KR100737246B1 - Bisphenol detection biosensor containing anti-bisphenol antiserum and bisphenol detection method - Google Patents

Abstract

The present invention relates to a biosensor for detecting bisphenol and a method for detecting bisphenol, including an anti-bisphenol antiserum for detecting bisphenol (A, BPA), which is one of endocrine disruptors. Preparing a bisphenol detection biosensor comprising an anti-bisphenol antiserum isolated from a mouse immunized with a conjugate of protein and a combination of bisphenol and protein; Preparing an anti-bisphenol antiserum by injecting the conjugate into a mouse and immunoreacting it; And when the bisphenol detection method comprising the step of antigen-antibody reaction of the marine organic pollutant containing the anti-bisphenol antiserum and bisphenol, bisphenol in the chemical analysis of marine organic pollutants requiring expensive equipment There is an effect that can be confirmed accurately and simply whether the endocrine disruptor, such as containing.

Bisphenol, Biosensor, Antigen Antibody Reactions

Description

Biosensor for Detection of Bisphenol-A Having Anti-BPA Antiserum and Detecting Process of Bisphenol-A}

1 is a graph measuring the change in titer of anti-bisphenol-A 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-bisphenol antiserum using an ELISA according to an embodiment of the present invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor for detecting bisphenol (A, BPA), and more particularly, to a biosensor for detecting bisphenol and a method for detecting bisphenol including anti-BPA antiserum. More specifically, anti-bisphenol antisera isolated from mice immunized with a combination of bisphenol and protein is used for antigen bisphenol detection.

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, to provide a biosensor for detecting bisphenol and a bisphenol detection method comprising an anti-bisphenol antiserum (anti-BPA antiserum). Through this, it is an object of the present invention to accurately and simply determine whether the endocrine disruptors such as bisphenol are included in the chemical analysis of marine organic pollutants requiring expensive equipment.

To achieve the above object, a biosensor for detecting bisphenol (bisphenol-A) of the present invention comprises an anti-bisphenol antiserum isolated from a mouse immunized with a combination of bisphenol and protein. It features. Herein, the protein is preferably ovalbumin (OVA) or cationized bovine serum albumin (cBSA).

Bisphenol detection method according to the present invention for achieving the above another object, the step of preparing a combination of bisphenol and protein; Preparing an anti-bisphenol antiserum by injecting the conjugate into a mouse and immunoreacting it; And antigen-antibody reaction of the marine organic pollutant containing the anti-bisphenol antiserum and bisphenol. The antigen antibody reaction may be a bisphenol 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 bisphenol (bisphenol-A, BPA), which is one of natural female hormones, and a method of detecting marine organic pollutants using the same, and more particularly to marine organic pollutants such as bisphenol. It is a method of detecting bisphenol by injecting a mouse to produce an antibody from serum, and measuring the degree of color development by the reaction of the produced antibody with an organic organic pollutant.

Bisphenol is a chemical that shows estrogen activity and is used to make polycarbonate plastics. It is present as a component of soft drink cans, dental sealants, and many plastics. Bisphenol is eluted from plastics because the polymer is destroyed or incompletely polymerized in an autoclave. Bisphenols are also used as plastic tooth fillers, especially polycarbonate plastics, which are children's tooth coatings.

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

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

In addition, when the color development was measured using a marine organic pollutant containing bisphenol as a substrate according to the indirect ELISA method described above, the sensitivity of the anti-bisphenol antiserum to bisphenol according to the present invention was also very high. When bisphenol antiserum was applied in situ, it was confirmed that bisphenol and protein conjugates can 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: bisphenol ( bisphenol Preparation of -A) -protein Conjugates

Purified bisphenol was purchased from Sigma, and binding to protein was performed by 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, and bisphenol is bound to the buffer. (conjugation buffer; 0.1 M MES, 0.15 M NaCl, pH 4.7) to prepare a concentration of 0.5 mg / ㎖.

500 μl of bisphenol and 200 μl of the delivery protein solution were mixed for conjugation, 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, the unbound bisphenol and EDTA remaining in the delivery protein solution were removed using a D-SaltTM desalting column.

Bisphenol-delivered protein conjugate was confirmed by SDS-PAGE, and after titration of protein using BCA protein assay reagent (Pierce), the concentration was adjusted to 1 μg / ml by using acetone precipitation method. Aliquoted 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-BPA 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-BPA Conjugate

Immobilization of the OVA-BPA conjugate was performed by diluting 200 μl of the OVA-BPA conjugate diluted to 0.5 μg / ml with 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-BPA conjugate

Antisera titer was measured using OVA (1 μg / well) and cBSA (1 μg / well) as a control of the OVA-BPA conjugate coated wells. Antiserum collected from mice immunized with cBSA-BPA 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-bisphenol antisera BPA  antiserum) Titer  Measure

In order to determine the titer of antiserum obtained from mice immunized with cBSA-BPA, indirect ELISA was performed using microtiter plates coated with OVA-BPA, cBSA and OVA. 1 is a graph measuring the titer change of anti-bisphenol-A 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-bisphenol antiserum according to the present invention reacts specifically with bisphenol-bound OVA-BPA as compared to the OVA or cBSA protein alone, and the OVA-BPA is diluted at concentration. Overall, it can be seen that the anti-bisphenol antiserum according to the present invention is significantly stronger than the OVA or cBSA protein (see FIG. 1).

Result example  2: anti-bisphenol antiserum for field application BPA  antiserum)

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

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

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

 Blank  OVA-bisphenol  cBSA  OVA 100 0.038 0.223 0.199 0.034 500 0.038 0.246 0.192 0.036 1000 0.036 0.237 0.145 0.036 5000 0.035 0.208 0.087 0.034 10000 0.035 0.179 0.048 0.034 50000 0.038 0.121 0.046 0.036 100000 0.036 0.085 0.044 0.038 500000 0.036 0.046 0.041 0.036 0 0.035 0.038 0.038 0.037

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

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

Such a biosensor for detecting bisphenol according to the present invention and a bisphenol assay using the same are new technologies that have not been systematically attempted or developed in the world yet, and are used in the chemical analysis of marine organic pollutants requiring expensive equipment. There is an effect that can be confirmed accurately and simply whether the endocrine disruptor, such as containing. 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. buffer) to prepare a concentration of 0.5 mg / ml, 500 μl of the bisphenol and 200 μl of the delivery protein were mixed, and then a binding reaction was performed at room temperature for 2 hours by adding a 50 μl coupling solution. Preparing a conjugate of bisphenol and protein at a concentration of 1 μg / ml; Preparing an anti-bisphenol antiserum by injecting the conjugate into the mouse to perform an immune reaction, collecting blood, and then centrifuging the blood; Diluting the anti-bisphenol antiserum 100,000-fold with 0.1% (w / v) skim milk / PBST; Antigen antibody reaction of the diluted anti-bisphenol antiserum with 100 μl of marine organic pollutant containing bisphenol; And Bisphenol detection method comprising the step of measuring the absorbance at 490 nm using a microplate reader. delete

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