KR100742115B1 - Method for deciding maturity using polyclonal antibody of rockfish - Google Patents

Abstract

A method for diagnosis of maturity by using a polyclonal antibody of rockfish(Sebastes schlegeli) is provided to improve diagnosis accuracy of rockfish maturity by measuring the amount of luteinizing hormone(LH) in rockfish. The method for diagnosis of maturity by using a polyclonal antibody of rockfish(Sebastes schlegeli) comprises the steps of: collecting the pituitary gland of rockfish and purifying it to obtain the rockfish luteinizing hormone; separating rockfish glycoprotein alpha(GPalpha) and rockfish luteinizing hormone beta(LHbeta) from the rockfish luteinizing hormone, and independently obtaining polyclonal antibodies against the rockfish glycoprotein alpha(GPalpha) and rockfish luteinizing hormone beta(LHbeta); and deciding maturity of rockfish through enzyme-linked immunoassay method by using the polyclonal antibody against rockfish luteinizing hormone beta(LHbeta).

Description

Method for deciding maturity using polyclonal antibody of rockfish}

1 is a process for purifying the luteinizing hormone of the gecko rock rock according to the present invention.

FIG. 2 is a peak graph of an extract (S3) eluted with 0.05 mol ammonium acetate in a gel chromatography column connected to a protein purification apparatus by extracting the pivolac pituitary gland in the embodiment of the present invention.

3 is an aliquot eluted while increasing the concentration of ammonium acetate to 0.05, 0.1, 0.2, 0.4, 1.0 mole in an anion chromatography column connected to a protein purification apparatus in an embodiment of the present invention (S3) S3DE5) is a peak graph of the two subunits of the Zopovolak luteinizing hormones (GPα and LHβ) combined into dimers.

Figure 4 is a graph of peaks eluted with a concentration gradient of acetniter by adsorption on reverse phase HPLC after treatment of aliquot (S3DE5) with lyophilization and TFA in the embodiment of the present invention.

Figure 5 is an electrophoretic and Western blotting picture of the aliquots (S3DE5W9 and S3DE5W12) in an embodiment of the present invention.

Figure 6 is a photograph showing the distribution of luteinizing hormone secretion cells in the blood pituitary pituitary pituitary gland using a zodiac rock luteinizing hormone β subunit antibody in the embodiment of the present invention.

Figure 7 is a recovery rate verification graph for verifying the accurate measurement of the concentration of luteinizing hormone in the blood by the blood gel formation luteinizing hormone enzyme immunoassay developed in the embodiment of the present invention.

8 is a parallel assay graph of the standard curve of the standard product and the mature pipivolak female pituitary extract and the ancestor dilution solution of the same individual plasma in the zodiac rock luteinizing hormone enzyme immunoassay system in an embodiment of the present invention.

9 is a graph showing the feasibility of measuring luteinizing hormone concentration in blood by means of a Zopovolak luteinizing hormone enzyme immunoassay system through an in vivo experiment in an embodiment of the present invention.

The present invention relates to a maturity determination method using a polyclonal antibody of the zodiac rock, and more specifically, the pituitary gland of rock zol (rockfish: hereinafter 'RF') by collecting the pituitary luteinizing hormone (hereinafter ' RF LH '), and glycoprotein alpha (hereinafter referred to as' RF GPα') and luteinizing hormone β (hereinafter referred to as RF) LHβ ') was isolated, polyclonal antibodies were obtained for each, and the enzyme immunoassay system using the polyclonal antibody of luteinizing hormone beta (RF LHβ) was used to determine sex maturity. Not only can it be used for diagnosing important sexual maturity, but also affects the reproductive endocrine system caused by endocrine disruptors And more economical management effort rating, also relates to a method for determining the maturity using a polyclonal antibody of rockfish to allow correct and easy diagnosis at an early stage.

In general, gonadotropin hormone (hereinafter referred to as 'GTH') in fish is called follicle-stimulating hormone (hereinafter referred to as 'FSH') and luteinizing hormone (hereinafter referred to as 'GTH'). LH is composed of two types of hormones with different physicochemical composition and endocrine function.FSH is mainly secreted during the early stages of immature maturation and acts on oocyte growth.LH is the final maturation and It is known to be mainly involved in ovulation.

In addition, as in vertebrates and other fish species, the luteinizing hormone produced and secreted by the pituitary gland is a glycoprotein hormone composed of heterozygotes of a common α chain and a specific β chain. It is the main hormone involved in sexual maturation, ovulation and pregnancy control.

Rockfish (RF) (Sebatis schelagilli) is a native fish that lives in Korea, Japan, and the coast of China. In Korea, it is produced as a stocked fish and aquaculture species for fishery enhancement, but it is mature in terms of parenting management. It is a fish species that is difficult to determine.

In particular, in the case of fish that is difficult to distinguish between male and female, most of the criteria for determining the maturity depend on the naked eye, and these criteria are different depending on individual and human criteria. It is important to accurately measure the amount of luteinizing hormone (LH) that is directly involved in LH.

Recently, it has been used as a biological indicator of environmental impact assessment on the reproductive endocrine system, which is connected to the hypothalamus-pituitary-gonad axis of fish in endocrine disruptors.

The present invention establishes a retrieval technique that can more easily and accurately determine biological indicators of environmental impact assessment for endocrine disruptors that are socially problematic as well as studies of physiological and endocrine rhythms related to fish maturation. It is a technical problem.

The present invention is obtained by purifying the pituitary gland of the rockfish (rockfish: RF) to obtain the rubbing bovine luteinizing hormone (RF LH), the glycoprotein alpha (RF GPα) which is a subunit of the rupeebolak luteinizing hormone (RF LH) ) And polyclonal antibody for each of the luteinizing hormone beta (RF LHβ), and the maturity of the luteinizing hormone beta (RF LHβ) is determined by using the polyclonal antibody. .

Hereinafter, a method for determining the maturity level using the polyclonal antibody of the gecko rockac according to the present invention will be described in detail with the accompanying drawings.

In 1988, Kawauchi, a group of professors at the University of Kisato, Japan, for the first time in the world like fish and other vertebrates, two types of GTH, follicle-stimulating hormone (FSH) and luteinizing hormone (luteinizing hormone) Hormone (LH) was extracted and purified from salmon pituitary gland (freeze-dried weight 50g) by ethanol method by liquid chromatography and high pressure liquid chromatography.

In addition, specific antibodies to these hormones were produced to develop blood concentration meters by radioimmunoassay, which revealed that they have different endocrine functions.

However, the ethanol extraction method requires a large amount of the pituitary gland in the process of extracting the glycoprotein, and also because the glycoproteins tend to be denatured and lost as the extraction process takes a lot of time. In order to improve the protein extraction method.

Therefore, marine fishes of fish species such as zodiac rock (RF) not only have a small size of the pituitary gland (about 1/20 of the salmon pituitary gland) but also have a high price of fish, and thus economic support is needed to secure a large amount of pituitary gland.

In addition, various diagnostic methods that can directly quantify luteinizing hormone in blood have been studied, and can be classified into radioimmunoassay (RIA) and enzyme immunoassay (EIA).

Firstly, radioimmunoassay was established by Suzuki et a: 1990 and used in fishes such as rainbow trout, catfish and red snapper, but it is difficult to label radioactive substances directly on luteinizing hormone. It has the disadvantage of handling in a limited space with facilities.

Secondly, enzyme immunoassay, which can measure luteinizing hormone directly in the blood without labeling luteinizing hormone, is recently developed by foreign researchers and used in sea bass. Especially, enzyme immunoassay is a special facility in the field. Not only can it be measured without the need, but it can also be easily manufactured as a diagnostic kit (KIT). Therefore, in the present invention, an enzyme immunoassay (EIA) for luteinizing hormone was developed as a measuring method for determining the maturity level of zodiac rock.

The present invention is to collect the pituitary gland of the blood rock rock (RF) to purify the blood rock luteinizing hormone (RF LH), the glycoprotein alpha (RF GPα) and luteinizing hormone beta is a subunit of the blood corpus lutein forming hormone (RF LH) After separation of (RF LHβ), to obtain a polyclonal antibody for each, and then to determine the sex maturity as an enzyme immunoassay system using the polyclonal antibody of the luteinizing hormone beta (RF LHβ), first to determine the zobobolak luteinizing hormone ( The process of purifying RF LH) is as follows.

After performing pituitary gland collecting step (101), which collects the mature pizobolac pituitary gland (742, lyophilized 685 mg), the pituitary gland is subjected to 0.2 mol ammonium acetate (pH 6.1) homogenizer, and then 15,000 g of gravitational acceleration. In the pituitary hormone extraction step 102 to obtain a pituitary hormone is precipitate by centrifugation for 30 minutes.
Pituitary hormone obtained in the pituitary hormone extraction step 102 as a 0.05 mole ammonium acetate (pH 9.0) buffer for eluting in a gel chromatography column (Sepacryl-100, φ2.6 x70cm) connected to a protein purification apparatus (FPLC) After purification and homogenization, the purification step 103 (Fig. 2) is carried out by gel chromatography to elute at a flow rate of 3 ml / 200 s / tube to obtain an aliquot (S3).

After confirming the aliquot (S3) obtained through the purification step 103 by the gel chromatography method by electrophoresis, 0.05, on an anion chromatography column (DE-52, φ 10 x 100mm) connected to a protein purification device (FPLC) Purification step 104 by an ion chromatography column (0.1) to increase the concentration of ammonium acetate (pH 9.0), an elution buffer, by one step to 0.1, 0.2, 0.4, 1.0 mole (S3DE5) (Fig. 3). Will be performed.

When the purification step 104 by the ion chromatography column is completed, the mixture is adsorbed onto a reverse phase high performance liquid chromatography (rpHPLC) column (Wakosil II 5C18-HG, 0.46 × 25 cm) to form and elute a concentration gradient of acetonitrile. By performing a high purity purification step 105 (FIG. 4) by a liquid chromatography column, the process for purifying bovine rock luteinizing hormone (RF LH) 100 was completed.

The glycoprotein alpha (RF GPα), which is a subunit of each reported subunit of zosterbolac luteinizing hormone, was prepared to clearly verify the aliquot (S3DE5) obtained through the purification step 104 by the ion chromatography column and to produce specific antisera. And RF GPα and RF LHβ subunits were separated by high pressure liquid chromatography (rpHPLC) to confirm and verify the amino acid sequence results deduced from nucleotide results for luteinizing hormone beta (RF LHβ).

Next, the verification of purified material by electrophoresis and verification of purified material by electrophoresis, verification of purified material by amino acid sequence analyzer, and immunohistochemistry The luteinizing hormone verification process of zodiac rock to verify the antibody was performed.
That is, the purified material obtained through the high purity purification step 105 by the high performance liquid chromatography column is subjected to the verification of the purified material by electrophoresis to check the peaks of reverse phase high performance liquid chromatography by electrophoresis, and then the peak 9 (S3DE5W9) and 12 (S3DE5W12) through the N-terminal amino acid sequencer to verify the purified material by the amino acid sequence analyzer that reveals about 9 amino acid sequences from the N- terminal as shown in Table 1 The amino acid sequence was identified and confirmed and verified by comparison with the amino acid results deduced from the nucleotides results for the previously reported glycoprotein alpha (RF GPα) and luteinizing hormone beta (RF LHβ). .

The following is a process for preparing polyclonal antibodies and Western polyclonal antibodies to verify antibody specificity against glycoprotein alpha (RF GPα) and luteinizing hormone beta (RF LHβ), which are subunits of purified zosterbolac luteinizing hormone (RF LH). Antibody validation was performed by immunoblotting and immunocytochemistry.

In other words, the preparation of polyclonal antibodies was carried out to make specific antibodies against glycoprotein alpha (RF GPα: S3DE5W9) and luteinizing hormone beta (RF LHβ: S3DE5W12), which are subunits of zosterbolac luteinizing hormone (RF LH). After a first immunization step, immunized by injecting 50 μg of RF GPα and RF LHβ with respective equivalent amounts of Freund's composite adjuvant into the lymph nodes of two New Zealand white rabbits, the RFPα and A second tablet immunization step was performed subcutaneously, injected four times subcutaneously with 50 μg of RF LHβ and the same amount of Freund's Incorporate Adjuvant.

After the final injection of the subunits RF GPα and RF LHβ of the zodiacolac luteinizing hormone (LH) into the rabbit through the second immunization step, the serum was separated from the rabbit blood for immunization and antibody specificity by Western immunoblotting. Was investigated.
In other words, the rabbit carotid artery is cut to perform a blood collection step, and then the blood is boiled at 4 ° C. for 16 hours and then the antisera separation step of separating antisera by centrifugation at 10,000 g for 30 minutes. Each of the separated antiserum was dispensed and lyophilized by 1 ml, and then stored at -80 ° C until use.
In addition, the antibody specificity was reconfirmed through the distribution of luteinizing hormone (RF LH) secretory cells in the pituitary pituitary gland by immunohistochemistry (FIG. 6).

Next, an enzyme immunoassay system (ELISA) was developed and verified using a polyclonal antibody prepared as an aliquot (S3DE5) through the preparation of the polyclonal antibody.

In other words, the development of enzyme immunoassay system using antibody and standard to quantify blood luteinizing hormone (LH) concentration of zodiac rock (FIG. 7) is a competitive method using RF GPα and RF LHβ and polyclonal antibodies to The enzyme immunoassay system was used, and the verification process of the enzyme immunoassay system is as follows.

(1) coating and reaction of the antibody with the sample on a well plate of the antigen (luteinizing hormone standard);

The prepared luteinizing hormone (LH) standard was diluted by 20 ng / ml concentration with 0.5 mole carbonate buffer (pH 9.6), and then, 100 µl was dispensed into a 96-well well plate and reacted at 4 ° C for 18 hours. Also, the same amount of luteinizing hormone beta subunit (LHβ) antibody (2,000-fold dilution) and the same volume of luteinizing hormone beta subunit (LHβ) antibody (2,000-fold dilution) in a polyethylene tube (φ10x70 mm) ) And each of the Zhipovolak luteinizing hormone standards diluted to 100-0.2 ng / ml were prepared and reacted at 4 ° C. for 18 hours.

(2) sublocking;

In order to prevent nonspecific binding of the well plate and the antigen, 200 μl of 2% bovine serum albumin (BSA) was added to the well plate and reacted at 37 ° C. for 1 hour, followed by phosphate buffer (0.05% Tween 20 in PBS). Washed twice.

(3) competitive binding reactions with coated antigens;

Into a well plate washed with phosphate buffer solution, 100 μl of the reaction solution reacted with the antibody, the blood sample and the standard product was dispensed into each well and reacted at 37 ° C. for 1 hour. After the reaction, the mixture was washed three times with phosphate buffer (0.05% Tween 20 in PBS).

(4) a second antibody reaction;

Horseradish peroxidase binding-rabbit immunoglobulin G antiserum (goat anti-rabbit IgG) was diluted 1,000-fold in phosphate buffer solution to the washed well plate, and 100 μl of each well was dispensed at 37 ° C. The reaction was carried out for 30 minutes at.

(5) color reaction;

After washing the plate three times in the same manner, 33 μl of 33 ', 55'-tetramethylylbenzidine was dispensed into each well and allowed to react at room temperature for 20 minutes, and then 100 μl of 1N H ₃ PO ₄ was added thereto. The reaction was stopped.

(6) measurement;

After 5 minutes of color stop, absorbance was measured with an enzyme immunoassay device at 450 nm.

Hereinafter, a method for purifying luteinizing hormone and a method for determining maturity using antiserum obtained by the method for purifying bovine volac according to the present invention will be described.

(Example)

First, in order to obtain a luteinizing hormone pituitary product (standard) from the zodiac rock pituitary gland, homogenizing the zodiac rock pituitary gland (742 rats, lyophilized 685 mg) with 0.2 mol ammonium acetate (containing 20 mM PMSF, 50 mM MEDTA, pH 6.1), After 18 hours of incubation at 4 ° C, centrifugation was performed at 15,000 g for 30 minutes, and the supernatant was eluted in a gel chromatography column (Sepacryl-100, φ 2.6 x 70 cm) connected to a protein purification device as shown in FIG. An aliquot (S3) (a luteinizing hormone standard) was obtained by elution with 0.05 moles of ammonium acetate buffer (pH 9.0).

(Figure 2 is a gel chromatography column (sepacrylic-100, φ 2.6 x 70 cm) connected to a protein purification device with elution buffer 0.05 mol ammonium acetate (pH 9.0), the elution flow rate is 3ml / 200 seconds / tube, aliquots (S3) is a peak graph containing zodiac rock luteinizing hormone.)

The aliquot (S3) was eluted with 0.05, 0.1, 0.2, 0.4, and 1.0 mol of ammonium acetate (pH 9.0) as an elution buffer in an anion chromatography column (DE-52, φ 10 x 100 mm) connected to a protein purification device. An aliquot (S3DE5) was obtained, and the luteinizing hormone (LH), which is the aliquot (S3DE5), is a glycoprotein alpha (RF GPα) and luteinizing hormone beta, as shown in FIG. (RF LHβ) is a dimer bound state.

(FIG. 3 shows the concentration of ammonium acetate (pH 9.0) as an elution buffer in an anion chromatography column (DE-52, φ 10 x 100 mm) in which aliquot (S3) is connected to a protein purification apparatus, 0.05, 0.1, 0.2, 0.4, 1.0 The elution flow rate is 3 ml / 200 sec / tube with stepwise increase in moles, and S3DE5 is a peak graph in which dimers of two subunits of Zibobolac luteinizing hormone, RF GPα and RF LHβ, are combined.)

Then, an aliquot (S3DE5) eluted by anion chromatography for separation of RF GPα and RF LHβ subunits by reverse phase high pressure liquid chromatography (rpHPLC) and amino acid sequencing was lyophilized, followed by reverse phase high pressure liquid chromatography. By adsorbing on the chromatography to elute the concentration gradient of acetnitrile (acetnitrile) as shown in Figure 4, the peaks are confirmed by electrophoresis as shown in Figure 5, peaks 9 (S3DE5W9) and 12 (S3DE5W12) Was confirmed by N-terminal amino acid sequencer as in Table 1 below.

Table 1

Table 1 shows the amino acid sequence of each subunit of the luteinizing hormones purified from the pipiolac pituitary gland by the N-terminal amino acid sequencer, and was previously published by the applicant (Published Patent Application 2006- No. 77227, published date: July 05, 2006) A comparison table with amino acids deduced from cDNA nucleotides for each subunit (RF GPα and RF LHβ) of the zodiac rock luteinizing hormone.

Here, the predicted peptide of the upper (RF GPα) is 100% of the amino acid sequence and native peptide deduced from the cDNA nucleotide of RF GPα and the native peptide (RF GPα) sequence 100%. It can be seen that, the lower (RF LHβ) predicted peptide (predicted peptide) is the amino acid sequence and native peptide deduced from cDNA nucleotides of RF LHβ is a product according to the present invention (RF It can be seen that 100% coincident with that previously published by the applicant (L Patent Publication No. 2006-77227) with the sequence of LHβ).

(FIG. 4 is a peak grapher eluted with an acetic acid concentration gradient after adsorption (S3DE5) by lyophilization and TFA, followed by reverse phase HPLC (Wakosil II 5C18-HG, 0.46 × 25 cm). (S3DE5W9) is the α subunit of zosterbolac luteinizing hormone (RF GPα), and peak number 12 (S3DE5W12) is the β subunit of zosterbolac luteinizing hormone (RF LHβ).)

Next, 50 µg / ml of each of the GP GPα (S3DE5W9) and RF LHβ (S3DE5W12) was prepared in the same amount of Freund's composite adjuvant for the preparation of polyclonal antibody and verification of antibody specificity by Western immunoblotting and immunohistochemistry. And injected into rabbit lymph nodes (primary injection) in an emulsion state, and mixed with 50 µg of genuine products (RF GPα and RF LHβ) and the same amount of Freund's Incorporate adjuvant at 10-day intervals, respectively. 4), serum was isolated from rabbit blood for immunoassay after the last 4 injections, and antibody specificity was examined as shown in FIG. 5 by Western immunoblotting.

In addition, one week later, the rabbit carotid artery was cut and pre-bleeded. The blood was boiled at 4 ° C. for 16 hours, and then the antisera was separated by centrifugation at 10,000 g for 30 minutes. After 1 ml aliquots and freeze-dried, stored at -80 ° C until use, the antibody specificity was reconfirmed through the distribution of luteinizing hormone secretory cells in the pituitary pituitary gland by immunohistochemistry (Immunocytochemistry) as shown in FIG. It was.

That is, FIG. 5 is an electrophoresis and Western blotting photograph of an aliquot (S3DE5), where A is the peak of peak 9 (S3DE5W9), peak 10 and peak 12 (S3DE5W12) of FIG. 4. It is an electrophoresis picture, staining was made with Coomassie blue (CBB).

In addition, B and C are the peak 9 (S3DE5W9), the peak 10 and the peak 12 (S3DE5W12) using the tuna GPα and LHβ antiserum, respectively, the aliquots were assayed by Western blotting method.

D and E prepared respective Zopobolac (RF) antiserum (polyclonal antibody) for peak 9 (S3DE5W9; RF GPα) and peak 12 (S3DE5W12; RF LHβ), and the specificity of each antibody was determined by Western blotting. It is black.

6 shows the distribution of luteinizing hormone secreting cells in the pituitary gland pituitary gland using the zosterbolac luteinizing hormone β subunit antibody (Anti-RF LHβ serum), (A) as a control (Anti-RF LHβ serum) The photo was treated with a phosphate buffer containing no phosphate buffer, and (b) is a 1,000-fold dilution of an anti-RF LHβ serum subunit antibody, and the brown part is the luteinizing hormone in the pituitary gland. Refers to secretory cells.)

Next, the enzyme immunoassay system according to the example was examined to see if the concentration of luteinizing hormone in blood was measured correctly. The correlation between the recoveries was Y = 0.980X + 0.069 (r ² = 0.9958), and as shown in FIG. 7, 0.76ng / ml of the sample was 0.94 ± 0.23ng / ml, and 6.2ng / ml was 6.1 ± 0.26ng. / Ml, 50ng / ml was measured at 50.5.50 ± 1.8ng / ml, and 400ng / ml was measured at 425 ± 51.3ng / ml, with recovery rates of 81%, 99%, 101%, and 106%, respectively. The average recovery was found to be 96.8%, which is accurate.

(FIG. 7 is a recovery rate verification graph for verifying whether the concentration of luteinizing hormone in the blood is accurately measured by the zophyllolac luteinizing hormone enzyme immunoassay developed in the present invention, and the recovery rate is 96.8% on average. )

In the enzyme immunoassay system for the physiological testosterone luteinizing hormone, the parallel dilution of the same pituitary extract of the female pituitary pituitary gland and the plasma dilution of the same individual with the standard dilution dilution solution by the 2 x 2 test method as shown in FIG. 8. The sample dilution liquid and the standard dilution liquid were parallel to each other, and it was verified that the luteinizing hormone in the pituitary gland and plasma can be accurately measured.

Fig. 8 shows the parallel curve between the standard curve of the standard product (●-●), the adult pituitary rock female pituitary extract (○-○), and the ancestor dilution solution of the same individual plasma (▲-▲). As a graph, it was verified that the luteinizing hormone in the pituitary gland and plasma can be accurately measured.)

In addition, as shown in FIG. 9, the results of investigating the intra-assay coefficient of variation and the inter-assay coefficient of variation in the enzyme immunoassay system for the zodiac rock luteinizing hormone were 5.2%. (n = 6), 7.8% (n = 12), which proved to be very stable.

In practice, it was attempted to measure the blood luteinizing hormone concentration through in vivo experiment as shown in FIG.

First, the yolk forming group of female yolkbolak (5) and yolk forming group injected with physiological saline for marine fish were injected with gonadotropin-releasing hormone, which secretes luteinizing hormone, at a concentration of 0.1 µg / g of fish body. After dividing into female zosterbolak group (control group; 5 mice), blood was collected in real time and measured by enzyme-linked immunoassay assay for the zosterbolol luteinizing hormone of the present invention. After 12 hours, the highest value was obtained and then gradually decreased. In contrast, the control group did not show any significant changes during the experiment.

(FIG. 9 is a graph showing the feasibility of measuring luteinizing hormone concentration in blood by using a blood-derived luteinizing hormone enzyme immunoassay system of the present invention through an in vivo experiment, wherein GnRHa is a gonadotropin-releasing hormone of gonadotropin-releasing hormone. As an analog, it is a hormone that secretes and promotes luteinizing hormone (LH).)

As described above, in the present invention, the pituitary gland is collected from the pituitary gland to purify the bovine rock lactose-forming hormone (RF LH), and the glycoprotein alpha (RF GPα) and the corpus luteum which are subunits of the zodiac rock luteinizing hormone (RF LH) After isolation of forming hormone beta (RF LHβ), polyclonal antibodies were obtained for each, followed by determination of sex maturity as an enzyme immunoassay system using polyclonal antibodies against luteinizing hormone beta (RF LHβ). In addition to being used for the purpose of diagnosing important sexual maturity, the environmental impact assessment of the reproductive endocrine system caused by endocrine-disruptive substances in the domestic coastline, which is a major social problem, is more economic, accurate and It is easy to diagnose early.

The present invention is the only enzyme immunoassay system developed for the luteinizing hormone of marine fish so far, and is an important endocrine search system for the species with high economic value. Not only can it be used for diagnostic purposes, but it is also more economical, accurate and easy to diagnose the environmental impact on the reproductive endocrine system caused by endocrine disruptors in the Korean coast, which is a major social problem. There is.

Claims (5)

delete delete The pituitary gland of rockfish (rockfish: RF) is collected and purified to obtain a rubellabolac luteinizing hormone (RF LH), After separating the glycoprotein alpha (RF GPα) and the luteinizing hormone beta (RF LHβ), which are subunits of the Zopovolak luteinizing hormone (RF LH), to obtain a polyclonal antibody for each, A maturity determination method using a polyclonal antibody of zosterbolac luteinizing hormone, characterized in that to determine the maturity as an enzyme immunoassay system using a polyclonal antibody against luteinizing hormone beta (RF LHβ). The method according to claim 3, wherein the zodiac rock luteinizing hormone (RF LH), A pituitary gland collecting step of collecting pituitary gland of the mature zodiac rock (RF), Pituitary hormone extracting step of obtaining pituitary hormone which is a precipitate by centrifuging the pituitary gland collected in the pituitary gland collecting step for 0.2 mole of ammonium acetate (pH 6.1) and centrifuging at 15,000 g of gravity for 30 minutes; Purification step by gel chromatography to obtain an aliquot (S3) by purifying the pituitary hormone obtained in the pituitary hormone extraction step with 0.05 mole of ammonium acetate (pH 9.0) buffer on a chromatography column, The aliquot (S3) obtained in the purification step by the gel chromatography method was purified by 0.05, 0.1, 0.2, 0.4, 1.0 moles of ammonium acetate (pH 9.0) buffer in an anion chromatography column to obtain an aliquot (S3DE5). Purification step by the ion chromatography column obtained, The aliquot (S3DE5) obtained in the purification step by the ion chromatography column is adsorbed on a reversed phase high performance liquid chromatography column and extracted in a high purity purification step by a high performance liquid chromatography column which is purified by a concentration gradient of acetamide. A maturity determination method using a polyclonal antibody of a zephyrvolak. According to claim 3 or 4, wherein the polyclonal antibody against luteinizing hormone beta (RF LHβ) of the Zopovolak luteinizing hormone (RF LH) subunit, A first injecting 50 μg of glycoprotein alpha (RF GPα) and luteinizing hormone beta (RF LHβ) of the Zopovolak luteinizing hormone (RF LH) subunit into a rabbit lymph node mixed with the same amount of Freund's composite adjuvant A genuine immunity stage, After the first immunization step, a second regular immunization step, which is injected 4 times subcutaneously, with 50 μg of RF GPα and RF LHβ and the same amount of Freund's incompetent adjuvant at 10-day intervals; A blood collection step of collecting blood of a rabbit after the second tablet immunization step, After the blood is boiled at 4 ° C. for 16 hours, a method for determining maturity using a polyclonal antibody of zodiac rock, characterized in that the antiserum separation step of separating antisera by centrifugation at 10,000 g for 30 minutes is performed. .

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