JP2994074B2 - Determination of cedar and cypress pollen - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a monoclonal antibody that specifically binds to a cedar pollen antigen or a common antigen of cedar pollen and hinoki pollen, and a method of using the same.

[0002]

2. Description of the Related Art Pollinosis is a method in which pollen released during the flowering season is blown off by the wind and drifts in the air. Allergic rhinitis is inhaled by inhaling the pollen from the nose, and allergic conjunctivitis is caused by adhering to the eyes. Is what causes it. In addition to physical distress such as nose and eyes, the patient receives mental stress,
It is a social problem at present because it has a great influence on daily life.

[0003] The number of hay fever patients has increased remarkably in recent years, and treatment and preventive measures are urgently required. Not get. However, many of the drugs used for symptomatic treatment are accompanied by side effects such as drowsiness and malaise, causing pain to patients, and are not always satisfactory.

As a preventive measure, it is best to keep hay fever patients away from the causative agent, since the causative agent of pollinosis is very clear as pollen and its debris. Not all pollen is the causative agent, and pollen of plants of particular concern are cedar, cypress, gramineae (spodoptera, sorghum), ragweed, mugwort and kanagura.

Particularly, Japanese cedar is attracting the most attention as a causal forest of hay fever because Japan has a large area for planting cedar and the amount of scattered pollen is enormous. In addition, since Japanese cedar pollinosis patients also have allergic symptoms to cypress pollen, it is presumed that a common antigen exists between cedar and cypress.

[0006] The need for pollen information is increasing as a part of preventive measures against such hay fever. Based on the pollen information, the patient can take self-care for pollen defense measures, and the doctor can take measures such as medical care such as administration of an antiallergic agent. In fact, various organizations have started measuring pollen counts in the air only for cedar pollen and providing pollen information via newspapers and television.

[0007]

At present, the method of measuring the number of pollen based on the above-mentioned pollen information is generally a method of morphologically observing under a microscope and counting the number. However, since pollen forms are various, identification requires a certain amount of skill, and the measured values vary from person to person. Furthermore, such a method that relies on humans based on a subjective criterion requires a great deal of labor and time, so that the number of samples that can be processed is naturally limited.

Therefore, in order to collect a large amount of highly accurate data from a wide area, a new measurement method needs to be developed. Therefore, as a result of conducting various studies in order to impart objectivity to the measurement results, it was concluded that a measurement method by an immunological technique using an antibody having a specific specificity was preferable.

As an immunological method for measuring the number of cedar pollen, there has been reported an immunoblot method using an antiserum against a major allergen of cedar pollen (allergy).
39, 12, 1612-1620). However, immunization with pollen or pollen antigen (polyclonal antibody)
Is produced, an antibody against a common antigen with a heterologous pollen contained in the pollen antigen is also produced at the same time, so that the number of a specific pollen cannot be measured using this antibody. Therefore, it is necessary to produce a monoclonal antibody with high antigen specificity, that is, a monoclonal antibody that specifically recognizes cedar pollen antigen or a common antigen of cedar pollen and hinoki pollen, and establish a method for measuring specific pollen using this antibody. is there.

The present invention provides a monoclonal antibody recognizing cedar pollen, or cedar pollen and cypress pollen, and a method for measuring pollen or pollen antigen using the same.

[0011]

Means for Solving the Problems The present inventors fused monoclonal antibodies against cedar pollen antigens and common antigens of cedar and cypress pollen with antibody-producing cells of animals immunized with cedar pollen antigens and myeloma cells. The present invention is intended to solve the above problems by using the hybridomas obtained as described above and using the same in a method for quantifying the numbers of cedar pollen and hinoki pollen by an immunological method.

That is, the present invention provides a monoclonal antibody recognizing a cedar pollen antigen, a monoclonal antibody recognizing a common antigen of cedar pollen and hinoki pollen,
A hybridoma obtained by fusing a splenocyte capable of producing an antibody recognizing cedar pollen or an antibody recognizing cedar pollen and hinoki pollen obtained with a cedar pollen antigen as an immunogen, and a myeloma cell, The above monoclonal antibodies are produced using these monoclonal antibodies, and the cedar pollen or cedar pollen and
And the number of pollen in cypress pollen
Recognize cedar pollen antigen and recognize cypress pollen antigen
No monoclonal antibody, no cedar pollen and cypress pollen
Use monoclonal antibodies that recognize common antigens
And a method characterized by the following.

Hereinafter, the present invention will be described in detail. Cell fusion technology for producing monoclonal antibodies is described in Koehler and Milstein (Nature, pp. 495-492, 19).
(1975). That is, it is known that hybrid cells (hybridomas) obtained by fusing mammalian spleen cells and myeloma cells produce various antibodies depending on the properties of the fused spleen cells. Attempts have been made to produce fusion cells that produce monoclonal antibodies against various proteins and carbohydrates by utilizing the properties of the hybridoma and cloning it, and to produce monoclonal antibodies (E. Dale Servier). e
t al .Clinical Chemistry Vol.27.No.11,1979-1806,19
81).

A hybridoma producing a monoclonal antibody specific to a cedar pollen antigen or a common antigen of cedar and cypress pollen is prepared, for example, as follows. That is, (1) cells were prepared from an antibody-producing cell-rich spleen from an animal immunized with a protein crudely purified from cedar pollen as an antigen, and (2) a hybridoma was prepared by fusing the spleen cells and separately prepared myeloma cells. Prepared, (3) hybridomas are selectively grown from the fused cells, and whether the monoclonal antibody in the culture medium of the antibody-producing hybridoma recognizes the cedar pollen antigen or the cypress pollen antigen And (4) Cloning a hybridoma producing the desired monoclonal antibody.

In order to prepare a monoclonal antibody using the obtained hybridoma and to quantify the cedar or cypress pollen using this, an immunoassay method using a monoclonal antibody labeled with an enzyme or the like is employed. You. Further, the pollen antigen can be purified by so-called immunoadsorption chromatography using the monoclonal antibody bound to a carrier.

The above steps will be described.

1. Preparation of antibody-producing cells Cells that produce antibodies to cedar pollen may be obtained by immunizing an animal with an antigen of cedar pollen and obtaining antibody-producing cells of the animal. Animals to be immunized include mice, rats, rabbits, guinea pigs, sheep, and the like. Sources of antibody-producing cells include spleen, lymph nodes, and peripheral blood.

As an immunization method, a cedar pollen antigen is emulsified in, for example, Freund's complete adjuvant, and when a mouse is used, the antigen is administered subcutaneously or intraperitoneally, and is administered at intervals of 1 to 3 weeks. A method in which administration is continued 2 to 4 times to complete immunity is preferred. Before the final immunization,
It is desirable to confirm an increase in blood antibody titer by enzyme immunoassay or the like.

2. Preparation of Hybridoma Antibody-producing cells are collected from the immunized animal, and cell fusion with myeloma cells is performed. The myeloma cells used for the fusion cells are not particularly limited, and many mammalian cell lines can be used. However, it is preferable to use the same animal cell line as the animal used for preparing the antibody-producing cells.

The myeloma cell line used should be capable of distinguishing unfused and fused cells after cell fusion.
Those having a specific drug marker for selection are preferable so that unfused myeloma cells cannot survive and only hybridomas can proliferate. For example, myeloma cells resistant to 8-azaguanine cannot grow in a medium (HAT) supplemented with hypoxanthine, aminopterin and thymidine, but a fusion cell of these cells and normal cells can grow in a HAT medium, It is preferably used because it has the property of being distinguishable from unfused cells.

Specifically, mouse myeloma cell line P3-
Ag8-γ, P3-X63-Ag8, P3-X63-A
g8-U1, NSI-Ag4 / 1, X63-Ag8.6
・ 5.3, SP2 / 0-Ag14, MPC11-45.
6TG1.7, S194 / 5XX0. BU. No. 1 is used, but other new strains have been established and can be used for cell fusion.

Cell fusion is usually performed using MEM, RPMI164.
0, antibody-producing cells and myeloma cells in a medium such as IMDM (these can be purchased from Gibco) from 10: 1 to 1: 1.
By mixing at a mixing ratio of At this time, it is preferable to use polyethylene glycol having an average molecular weight of 1,000 to 6,000 as the fusion accelerator. The use concentration of polyethylene glycol is usually 30 to 50%.

Also, fusion by electric pulse is effective. For example, an initial high-frequency voltage of 20 to 40 V, a pulse width of 1
The fusion can be performed by an electric pulse of 0 to 20 μS, an AC voltage of 200 to 300 V, a pulse voltage of ^{2 to 3} kV / cm ² , and a high frequency electric field of 20 to 40 V.

The cells after cell fusion are appropriately diluted with, for example, IMDM medium containing fetal bovine serum and centrifuged. The precipitate is suspended in a selection medium (for example, HAT medium), dispensed into a 96-well microtiter plate, cultured, and only hybridomas are grown.

3. Search for antibody-producing hybridomas The search for antibody-producing hybridomas is carried out by collecting a culture medium in which hybridomas have grown, using an immunoassay method using a cedar pollen antigen, and a second antibody labeled with an enzyme, a fluorescent substance, a luminescent substance, or the like. Search for. In this, what is called enzyme immunoassay (ELISA) which uses an enzyme as a thing which labels a 2nd antibody is preferably employ | adopted.

In the ELISA method, an antigen is immobilized on a plate or the like, and a hybridoma culture solution is added thereto, and further reacted with an enzyme-labeled second antibody. Alternatively, the antibody is immobilized, and the antigen is added. This is a method of measuring the amount of antibody by sequentially incubating with an enzyme-labeled second antibody and measuring the enzyme activity of the enzyme-labeled second antibody bound to the antigen.

4. Cloning of antibody-producing hybridoma Cloning is performed by a limiting dilution method or the like from cells in the culture wells confirmed to contain the antibody-producing hybridoma, to obtain a monoclonal antibody-producing hybridoma. By the above operation, the monoclonal antibody-producing hybridoma of the present invention specific to the cedar pollen antigen can be obtained.

5. Preparation of Monoclonal Antibody A monoclonal antibody specific to the cedar pollen antigen can be obtained from the hybridoma thus obtained as follows. That is, the monoclonal antibody of the present invention can be obtained by culturing the antibody-producing hybridoma in an appropriate medium and collecting the culture supernatant.

In order to produce a large amount of monoclonal antibodies, a mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) is intraperitoneally injected into an animal of the same species as the animal from which the myeloma cells were derived. And then inoculating the antibody-producing hybridoma and intraperitoneally growing the antibody-producing hybridoma in a large amount. According to this method, high concentrations of monoclonal antibodies are generated in the serum and ascites of the inoculated animals.

The monoclonal antibody can be separated and purified by salting out with ammonium sulfate, ion exchange chromatography, affinity chromatography using immobilized protein A or protein G, or immunoadsorption chromatography using immobilized antigen. it can.

6. Method for measuring cedar pollen The number of cedar pollen is measured using the monoclonal antibody according to the present invention, for example, by the following method.

The monoclonal antibody according to the present invention may be an enzyme,
It is labeled with a labeling agent such as various isotopes and fluorescent substances, and the labeled antibody is reacted with a solidified cedar pollen antigen. The number of cedar pollen or the numbers of cedar pollen and hinoki pollen can be measured by measuring the amount of labeled antibody bound to the cedar pollen antigen by immunoreaction.

For immobilization of the antigen, a nitrocellulose membrane,
Durapore (PVDF) membranes, nylon membranes and the like are preferred.

The antigen amount of cedar pollen can be measured by a sandwich ELISA method using two types of monoclonal antibodies that recognize different antigenic determinants. That is, the first monoclonal antibody is immobilized, and then the cedar pollen or cedar pollen antigen to be measured and the labeled second monoclonal antibody are added, and the mixture is incubated sequentially and labeled amount of the labeled second antibody bound by the immune reaction. By measuring, the amount of antigen can be measured.

For the immobilization of the antibody, agarose, latex particles, wells of a microplate, and the like can be used, but wells of a microtiter plate are preferable.

Enzymes are preferred for labeling the antibody, and alkaline phosphatase, peroxidase, etc. can be used as the enzyme.

7. Purification of cedar pollen antigen The antigen of cedar pollen or hinoki pollen can be purified by using the monoclonal antibody according to the present invention as an immunoadsorbent. For example, the pollen antigen is purified by binding the monoclonal antibody according to the present invention to an insoluble carrier such as dextran gel, agarose gel, or polyvinyl gel, and subjecting the monoclonal antibody-bound carrier as an immunosorbent to column chromatography.

The binding between the insoluble carrier and the monoclonal antibody can be carried out by the Bromcian method or a method of binding via an epoxy group, an amino group, a carboxyl group or a formyl group.

After adding a pollen extract or a solution obtained by artificially synthesizing a cedar or cypress antigen to the column comprising the monoclonal antibody-bound insoluble carrier, the antigen adsorbed on the column is eluted to obtain a highly purified purified antigen. Is obtained.

[0040]

The present invention will be described in more detail with reference to examples.

1. Preparation of Antibody-Producing Cells A cedar pollen antigen (SBP. Cry j I) was extracted and purified from cedar pollen as follows according to the method of Yasue et al. (J. Allergy Clin. Immunol. 71.77-86 1983).

0.125 M of cedar pollen (100 g)
It was suspended in a solution of _{(NH 4) HC O 3 (} pH8.3), 4 ℃
The antigen was extracted with. After removing pollen from the suspension by centrifugation and salting out with 80% ammonium sulfate, the flow-through fraction of anion exchange column chromatography (DEAE-cellulose column) is adsorbed to cation exchange column chromatography (CM cellulose). After that,
Elution with 3M NaCl and finally Sephacryl S-2
SBP was purified by gel filtration using 00.

200 μg of SBP thus purified
Was emulsified in 0.2 ml of Freund's complete adjuvant, and the whole amount was subcutaneously administered to male BALB / C mice. Thereafter, administration was performed three times at two-week intervals, and finally 200 μg of SBP dissolved in physiological saline was intravenously administered.

Three days after the last administration, the spleen was removed from the mouse, and the mouse was subjected to Iscove's Modified Dulbecco's Medium (IMD
M) (supplied from Gibco) to loosen spleen cells
Single cells were obtained by passing through a 0 mesh nylon net.

2. Preparation of hybridoma Hypotonic solution (15
After adding 5 mM ammonium chloride) to lyse the red blood cells, the cells were washed again with IMDM.

On the other hand, mouse myeloid cells (P3-Ag8-
γ) added to 10% fetal calf serum (FCS)
M. Subcultures are performed at 3 day intervals and fresh I.P.
The medium was exchanged with MDM, and cells with a high viability were used for cell fusion the next day.

The numbers of spleen cells and myeloid cells were counted, mixed at a ratio of 5: 1 and centrifuged. After discarding the supernatant and resolving the cell sediment, fusion buffer (Mannitol 0.25 M, CaCl ₂ 0.1 mM, MgCl ₂ 0.1 mM, Tris-HC
l 0.2 mM, pH 7.2).

After re-centrifugation, the cells were suspended in a small amount of a fusion buffer, and electro-cell fusion (Shimazu Seisakusho, SSH-1)
Using an electric pulse having an initial high-frequency voltage of 20 to 40 V, a pulse width of 10 to 20 μS, an AC voltage of 200 to 300 V, a pulse voltage of ^{2 to 3} kV / cm ² , and a high-frequency electric field of 20 to 40 V.

The cells subjected to the fusion treatment were subjected to HAT (4 × 10 ⁻⁷ each).
M) The suspension was suspended in IMDM supplemented with 10% FCS at a concentration of 5 × 10 ⁵ / ml and dispensed 100 μl at a time into a 96-well microplate. The same medium is added to the medium every 3 to 4 days.
The hybridomas alone were grown by selective culture in this HAT medium by adding 0 μl each.

3. Search for antibody-producing hybridoma Collect the culture solution from each well in which the hybridoma has grown,
Wells containing antibodies to SBP were searched for by the indirect ELISA using a secondary antibody as follows.

A 3 μg / ml SBP solution prepared with a coating buffer (0.05 M sodium carbonate buffer, pH 9.6) was dispensed at 100 μl / well in a 96-well microplate (NUNC, Immunoplate II ). The mixture was poured and left at 25 ° C. for 2 hours to coat each well with SBP.

Next, 10 m containing 0.05% Tween 20 (polyoxyethylene sorbitan monolaurate) was used.
After washing with M-phosphate buffered saline (PBS-T), 25
200 μl of% Block Ace (purchased from Dainippon Pharmaceutical)
Per well, and blocked at 4 ° C. overnight.

After the plate was washed with PBS-T, 100 μl of each culture was added, and the plate was allowed to stand at 25 ° C. for 2 hours.

Subsequently, after washing with PBS-T, a 2000-fold diluted alkaline phosphatase (Alp) -labeled goat anti-mouse IgG antibody (TAGO, No. 6550) 10
After adding 0 μl, the mixture was allowed to stand at 25 ° C. for 2 hours.

After washing with PBS-T, 1 mg of a substrate solution (Sigma, 104 phosphatase substrate) was added.
/ M to 0.05M NaHCO ₃ -10mM
MgCl ₂ (dissolved in pH 9.8)
The reaction was performed at 30 ° C. for 30 minutes. After stopping the reaction by dispensing 5 M NaOH (50 μl / well), the absorbance at 415 nm was measured.

As a result, 150
Of the 0-well cultures, 141-well cultures showed an absorbance of 0.3 or more, and reacted strongly with SBP.

Subsequently, the cells (hybridomas) in the wells that had reacted strongly were transferred to the wells of a 24-well plate, and the culture was scaled up by adding a new culture medium.

The culture was screened by the ELISA method using pollen immobilized as follows.

3 mg / ml of cedar or cypress pollen prepared with a coating buffer in a 96-well microplate
The solution was dispensed at 100 μl / well and reacted at 4 ° C. overnight to coat the plate with pollen proteins. Less than,
ELISA was performed in the same manner as above.

As a result, 37 wells strongly reacted with cedar pollen, and 11 wells strongly reacted with both cedar and hinoki pollen.

That is, the monoclonal antibodies were divided into two groups: a group that reacted only with cedar pollen and a group that reacted with both cedar and cypress pollen.

4. Cloning of monoclonal antibody-producing hybridomas Monocloning of each antibody-producing hybridoma was performed as follows.

Peritoneal cells collected by injecting IMDM into the peritoneal cavity of normal mice were used as feeder cells. This peritoneal cell was added to IMDM supplemented with 10% FCS at 1 × 10 ⁵ cells / m 2.
l and suspended in 50 μl aliquots.

The next day, 5 antibody-producing hybridomas / m
and 100 μl was dispensed into each well. The culture medium was added or replaced every three days, and the culture supernatant was collected in order from the well in which the cells had grown, and the production of the antibody was confirmed by the same operation as in the indirect ELISA method described above. Cells in the wells in which antibody production was observed were cloned again to obtain anti-SBP monoclonal antibody-producing hybridomas.

The anti-SB monocloned as described above
2. The culture supernatant of the P monoclonal antibody-producing hybridoma was The SBP described in the column was assayed by an indirect ELISA method that coats microplates, and 14 hybridoma strains producing a monoclonal antibody highly reactive to SBP were selected.

Next, the culture solution of these hybridomas was used for 3. With immobilized cedar or cypress pollen as described in
Method A was performed. As a result, the reactivity of the obtained monoclonal antibody with cedar pollen and cypress pollen was found. Some of the monoclonal antibodies that reacted with cedar pollen were those that also reacted with cypress pollen.

That is, eight hybridomas producing a monoclonal antibody reactive only to cedar pollen, and six hybridomas reactive to both cedar and hinoki pollen were obtained. Therefore, it is presumed that there is a common antigenic determinant site contained in both cedar pollen and hinoki pollen.

5. Preparation of Monoclonal Antibody BALB / C mouse 7 weeks old or older
The mice were intraperitoneally administered, and about one week later, 1 × 10 ⁵ cloned hybridomas were inoculated intraperitoneally into each mouse.

After about 10 days, the ascites of the mouse was collected and
After centrifugation at 000 rpm for 10 minutes, the supernatant was collected.

To this supernatant, an equal volume of PBS was added.
Through a column packed with 5 ml of Protein G Sepharose 4FF (Pharmacia) equilibrated in
After sufficient washing with, elution was carried out with a 0.1 M glycine-hydrochloric acid buffer solution (pH 2.7).

The eluate was 1 M Tris-HCl buffer (pH
7.5) was collected in a test tube, and A ₂₈₀ was measured to collect the antibody fraction, which was dialyzed against PBS to give S.
A purified monoclonal antibody against BP was obtained.

6. Selection of monoclonal antibody-producing hybridoma The antigen recognition site (epitope) of the monoclonal antibody thus purified was examined by the following method.

The purified monoclonal antibodies were labeled with alkaline phosphatase, and competition experiments were performed with various unlabeled monoclonal antibodies, and the monoclonal antibodies were grouped by epitope.

The labeling of the monoclonal antibody was performed as follows. Monoclonal antibody purified with protein G
33 mg and alkaline phosphatase (1.15 mg,
Sigma), and PBS was further added to make up to 300 μl. Add 25% glutaraldehyde at a final concentration of 0.2
%, And allowed to react for 2 hours.
It was dialyzed overnight at 4 ° C. against PBS containing Cl ₂ .

After dialysis, NaN ₃ and ZnCl ₂ were added to a final concentration of 0.02% and 1 mM, and stored at 4 ° C. This solution was used as an alkaline phosphatase-labeled monoclonal antibody to perform monoclonal antibody grouping. Competition experiments between various unlabeled monoclonal antibodies and enzyme-labeled monoclonal antibodies were performed by the following method.

100 μl / well of SBP adjusted to 0.25, 0.5 and 1.0 μg / ml with a coating buffer was dispensed into a 96-well microplate, and
The reaction was carried out at ℃.

Two hours later, the plate was washed three times with PBS-T, and 100 μl of 25% Block Ace was dispensed and left standing at 4 ° C. overnight to perform blocking. After that, PB
After washing three times with ST, 0, 1, 10, 100 μg / m
l of unlabeled monoclonal antibody at each concentration of 100 μl /
Each well was added and reacted at 25 ° C. for 2 hours.

Next, after washing with PBS-T three times, a 500-fold diluted solution of alkaline phosphatase-labeled monoclonal antibody was added at 100 μl / well, and the mixture was reacted at 25 ° C. for 2 hours. (Sigma 104 phosphatase substrate) 100 μl
/ Well and reacted at 25 ° C. for 30 minutes.

Further, a 5 M NaOH aqueous solution was added to 50 μl /
After addition in the wells, the absorbance at 415 nm was measured.

As a result, eight types of antibodies produced by eight strains of hybridomas reacting only with cedar pollen could be divided into five epitopes, and antibodies produced by six strains reacting with both cedar and cypress pollen could be divided into five types of epitopes. . The antibody subtypes were G1 12 strains, G2b 1 strain, and G2a 1 strain.

Next, a monoclonal antibody-producing hybridoma that reacts with pollen was confirmed by immunoblotting (indirect method) using an enzyme-labeled anti-mouse antibody.

Blotting buffer (0.05M T
ris-HCl (pH 7.6), 0.04M glyc
and a Durapore membrane (PVDF) (manufactured by ATTO) immersed in the same solution was placed on a filter paper immersed in the same liquid. After spraying cedar or cypress pollen on this membrane, a Durapore membrane soaked in the same solution is overlapped so as to prevent air bubbles from entering, and a filter paper soaked in the same solution is further stacked and allowed to stand at room temperature for 1 hour, and the pollen protein is removed. Was adsorbed on the membrane.

After washing the membrane twice with PBS-T for 5 minutes,
Immerse in a 1 μg / ml solution of monoclonal antibody, and
Allowed to react for hours.

After washing with PBS-T, 3 times with PBS-T
Alkaline phosphatase-labeled anti-mouse IgG antibody (TAGO, No. 6550) diluted 000-fold was added and reacted at room temperature for 1 hour. 3 minutes with PBS-T for 10 minutes
After washing twice, an NBT / BCIP substrate solution (NBT 3
30 μg and 1650 μg of BCIP were added to 1 M Tris-H.
Cl (pH 9.5), 0.1 M NaCl, 5 mM Mg
(A solution of Cl _{2 at} a concentration of 1 ml) for 30 minutes, and then washed with water and dried.

The reactivity of the monoclonal antibody to cedar pollen and cypress pollen in the indirect immunoblot was consistent with the results obtained using the microplate.

Next, a monoclonal antibody-producing hybridoma that reacts with pollen was confirmed by an immunoblot method (direct method) using an alkaline phosphatase-labeled anti-SBP monoclonal antibody.

Durapore (PVD) sprayed with cedar or cypress pollen in the same manner as in the immunoblot (indirect method) described above.
F) Pollen protein was transferred to the membrane. Put this membrane in PBS
After washing twice for 5 minutes with -T, 500 times with PBS-T,
The membrane was immersed in a 1000- or 2000-fold diluted alkaline phosphatase-labeled monoclonal antibody solution and incubated at room temperature for 1 hour.
Allowed to react for hours.

After washing with PBS-T, the plate was washed with a substrate solution for 30 minutes.
After coloring for minutes, it was washed with water and dried. As a result, among the monoclonal antibodies that react only with cedar pollen, KW-
Among the monoclonal antibodies produced by the hybridoma named S10, those produced by the hybridoma named KW-S91 among the monoclonal antibodies reactive to cedar and cypress pollen had the strongest reaction. KW-S10 and KW-S9
The monoclonal antibody produced by 1 was selected.

Next, alkaline phosphatase-labeled KW-
Sandwich ELI using S10 monoclonal antibody
A combination of monoclonal antibodies for coating, which can measure the amount of SBP by the SA method, was studied.

As a result of the competition experiment previously performed with the KW-S10 monoclonal antibody, a monoclonal antibody solution having a different epitope (10 μg / ml coating buffer) was used.
Dispense 100 μl / well into a 6-well plate,
Coated at 5 ° C for 2 hours.

After washing with PBS-T, blocking was performed by dispensing 100 μl of 25% Block Ace at 4 ° C. overnight.

After washing with PBS-T, an SBP solution diluted with PBS-T was added (100 μl / well) and reacted at 25 ° C. for 2 hours. After washing with PBS-T, alkaline phosphatase-labeled KW diluted 500-fold with PBS-T
Dispensing 100 μl of the S10 monoclonal antibody,
The reaction was performed at 25 ° C. for 2 hours.

After washing with PBS-T, the substrate solution was
1 / well was dispensed and reacted at 25 ° C. for 30 minutes.
After stopping the reaction by adding 50 μl / well of 5N NaOH, the absorbance at 415 nm was measured.

As a result, the reactivity when the monoclonal antibody named KW-S131 produced by the hybridoma was used for coating was the highest. Therefore, a monoclonal antibody produced by KW-S131 was selected as an unlabeled monoclonal antibody for coating in the sandwich ELISA method.

KW-S10, KW-S91, KW-S1
Since 31 is a novel cell that produce a monoclonal antibody specific to an antigen of cedar pollen, respectively, each of these cells Agency of Fermentation Research Institute F
ERM P-12137, P-12138, P-121
The deposit number was 39.

The properties of the monoclonal antibodies produced by these hybridomas are shown below. The molecular weight is SD
Isoelectric point is determined by S-polyacrylamide gel electrophoresis. Isoelectric focusing method (LKB, isoelectric focusing apparatus)
And the immunoglobulin subclass was determined by a membrane immunoassay (using a mouse monoclonal antibody isotyping kit manufactured by Amersham). (1) Monoclonal antibody produced by KW-S10 (a) molecular weight; 148,000 ± 5,000 (b) IgG subclass; IgG1, κ (c) isoelectric point; 6.20 to 6.40 (d) cedar It specifically recognizes pollen antigens and does not react with cypress pollen antigens. (2) Monoclonal antibody produced by KW-S91 (a) molecular weight; 150,000 ± 5,000 (b) IgG subclass; IgG1, κ (c) isoelectric point; 7.35 to 7.55 (d) cedar It recognizes pollen antigens and cypress pollen antigens. (3) Monoclonal antibody produced by KW-S131 (a) molecular weight; 145,000 ± 5,000 (b) IgG subclass; IgG1, κ (c) isoelectric point; 6.50 to 6.70 (d) cedar It specifically recognizes pollen antigens and does not react with cypress pollen antigens.

7. Methods for measuring the number of pollen and the amount of pollen antigen The number of cedar pollen, the number of cedar or cypress pollen, and the amount of antigen and SBP of cedar pollen were measured using an alkaline phosphatase-labeled anti-SBP monoclonal antibody. (1) Reactivity of Enzyme-Labeled Monoclonal Antibody with Pollen Antigen (Immunoblot Method) The protein of cedar pollen or cypress pollen was converted to durapore (PV
DF) It was adsorbed on a membrane, immersed in a 500-2000-fold diluted alkaline phosphatase-labeled monoclonal antibody solution, reacted for 30 minutes, washed and dried, and immersed in a dye solution to develop color.

The results are shown in Table 1.

[0099]

[Table 1]

The monoclonal antibody produced by KW-S10 reacted strongly only with cedar pollen to the same extent as in the indirect immunoblot method. The monoclonal antibody produced by KW-S91 reacted strongly with cypress pollen as well as cedar pollen. (2) Measurement of the amount of antigen in cedar pollen by sandwich ELISA method The amount of antigen in cedar pollen was measured by an ELISA method using an alkaline phosphatase-labeled anti-SBP monoclonal antibody as follows.

A 10 μg / ml solution of the unlabeled monoclonal antibody prepared with the coating buffer was dispensed at 100 μl / well into a 96-well plate and coated at 25 ° C. for 2 hours. After washing with PBS-T, 25% Block Ace was dispensed at 100 μl / well at 25 ° C.
Let stand for hours. After washing with PBS-T, the cedar pollen suspension (suspending the pollen in a 0.125 M NH ₄ HCO ₃ solution,
(At room temperature for 2 hours) was added in PBS-T at 100 μl / well, and the mixture was reacted at 4 ° C. for 18 hours.

After washing with PBS-T, an alkaline phosphatase-labeled monoclonal antibody diluted with PBS-T was added at 100 μl / well and reacted at 25 ° C. for 2 hours. After washing with PBS-T, the substrate solution (Sigma, 104
(Phosphatase substrate) was added at 100 μl / well, and reacted at 25 ° C. for 20 minutes. 5N N
After stopping the reaction by adding 50 μl of aOH, 415
The absorbance at nm was measured.

FIG. 1 shows a calibration curve obtained when an antibody produced by KW-S131 was used as a coating monoclonal antibody and an antibody produced by KW-S10 was used as an enzyme-labeled monoclonal antibody. The measurement sensitivity of cedar pollen by this measurement system is 20 ng / ml, and the measurement range is 0 to 250.
It was 0 ng / ml. Good linearity was observed between cedar pollen and absorbance in this measurement range. (3) Measurement of SBP amount by sandwich ELISA method Instead of pollen, SBP extracted from cedar pollen was used to examine the measurement sensitivity and the measurement range by this measurement system.

FIG. 2 shows a calibration curve when an antibody produced by KW-S131 was used as the coating monoclonal antibody and an antibody produced by KW-S10 was used as the enzyme-labeled monoclonal antibody. SBP by this measurement system
Has a measurement sensitivity of 0.2 ng / ml and a measurement range of 0 to 3 ng.
/ Ml. Within this measurement range, good linearity was observed between the SBP concentration and the absorbance.

8. Purification of cedar pollen antigen by immunoadsorption chromatography (1) Production of antibody column 0.15 g of Bromcian-activated Sepharose 4B (Pharmacia) was swollen with 1 mM hydrochloric acid, washed, and then aspirated on a glass filter to obtain 1 mM hydrochloric acid. Was removed. This was suspended in 1 ml of the above-mentioned 0.1 M sodium bicarbonate buffer (pH 8.3) containing 0.5 M sodium chloride in which 1 mg of the KW-S10 monoclonal antibody was dissolved,
The mixture was shaken at room temperature for 2 hours and sucked on a glass filter to dehydrate.

Further, a 0.2 M glycine solution (pH 8.
0) 5 ml was added and suspended, shaken at room temperature for 2 hours, dehydrated on a glass filter, and then 0.1 M acetate buffer (pH 4.0) containing 0.5 M sodium chloride and 0.1 ml.
After washing several times alternately with 0.1 M sodium bicarbonate buffer (pH 8.3) containing 5 M sodium chloride,
After equilibration with BS, an antibody-bound carrier was obtained, which had a diameter of 0.9.
The column was packed into a 0.8 cm long column to form an antibody column. (2) Purification of cedar pollen antigen using an antibody column
50 μg of BP was adsorbed and washed with PBS. This was eluted with a 0.1 M glycine-hydrochloric acid buffer (pH 2.7).

At this time, the recovered amount of SBP was 93.2% as a result of measurement by a sandwich ELISA method.

In addition, 0.125 M NaH was obtained from cedar pollen.
The above antibody column KW-S10 was extracted from the solution roughly extracted with CO _3.
The purity of the purified SBP adsorbed on the monoclonal antibody-Sepharose and eluted was examined by 12.5% SDS-polyacrylamide gel electrophoresis. No protein other than SBP was found, and the antibody was directly detected from the crude extract of cedar pollen. It was found that SBP could be purified by the column.

[0109]

The monoclonal antibody prepared using the hybridoma according to the present invention specifically binds to cedar pollen or antigens of cedar pollen and hinoki pollen. By labeling the monoclonal antibody with an enzyme or the like, an immunoassay system for measuring the total number of cedar pollen alone or cedar and cypress pollen without relying on morphological classification that is difficult to distinguish can be established.

A monoclonal antibody recognizing a common antigen of cedar pollen and cypress pollen can form an immunoassay system for measuring the total amount of antigens related to cedar pollen.
All of these will lead to the development of socially useful measuring instruments such as the forecast of cedar pollen scattering. Furthermore, when this monoclonal antibody is used in the step of separating and purifying the antigen of cedar or cypress pollen, it can be purified to an extremely high purity, and the insoluble carrier bound to the monoclonal antibody can be reused by washing, and purified. It is extremely useful industrially because it is economical as well as shortening the process.

[Brief description of the drawings]

FIG. 1. Measurement of antigen content of cedar pollen by sandwich ELISA method

FIG. 2 Measurement of SBP amount by sandwich ELISA method

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ identification symbol FI C12P 21/08 C12N 15/00 C (C12P 21/08 C12R 1:91) (58) Fields surveyed (Int.Cl. ⁶ , DB name) C12N 15/00-15/90 BIOSIS (DIALOG) WPI (DIALOG) JICST file (JOIS)

Claims (3)

(57) [Claims] 1. A method for quantifying the amount of antigen or the number of pollen in cedar pollen or cedar pollen and cypress pollen by an immunological method, comprising: a monoclonal antibody that recognizes cedar pollen antigen and does not recognize cypress pollen antigen; A method comprising using pollen and a monoclonal antibody that recognizes a common antigen of cypress pollen. 2. The method according to claim 1, wherein the monoclonal antibody that recognizes the cedar pollen antigen but does not recognize the cypress pollen antigen is a monoclonal antibody having the following property (1) or (2). (1) (a) molecular weight; 148,000 ± 5,000 (b) IgG subclass; IgG1, κ (c) isoelectric point; 6.20-6.40 (2) (a) molecular weight; 145,000 ± 5,000 (b) IgG subclass; IgG1, κ (c) isoelectric point; 6.50-6.70 3. The method according to claim 1, wherein the monoclonal antibody recognizing a common antigen of cedar pollen and cypress pollen is a monoclonal antibody having the following properties. (a) molecular weight; 150,000 ± 5,000 (b) IgG subclass; IgG1, κ (c) isoelectric point; 7.35 to 7.55