenin.

FIG. 7 is a graph showing the ELISA antibody titer of the produced monoclonal antibody that specifically binds to vitellogenin derived from Triggered turtle.

FIG. 8 is a drawing-substituting photograph showing the results of an experiment in which the reactivity of the produced monoclonal antibody that specifically binds to vitellogenin derived from Triggered turtle and the reed turtle vitellogenin was confirmed by Western blotting.

FIG. 9 is a graph showing an experimental result of the system 1.

FIG. 10 is a graph showing an experimental result of the system 2 described above.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Masahiro Saka             1-9 Minamiyuda, Iseta Town, Uji City, Kyoto Prefecture F-term (reference) 4B064 AG27 CA10 CA20 CC24 DA13                       DA16                 4G057 AB38 AF13                 4H045 AA11 AA20 AA30 CA40 DA75                       DA76 EA50 FA74

Claims (16)

[Claims] 1. An immunoreaction step of reacting a vitellogenin antibody that specifically recognizes vitellogenin derived from freshwater turtle with a body fluid of a freshwater turtle to be the target of vitellogenin detection, and immunity for determining the presence or absence of an immune reaction by the vitellogenin antibody. A method for evaluating endocrine disruptor contamination, comprising a reaction determination step. 2. The endocrine disruptor contaminant according to claim 1, further comprising a vitellogenin quantification step of quantifying the amount of vitellogenin produced in the freshwater turtle body based on the immune reaction caused by the vitellogenin antibody. Evaluation method. 3. The endocrine disruptor contamination according to claim 2, wherein the vitellogenin quantification step is quantified by using a calibration curve for estimating the amount of vitellogenin based on the measured absorbance. Evaluation method. 4. The method for evaluating endocrine disruptor contamination according to any one of claims 1 to 3, wherein the vitellogenin antibody recognizes vitellogenin derived from the Japanese turtle as an antigen. 5. The method for evaluating endocrine disrupter contamination according to any one of claims 1 to 4, wherein the vitellogenin antibody is a polyclonal antibody. 6. The method for evaluating endocrine disruptor contamination according to any one of claims 1 to 4, wherein the vitellogenin antibody is a monoclonal antibody. 7. The method for evaluating endocrine disruptor contamination according to claim 6, wherein the immunoglobulin class of the vitellogenin antibody is an IgG monoclonal antibody. 8. The method for determining an immune reaction in the immunological reaction determining step, wherein any one of an ELISA method, an immunoprecipitation method, a Western blotting method, and an affinity chromatography method is used. 7
The method for evaluating endocrine disrupter contamination according to any one of 1. 9. A monoclonal antibody used in the method for evaluating the endocrine disruptor contamination according to claim 1, which is a mouse spleen lymphocyte immunized with fresh water turtle-derived vitellogenin and a mouse myeloma. A monoclonal antibody produced by a hybridoma formed by fusing with cells, which specifically binds to the above-mentioned freshwater turtle-derived vitellogenin. 10. A polyclonal antibody used in the method for evaluating the endocrine disruptor contamination according to any one of claims 1 to 8, which is purified from immune serum of a rabbit immunized with vitellogenin derived from freshwater turtle, A polyclonal antibody that specifically binds to vitellogenin derived from the freshwater turtle. 11. A monoclonal antibody which specifically binds to vitellogenin derived from freshwater turtle. 12. The monoclonal antibody according to claim 11, wherein the vitellogenin is vitellogenin derived from a turtle. 13. A polyclonal antibody which specifically binds to vitellogenin derived from freshwater turtle. 14. The polyclonal antibody according to claim 13, wherein the vitellogenin is a turtle-derived vitellogenin. 15. A reagent for detecting vitellogenin, which comprises at least one of the monoclonal antibody according to claim 9, 11 or 12 and the polyclonal antibody according to claim 10, 13 or 14. 16. A kit for evaluating endocrine disruptor contamination, which comprises the detection reagent according to claim 15.