JPH1057055A - Identification of microcystis as microorganism causing water-bloom by using monoclonal antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately conduct an interspecies identification of Microcystis as microorganisms as a cause of water-bloom even in case of incomplete formation of the microorganims' population, by using a monoclonal antibody capable of specifically identifying only Microcystis aeruginosa f. aeruginosa. SOLUTION: An interspacies identification of Microcystis as microorganisms as a cause of water-bloom by using a monoclonal antibody capable of specifically identifying only Microcystis aeruginosa f. aeruginosa. It is preferable that the reactivity of this monoclonal antibody is assessed by the fluorescent antibody indirect method using Goat anti-mouse immunoglobulin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for discriminating among species of the genus Microcystis among cyanobacteria constituting blue-green algae (water bloom) in freshwater bodies such as lakes using a monoclonal antibody. .

[0002]

2. Description of the Related Art In recent years, in freshwater areas such as rivers and lakes, water pollution and eutrophication have progressed, and large-scale occurrence of blue-green algae (water blooms) has often been observed. This blue-green algae is a cyanobacterium, Microcystis (Microctis)
It is composed of microorganisms such as the genus ystis, the genus Osillatoria, and the genus Anabeana. It occurs in large quantities from spring to autumn, and not only impairs the aesthetics of rivers and lakes, but also generates unpleasant odors and is unsanitary Giving a feeling.

[0003] Among the above-mentioned microorganisms that cause water bloom, the genus Microcystis is a representative microorganism, and there are species having microcystin, which is a liver poison, and there is a concern that humans and animals may be adversely affected. ing. This genus of Microcystis is characterized by its morphological characteristics such as the size of the cells constituting the colony, the morphology of the colony, the clarity and shape of the outer green of the mucous body, and Microcystis f. Aeruginosa (Microcystis). aeruginosa f.
aeruginosa), Microcystis aeruginosa f. flos-aqua
e), Microcystis virid
is), Microcystis Besen Belgium (Microcysti
s wesenbergii).

[0004] In order to ascertain the actual condition of the blue-green algae caused by the genus Microcystis, it is necessary to classify and identify the microorganisms in the sample, and to investigate their distribution and their progress. Is required. In this case, since the conventional identification method is performed by recognizing the morphological features described above with a stereoscopic microscope, the following problems occur.

[0005] That is, when species having similar morphologies are identified, when two or more species of near green species are mixed, and before the formation of the colony or after the collapse of the colony, the species is correctly classified and classified. The problem is that it becomes difficult to identify. In addition, since the conventional identification method requires an examiner having a special appraisal eye, there is also a problem that the identification result is often inevitably confused depending on the skill of the examiner.

[0006]

SUMMARY OF THE INVENTION The present invention is based on the recognition of morphological features.
Resolving the above-mentioned problems that occur when performing interspecies identification of genera,
By using a monoclonal antibody described below, it is possible to provide an identification method capable of reliably performing inter-species identification in the genus Microcystis, which is a microorganism that causes blue-green algae, even when colony formation is incomplete. Aim.

[0007]

Means for Solving the Problems In order to achieve the above-mentioned object, the present invention provides a method for discriminating between species of the genus Microcystis, which is a microorganism that causes blue-green algae, using a monoclonal antibody. As the monoclonal antibody, Microcystis aeruginosa f. Aerugi
The present invention provides a method for distinguishing the genus Microcystis, which is a microorganism that causes blue-green algae, with a monoclonal antibody, which comprises using a monoclonal antibody that specifically recognizes nosa) alone.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody used in the present invention is the aforementioned Microcystis aeruginosa f. It is a monoclonal antibody that specifically reacts only against alginosa (Microcystis aeruginosa f. Aeruginosa; hereinafter, referred to as M. aeruginosa f. Aeruginosa).

[0009] Therefore, by using this monoclonal antibody, microcystis (Mis
crocystis) of the genus M.aeruginosa f. aeruginosa
And other species, namely Microcystis aeruginosa f. Floss-acue (Microcystis aeruginosa f. Flos
-aque, hereinafter referred to as M.aeruginosa f. flos-aquae),
Microcystis viridis,
M. viridis) and Microcystis wesenbergii (hereinafter M. wes)
enbergii).

[0010] This monoclonal antibody can be produced as follows. It is described below according to the procedure. (1) Preparation of antigen and immunization process National Institute of Environment Science (NIES)
Strains such as M.aeruginosa f. Aeruginosa (NI
ES-87) is cultured, and a cell suspension having a predetermined cell concentration is prepared from the obtained medium, and this is used as an antigen suspension.

Then, the antigen suspension is administered to, for example, a mouse to immunize the mouse with the antigen. (2) Cell fusion step The antibody of the above antigen is produced in mouse spleen cells, but since the spleen cells lack the proliferative ability,
Subsequent to (1), a mouse myeloma cell (myeloma cell) is fused with this to provide a proliferation ability.

[0012] This procedure is described in "Introduction to Monoclonal Antibody Experimental Procedures" (T. Andoh, Takeshi Chiba, Kodansha Publishing, 1991)
), "New Course on Experimental Chemistry 12, Molecular Immunology 1, Immune Cells / Cytokines, 7, How to Make Monoclonal Antibodies against Leukocyte Antigen" (written by Hiroyuki Iwaki, Shoji Saito, Kokichi Kikuchi, edited by The Chemical Society of Japan, Inc.) Tokyo Chemical Dojinsha Publishing, 1989
Year)).

As the cell fusion promoter at this time, for example, polyethylene glycol having a molecular weight of 4000 is used. (3) Selection of fused cells (hybridoma) In the culture after the above-mentioned cell fusion, in addition to the fused cells (hybridoma), cells with incompletely fused cells or cells that have not been fused at all are mixed. ing. As described above, spleen cells do not have the proliferation ability, and thus die in 1 week to 10 days. In the case of myeloma cells, growth inhibition may occur with hybridomas.

Therefore, in this step, incompletely fused cells or completely unfused cells (myeloma cells) are removed, and only the desired hybridoma is selectively grown. In that case, the following knowledge about myeloma cells is used. That is, first, cells have two pathways called the salvage pathway and the neonatal pathway.
There are two nucleic acid synthesis pathways. Since myeloma cells lack an enzyme essential for nucleic acid synthesis in the salvage pathway, that is, hypoxanthine-guanine phosphoribosyltransferase, the myeloma cells undergo nucleic acid synthesis only through the nascent pathway. Become.

On the other hand, in the case of the nascent pathway, as shown in FIG. 1, there are two places in the pathway where an enzyme called dihydrofolate reductase is involved. And it is known that the function of the said enzyme is inhibited by the binding of aminopterin. Therefore, when the harvest obtained in the cell fusion step described above is cultured in a HAT (hypoxanthine-aminopterin-thymidine) medium containing aminopterin, incompletely fused cells or myeloma cells not fused at all are obtained. They are removed because nucleic acid synthesis by the nascent pathway does not proceed. Then, only the hybridomas having the salvage pathway derived from spleen cells will proliferate.

Specifically, the harvest obtained in the cell fusion step may be spread on a culture dish, and the above-mentioned HAT medium may be added thereto and cultured for about 10 days. Then, after confirming the growth of the hybridoma, the medium may be replaced with an HT medium containing no aminopterin. By exchanging the medium, cells other than the target hybridoma are killed. (4) Cloning Step A plurality of types of clones may exist in the hybridomas selectively cultured in the above step.

Therefore, the hybridomas that have been selectively cultured are cloned by the limiting dilution method.
Then, a monoclonal clone of a hybridoma producing a monoclonal antibody that specifically reacts only with M.aeruginosa f.aeruginosa is obtained. (5) Cultivation of hybridoma The desired monoclonal antibody can be produced by culturing the hybridoma obtained by the above-mentioned cloning in vitro or in vivo.

In the case of in vitro culture, for example, a 96-well plate culture plate may be used and cultured in, for example, FCS-RPMI 1640 medium containing mouse spleen cells as feeder cells. At this time, the target monoclonal antibody is secreted and accumulated in the culture supernatant. In the case of in vivo culture, for example, by inoculating the hybridoma into the peritoneal cavity of a mouse, the target monoclonal antibody is accumulated in the ascites fluid.

By using the monoclonal antibody produced in this manner, Microcysti (Microcysti
s) The genus is identified as follows: Goat anti-mouse imm
It is performed by applying an indirect fluorescent antibody method using unoglobulin. That is, the above-described monoclonal antibody is mixed with a specimen to be identified containing the genus Microcystis, and the mixture is mixed with a secondary antibody Goat anti-labeled with a fluorescent dye such as fluorescein isothiocyanate (FITC). -Mouse A solution obtained by diluting IgM + IgG with a phosphate buffer is added to prepare a cell suspension, which is observed with an epifluorescence microscope.

This monoclonal antibody is a member of the genus Microcystis belonging to the genus M. aeruginosa f. Aerug.
It reacts specifically with inosa only and does not react with the other three species. Then, when reacted with M. aeruginosa f. Aeruginosa, fluorescence is observed. Therefore, when fluorescence is observed during observation with an epi-illumination fluorescence microscope as described above, M. aeruginosa
f. aeruginosa is present and the species can be identified.

[0021]

【Example】

1. Production of Monoclonal Antibody (1) Preparation of Antigen and Immunization of Mice First, an MA medium having the following composition was prepared. Composition of MA medium: Ca (NO ₃ ) ₂ .4H ₂ O 50 mg KNO ₃ 100 mg NaNO ₃ 50 mg Na ₂ SO ₄ 40 mg MgCl ₂ .6H ₂ O 50 mg β-Na ₂ glycerophosphate 100 mg MA soln 1 ml Bicine 500 mg distilled water 1 l MA soln has the following composition:

MA soln: Na ₂ EDTA 5 mg / ml FeCl ₃ .6H ₂ O 0.5 mg / ml MnCl ₂ .4H ₂ O 5 mg / ml CoCl ₂ .6H ₂ O 5 mg / ml NaMoO ₄ .2H ₂ O 0.8 mg / ml ZnCl ₂ 0.5 mg / ml H ₃ BO ₃ 20 mg / ml Next, four strains which are stock strains of NIES were prepared. That is, M.aeruginosaf. Aeruginosa (NIES-87), M.ae
ruginosa f. flos-aque (NIES-98), M.viridis (NIES-
102) and M.wesenberugii (NIES-111).

These four strains were cultured in the above-mentioned MA medium under the conditions of a temperature of 25 ° C. and a luminous intensity of 20 μE / m ² · sec. The number of cells of the NIES-87 strain in the culture was measured using an EKDS hemocytometer (manufactured by Kayagaki Medical Science Co., Ltd.), and then 3600 rp.
The MA medium was removed by centrifugation at 15 m for 15 minutes.

Next, the cells in the form of pellets are dispersed,
After washing with a phosphate buffer, the cells were suspended in a phosphate buffer so that the number of cells became 10 ⁸ cells / ml, to prepare a cell suspension. This is used as an antigen suspension. 1 ml of this antigen suspension was intraperitoneally injected into BALB / c mice (6 weeks old, female).
The mice were immunized every 8 weeks every other week for 8 weeks.

(2) Measurement of antibody titer in mouse antiserum In order to determine whether or not an antibody against the NIES-87 strain is produced in the above-mentioned mouse body, the mouse antiserum was determined as follows. Was examined for antibody titer. First, the tail vein of a mouse immunized for 8 weeks was incised, and about 100 μM of blood was obtained.
l was collected in hematocrit tubes. Then, the blood was transferred to an Eppendorf tube, centrifuged at 1000 rpm for 10 minutes to separate serum and blood cells, and the obtained supernatant was transferred to another Eppendorf tube.
The sample was diluted 25-fold with MA medium and used for antibody titer search.

In 100 μl of the diluted serum, NIES-87 strain, NI
100 μl of each suspension of ES-98, NIES-102 and NIES-111
Were mixed, and each mixture was reacted in a dark place at a temperature of 20 ° C. for 2 hours. Furthermore, the secondary antibody Goat anti-mous labeled with the fluorescent dye fluorescein isothiocyanate (FITC)
e 10 μl of a solution obtained by diluting IgG + IgM (manufactured by Caltag Laboratories) 100-fold with a 0.01 M phosphate buffer was added to each of the above culture solutions, and the mixture was allowed to react in a dark place at a temperature of 20 ° C. for 2 hours.

The fluorescence intensities of the four cell suspensions thus obtained were measured using an epi-fluorescence microscope (BHS-RFC-A2, manufactured by OLYMPUS, U excitation, using G520 filter). . The results are shown in Table 1.

[0028]

[Table 1]

As is apparent from Table 1, mice immunized for 8 weeks contained Microcystis in the body.
Among the genera, it was found that antibodies specifically reacting with M. aeruginosa were produced. That is, M.aerugino
It is possible to immunize mice using sa as an antigen,
Weekly immunization can provide sufficient immunity,
Furthermore, the reactivity of the antibody is determined by Goat anti-mouse immunoglobul
It was found that determination could be made by the indirect fluorescent antibody method using in.

(3) Production of hybridoma Prior to cell fusion, myeloma cells were cultured as follows. First, a medium for myeloma cells was prepared as follows. That is, the powder RPMI1640 (GIBC
The O-made Co.), distilled water: 1l, NaHCO _3: 2g, manufactured by penicillin G to prevent the growth of Gram-positive bacteria in the culture medium (Wako Pure Chemical (Co.)): 50 U / ml, gram-negative in the medium Streptomycin (Wako Pure Chemical Industries, Ltd.) to prevent bacterial growth
): 100 µg / ml was added to prepare a basal medium.

Then, the basal medium is supplemented with fetal bovine serum (GI
A medium for myeloma cells was prepared by adding 10% of BCO (Lot No. 37K2043). Next, in a carbon dioxide incubator at a temperature of 37 ° C. and a carbon dioxide concentration of 5.0%, the above-mentioned medium for myeloma cells was used, and P3 / X63-A derived from BALB / c mouse was used as myeloma cells.
Myeloma cells were cultured using g6.5.3.

On the other hand, on the third day after the end of the final immunization, the immunized mouse was dissected and the spleen was excised, placed on a sterilized mesh to disperse the spleen cells, and filtered with the above-described basal medium. Spleen cells were prepared by centrifugation at 1600 rpm for 6 minutes, and the number of cells was counted. And
The same treatment as described above was performed on the cultured myeloma cells, and the number of myeloma cells was counted.

Next, 2 × 10 ^{8 of} the prepared spleen cells
The cells were mixed at a ratio of 2 × 10 ⁷ myeloma cells per cell, the mixture was washed by centrifugation at 1600 rpm for 6 minutes, and the cell pellet formed after aspirating and removing the supernatant was loosened. Here, 1 ml of polyethylene glycol, which had been preheated to a temperature of 37 ° C., was added dropwise over 1 minute.

Using a pipette, 1 ml of the above-mentioned basic medium is dropped over 1 minute while the cells are well stirred at the tip of the pipette, and 1 ml of the basic medium is gently dropped over 1 minute. After gently dropping 8 ml over 3 minutes, centrifugation was performed at 1000 rpm for 5 minutes.
The supernatant was removed. To the obtained pellet, 18 ml of the above-described medium for myeloma cells was gradually added, and 100 μl of the medium was added.
Each was added to the inner 60 wells of three 96-well plate culture plates numbered No. 1, No. 2, and No. 3, and cultured under the same conditions in the above-described carbon dioxide incubator.

After the start of the culture, on the first day, the third day, the sixth day,
On each of day 0, the above-mentioned medium 5 for myeloma cells
HAT consisting of 100ml of HAT supplement100X10ml
Medium was added to each well in 100 μl. After 10 days from the start of the culture, after confirming the growth of the hybridoma, the medium was replaced with an HT medium containing no aminopterin.

(4) Cloning By limiting dilution, one well of a 96-well plate culture plate was theoretically adjusted to contain one hybridoma, and BALB / c
FCS-RPMI16 containing mouse spleen cell suspension: 10% by volume, Briclones (trade name, cell growth factor-containing medium manufactured by Dainippon Pharmaceutical Co., Ltd.): 10% by volume, pyruvate: 1 mM
Culture was performed using 40 media. The cultivation was performed in a carbon dioxide incubator at a temperature of 37 ° C. and a carbon dioxide concentration of 5.0%.

After confirming the hybridoma colonies, the medium was replaced with FCS-RPMI1640 medium, and the culturing was continued under the same conditions as described above. After culturing for 20 days, it was confirmed that the colony had grown sufficiently large, and
0 strains were measured. 2. Identification of the genus Microcystis The culture supernatant was collected, and 100 µl of the supernatant was added to the NIES-87 strain, NIES
100 μl of each suspension of -98 strain, NIES-102 strain and NIES-111 strain were mixed, and each mixed solution was cultured in a dark place at a temperature of 20 ° C. for 2 hours.

Next, a secondary antibody Goat anti-mouse Ig labeled with a fluorescent dye fluorescein thiocyanate (FITC)
10 μl of a solution obtained by diluting G + IgM (manufactured by caltog Labpratories) 100-fold with a 0.01 M phosphate buffer was added to each of the above culture solutions, and the mixture was further reacted in a dark place at a temperature of 20 ° C. for 2 hours. Using the four types of cell suspensions thus obtained, an epifluorescence microscope (BHS-RFC-A2, manufactured by OLYMPUS, U.S.A.)
Excitation, using a G520 filter). The results are shown in Table 2.

[0039]

[Table 2]

As is clear from Table 2, the hybridomas produced through the above-mentioned steps are M. aeruginosa f. Aerug among the four species of Microcystis.
It can be seen that a monoclonal antibody specifically reacting with inosa was produced. Therefore, by using this monoclonal antibody, microcystis (Micr
ocystis), it is possible to reliably classify and identify only M.aeruginosa f.aeruginosa.

[0041]

As is apparent from the above description, the present invention relates to M.aer of the genus Microcystis.
uginosa f. Aeruginosa only reacts specifically,
Other species, namely M. aeruginosa f. Flos-aquae, M. vi
ridis and M. wesenbergii using a monoclonal antibody that does not react with the three species of M. aeruginosa f. aerginos
a can be reliably identified.

The present invention utilizes the above-described specific antigen-antibody reaction. Therefore, even if the formation of colonies is incomplete, which was difficult to identify by the conventional method of classification and identification based on morphological features, M.
f. aeruginosa can be identified.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram showing the nascent pathway and the site of action of aminopterin.

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Claims (2)

[Claims] 1. A method for distinguishing between species of the genus Microcystis, which is a microorganism that causes blue-green algae, using a monoclonal antibody, wherein the monoclonal antibody is Microcystis aeruginosa f. aeruginosa), wherein a monoclonal antibody specifically recognizing only aeruginosa is used to identify the genus Microcystis, a microorganism that causes blue-green algae. 2. The method according to claim 1, wherein the reactivity of the monoclonal antibody is Go
The identification method according to claim 1, which is determined by an indirect fluorescent antibody method using at anti-mouse immunoglobulin.