JP2002058476A - Anti-streptococcus sobrinus monoclonal antibody and cell strain for producing the same or kit for detecting streptococcus sobrinus containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means for accurately and simply determining Streptococcus sobrinus. SOLUTION: The cell strain produces a Streptococcus sobrinus specific monoclonal antibody and is obtained by a method comprising a process for subjecting a mammalian spleen cell immunized against Streptococcus sobrinus and a mammalian myeloma-derived cell to cell fusion to obtain a hybridoma and a process which is a process for cloning a cell strain capable of producing a Streptococcus sobrinus specific monoclonal antibody from the hybridoma and which assays the antibodies produced from the hybridoma with respect to combining ability with Streptococcus sobrinus and other bacteria of the genus Streptococcus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a Sobrinus bacterium (S
Treptococcus sobrinus, hereinafter simply referred to as sobrinus)
And a cell line producing the monoclonal antibody, and a kit containing the monoclonal antibody.

[0002]

2. Description of the Related Art Sobrinus is a mutans strain (Str.
eptans (Eptococcus mutans, hereinafter simply referred to as mutans), and bacteria that inhabit the oral cavity of animals and cause dental caries. Therefore, the examination of sobrinus is very useful from the viewpoint of dental caries prevention. For example, if an antibody specific to Sobrinus is obtained, a caries preventive test, pathological screening by quantifying the amount of Sobrinus,
It is possible to confirm the therapeutic effect and determine the prognosis. However, hitherto, no antibody specific to sobrinus has been obtained, and there is no method for specifically detecting sobrinus. (S. mutans, Sc
ricetus, S. et al. rattus and the like, and a test method of identifying and detecting sobrinus present in the bacterial population of the mutans group that has grown has been carried out.

[0003] However, detection of sobrinus using a culture method requires 24 hours or more, which makes the operation complicated and quantification of sobrinus not easy. Therefore, it has been desired to perform the quantification of Sobrinus more accurately and simply.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a means for accurately and simply quantifying Sobrinus. The present invention provides a monoclonal antibody specific to Sobrinus bacterium, and provides a simple method for quantifying Sobrinus by sandwich immunochromatography using the monoclonal antibody.

[0005] Sobrinus is a bacterium and it is relatively easy to obtain a pair of antibodies that can bind in a sandwich via sobrinus. However, when a monoclonal antibody is used, the cross-reactivity of this monoclonal antibody is very important because the antibody is homogeneous. Among the monoclonal antibodies against Sobrinus, there may be antibodies having high binding ability not only to Sobrinus but also to other Streptococcus bacteria. If such an antibody is used in the sandwich method, accurate quantification of Sobrinus may be hindered. Therefore, it is necessary to use an anti-sobrinus monoclonal antibody having low cross-reactivity to other Streptococcus bacteria.

The present invention is intended to solve the above problems, and has a high binding ability to Sobrinus.
An object of the present invention is to provide an antibody having low cross-reactivity to other Streptococcus bacteria, a cell line producing the same, and a kit for detecting sobrinus containing the antibody.

[0007]

SUMMARY OF THE INVENTION The present invention relates to a cell line that produces a monoclonal antibody specific to Sobrinus,
This cell line is obtained by fusing mammalian spleen cells immunized with sobrinus with cells derived from mammalian myeloma,
Obtaining a fused cell; and cloning a cell line producing a monoclonal antibody specific to Sobrinus from the fused cell, wherein the antibody produced from the fused cell is isolated from Sobrinus and other streptomyces. It can be obtained by a method including a step of assaying the ability to bind to a bacterium of the genus Tococcus by an immunoassay.

[0008] Preferably, the monoclonal antibody-producing cell line is a deposit number FE of the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.
RM BP-7202 or FERM BP-72
No. 03.

Preferably, the cell derived from the mammalian myeloma is P3X63 Ag8.653 derived from a mouse myeloma.
Cells of the strain. Preferably, the immunoassay is an enzyme-linked immunosorbent assay (ELISA).

[0010] Preferably, the immunized mammal is a mouse or a rat.

[0011] Preferably, the immunized mammal comprises B
ALB / C strain mouse.

[0012] The present invention, in one aspect, relates to an anti-sobrinus monoclonal antibody produced by the above cell line,
This monoclonal antibody has high binding ability to Sobrinus and does not have binding ability to other Streptococcus bacteria.

[0013] The present invention, in one aspect, relates to a kit for detecting sobrinus, the kit comprising a first antibody and a second antibody bound to a solid phase.
Is the monoclonal antibody and the second antibody is a labeled antibody that specifically binds to Sobrinus.

The second antibody may be an anti-sobrinus monoclonal antibody produced by the cell line;
Alternatively, it may be another antibody having high binding ability to Sobrinus.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically.

In the present invention, unless otherwise specified.
Protein separation and analysis methods and immunological techniques known in the art may be employed. These techniques can be performed using commercially available enzymes, kits, antibodies, labeling substances and the like.

The preparation of the anti-sobrinus monoclonal antibody-producing cell line of the present invention will be described below along the preparation procedure.

(Immunization) First, antibody-producing cells are prepared in an animal body by immunizing a mammal with sobrinus.

Examples of "mammals" include mice, rats, cows, rabbits, goats, sheep, and guinea pigs. Mammals are preferably mice and rats, and more preferably mice. As an example of a mouse,
A / J system, BALB / C system, DBA / 2 system, C5
7BL / 6 system, C3H / He system, SJL system, NZ
B strain and CBA / JNCrj strain mice. Since BALB / C strain mice show high antibody titers in serum after immunization, it is possible to obtain monoclonal antibodies having extremely high affinity for sobrinus. It is known that blood antibody titers are associated with the likelihood of specific hybridomas. In addition, BALB / C strain mice are generally used for large-scale production of antibodies using ascites after cell lines have been established. From the above, BALB /
Mice of strain C are a preferred example for immunization of Sobrinus. The age of the experimental animal is not particularly limited, but is typically about 4 weeks to about 12 weeks, preferably about 6 to about 10 weeks.
The mouse or rat is about 7 weeks old, more preferably about 7 weeks old.

The sobrinus used for immunization may be isolated from the human oral cavity or commercially available (for example, available from Parklone Drive 12301, Rockville, MD 20852, American Type Culture Collection, USA). May be used. From the human oral cavity, it can be isolated as a mucoid-type colony on MS agar medium according to a standard method.

Prior to immunization, sobrinus may be mixed with an adjuvant to enhance the immune response. Examples of adjuvants include water-in-oil emulsions (eg, incomplete Freund's adjuvant), water-in-oil-in-water emulsions, oil-in-water emulsions, liposomes, aluminum hydroxide gels, silica adjuvants, powdered bentonite, and tapioca adjuvants. In addition, BCG, Propionibacteri
um acnes, cell walls and cell components such as trehalose dicholate (TDM); lipopolysaccharide (LPS) and lipid A, endotoxins of Gram-negative bacteria
Fraction; β-glucan (polysaccharide); muramyl dipeptide (MDP); bestatin; synthetic compounds such as levamisole; proteins or peptide substances derived from biological components such as thymic hormone, thymic hormone humoral factor and tuftsin; Mixtures (eg, complete Freund's adjuvant) and the like.

These adjuvants are effective in enhancing or suppressing the immune response depending on the administration route, dosage, administration timing and the like. Furthermore, depending on the type of adjuvant, there are differences in the production of antibodies to blood against antigen, induction of cellular immunity, immunoglobulin class, and the like. Therefore, it is preferable to select an adjuvant appropriately according to the intended immune response. Handling of selected adjuvants,
For example, a method for mixing with Sobrinus is known in the art.

Immunization of a mammal is performed according to a method known in the art. For example, the antigen Sobrinus
It can be injected subcutaneously, intradermally, intravenously, or intraperitoneally in a mammal. Since the immune response depends on the type and strain of mammal being immunized, the immunization schedule can be modified appropriately for the animal used. The challenge is repeated several times after the first immunization. Booster immunizations can be performed, for example, 4 weeks, 6 weeks, and 6 months after the initial immunization.

(Confirmation of antibody production) After immunization, blood is collected from a mammal, and the obtained blood is assayed for the presence of sobrinus binding activity, so that the antibody to sobrinus is produced in the body of the mammal; and Confirm that class switching from IgM to IgG occurs at the end of immunity (for example, Harlow and La
ne, ANTIBODIES: A LABORATORO
RY MANUAL, COLD SPRING HAR
BOR LABORATORY, New York
(1988)). Examples of suitable assay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent antibody method. In the present invention, it is desirable to obtain an anti-sobrinus monoclonal antibody having high affinity for sobrinus. In order to obtain a high-affinity monoclonal antibody-producing cell, it is necessary to show a high antibody titer at the time of antiserum.

(Boost) After confirming the production of sobrinus binding antibody, a boost (boost injection of immunogen) can be performed to enlarge the spleen. The amount of Sobrinus administered in the boost is preferably about 4 to 5 times the amount of Sobrinus to be immunized first, but is not limited thereto.

The boost is typically performed using an emulsion of sobrinus and incomplete Freund's adjuvant. However, as the sobrinus to be administered in the final immunization (additional injection of the immunogen several days before the cell fusion), it is preferable to use a pure product without adding an adjuvant. The administration route is appropriately determined in consideration of the possibility that an antibody recognizing a different site in Sobrinus may be obtained by subcutaneous, intradermal, intravenous, or intraperitoneal administration.

(Cell fusion) After the final immunization, spleen cells are excised from the immunized mammal and fused with cells of a cell line derived from myeloma.

Since the proliferation ability of the fused cells depends on the type of myeloma-derived cell line used at the time of cell fusion, it is preferable to use a cell line having excellent proliferation ability for cell fusion. Preferably, the cell line derived from myeloma is compatible with the mammal from which the spleen cells to be fused are derived. A myeloma-derived cell line may be newly prepared or a commercially available cell line may be used. Cell lines derived from mouse myeloma include P3X63Ag8.653, Sp2 / O Ag
14, FO · 1, S194 / 5. XX0 BU. l, P
3 / NS1 / 1 Ag41. Use of P3X63 Ag8.653 is preferred because it does not produce antibody fragments and the fusion cells have excellent growth ability. Rat myeloma-derived cell lines include 210, R
CY3. Ag. 1.2.3, YB2 / 0 and the like.

Cell fusion is performed according to methods known in the art (Koehler and Milstein,
Nature 256: 495 [1975], Kosb
or et al., 1983, Immunol. Today
4:72, Cote et al., 1983, Proc. Nat
l. Acad. Sci. USA, 80: 2026, Co
le et al., MONOCLONAL ANTIBODIES
AND CANCERHERAPY, Alan R
Liss Inc. , New York, NY, 77
-Page 96 [1985]). Examples of the cell fusion method include a polyethylene glycol method, a method using Sendai virus, a method using electric current, and the like. The polyethylene glycol method is preferred because of relatively low cytotoxicity, easy fusion operation and high reproducibility.

The obtained fused cells can be grown according to conditions known in the art. Desired fusion cells can be selected based on the binding ability of the produced antibody.

(Cell Sorting and Cloning) The binding ability of the antibody produced from the fusion cell can be assayed based on a method known in the art. In the present invention, in order to obtain a fusion cell that has high binding ability to Sobrinus and does not have binding ability to other Streptococcus bacteria, or that produces an antibody having low binding ability, The cell line of interest is cloned using the selection based on the binding ability of Streptococcus to bacteria of the genus Streptococcus. Therefore,
As used herein, the term `` monoclonal antibody specific for sobrinus '' has a high binding ability to sobrinus,
Antibodies that have no or low binding ability to other Streptococcus bacteria. Antibodies that have a high binding ability to Sobrinus and “no binding ability” to other Streptococcus bacteria have no cross-reactivity.

The term "having a high binding ability" means that the inhibition occurs in the measurement under substantially the same conditions as in the inhibition ELISA method described in the following Examples, and the half value of the inhibition is reduced. It means less than about 10 ^-6 M. The term "having a low binding ability" means that the inhibition occurs in the same measurement, but the half value of the inhibition is about 10 ^-5 M or more (for example, 10 ^-4 M, 10 ^-3 M, etc.). Say. The term "no binding capacity" refers to no inhibition in the same measurement. The term “inhibited” refers to a decrease in the amount of antibody that binds to Sobrinus immobilized on a solid phase in the presence of a competitor (inhibitor) compared to the absence of the inhibitor. . The term "inhibition free" means that the amount of antibody that binds to Sobrinus immobilized on a solid phase is substantially equivalent in the presence and absence of the inhibitor. The “half value of the inhibitor” refers to the concentration of the inhibitor at which half the absorbance (reflecting the amount of antibody binding) in the absence of the inhibitor is measured.

The binding ability of the antibody is assayed using a method such as an ELISA method, an RIA method, or a fluorescent antibody method as described above for confirming the production of the antibody. The ELISA method is preferable because the antibody can be easily detected with high sensitivity.

For cloning of the fused cells, a method known in the art can be used. Examples of the cloning method include a limiting dilution method and a soft agar method. The limiting dilution method is preferable because the operation is easy, there are many results, and the reproducibility is high.

In order to efficiently select useful cells from among a number of fused cells obtained by cell fusion, it is preferable to carry out cell selection from an early stage of cloning.

Thus, a fusion cell line producing an antibody having a desired binding ability is finally selected. Sorted cell lines can be stored semi-permanently in liquid nitrogen.

(Purification of Antibody) By culturing the monoclonal antibody-producing cell line selected as described above in a large amount, a monoclonal antibody specific to Sobrinus can be produced in a large amount. Methods for mass culture of monoclonal antibody producing cell lines include in vivo and in vitro culture. Examples of large-scale in vivo culture include:
The method includes injecting the fused cells into the peritoneal cavity of a mammal to grow the cells, and producing antibodies in ascites. In in vitro culture, the fused cells are cultured in a medium and the antibodies are produced in the medium.

[0038] The monoclonal antibody of the present invention can be purified from ascites or culture supernatant obtained by mass culture using a method known in the art. For purification, for example, DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation, PEG fractionation, ethanol fractionation and the like are used in an appropriate combination. The antibodies of the present invention are generally purified to a purity of about 90%, preferably about 95%, more preferably about 98%.

(Evaluation of Antibody) By evaluating the binding ability of the purified monoclonal antibody, a combination of antibodies recognizing different epitopes on Sobrinus cells can be selected from several obtained antibodies. Any combination of two antibodies that recognize different epitopes is useful for the sandwich method. On the other hand, since many identical proteins are present on the bacterial surface, the sandwich method can be performed using one kind of monoclonal antibody.

In the present invention, a kit for detecting sobrinus is provided. The kit of the present invention can be provided, for example, for performing immunochromatography for detecting an antigen in an aqueous sample based on an antigen-antibody binding reaction. The kit of the present invention comprises a first antibody bound to a solid phase and a second antibody used in a mobile phase.

As the first antibody bound to the solid phase, accession number FERM BP-7202 or accession number FE
Monoclonal antibodies produced by the cell line RM BP-7203 can be used. Preferably, the accession number F
A monoclonal antibody produced by the cell line of ERM BP-7202 can be used as the first antibody.

As the second antibody, any antibody can be used as long as it has a high binding ability to Sobrinus, and it may be a polyclonal antibody or a monoclonal antibody. Preferably, a monoclonal antibody is used. Accession number FERM BP-7 as the second antibody
A monoclonal antibody produced by the cell line No. 202 or the accession number FERM BP-7203 is preferably used. In this case, the accession number FERM BP-720
Monoclonal antibodies produced by the cell lines No. 2 and accession number FERM BP-7203 can be used as a pair of first and second antibodies to perform a sandwich assay.

[0043] The second antibody can be labeled with any label by methods known in the art. Examples of signs include:
Examples include enzyme labels, dye labels, magnetic labels, radioactive labels, labels with colored particles (colloidal gold, latex, etc.).

[0044] The kit of the present invention can be appropriately prepared by a method known in the art. The kit of the present invention comprises the first antibody and the second antibody in one or more containers.
Antibodies. The kit may also include instructional materials teaching the use of the antibody in a Sobrinus sandwich assay. The kit may include appropriate reagents, washing solutions, dilution buffers, and the like, for detecting the label or for detecting the positive and negative controls.

As described above, the first antibody is usually immobilized on a solid phase, and the second antibody is labeled. In measuring Sobrinus, first, the second antibody is reacted with Sobrinus in a liquid phase to form a label-antibody-Sobrinus complex. Then, the reaction solution containing the complex is reacted with the solidified first antibody as a mobile phase. As a result, the first antibody and the second antibody bind in a sandwich manner via sobrinus. Therefore, the label is immobilized on the solid phase via sobrinus only when sobrinus is present.

The anti-sobrinus monoclonal antibody produced by the cell line of Accession No. FERM BP-7202 has high binding ability to sobrinus and has no binding ability to other Streptococcus bacteria. Accession number FERM
Similarly, the anti-sobrinus monoclonal antibody produced by the BP-7203 cell line has a high binding ability to sobrinus and no binding ability to other Streptococcus species.

The "anti-Sobrinus monoclonal antibody" according to the present invention also includes a functional fragment that retains its binding characteristics. These fragments can compete with the intact antibody from which they are derived for specific binding to Sobrinus, at least 10 ⁷ , 10 ⁸ , 10 ⁹ M ⁻¹ , or 1
0 ¹⁰ may bind with an affinity of M ^-1. Antibody fragments include immunoglobulin heavy and light chains, Fab, Fab ′, (ab ′)
₂ , Fabc and Fv. Antibody fragments can be generated by enzymatic or chemical separation of intact immunoglobulins. For example, the F (ab ') ₂ fragment is Ha
rrow and Lane, ANTIBODIES: A
LABORATORY MANUAL, COLD SP
RING HARBOR LABORATORY, Ne
It can be obtained from an IgG molecule by protein digestion with pepsin at pH 3.0-3.5 using standard methods as described in w York (1988). Fab fragments are converted to F (a
b ') from ₂ fragments, or may be obtained from whole antibody by the presence papain digestion of a reducing agent (Paul,
W. Hen, FUNDAMENTAL IMMUNOLOG
Y second edition RavanPress, N.M. Y. , 198
9, see Chapter 7).

[0048]

EXAMPLES Hereinafter, the production of the monoclonal antibody-producing cell line of the present invention will be described more specifically. The present invention is not limited by the following examples.

<Enzyme-linked immunosorbent assay (ELISA)> In the following examples, the obtained antisera, culture supernatants and monoclonal antibodies were evaluated by ELISA. The operation method is described below.

[0050] The coating sobrinus of (A) an antigen (sobrinus) were prepared at a concentration of 10 ⁸ cells / mL. Microplate (polystyrene high binding type flat bottom # 258
0, Costar Co., Ltd.), and the antigen solution was injected at 100 μl / well and stored overnight in saturated steam at room temperature. Immediately before the experiment, the antigen solution was removed with an aspirator.

(B) Blocking 1% by weight of BSA-PBS-Az (Az: azide sodium salt) was injected at 200 μl / well and left at room temperature for 30 minutes. Then, 1% by weight BSA-P with aspirator
BS-Az was removed. When the subsequent experiments were not carried out on the same day, they were stored at 4 ° C. in saturated steam in this state.

(C) Reaction of Antibody 50 μl of an antibody solution (antiserum, culture supernatant, purified antibody, etc.) diluted to various concentrations with 1% by weight of BSA-PBS-Az was used.
/ Well, and 50% 1% by weight BSA-PBS-Az
Injected in μl / well. After one and a half hours at room temperature,
After washing three times with PBS, residual PBS was removed with an aspirator.

(D) Reaction of Second Antibody 0.2 μg / mL peroxidase-labeled goat-derived anti-mouse IgG antibody (manufactured by KPL) was added to 1% by weight BSA.
Dissolved in PBS solution, or 0.2μg / mL
Peroxidase-Labeled Goat-Derived Anti-Mouse IgM
A solution prepared by dissolving an antibody (manufactured by KPL) in a 1% by weight BSA solution in PBS was injected at 50 μl / well, and left at room temperature for 30 minutes. After washing three times with PBS, the remaining PBS was removed with an aspirator.

(E) Substrate reaction and termination 10 mg of O-phenylenediamine (for biochemistry)
L in citrate-phosphate buffer (pH 5)
Immediately before use, a solution (substrate solution) to which 4 μL of a 30% by weight hydrogen peroxide solution was added was injected at 100 μl / well and left at room temperature. After about 3 minutes, the reaction was stopped by injecting 25 μl / well of 4N sulfuric acid.

(F) Measurement 4 Using a microplate reader (manufactured by Toyo Soda Co., Ltd.)
The absorbance at 92 nm was measured.

In this example, an enzyme immunoassay was used as an immunoassay, but alternatively, an RIA method, a fluorescent antibody method, or the like may be used.

<Examples> In this example, the BALB / C strain mice were most often used in the laboratory of the present inventors and in the mass cultivation of antibodies using ascites after establishing a monoclonal antibody-producing cell line. BALB / C strain mice were used for immunization, taking into account their use.

(Immunity) Sobrinus, which is an immunogen, was treated with 10 ⁸ cells / phosphate buffered saline (PBS).
It was adjusted to mL. The same volume of an adjuvant (complete Freund's adjuvant containing killed Mycobacterium tuberculosis, H37Rv, manufactured by Wako Pure Chemical Industries, Ltd.) was added to the PBS solution of Sobrinus, and the mixture was sufficiently emulsified with a homogenizer at 1,000 rpm to obtain an adjuvant containing an immunogen. An emulsion was obtained.

About 7 weeks old female mice (BALB /
C) 15 mice were injected intraperitoneally or subcutaneously with 100 μl of the adjuvant emulsion containing the immunogen. 2
One week later, a Sobrinus solution adjusted to 10 ⁸ cells / mL using PBS and an incomplete Freund's adjuvant of the same volume were emulsified with a homogenizer, and this emulsion was injected into BALB / C mice at the same site as the previous time by 100 μl. .

Thereafter, 4 weeks, 6 weeks, and 6 months after the start of immunization, an incomplete Freund's adjuvant emulsion containing sobrinus having the same composition and concentration as the immunization 2 weeks after immunization was applied to the mice at the same site as the previous site in 100 μl portions. Injected. One week after the second injection and one after the fifth injection
Blood was collected after each week, and the following antibody production was confirmed.

(Confirmation of Antibody Production) Serum was separated from the collected blood, and the obtained serum was used for enzyme immunoassay (E
LISA method) was used to confirm antibody production. As a solid phase, 10 ⁸ cells / mL sobrinus prepared with PBS-Az was dispensed at 100 μl / well, and a microplate coated overnight at room temperature was used. As a second antibody, a peroxidase-labeled anti-mouse IgG antibody or a peroxidase-labeled anti-mouse IgM antibody was used. The color development in the wells confirms the presence of antibody binding to Sobrinus in the antibody sample.

As a result, production of anti-sobrinus antibody was observed in all 15 mice. Furthermore, in all mice, the antibody production after the second injection was Ig
The shift from G to IgM was confirmed, and after the fifth injection, the IgG / IgM ratio was 300 or more, and it was confirmed that class switching occurred sufficiently.

(Cell fusion) A final immunization was performed to enlarge the spleen of three of the immunized mice, which were particularly high in titer. Six months after the start of immunization, the immunogen sobrinus was adjusted to a concentration of 10 ⁸ cells / mL using PBS, and the mice were injected in an amount of 100 μl without adjuvant.

One of the mice 3 days after the last immunization,
The spleen cells of the animals were removed. Using polyethylene glycol having an average molecular weight of 1,500, a spleen cell and a mouse myeloma-derived cell line (P3X63 Ag 8.65) were obtained by a conventional method.
3) to obtain a fused cell.

The fused cells were suspended in hypoxanthine / aminopterin / thymidine (HAT) medium prepared in Ishikov medium containing 15% by weight of fetal calf serum (hereinafter referred to as FCS) and spread on one 96-well plate. (20
0 μl / well). At this time, spleen cells of the same mouse individual were used as feeder cells (cells supplying a growth factor at the start of culture). Culture was started in a CO ₂ incubator (CO ₂ concentration: 5% by volume, temperature: 37 ° C., humidity: 95%). In the following cultures, cultures were performed under the same conditions unless otherwise indicated.

(Cell selection and cloning) One week later, 100 μl of the culture supernatant of the fused cells was collected, and the remaining culture solution containing the fused cells was subcultured into four 24-well plates, and 1 ml was added to each well. A hypoxanthine / thymidine (HT) medium containing 15% by weight of FCS was added.

Four days after passage of the fused cells to a 24-well plate, cell culture supernatants were collected at 150 μl / well. Using this culture supernatant and the culture supernatant collected one week after the start of the culture, the binding ability to sobrinus was measured by the following ELISA method.

0.1 mg / mL BSA-P as solid phase
Sobrinus adjusted to a concentration of 5 μg / mL with BS-Az was used at 100 μl / well. As an antibody solution,
Cell culture supernatant was used. A peroxidase-labeled anti-mouse IgG antibody was used as the second antibody.

The results of the ELISA method of the culture supernatant collected twice were combined to show that they have high binding ability to Sobrinus.
20 wells with good growth conditions were identified. As a first step selection, the cells in these wells were all passaged into four 6-well plates, and 4 ml of HT medium containing 15% by weight of FCS was added to each well.

Two days after the first stage of cell selection, the culture supernatant was collected, and its binding ability to Sobrinus was measured by the following ELISA method.

10 ⁸ cells / m in PBS-Az as solid phase
100 μl / well of sobrinus adjusted to a concentration of L was used. The cell culture supernatant was used as the antibody solution.
As a second antibody, a peroxidase-labeled anti-mouse IgG
Antibodies were used.

As a result, 10 wells having high binding ability to Sobrinus were selected. The cells in each of these wells were passaged into medium flasks (50 ml volume). As the medium, 45 ml of an HT medium containing 15% by weight of FCS was added.

Three days after the passage of the cells selected in the second stage, the culture supernatant is collected, and the binding ability to Sobrinus and other Streptococcus species (Streptococcus mutans) is measured by the following ELISA method. did.

As a solid phase, 10 ⁸ cells / PBS-Az was used.
Mutant prepared to a concentration of mL was used in an amount of 100 μl / well. The cell culture supernatant was used as the antibody solution. As the second antibody, peroxidase-labeled anti-mouse I
The gG antibody was used. If an antibody that binds to mutans is present in the cell culture supernatant, a color develops on the well.

Two wells that showed binding ability only with Sobrinus and did not show binding ability with mutans, another Streptococcus bacterium, were selected.

15% by weight of the cells in the two wells
Was diluted (limited dilution) to a concentration containing two cells per well using HT medium containing FCS, and dispensed into each of two 96-well microplates. Thymocytes of 4-week-old female mice (BALB / C) were used as feeders to promote initial proliferation. Culture was performed while increasing the size of the plate.
Screening by the ISA method was repeated. Cell lines showing high titers against S. sobrinus and showing good growth were finally selected and 5 × in 200 mL of medium.
Culture was advanced to a concentration of 10 ⁵ cells / mL. Finally, two strains were selected which had high binding ability to Sobrinus and did not cause cross-reaction with other Streptococcus bacteria.

One of the strains showing high binding ability to Sobrinus was named cell line name: SS-1 and deposited with the National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology on June 30, 2000. (Accession number FERM BP7202).

Similarly, another strain having a high binding ability to Sobrinus was designated as a cell strain name: SS-2, and deposited with the National Institute of Bioscience and Human Technology on June 30, 2000. (Accession No. FERM BP7203).

The antibodies produced by the SS-1 strain and the SS-2 strain are called SS1 antibody and SS2 antibody, respectively.

(Preservation of cells) The cell lines finally selected were centrifuged to remove the supernatant, and 1 × 10 ⁷ cells / m
FCS: dimethylsulfoxide = 9: 1 at L concentration
After suspending in 1 mL of the solution (volume ratio) and pre-freezing at -80 ° C, it was transferred to liquid nitrogen and kept in a long-term storage state.

(Purification of Antibody) Each of the selected two strains was cultured in large amounts in an Ishikov medium containing 15% by weight of FCS, and the supernatant was centrifuged. In addition, the selected two strains,
Each was injected intraperitoneally into female BALB / C mice and allowed to proliferate and accumulate ascites. The accumulated ascites was collected. The culture supernatant or ascites of each strain is subjected to affinity chromatography using a protein A binding gel (Protein A Sepharose 4FF, manufactured by Pharmacia),
Each monoclonal antibody (SS1 antibody and SS2 antibody) was purified under the following conditions.

The column filled with the protein A binding gel was washed with a binding buffer (1.5 M glycine / 3 M NaC).
1, pH 8.9). The culture supernatant or ascites was diluted about 3 times with a binding buffer, and then applied to an equilibrated column. The column was washed with binding buffer while monitoring the eluate from the column at 280 nm until the elution of the impurities was complete. After washing, elution buffer (10
(0 mM citric acid, pH 4) was applied to the column (linear flow rate: about 20 cm / hour), and an IgG-containing eluate was collected. The absorbance at 280 nm of the collected IgG-containing eluate was measured with an absorptiometer, and the measured absorbance was converted into an extinction coefficient to determine the antibody concentration.

From the comparison with the standard protein by SDS polyacrylamide gel electrophoresis, the purified fractions of these monoclonal antibodies (SS1 and SS2) were all H chain having a molecular weight of about 50,000 and L chain of about 25,000. It was confirmed to be an IgG consisting of In addition, contamination on the electrophoresis was below the detection limit.

(Evaluation of Antibodies) For the two types of monoclonal antibodies purified by the affinity chromatography, the second
Antibody evaluation was performed under the same conditions as in the inhibition ELISA method at the stage selection.

FIG. 1 is a graph showing the results obtained by measuring the binding ability of the SS1 antibody to (a) Sobrinus (open circles) and (b) the mutans (black circles) of the SS2 antibody. .

As shown in FIG. 1, for the SS1 antibody, × 1
While binding to sobrinus was observed with ~ × 1/10 dilution, binding to mutans was not observed with any dilution, indicating that Sobrinus could be specifically detected by the SS1 antibody. Indicated.

Similarly, as shown in FIG. 2, for SS2 antibody, binding to Sobrinus was observed with the × 1 to × 1/100 dilution, whereas for the mutans, any of the dilutions was used. No binding was observed, SS2
It was shown that Sobrinus could be specifically detected by the antibody.

(Sandwich reaction) In the ELISA method, the SS1 antibody was coated on a plate, sobrinus was bound, the enzyme-labeled SS2 antibody was allowed to react, the excess antibody was removed, and a chromogenic substrate was added and incubated. Sufficient color development was obtained. That is, it was confirmed that the combination of the SS1 antibody and the SS2 antibody or the polyclonal antibody was useful for an inspection method using a sandwich reaction such as immunochromatography.

(Detection Sensitivity in Immunochromatography) The SS1 antibody was immobilized on filter paper according to a conventional method, and the SS2 antibody labeled with colloidal gold was placed in a mobile phase to prepare an immunochromatography apparatus. When samples containing sobrinus at various concentrations were applied, the sensitivity of the immunochromatography apparatus was about 10 ⁶ cells / ml.

In general, it is known that the level of sobrinus in the oral cavity of a healthy person is about 10 ⁴ to 10 ⁷ cells / ml. Therefore, the immunochromatography device prepared using the antibody of the present invention can detect sobrinus in the oral cavity.

[0091]

According to the method for producing a monoclonal antibody-producing cell line of the present invention, in the cloning of the cell line,
The antibody produced from the fused cells is tested for its ability to bind to Sobrinus and other Streptococcus species, and cells of interest are selected. Therefore, useful cells can be efficiently selected from many cells existing at the beginning after cell fusion. Then, a cell line that produces a highly specific antibody while achieving high affinity for Sobrinus can be produced.

The immunoassay was performed by enzyme immunoassay (ELISA).
Method), the antibody binding ability can be assayed simply and with high sensitivity.

When the mammalian myeloma-derived cell line is mouse myeloma-derived P3X63Ag8.653, it does not produce an antibody fragment, and furthermore, the obtained fused cells have particularly excellent proliferation ability. Many cells can be assayed at a time.

When the mammal to be immunized is a mouse or a rat, it is convenient in terms of handling of the animal and immunization. When the mammal to be immunized is a BALB / C strain mouse, it becomes possible to obtain a monoclonal antibody having extremely high affinity for sobrinus.

According to the monoclonal antibody-producing cell line of the present invention, by culturing it, it is possible to semipermanently provide an anti-sobrinus monoclonal antibody having high affinity for sobrinus and high specificity.

The monoclonal antibody of the present invention, SS
If one antibody and SS2 antibody are used in combination or the monoclonal antibody of the present invention is used in combination with an anti-Sobrinus polyclonal antibody in a sandwich method,
A highly specific sobrinus detection kit can be provided.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of an ELISA method using an SS1 antibody. The data in the graph show the binding ability of the anti-sobrinus monoclonal antibody (SS1 antibody) of the present invention to sobrinus and mutans.

FIG. 2 is a graph showing the results of an ELISA method using an SS2 antibody. The data in the graph shows the binding ability of the anti-sobrinus monoclonal antibody (SS2 antibody) of the present invention to sobrinus and mutans.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) G01N 33/577 C12N 15/00 C

Claims (8)

[Claims] 1. A cell line that produces a monoclonal antibody specific to sobrinus, wherein the cell line is fused with a mammalian spleen cell immunized with sobrinus and a cell derived from a mammalian myeloma. And cloning a cell line producing a monoclonal antibody specific for Sobrinus from the fusion cells, wherein the antibody produced from the fusion cells is isolated from Sobrinus and other Streptococcus. A cell line obtained by a method including a step of assaying the ability to bind to a genus bacterium by an immunoassay. 2. The accession number FERM BP-7202 or FERM B of the National Institute of Advanced Industrial Science and Technology
The monoclonal antibody-producing cell line according to claim 1, which is P-7203. 3. The cell line according to claim 1, wherein the mammalian myeloma-derived cell is a mouse myeloma-derived P3X63 Ag8.653 cell. 4. The method according to claim 1, wherein the immunoassay is an enzyme immunoassay (E
The cell line according to claim 1, which is a LISA method). 5. The cell line according to claim 1, wherein the immunized mammal is a mouse or a rat. 6. The method according to claim 6, wherein the immunized mammal is BALB / C.
2. The cell line according to claim 1, which is a strain mouse. 7. An anti-sobrinus monoclonal antibody produced by the cell line according to claim 1, which has high binding ability to sobrinus and has no binding ability to other Streptococcus bacteria. antibody. 8. A kit for detecting sobrinus, comprising a first antibody and a second antibody bound to a solid phase, wherein the first antibody is a monoclonal antibody according to claim 7. And wherein the second antibody is a labeled antibody that specifically binds to Sobrinus.