KR20040027222A - Biomarker Reagent for the detection of Endocrine Disruptors - Google Patents

Abstract

PURPOSE: A biomarker reagent for detecting environmental endocrine disruptors is provided, thereby easily detecting the environmental endocrine disruptors with reduced time, cost and labor for the detection. CONSTITUTION: A method for preparing a biomarker reagent for detecting environmental endocrine disruptors such as vitellogenin from a fish comprises the steps of: injecting estrogen into the abdominal cavity of a male flatfish, collecting a blood sample from the flatfish; isolating vitellogenin from the blood sample by centrifugation or sepharose gel; injecting the vitellogenin into the abdominal cavity of a rabbit; collecting a blood sample from the rabbit; isolating a rabbit anti-vitellogenin antibody from the blood sample; attaching the anti-vitellogenin antibody to gold particles to produce a gold and anti-vitellogenin conjugate; adsorbing the gold and anti-vitellogenin conjugate to a matrix such as polyester or glass fiber; immunizing a mouse with the vitellogenin antigen; fusing a spleen cell of the immunized mice with myeloma cell to obtain mouse anti-vitellogenin antibody; and adsorbing the mouse anti-vitellogenin antibody to a membrane.

Description

Biomarker Reagent for the detection of Endocrine Disruptors}

The present invention relates to an immunochromatography detection reagent for detecting vitelogenin in plasma, serum and hepatocytes culture supernatant of olive flounder for the purpose of detecting endocrine disruptors.

Vitelogenin is a yolk precursor that is synthesized in the liver of fish and other vertebrates.It is synthesized in the liver according to the secretion of estrogen, a female hormone. Accumulate in eggs after separation into tin and beta prime-components.

Vitelogenin is not synthesized in normal males, but when exposed to estrogen or exposed to estrogen-like endocrine disruptors, it is also synthesized in male liver and detected in blood. Therefore, the presence of endocrine disruptors can be confirmed by examining the presence or absence of vitelogeny synthesis in blood after growing or artificially breeding in suspected endocrine disruptors. In addition, the test substance is added to the cultured hepatocytes and cultured, followed by irradiation of the vitelogenin in the culture supernatant to determine whether the target substance has endocrine disruption.

As a diagnostic reagent for measuring vitelogenin in serum, the EIA method using enzymes has been introduced in Japan, but these methods require specially designed test equipment, take a long time to test, and cannot be applied immediately at contaminated sites. Therefore, there is an urgent need for a simple inspection method applicable to the field. In particular, the vitelogenin test reagent introduced in Japan is a method designed for carp and trout.

The present invention relates to the development of a diagnostic reagent that detects vitelogenin in olive flounder by applying immunochromatography, an ultra-fast diagnostic method that can be seen in about 3 minutes. In order to detect the vitelogenin by immunochromatography, the antibody against the vitelogenin is adsorbed to the nitrocellulose membrane at a predetermined position, and the antibody to each of the gold particles is conjugated and dried on a pad. The test strips were prepared by overlapping the dried gold pads and the pads to which the sample was applied and covering them on the nitrocellulose membrane and attaching the hygroscopic pads to the opposite positions (FIG. 2).

In order to obtain antibodies to vitelogenin, a biomarker of environmental hormones, purified vitelogenin must be present. Estrogen was inoculated into the abdominal cavity of the flounder to obtain vitelogenin, and vitelogen synthesis was induced from the liver, and blood was collected. The serum was separated, followed by ice-cold water precipitation and ion exchange chromatography. Pure bitellogenin was obtained by ion-exchange chromatography) and gel chromatography.

Antibodies against Vittelogenin develop a hybrid cell line that immunizes each purified antigen with blab / c mice, isolates splenocytes and fuses them with myeloma cells to secrete monoclonal antibodies against Vittelogenin. . Monoclonal antibodies were prepared using this cell line. In addition, the antibody attached to the gold particles was prepared by immunizing each of the purified antigens to rabbits and only specific antibodies were purified by affinity chromatography.

The test strip prepared by the above method is placed in a plastic device with a sample application hole and a display window. When about 100ul (2 drops) of flounder blood or hepatocyte culture supernatant is put into the sample application hole, vitelogenin present in the sample reacts with each antibody attached to the gold particles, Developed. The monoclonal antibody to vitelogenin is adsorbed on the membrane at a certain position. When vitelogenin is present in the serum, the vitelogenin reacted with the antibody conjugated to the gold particles reacts with the antibody adsorbed on the membrane again. To form a purple band with gold particle color. Anti-rabbit IgG was adsorbed at the control line so that the rabbit IgG-adsorbed gold particles always reacted to show purple bands regardless of the presence of the vitelogenin marker in the serum. The unreacted contents soaked into the absorbent pads so that the membrane of the inspection window appeared clean white so that the formed band could be easily read. In other words, if vitelogenin is present in the serum, a purple band appears at the position where the monoclonal antibody is adsorbed against the vitelogenin and the control line.

Since vitelogenin is extremely unstable and has a rapid protein denaturation and degradation as a lower protein, the present invention is refrigerated and frozen by constructing a system of antibodies against the antigenic determinants in the denatured and degraded state. Detection can also be carried out using some denatured or degraded samples.

In addition, the present invention can be basically used for the determination of endocrine disruption, but considering the characteristics of vitelogenin is a female (female) and difficult to discriminate between male and female, It can be easily used for discrimination. In fish experiments, it is possible to easily distinguish between sexes in living conditions (determination of sex ratios, sex change experiments, etc.). The discrimination of male and female is almost impossible, especially in the young fishes of juveniles. The differentiation method has been possible only through the dissection of the gonad after dissection. The present invention has a feature that can discriminate between male and female by collecting and reacting secretion mucus material of fish. However, the target fish is limited to those at the time of maturation of endocrine hormones (sex hormones) during the maturation stage and the synthesis of blood vitelogenin.

The present invention develops a rapid diagnostic reagent that can more quickly and accurately determine the detection of environmentally polluted endocrine disruptors, also known as environmental hormones, thereby preventing exposure to endocrine disrupting chemicals and helping to promote human health. The purpose is to give. As a result of intensive studies in view of the above problems, the present inventors have found that the use of the vitelogenin rapid diagnostic immunochromatography method enables the detection of endocrine disrupting environmental hormones more quickly and simply to complete the present invention. Was done.

That is, an object of the present invention is to produce a rapid diagnostic reagent that produces monoclonal antibodies of vitelogenin and determines endocrine disruption through rapid qualitative analysis of vitelogenin in serum, plasma and hepatocyte culture medium.

Figure 1a is a manufacturing process showing the pure separation process of bitellogenin of the biomarker manufacturing process according to an embodiment of the present invention.

Figure 1b is a manufacturing process showing a process for producing a rabbit darkon specific antibody in the manufacturing process of the biomarker reagent according to an embodiment of the present invention.

Figure 1c is a manufacturing process showing a process for producing a mouse monoclonal antibody specific manufacturing process of the biomarker reagent according to an embodiment of the present invention.

Figure 1d is a manufacturing process showing the formulation of the product of the biomarker reagent manufacturing process according to an embodiment of the present invention.

2 is a basic configuration of a test strip to be used in the invention.

Figure 3 is an electrophoretic picture of the protein induced in estrogen.

(HMM: molecular weight marker, 1,3: male serum, 2,4: estrogen-treated male serum)

Figure 4 is an electrophoresis picture of serum protein before purification by Ice-Cold method.

Figure 5 is an electrophoretic picture of the bitelogenin purified by Ice-Cold method.

FIG. 6 is an absorbance image of protein isolated serum by Sepharose 6 non-column chromatography.

Figure 7 is an electrophoresis picture of vitelogenin separated and purified by Figure 7.

8 is an electron micrograph of a colloid (left: 20 nm, right; 40 nm aperture).

Figure 9 is a photograph of the results of the immunochromatography developed by dividing the buffer and adsorption concentration.

10 illustrates test results for various concentrations of vitelogenin serum.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

Referring to the present invention, a method for preparing the vitelogenin rapid diagnostic reagent is as follows.

The bitelogenin used in the present invention was injected into the abdominal cavity with 1 mg of estrogen per kilogram of body weight after anesthetizing halibut males (500-600 g), and re-injected the same amount for each fish 5 days later. After 1 day, the tail tails were cut to collect blood, 0.01% sodium azide and 1 mM Phenylmethylsulfonyl fluoride (PMSF) were added to allow sufficient coagulation in the refrigerating chamber, followed by centrifugation to obtain serum.

The serum was refrigerated for 12 hours by adding 80 ml of distilled water per 8 ml, and then centrifuged at 15,000 rpm / min for 15 minutes, and the precipitate was washed by adding 50 ml of distilled water. Centrifuge for 15 minutes at 15,000 rpm / min to dissolve the precipitate in a solution of 2 ml 0.02M Tris-HCl, 0.1% NaN3, 2% NaCl pH 8.0, and the dissolved solution is again centrifuged for 15 minutes at 10,000 rpm / min. After separation, the supernatant was diluted with 20 ml and distilled water at 4 ° C. The diluted solution was centrifuged at 15,000 rpm / min for 15 minutes, and then 1 ml of 0.02M Tris-HCl was added to the diluted solution and centrifuged at 5,000 rpm / min for 5 minutes. Isolate to give a precipitate of the final bitellogenin (FIGS. 4, 5).

As another method, it is obtained by pure purification through filtration using Sephaose gel (Sephalose CL4B gel, 30cm, 5ml / cm) (Fig. 6, 7).

Using purified vitelogenin, animals (i.e. rabbits) and mice (Balb / c 2 mice) for immunization were prepared, and the rabbits were emulsified with complete adjuvant solution 1: 1 (protein amount = 100 µg). After 2 weeks of inoculation, the mice were inoculated by intraperitoneal administration after complete adjuvant and emulsion with 100 μg of the first protein, and the second immunization after incomplete adjuvant and emulsion with 100 μg of protein after 2 weeks. After intravenous administration, inoculate three times at a daily interval with 100 μg of protein only three times in a row at intervals of one week, followed by fusion on the following day.

About 50­100 μl of blood of mice (Balb / c; 2 males) immunized with vitelogenin was collected and measured for antibody titer by ELISA, and then cell fusion was performed when antibody formation was confirmed. The spleens of euthanized mice are aseptically removed, and then, the cells are pulverized into a sterile wire mesh, fused with previously prepared SP2 bone marrow cancer cells, and PEG 1500 is used for fusion.

On the basis of the data obtained through the ELISA values, clones with high antibody titers to vitelogenin were isolated and cultured so that 1 cell per well was entered. When clones are incubated at the bottom of 2/3, ELISA is performed to separate the wells with the highest titers and incubate them separately.

One week after treatment with the intraperitoneal pristane of the mouse, cultured monoclonal cells were collected and intraperitoneally administered. After one week, the ascites of the antibody was amplified in the abdominal cavity of the mouse, and the ascites were cryopreserved (-70 degrees).

100 nm of 20 nm gold was prepared to prepare 40 nm plain gold through nucleation (FIG. 8). The superfluid was used after washing thoroughly. All water used pure water of 18.2 MOhm. 1% gold chloride was prepared by dissolving 0.1 g tetrachloroauric (III) acid trihydrate. 1% tri-Sodium citrate was 0.5g tri-sod. The citrate was prepared by dissolving in 50 ml.

89 ml of water was heated to 100 degrees, 1% gc and 1 ml were added to make 0.01%. Then, 6 ml of water was mixed with 4 ml of 1% sc and boiled for an additional 15 minutes. After adding water to the 20nm gold and then boiled to prepare a 40nm plain gold. 1% sc 2ml was added to make 0.01%, and 1% gc 2ml was mixed with 18ml of water, dropped and boiled for 20 minutes, and allowed to stand at room temperature to cool. NaN ₃ was added to 0.01% as a preservative.

Example 1

* Preparation of vitelogenin diagnostic immunochromatography reagent

1. Pure Separation of Olive Flounder

After anesthetizing males (500-600 g) of the flounder, estrogen was injected intraperitoneally at 1 mg / kg body weight. After 5 days, estrogen was re-injected for each fish, and after 10 days, the tail was cut and collected. 0.01% (sodium azide) and 1mM Phenylmethylsulfonyl fluoride (PMSF) were added to allow sufficient coagulation in the refrigerating chamber, and then purified by chromatography by centrifugation at 1,500g for 10 minutes at 4 ° C.

First dissolve 60 ml per hour in a Spharose 6B column (850 x 16 mm) with 100 mM Tris-buffer (pH7.8) containing 1 mM PMSF. Recover the fraction containing Xenin, add it to DEAE-Sepharose column (350 x 16mm), and elute 36ml per hour with 150mM Tris buffer (pH7.8) containing 1mM PMSF. The fraction obtained by eluting and recovering the fraction eluted with vitelogenin was used as an immunogen for the development of rabbit anti-bitelogenin antibody and monoclonal antibitelogenin.

2. Preparation and Purification of Specific Antibodies

Rabbit anti-bitellogenin antibody was inoculated four times under the rabbit subcutaneous 4 times per month with freund's adjuvant at 20 ug purified bitelogenin at a time to obtain antiserum. The antiserum was purified using only the rabbit antibitellogenin in the same manner using a sepharose gel attached to the vitelogenin. This purified antibody was used for adsorption on gold particles. That is, gold chloride was reduced to sodium citrate solution to prepare gold particles having a size of 40 nm with absorbance of 10 +/- 1 at 532 nm. Purified rabbit antibitelogenin was attached to 10ug / ml and stabilized gold particles with PEG solution. The prepared gold conjugate was dipped in a matrix such as polyester or glass fiber and dried together with the other gold conjugate. Isolated purified vitelogenin antigen to blab / c mice, splenocytes were isolated and conjugated with bone marrow cancer cells to select hybrid cell lines that secrete monoclonal antibodies against vitelogenin by ELISA and establish mononuclear cell population by limiting dilution. It was. The cell lines were cultured in large quantities and inoculated to balb / c mice intraperitoneally to obtain ascites containing a large amount of antibody, and IgG antibodies were purified using protein A gel. Purified antibody was diluted with PBS at a concentration of 1 mg / ml and adsorbed onto the membrane.

Formulation of Diagnostic Reagents

Plain gold for the production of conjugates with proteins is commonly used in 20nm or 40nm diameter. Gold's performance is close to spherical and uniform in size. The colloidal gold 20 nm and 40 nm electron micrographs developed in this study are shown in Figure 8. It is believed that gold with satisfactory quality has been developed since it has an even shape for each size.

A gold conjugate was prepared by conjugating a monoclonal antibody obtained by fusing mouse spleen cells and bone marrow cancer cells with a colloidal gold having a diameter of 40 nm (FIG. 8). The format of the immunochromatography kit for detecting vitelogenin was completed by examining the conditions of adsorption on the NC paper, conjugation conditions, and buffer conditions at each stage. 9 is different in sensitivity and specificity according to the difference between the goldconjugate diluent, NC paper coating concentration, serum diluent. The constructed strips were bonded to commercially available devices.

[Test Example 1]

* Inspection method and judgment method

Serum, plasma or whole blood of the fish to be tested or hepatocyte culture supernatant is placed in an elliptical sample hole and observed for about 20 minutes to see if purple bands appear at positions 1 and C. That is, in all cases, a purple line should appear at the C position and a purple line at the C position is determined as negative for the vitelogenin, and if a purple line appears at the position 1, the vitelogenin is determined to be positive.

Judgment example)

* Comparison data between the present invention and existing products

end. Comparative test result of olive flounder bitellogenin detection reagent

As a result of the comparison with the sandwich ELISA method developed by the veterinary department of Cheju National University, it is as follows.

division ELISA reagent results positivity voice invention voice 0 88 positivity 95 0

* Positive criteria: more than 20ng / ml of bitellogenin in the sample

According to the above results, the present invention is able to confirm the presence of vitelogenin synthesized in plasma, serum and liver cell culture supernatant of marine fish flounder (flatfish, Olive Flounder, Paralichthys olivaceus ) by endocrine disrupting environmental hormones. As a rapid detection method, it was confirmed that it is a diagnostic reagent with both specificity and sensitivity.

As described above, the effects obtained by using the flounder vitelogenin detection reagent according to the present invention are as follows.

① endocrine disruption can reduce the time, cost and manpower for the detection of chemicals,

② Endocrine disruption can be used to verify the toxicology of new synthetic chemicals by rapid diagnosis of chemicals,

③ It can enable early sex discrimination of fry and enable cultivation of cultivation of aquaculture industry.

Claims (1)

Rapid Diagnosis Method for Detection of Endocrine Disrupting Chemical Substances Using a Vitellosenin Detection System of Olive Flounder (Flounder) Using Immunochromatography