KR100436027B1 - The quantification method of crassostrea gigas's reproductive output using immunological technique - Google Patents

Abstract

The present invention relates to a method for quantitative determination of Crassostrea gigas Reproductive Output using an immune response.

Antiserum was obtained by injecting the extract of Sesame oyster into the rabbit as an antigen, and then prepared an immunosorbent using glutaraldehyde as an antibody that cross-reacts with somatic tissues other than the Sesame egg. Antibodies specific to the oyster eggs were prepared by removing the antibody causing the scab, and quantitatively measuring the propagation amount of the oyster using the enzyme-linked immunosorbent assay (ELISA). A method is provided.

Description

QUANTIFICATION METHOD OF CRASSOSTREA GIGAS'S REPRODUCTIVE OUTPUT USING IMMUNOLOGICAL TECHNIQUE}

The present invention relates to a method for quantitatively measuring the breeding capacity of sesame seeds by preparing specific antibodies against sesame eggs, which are a type of dissolved bivalve, using the specific reaction principle of the antigen-antibody.

Recently, it has been found that the reproduction of marine life is mainly controlled by the physical environment or food such as water temperature and salinity, and is affected by parasites and various diseases.

Since organisms express their influences from the surrounding environment or disease in the form of breeding cycle and breeding quantity, quantitative analysis of breeding cycle and breeding quantity shows the status of the habitat or the health status of living organisms. Can be used as an index to express directly or indirectly.

However, most of the marine molluscs except the scallop protruding out of the gonad are unable to separate the gonads because the gonads are surrounded or interspersed with somatic tissues.

As far as is known, the quantitative method is a volume fraction measuring method using a histological method.

It has been widely used to indicate the degree of gonad development by producing a tissue section for the target organism and counting the overlapping part of the gonad and lattice in the section on the microscope to express the gonad part as a percentage.

However, since the gonads of the molluscs of marine molluscs have a great variation in the degree of development or the amount of gonads depending on the section, the method has a large margin of error, and only relative evaluation is possible.

In addition, there is a method of subjectively evaluating each developmental stage and then formulating it after histologically observing the gonad development, but this also requires the observer's skill and can only be limited to relative evaluation. .

In addition, by injecting chemicals or irradiating with ultraviolet light to stimulate the sample to induce the release of artificial gonads and the amount and by measuring the wet weight of the gonads or whole tissue before and after spawning the difference is estimated as the amount of scattering There is a way.

However, since organisms do not release all germ cells at once, it is impossible to measure the remaining germ cells in the body.

The present invention, in order to solve the above problems, in order to more accurately and easily determine the reproduction amount of the mollusk mollusk, to develop an antibody that specifically reacts to the oyster egg protein of one of the mollusk mollusk, and then the antibody It is to provide a method for quantitatively measuring the breeding amount of oyster using.

1 is a whole process diagram of the present invention.

Figure 2 is a manufacturing process of the immunosorbent using glutaraldehyde.

Figure 3 shows the results of the specificity of the antibody by immunodiffusion test (immunodiffusion test).

A: Before immunosorbent treatment B: After immunosorbent treatment

Figure 4 is histological observation using the immunofluorescence of the present invention oyster egg specific antibody (immunofluorescence).

O: oyster egg DD: digestive system CT: connective tissue

5 is a calibration curve of enzyme-linked immunosorbent assay (ELISA) for quantitative determination of oyster eggs.

Figure 6 is an enzyme-linked immunosorbent assay for determination of breeding oyster eggs of the present invention. (ELISA)

In the present invention, the anti-serum is obtained by injecting a oyster egg extract into a rabbit as an antigen, and then, an antibody that cross-reacts with somatic tissues other than the oyster egg is removed with an immunosorbent using glutaraldehyde, which is specific to the oyster egg. It is composed of a method of preparing an antibody and quantitatively measuring the reproductive output of the oyster by the Enzyme Linked Immunosorbent Assay (ELISA) using the antibody (see FIG. 1).

Hereinafter, the present invention will be described in detail with reference to Examples, but the Examples do not limit the content of the present invention.

<Example 1> Preparation of oyster egg antigen

Mature gourds were collected from the spawning season, and the female gonads were extracted and washed several times with phosphate buffered solution (PBS, Phosphate Buffered Saline) to purely separate eggs.

The optical microscope confirmed that there were no impurities other than eggs.

Purely separated oyster eggs were homogenized using an ultrasonic mill, centrifuged (10,000 rpm, 20 min, 4 ° C.), and the supernatant was used as an antigen.

Example 2 Preparation of Antisera to Obtain Antibody Against Tunnel Egg

0.5 ml (concentration 0.5 mg / ml, in PBS) and 0.5 ml of Complete Freund's Adjuvant obtained from Example 1 were mixed, and then injected subcutaneously into a rabbit (New Zealand White rabbit). It was.

Two weeks after the injection, 0.5 ml of the Sesame Seed Protein extract (concentration 0.5 mg / ml, in PBS) obtained in Example 1 and 0.5 ml of InComplete Freund's Adjuvant were mixed, followed by rabbit (New Zealand White). Rabbits were injected three times at biweekly intervals.

Two weeks after the last injection, blood was drawn from the ear artery and serum was isolated.

The antiserum manufacturing process for obtaining antibodies to oyster eggs is summarized in Table 1 below.

Table 1. Antiserum manufacturing process for oyster eggs

term Treatment method Dosage 1 week Initial injection [1] 500 μg antigen in 500 μl + 500 μl FAC 2 weeks None 3 weeks Booster [2] 500 μg antigen in 500 μl + 500 μl FAI 4 Weeks Blood test 5 Weeks Booster [3] 500 μg antigen in 500 μl + 500 μl FAI 6 Weeks None Week 7 Booster [4] 500 μg antigen in 500 μl + 500 μl FAI 8 Weeks Blood test FAC: Freund's adjuvant complete FAI: Freund's adjuvant incomplete

<Example 3> Preparation of oyster egg specific antibody

Since the cross-reaction of the antibody in the antiserum obtained in Example 2 with somatic cells, which are non-specific cells other than the oyster eggs, acts as a factor of overestimation when quantitatively measuring the oyster breeding amount, the antibody that cross-reacts with the somatic cells is glued. Removal was performed using a taraldehyde (glutaraldehyde) immunosorbent.

The process will be described with reference to FIG.

The oyster extract (50 mg / ml in PBS) and 2M NaOAc buffer (pH 5.0) collected in winter were mixed and 25% glutaraldehyde was added.

To remove residual protein that did not bind glutaraldehyde, washed with 0.01 M PBS (containing 0.5 M NaCl), eluted with 0.2 M glycine buffer and adjusting the pH with 1 M K ₂ HPO ₄ to obtain an immunosorbent. Prepared.

After reacting the prepared immunosorbent with the same amount of antiserum obtained in Example 2 at room temperature for 3 hours, the supernatant was collected by centrifugation (3,000 rpm, 5 min, 4 ℃), and the cross-reaction was confirmed.

Anti-serum (NH ₄ ) ₂ SO ₄ in 25% saturation confirmed that no cross-reaction by immunoglobulin (immunoglobulin) was isolated to prepare a specific antibody to the oyster eggs of the present invention.

<Experimental Example 1> Confirmation of the oyster egg specificity of the antibody of the present invention by immunodiffusion

The antiserum before glutaraldehyde immunosorbent prepared in Example 2 and the antibody specific to the oyster eggs after glutaraldehyde immunosorbent prepared in Example 3 were reacted with antigens of the same conditions.

The result is shown in FIG.

Antibodies prior to immunosorbent treatment include gills (1,2), mantle membranes (3,4), shell muscles (5,6), tentacles (7,8), and oyster eggs (9-12), as shown in FIG. 3A. The reaction precipitation band appeared, but the antibody after the immunosorbent treatment showed the reaction precipitation band only in the oyster eggs (9-12) as shown in FIG. 3B.

Experimental Example 2 Confirmation of Oyster Egg Specificity of Antibody of the Present Invention Using Immunofluorescence

In order to confirm the oyster egg specificity of the antibody of the present invention, it was investigated using immunofluorescence.

The histological observation result is shown in FIG.

The oyster egg specific antibody prepared in Example 3 of the present invention specifically reacted with the cytoplasm of egg cells, and did not react with other site tissues such as nucleus, digestive duct, stomach, and intestine.

Example 4 Preparation of Absorbance Calibration Curves for the Oyster Egg Protein

The number of purely isolated oyster eggs were counted and lyophilized, and the weight was calculated to calculate the protein content of each egg.

As a result, about 5.3 ng of protein was tested per oyster egg, and the dry weight of each egg was about 13 ng.

Based on this, a standard section in the range of 5 to 300 ng / ml of the al protein was set, and a calibration curve of the absorbance of the al protein was obtained as shown in FIG. 5.

Example 5 Quantitative Measurement of Breeding Eggs

Enzyme-linked immunosorbent assay (ELISA) was used to quantitatively measure the amount of egg protein in sesame extract.

The sesame oyster extract was diluted to about 1-5 μg / ml and used as an antigen.

8 μg / ml of the oyster roe specific antibody prepared in Example 3 and the secondary binding antibody (Alkaline Phosphatase-conjugated goat anti-rabbit IgG) purchased from SIGMA Co. The reaction was carried out by injecting and emitting light with p-nytrophenyl phosphate, and the absorbance was measured at a wavelength of 405 nm.

The measured absorbance values were substituted for the standard calibration curve of the oyster eggs prepared in Example 4 to quantify the egg protein in the oyster extract.

Egg value was calculated by dividing this value by the amount of egg protein contained in the oyster individuals (5.3 ng), and multiplying it by the dry weight value of one egg (13 ng) to calculate the dry weight of eggs per individual oyster (Equation 1) Reference).

<Calculation Formula 1>

Dry weight of eggs contained in oysters = K × amount of protein contained in oysters

[K: dry dry weight / protein mass = 13 ng / 5.3 = 2.45]

Number of grains contained in the oyster object = dry weight of egg contained in the oyster object / one dry weight

Table 2 shows the results of measurement of the degree of entrapment of the oyster eggs.

Table 2 Results of measurement of the degree of porridge of oysters according to the present invention

month. Work Average GSI (mg / mg) Top GSI Lowest GSI 1.26 - - - 2.25 - - - 3.30 0.002 - - 4.26 0.244 0.433 0.079 5.30 0.265 0.34 0.112 6.15 0.423 0.667 0.017 6.28 0.129 0.482 0.011 7.12 0.145 0.247 0.044 7.27 0.268 0.454 0.052 8.10 0.189 0.414 0.034 8.29 0.068 0.256 0.012

GSI: Total dry weight of oyster eggs (mg) / Dry weight of whole oysters (mg)

As in Table 2, no pores were measured in January-February.

From the end of March, the phurando began to appear, peaking around June 15, then decreasing and increasing at the end of July.

The present invention relates to a method for quantitatively measuring the eggs contained in the production of the sesame oysters, an antibody to the eggs of sesame oysters, which is a kind of dissolved bivalve shells, using the specific reaction principle of the antigen-antibody. Breeding can be measured and used to assess their physiological status and habitat environment.

In addition, by more accurately estimating the recruitment, it is possible to provide critical data in estimating the optimal environmental capacity using numerical modeling, which is expected to contribute greatly to the establishment of fisheries policy based on this. .

Claims (3)

After collecting the oyster eggs to obtain an antigen, using this antigen to obtain an antiserum, the antibody that cross-reacts to the oyster body tissue was removed by immunosorption to prepare an antibody specific to the oyster eggs, and then specific to this oyster eggs A method of quantitative determination of propagation amount of oysters using an immune reaction, characterized in that the antibody and whole extract of oysters are reacted as antigens, and the degree of reaction is measured to quantify the amount of protein. The method of claim 1, wherein the immunosorbent method is characterized by using glutaraldehyde. The method of claim 1, wherein the amount of protein of oyster roe is characterized by enzyme-linked immunosorbent assay (ELISA).