KR100737186B1 - Biosensor for Detection of estriol Having Anti-estriol Antiserum and Detecting Process of estriol - Google Patents

Abstract

The present invention relates to a biosensor for detecting estriol and an estriol detection method comprising an anti-estriol antiserum for detecting estriol, which is one of natural female hormones. Preparing a biosensor for detecting an estriol comprising an anti-estriol antiserum isolated from a mouse immunized with a conjugate of estradiol and a protein; Preparing an anti-estriol antiserum by injecting the conjugate into a mouse and immunoreacting it; And when the estriol detection method comprising the step of antigen-antibody reaction of the marine organic pollutant containing the anti-estriol antiserum and estriol, the marine organic pollutant that requires expensive equipment In chemical analysis, it is possible to accurately and simply determine whether natural female hormones such as estriol are included.

Estriol, Biosensor, Antibody Antibody Reactions

Description

Biosensor for detection of estriol having anti-estriol antiserum and detecting process of estriol}

1 is a graph measuring the change in titer of anti-estriol antiserum using an Enzyme-Linked Immuno Sorbent Assay (ELISA) according to a preferred embodiment of the present invention.

Figure 2 is a result of measuring the color development of anti-estriol antiserum using an ELISA according to an embodiment of the present invention.

The present invention relates to a biosensor for detecting estriol, and more particularly, to a biosensor for detecting estriol and an estriol detection method comprising anti-estriol antiserum. will be. More specifically, anti-estriol antisera isolated from mice immunized with a combination of estriol and protein is used to detect estriol using antigen antibody reaction.

In today's oceans, the presence of aquatic ecosystems is threatened by synthetic synthetic chemicals and wastewater from households and industries. Accordingly, countries around the world are preparing the present situation of environmental pollutants in the aquatic environment and various technical methods and institutional devices for managing them.

The development of biosensors that can detect environmental pollutants in Korea is being studied for water quality monitoring, soil toxicity monitoring, and gas toxicity monitoring. In particular, biological biosensors include water fleas, seaweeds, fish, and the like, and methods using recombinant microorganisms derived from microorganisms such as Escherichia coli, Bacillus, Pseudomonas, and Salmonella. Among them, the method of using water fleas, algae, fish, etc. has a disadvantage in that it can only provide a single detection function due to problems such as maintenance and storage of living organisms. On the other hand, in the method of using a recombinant microorganism, studies using biological light (bioluminescence) and green fluorescence (green fluorescence) as marker genes are actively underway. Recently, microarray techniques using DNA chips have also been applied.

However, although the development of bacterial DNA chips for marine organic pollutants and the development of biosensors using these bacterial genes and fluorescent or luminescent genes have been recently conducted, practically in situ ng or pg Biosensors capable of detecting marine organic pollutants at the level have not been developed yet. In addition, the research on marine organic pollutants until now is mainly conducted on the ecological risk assessment technique and monitoring of organic pollutants centered on fresh water, and the research on marine organic pollutants is rarely conducted. to be.

The conventional marine organic contaminants have been analyzed by HPLC, FPLC, or GC-MS in addition to complex sample preparation. However, it has been pointed out that expensive equipment, manpower, and a lot of analysis costs are required. Therefore, the development of biosensors that can detect marine organic pollutants easily and quickly is recognized as an important technology to reduce the cost of chemical analysis and increase the number of processed samples because it can screen large quantities of samples semi-quantitatively.

Currently, some molecular biological studies on marine organic pollutants have been initiated, but only a few studies on immunological qualitative quantification (Szekacs, et.al., 1999; Dickert, et.al., 2000; Kim, et.al., 2001; Scharnweber, et.al., 2001; Lee, et. Al., 2002), and these are mainly related to the analysis by ELISA, the study of Molecular imprints, and the development of monoclonal antibodies. . SDI (Newark, DE) or Biosense Co. Although some have been commercialized by other companies, such kits are commercially available and work on a wide range of organic contaminants at the same time. There is a problem that unknown.

Currently, the antigen-antibody method is most frequently used as a rapid detection method of toxins and other harmful substances. However, these harmful substances are not easy to produce antibodies, and thus only the early stages of research have been made. Therefore, there is a need for a technology that can specifically detect harmful substances contained in marine organic pollutants and accurately detect them, and for this purpose, it is time to develop antibodies for each component of marine organic pollutants.

The present invention has been made to solve the problems described above, and to provide a biosensor for detecting an estriol and an estriol detection method comprising an anti-estriol antiserum. Through this, in the chemical analysis of marine organic pollutants requiring expensive equipment, it is an object of the present invention to accurately and simply determine whether natural female hormones such as estriol are included.

The biosensor for detecting an estriol of the present invention for achieving the above object is an anti-estriol antiserum isolated from a mouse immunized with a combination of estriol and protein. It is characterized by including. Herein, the protein is preferably ovalbumin (OVA) or cationized bovine serum albumin (cBSA).

In accordance with another aspect of the present invention, there is provided a method for detecting an estriol, comprising: preparing a combination of an estriol and a protein; Preparing an anti-estriol antiserum by injecting the conjugate into a mouse and immunoreacting it; And antigen-antibody reaction of the marine organic pollutant including the anti-estriol antiserum and estriol. The antigen antibody reaction may be an intriol detection method using an indirect enzyme immunoassay (ELISA) using a microplate or a method of measuring color development by the indirect enzyme immunoassay.

The present invention relates to a biosensor capable of detecting estriol, which is one of natural female hormones, and a method for detecting marine organic pollutants using the same, and more particularly to marine organic pollutants such as estriol. It is a method of detecting estriol by measuring the degree of color development by the reaction of the produced antibody and marine organic pollutants by injection into the mouse and serum production.

Estriol is one of the female pregnancy hormones and one of the estrogens. It is a weakened form of estrogen produced in the fetal placental unit and is found primarily in pregnant women. This hormone is widely used in Europe and Asia to treat menopausal symptoms, but it is not recognized as a hormone replacement therapy in the United States because it does not prevent osteoporosis.

The biosensor for estriol detection according to the present invention is manufactured through the following process. First, conjugates of estriol and carrier proteins are prepared. Ovalbumin (OVA) or cationized bovine serum albumin (cBSA) may be used as the delivery protein. The cBSA-estriol conjugate thus prepared is injected subcutaneously and intramuscularly into mice to immunize, and then blood is collected to separate anti-estriol antiserum.

In order to determine the titer of the anti-estriol antiserum obtained from the mice immunized with cBSA-estriol, indirect ELISA was performed using an OVA-estriol-coated microtiter plate. Anti-estriol antiserum was shown to react more specifically with cBSA-estriol than cBSA protein or OVA protein used as a conjugate. That is, the anti-estriol antiserum according to the present invention specifically reacts with estriol, which is one of marine organic pollutants.

In addition, when the color development was measured using a marine organic pollutant containing estriol as a substrate according to the indirect ELISA method, the sensitivity of the anti-estriol antiserum according to the present invention to the estriol is also very high. It was found that when the anti-estriol antiserum was applied in situ, the estriol and protein conjugate could be effectively detected up to a concentration of about 0.025 ng.

Hereinafter, one preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings. The invention can be better understood by the following examples, which are intended for the purpose of illustration of the invention and are not intended to limit the scope of protection defined by the appended claims.

Example  1: Preparation of estriol-protein conjugate

Purified estriol was purchased from Sigma, and binding to protein was performed using a binding kit (Imject).^RPharmalink^TM Immunogen kit, Pierce) was used. That is, cationized bovine serum albumin (cBSA) and ovalbumin (OVA), which are to be used as carrier proteins, are dissolved in distilled water to a concentration of 10 mg / ml. A concentration of 0.5 mg / ml was prepared in conjugation buffer (0.1 M MES, 0.15 M NaCl, pH 4.7).

For conjugation, 500 μl of estriol and 200 μl of the delivery protein solution were mixed, and then 50 μl of coupling solution was added to carry out the binding reaction at room temperature for 2 hours. Although some precipitate was formed during the reaction, the precipitate was removed by centrifugation. After the reaction, EDTA remaining in the unbound estriol and delivery protein solution was removed using a D-SaltTM desalting column.

Estriol-delivered protein conjugate was confirmed by SDS-PAGE, and the protein was quantified using BCA protein assay reagent (Pierce). An appropriate amount was dispensed and stored at 20 ° C.

Example  2: Immunization

Polyclonal antiserum was prepared using 4 week old BALB / c mice. In the immune response, Injection ^R Alum (Pierce) was used as an adjuvant up to tertiary immunity and no adjuvant was used for the fourth immunity.

Each immune response was diluted in sterile PBS to 100 μg of cBSA-estriol conjugate per mouse and injected by subcutaneous and intramuscular (Subcutaneous, intradermal injection) route. The immune response was conducted once every 10 days. After a total of 4 immune reactions, blood was collected to determine antibody titers, and the blood was centrifuged to use only the serum of the upper layer.

Example  3: enzyme immunoassay (ELISA)

① Immobilization of OVA-estriol Conjugates

Immobilization of the OVA-estriol conjugate was performed by diluting 200 μl of the OVA-estriol conjugate diluted to 0.5 μg / ml with the coating buffer (0.2 M carbonate buffer, pH 9.4) in a 96-well mite titer plate at 37 ° C. The reaction was carried out for 2 hours. After the reaction, a 1% (w / v) non fat dry skim milk solution was prepared in PBST for blocking, and 100 μl of each well was dispensed at 37 ° C. for 1 hour. After blocking the microplates were washed three times with PBST.

② Indirect ELISA using OVA-estriol conjugate

Antisera titer was measured using OVA (1 μg / well) and cBSA (1 μg / well) as a control of wells coated with OVA-estriol conjugates. Antiserum taken from mice immunized with cBSA-estriol for primary antibody binding was serially diluted from 1/500 to 1/500000 using 0.1% (w / v) skim milk / PBST, and then 100 μl divided. The reaction was carried out at 37 ° C. for 2 hours. Two antigens were performed for each antigen, and the wells containing no antiserum were used as counter controls. After the reaction was completed, washed three times with PBST.

As a secondary antibody, goat anti-mouse IgG-HRP conjugate (goat anti-mouse HRP conjugate, Sigma) was used by diluting 1000 times with 0.1% (w / v) skim milk / PBST. 100 μl of each well was reacted at 37 ° C. for 2 hours, washed with PBST, and then 50 μl of substrate (4 mg OPD and 5 μl hydrogen peroxide in citrate-phosphate buffer) was added to each well for 10 minutes. After the color reaction was observed, the color reaction was stopped by using 100 µl of the suspension solution (1 N H ₂ SO ₄ ). Absorbance was measured at 490 nm using a Microplate reader.

Through the experiment conducted above, the following results were obtained.

Result example  1: Anti-estriol Antiserum Titer  Measure

In order to determine the titer of antiserum obtained from mice immunized with cBSA-estriol, indirect ELISA was performed using microtiter plates coated with OVA-estriol, cBSA and OVA. 1 is a graph measuring the titer change of anti-estriol antiserum using an Enzyme-Linked Immuno Sorbent Assay (ELISA) according to a preferred embodiment of the present invention.

As shown here, it can be seen that the anti-estriol antiserum according to the present invention reacts specifically with the OVA-estriol to which the estriol is bound, as compared with the OVA or cBSA protein alone. It can be seen that estriol reacts strongly with the anti-estriol antiserum according to the invention over OVA or cBSA protein throughout dilution concentrations (see FIG. 1).

Result example  2: Sensitivity measurement of anti-estriol antiserum for field application

According to the above-described indirect ELISA method, when the color development was measured using a marine organic pollutant containing estriol as a substrate, the sensitivity of the anti-estriol antiserum to the estriol according to the present invention was also very high. appear. Figure 2 is a result of measuring the color development of anti-estriol antiserum using an ELISA according to an embodiment of the present invention.

In other words, when the anti-estriol antiserum was applied in situ, the serum showed a very high absorbance value (OD490> 0.1) even under the dilution of 1 / 100,0000 of the estriol and the protein conjugate. It can be seen that it has high sensitivity to triol (see FIG. 2). Table 1 shows the change in sensitivity of anti-estriol antiserum according to the dilution ratio of estriol and protein conjugate.

Table 1: Changes in sensitivity of anti-estriol antiserum according to dilution ratio of estriol and protein conjugate

   Dilution factor  Blank  OVA-Estriol  cBSA  OVA 100 0.037 0.233 0.189 0.034 500 0.038 0.236 0.192 0.038 1000 0.036 0.222 0.141 0.036 5000 0.035 0.188 0.09 0.037 10000 0.035 0.159 0.058 0.038 50000 0.035 0.121 0.046 0.036 100000 0.038 0.095 0.04 0.035 500000 0.037 0.046 0.038 0.036 0 0.035 0.038 0.036 0.036

As described above, when the anti-estriol antiserum was applied in situ, it was confirmed that the separate formula can effectively detect the estriol and the protein conjugate up to a concentration of about 0.025 ng. From the above results, it can be confirmed that the anti-estriol antiserum prepared according to the present invention and the estriol detection method using the same are effective for the early precision assay of marine pollutants including estriol.

According to the present invention as described above, a biosensor for detecting an estriol comprising an anti-estriol antiserum isolated from a mouse immunized with a combination of estriol and protein and Preparing a combination of estriol and protein; Preparing an anti-estriol antiserum by injecting the conjugate into a mouse and immunoreacting it; And it is possible to provide an antistriol detection method comprising the step of antigen-antibody reaction of the marine organic pollutant containing the anti-estriol antiserum and estriol.

The estriol detection biosensor according to the present invention and the estriol assay using the same is a new technology that has not yet been systematically tried or developed in the world, and it is a chemical of marine organic pollutants requiring expensive equipment In the analysis, it is possible to accurately and simply determine whether an endocrine disruptor such as estriol is included. In addition, the method for detecting toxic substances using the antigen-antibody reaction according to the present invention is capable of screening a large amount of samples semi-quantitatively, so that it is also applicable to the early precise diagnosis of other harmful toxins, and its use is very high.

Claims (4)

delete delete Dissolve cationized bovine serum albumin (cBSA) or ovalbumin (OVA) in distilled water to a concentration of 10 mg / ml to be used as a carrier protein. Prepare a concentration of 0.5 mg / ml in the conjugation buffer, mix 500 μl of the estriol and 200 μl of the delivery protein, and add 50 μl of coupling solution to perform a binding reaction at room temperature for 2 hours. And then preparing a conjugate of estriol and protein at a concentration of 1 μg / ml; Preparing an anti-estriol antiserum by injecting the conjugate into the mouse to perform an immune reaction, collecting blood, and then centrifuging the blood; Diluting the anti-estriol antiserum 100,000-fold with 0.1% (w / v) skim milk / PBST; Antigen antibody reaction of the diluted anti-estriol antiserum with 50 μl of marine organic pollutant containing estriol; And Colorimetric reaction by the reaction comprising a step of measuring the absorbance at 490 nm using a microplate reader (Elateol detection method). delete

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