JPS63192398A - Monoclonal antibody to outer membrane protein c trimer of escherichia coli and hybridoma capable of producing said monoclonal antibody - Google Patents

Abstract

PURPOSE:To obtain the titled antibody, produced by a hybridoma prepared using an outer membrane protein C (OmpC) of Escherichia coli as an immunologi cal source and capable of directly detecting Gram-negative bacilli mixed in a sample, e.g. biological sample or food, etc., without pretreatment by heating. CONSTITUTION:A blend prepared by blending OmpC or a crude outer membrane fraction of Escherichia coli containing the OmpC with complete or incomplete Freund's adjuvant is injected into an animal, e.g. mouse, etc., to carry out sensitization and splenic cells or lymphocytic B cells extracted from the immunized animal are fused to myelomatous cells. The aimed specific antibody-producing cells are selected from the fused cells and cloned to provide a hybridoma capable of producing the aimed antibody. The above-mentioned antibody is cultivated in a normal culture medium for animal cells and reacted with OmpC trimer to afford a monoclonal antibody unreactive with the OmpC monomer and OmpC trimer. The afore-mentioned antibody is capable of linking to live cells of Escherichia coli and Citrobacter freundii.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel monoclonal antibodies produced by hybridomas.

More specifically, the present invention relates to a novel anti-Escherichia coli outer membrane protein C trimer antibody produced by a hybridoma, a hybridoma cell line producing the antibody, and a method for detecting Gram-negative bacilli using the antibody. Hereinafter, in this specification, E. coli outer membrane protein C trimer will be referred to as OmpC, and anti-E. coli outer membrane protein C trimer monoclonal antibody will be referred to as anti-OmpC.
It is written as monoclonal antibody.

OmpC is Escherichia coli
It is the main outer membrane protein of the Dalton OmpC monomer (Uemura et al., alHBio
chim, Biophys.

Acta, 413.163-176, 1975) is 3
It functions in vivo as an individually associated trimer. That is,
OmpC non-covalently bonds with the peptidoglycan layer in the outer membrane of E. coli to form the bacterial surface structure, and OmpC
With E. coli outer @ Fluorescent F, which has a very good composition and properties, it forms permeable pores for hydrophilic low-molecular substances in the outer membrane.
It plays an important role in both the structural maintenance and functional expression of E. coli.

The OmpC monomer gene has already been isolated, and the entire DNA
The base sequence has been determined. Omp from DNA base sequence
It was revealed that C monomer consists of 346 amino acids and its entire amino acid sequence (Mizuno
et, al; J, Biol, Chem, 258°6932-6940.1983).

The present inventor previously developed anti-Omp using OmpC monomer as an antigen.
A C monomer monoclonal antibody was prepared and its application was investigated (Japanese Patent Application No. 61-75188).

Among them, anti-OmpC monoclonal antibody is
It reacts only with OmpC monomer, and its trimer Omp
It has been revealed that this product does not react with C or living bacterial cells such as E. coli containing it, but does react with heated E. coli cells.

Therefore, when detecting E. coli in a biological sample or food, it is necessary to heat the bacterial cells or a sample containing them at 100° C. for 5 minutes. The need for some kind of pretreatment when detecting bacteria is a major drawback in practical terms. A method that directly reacts and detects bacteria has been desired.

Therefore, the present inventor conducted intensive research and established a monoclonal antibody that can detect bacteria such as E. coli without pretreatment and a detection method using the same, thereby completing the present invention. Specifically, we created a monoclonal antibody against OmpC, which is a trimer, and examined its application. The monoclonal antibody reacted with live bacteria that had not been pretreated by heating, and specifically and sensitively inhibited bacteria such as Escherichia coli. They discovered that bacteria can be detected.

By using the anti-OmpC monoclonal antibody disclosed by the present invention, Gram-negative bacilli present in biological samples such as urine, blood, sputum, and samples such as food,
In particular, Escherichia coli and Citrobacter freundii can be directly detected without pretreatment by heating.

Furthermore, by using the antibody in affinity chromatography, the OmpC antigen can be purified for purposes such as vaccine production, and the antibody is useful as a prophylactic or therapeutic agent for bacterial infection by binding to viable bacteria. It has high industrial value.

The present invention provides a monoclonal antibody that reacts with E. coli outer membrane protein C trimer (Ompc), a hybridoma cell line that produces the antibody, a method for producing the antibody, and a method for detecting bacteria using the antibody.

The present invention will be explained in detail below.

E. coli outer membrane protein C trimer (OpmC) as an antigen is derived from E. coli by Rosenbusch (J, Biol, C.
hem.

"■, 8019-8029.1974), Nakamu
ra et al. (J, Biochen+.

80.1411-1422.1976), Tokun
(Eur, J. Bioches+, 95
, 441-448.1979), and Yama
da et CEur, J. Bioches,, 103+
209-218+ 1980) refers to an outer membrane protein with a molecular weight of about 114,000 daltons, which is isolated and purified by the method of 209-218+ 1980). That is, the outer membrane protein/peptidoglycan complex extracted by reacting Escherichia coli cells in a sodium dodecyl sulfate (SDS) solution at 50°C is further treated in an SDS solution containing salt to extract the complex. Releases and removes butydoglycan.

It is obtained by further isolating and purifying the separated outer membrane protein fraction using column chromatography. In 5DS-polyacrylamide slab gel electrophoresis, this antigen preparation exhibits several bands over the apparent molecular weight range of 70 to -120 Daltons. After heat treatment for minutes, 5DS-
When analyzed by polyacrylamide slab gel electrophoresis, it is characterized by being obtained as a single band with an apparent molecular weight of 30 to -40 Daltons.

This antigen preparation is thought to be a trimer in which three outer membrane protein C monomers are assembled6. The outer membrane protein C monomer consists of 346 amino acids, and its entire amino acid sequence has already been revealed (Mizuno et al.;
J, Biol, Chem, 258 +6932-6
940°1983).

In addition, this antigen acts as a protein that forms the bacterial surface structure through non-covalent bonds with the peptidoglycan layer in the outer membrane of E. coli, and together with E. coli outer membrane protein F, which has a very similar compositional property to OmpC, this antigen forms a hydrophilic protein in the outer membrane of E. coli. It is defined from a functional point of view as a fluorescent substance that forms permeable pores in low-molecular-weight substances.

For the preparation of the antigen, any strain of E. coli, typified by E. coli K-12, can be used, but it is more desirable to use a mutant strain lacking outer membrane protein F.

The hybridoma of the present invention is produced by K″6hler et al.
It is established by a method known as the method of ure Ahi, 495-497.1975). That is, it consists of five steps: immunization, cell fusion, fused cell selection, assay, and cloning.

The immunization step is the step of teasing the animal with the desired antigen.

Animals such as mice are sensitized by injecting OmpC or a mixture of E. coli outer membrane proteins containing OmpC with Freund's complete or incomplete adjuvant one to four times at intervals of one week to several months.

It is desirable to additionally sensitize OmpC purified product 2 to 5 days before cell fusion.

The cell fusion step consists of fusing lung cells or lymphoid B cells isolated from the immunized animal with myeloma cells. Myeloma cells include HGP RT (h
ypoxanthine-guanine phosp
horibosyltransferase) deficient mouse myeloma cells, P3-X63-Ag8-Ul (P
3U1), P3-MS-1/1-Ag4-1 (NS-
1), P3-X63-Ag8-653 (653),
SP210-Ag 14 (SP210) is used as a representative example, but other non-immunoglobulin-producing or non-secreting cells derived from animals and humans that are commonly used in the field of cell engineering can also be used.

As the fusion method, the HVJ method (method using Sendai virus), polyethylene glycol method (PEG method), electrical fusion method, etc. are used.

The PEG method will be explained in more detail below.

That is, lymphocyte B cells and myeloma cells were mixed at a ratio of 10 to 1:1 and mixed with 30 to 50% (W/V) PEG1000.
Add ~6000 little by little over 0.5 to 1 minute,
Let stand for 1 to 10 minutes. Thereafter, dilute by adding serum-incompatible medium for 2 to 10 minutes.

Then add more medium to 10'~10-cells/m12
Cells were seeded at 2xlO' to 2xlO' per i well in 966 microplates and incubated at 5% C.
Incubate at 32°C to 37°C in an o2 incubator. Fusion efficiency can be increased by adding DMSo, poly-L-lysine, or the like when adding PEG.

The fused cell selection step is a step of selecting fused cells by culturing them in an appropriate selective medium in which only fused cells can grow. When HGPRT-deficient myeloma cells were used as fusion parental cells, HAT (Hypo
xanthine, A+winopterin.

Thymidine) medium and HA z (Hypoxa
nthine.

Azaserine) medium is used. Replace with fresh selective medium every 2-4 days and culture for 10-20 days. In addition, a combination method of actinomycin D and emetine, a diethylbirocarbonate method, and a known method known from Ouabain Sakyo are used.

The assay step is a step of selecting specific antibody-producing cells of interest from among the fused cells.

Antibody activity contained in the culture supernatant is usually measured by solid-phase or liquid-phase radioimmunoassay (RIA), Enzyme
Linked Immunosorbent As5a
y (HLISA), agglutination method, fluorescent antibody method, and other methods commonly used in the field of immunology. Purified OmpC is used as the antigen.

The cloning step is a step in which the target specific antibody-producing cells are made into a homogeneous cell population derived from one clone. Limit dilution method, soft agar method, single cell manipulation
) method to obtain a stable cell line, a so-called hybridoma, which produces an antibody that reacts with rapidly proliferating OmpC derived from a monoclonal clone.

The antibody of the present invention can be obtained by inoculating the hybridoma into an &11 tissue culture flask and culturing it in a serum-free medium or a normal animal cell culture medium containing serum, or by inoculating it into an animal such as a mouse that has been pre-administered with Bristan. and obtained by culturing in vivo. The obtained antibody is purified by biochemical techniques commonly used for protein purification, such as ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and affinity chromatography.

From a functional standpoint, this antibody is characterized by its ability to react with and bind to E. coli outer membrane protein C (Ompc) trimer.

These are classified into two types based on their reaction specificity.
That is, the first group is a group with very high reaction specificity, in that it does not react with OmpC monomers and OmpF triketa, which is similar to OmpC in both structure and function. The second group is O
The mpC monomer is a group that reacts, and the OmpF monomer is a group that does not react.

The present antibody is mainly immunoglobulin G (IgG), but may be other immunoglobulin IgM or gA. Furthermore, although the antibody may be derived from any animal species (including humans), it is generally a monoclonal antibody derived from a mouse. Taking mouse IgG as an example, depending on the amino acid sequence of its constant region, it can be divided into IgG1 and IgG1.
G2a.

It is classified into subclasses of IgG2b and IgG3. Both contain four polypeptides (two heavyweights and two
It is a glycoprotein with a molecular weight of approximately 150 Daltons, consisting of many light chains).

For detection of Gram-negative bacilli, especially Escherichia coli, using the antibody, a general technique normally used for detecting antigen-antibody complexes is used. An example of this would be as follows.

An appropriate amount of a food or biological sample that is assumed to be contaminated with E. coli is collected, and the appropriately diluted antibody is added to the sample and allowed to react without heat treatment.

After washing, E. coli is detected by reacting the antibody bound to the bacterial cells in the sample with radioisotope-labeled Staphylococcus-derived protein A and measuring the formed complex with a gamma counter. Furthermore, the number of E. coli in a specimen sample can be quantitatively measured by creating a calibration curve using a sample containing a known amount of E. coli and comparing it with the calibration curve.

An immunological or immunodiagnostic composition for detecting Gram-negative bacilli using the present antibody contains the antibody as at least one component and is composed of a carrier, auxiliary reagents, etc. that are commonly used as immunodiagnostic agents. For example, the antibody can be used by adsorbing it onto a carrier such as a polystyrene ball.

Furthermore, this antibody is applied to affinity chromatography and used to purify OmpC.

Furthermore, by administering this antibody to animals or humans via the parenteral route, it can be applied to prevent or treat bacterial infections caused by Gram-negative bacilli.

As described in detail above, the monoclonal antibody against E. coli outer membrane protein C trimer obtained by the present invention is
Specifically reacts and binds to live bacterial cells of Gram-negative rods such as Escherichia coli and Citrobacter freundii This means that monoclonal antibodies made against E. coli outer membrane protein C monomer bind to these living bacterial cells. A good contrast to what could not have been done. From the viewpoint of detection of bacteria and prevention and treatment of bacterial infections, monoclonal antibodies that directly bind to the specimen without pretreatment such as heat treatment have high practical value.

The present antibody can be applied to detect Gram-negative bacilli, especially E. coli and Citrobacter freundii, without pretreatment of specimens containing bacterial cells.

EXAMPLES The present invention will be explained in more detail by giving examples below.

The present invention is not limited to the following examples, but includes modifications and improvements that may ordinarily occur.

Example 1 Preparation of anti-OmpC monoclonal antibody (1) Preparation of antigen E. coli E, coli K-12 (ATCC10798;
IFO3301), Rosenbusch (J,
Biol.

Chew, 249.8019-8029.1974
),) lakamura et al (J, Bioc
hea+, 80.1411-1422.1976)
and Yamada et al (t!ur, J
, Biochem, 103+ 209-218,
A crude E. coli outer membrane fraction containing OmpC and a purified OmpC sample were prepared according to the method of (1980).

The method for preparing purified OmpC is shown below.

E. coli K-12 with 20% sucrose LB
Medium (20% sucrose, Bacto-trypto
ne 1%, yeast extract 0.5%,
The cells were cultured in NaCl 0.5%, pH 7°0) and harvested at the logarithmic growth phase. Bacteria are disrupted by ultrasound, 14.00
Unbroken bacterial cells were removed by centrifugation at 0xg for 10 minutes, and the supernatant was centrifuged at 100,000xg for 40 minutes to obtain a bacterial wall (cell envelope) fraction. Bacterial wall fractions were cultured in 10 mM lithium containing 2% sodium dodecyl sulfate (SDS), 10% glycerol, o.i.% 2-mercaptoethanol (2-ME).
Hydrochloric acid buffer (Tris-HCi; PH7,5)
suspended in water, heated at 50°C for 130 minutes, and then heated at 100°C.
．． The mixture was centrifuged at 000xg for 40 minutes, and the precipitate was used as the outer membrane crude fraction.

The outer membrane crude fraction was collected using 0.05M sodium chloride (NaCl).
), washed with 10mM Tris-HCl buffer (pH 7.5) containing 1% SDS and 0.01% 2-ME, then 0°5M
NaC! 11%S OS, 0.01%2-MES 1
Lis-HCl buffer (10mM) containing 0% glycerol (p
H7,5) and shaking at 37°C for 30 minutes, followed by centrifugation at 100.000xg for 40 minutes to obtain a supernatant. Next, Sephacryl S-300 (
The product was purified by gel filtration using (Pharmacia). 0.5
Fractions eluted with 25mM phosphate buffer (p) 17.2) containing % SDS and 0.04% NaN were collected to obtain purified OmpC.

The E. coli outer membrane crude fraction (7.8 mg protein/mA) containing OmpC obtained by the above method had an apparent molecular weight of 70 to 70 by 5DS-polyacrylamide slab gel electrophoresis.
120, several bands across Dalton's macromolecular region and Braun's 1ipoprotein and lipoprotein 1! A band due to (LPS) was observed. The content of LPS was 1.05% (W/W). On the other hand, purified O
The mpC preparation (1.9 mg protein/m2) exhibited only a few bands in the dalton region of apparent molecules N70-120 in 5DS-polyacrylamide slab gel electrophoresis, and did not contain LPS. In addition, the same specimen was used in SO3
After heat treatment at 100"C for 5 minutes in the presence of SDS, a single Dalton band with an apparent molecular weight of 30 to 40 was obtained when analyzed by 5DS-polyacrylamide slab gel electrophoresis. It is already known that OmpC (trimer) is converted into a monomer by .

(2) Preparation of immunized lung cells using Freund's complete adjuvant (Freund'sco
Crude E. coli outer membrane fraction 11001I containing Ompc mixed with
It was administered intraperitoneally to ALB/C mice (female, 4 weeks old). Freund's incomplete adjuvant was then added three times at two appropriate intervals.
100 μg of the E. coli outer membrane crude fraction containing OpmC mixed with juvant (FIA) was administered intraperitoneally. Approximately one month later, the mice were boosted by intravenous administration of 100 μg of purified Ompc dissolved in 50 mM phosphate buffer pH 7.0. Three days later, the lungs were removed and Eagle's MEM
Lung cells suspended in a medium (Gyudon Kagaku) were obtained.

(3) Cell fusion mouse myeloma cell P3X63-Ag8・Ul (P3U1
) (ATCC CRL1597) with 10% fetal bovine serum F
RPMI-1640 medium with CS (Gibco) (
The cells were cultured in Gyudon Kagaku), and the cells were collected in the logarithmic growth phase and used for cell fusion. The fusion method was described by K6hler et al.
re, 2 ¥6.495-497.1975). That is, lung cells and myeloma cells were suspended in serum-free RPMI-1640 medium at a ratio of 5=1, and centrifuged for 7 minutes at 1.00 Orpm (Mie Seiko CD-10OR). For the sedimentation, 45% (
W/W) Polyethylene glycol (PEG) 40
00.10χ (V/V) Dimethyl sulfoxide solution 1
m! ! , was added over 1 minute, and then heated to room temperature (23±3
℃) for 1 minute. Then serum-free RPM
2 d of I-1640 medium was added and suspended at a rate of 1Il11/min. Furthermore, serum-free RPMI-1640 medium 7
d was gradually added to dilute the PEG.

Centrifuge at 1,000 rpn+ for 7 min, then add 6 x 10' 7 ml of mouse myeloma cells. 10% FC3-RPMI-164 to obtain a cell concentration of
The cells were resuspended in 0 medium and dispensed into a 96-well microplate (Jinzen Bakelite MS-3096F) with 0.2 cells per well. The fused cells were cultured at 37°C in 5% CO□. One day after cell fusion, half the medium was added to fresh HA.
T medium (10-'M hypoxanthine, 4 x 10-'
RPMI-1640 medium containing M aminopterin, 1.6 x 10-'M thymidine). Thereafter, half-volume replacement with HAT medium was performed three times every two days.

After 10 days, cell proliferation was observed in 100% of the microplate holes.

(4) Assay for anti-OmpC antibody Eleven days after cell fusion, the presence of antibodies against OmpC in the culture supernatant of the hybridoma cells was screened by enzyme immunoassay, so-called ELISA. That is,
061M bicarbonate buffer (bicarbonate b
uffer) 100 μl of OmpC solution dissolved in pH 9.6 was added to a polyvinyl chloride 966 microplate (Falcon Microtest m fle).
xible assay plateHBeckton
After incubation at 37° C. for 2 hours, the solution was removed, and the PVC assay plate was then dried.

In order to prevent non-specific binding, bovine serum albumin (BSA) was adsorbed using the same procedure and used as an antigen plate. 1%BSA, 0.05%(V/V) Twee
Phosphate buffered saline pH 7 containing n20 (Gyudon Kagaku)
Add 100 μl of the culture supernatant appropriately diluted with PBST (hereinafter abbreviated as PBST) per well and incubate at 37°C for 2 hours.
It was cultured in Thereafter, it was washed 3 times with PBST, and then washed with calcium-magnesium-free phosphate buffered saline pH 7.2 (hereinafter abbreviated as PBS(-)) for 10'.
Double diluted alkaline phosphatase (alkali
ne phosphatase)-labeled anti-mouse immunoglobulin antibody (anti-mouseimmunoglobulin antibody)
obulin antibody (Neev Eng
land nuclear)) at 100 per well.
μl was added and cultured at 37°C for 2 hours. After washing 5 times with PBST, para-nitrophenyl pyrophosphate (para-nitrophenyl pyrophosphate)
sphate (P N P P ) as a substrate,
Color was developed by incubating for 15 minutes at 37°C, and multiscan (Titerteck Multiskan MCC,
Absorbance was measured at 405 nm using Flow Lab, ). As a result of the assay, 33 wells were positive.

Typical positive cases are listed in Table 1.

Table 1 Well 0Dsos 2 B 11 1.32 2 F 10 1.15 2 H41,31 2H91゜61 3 D 8 1.70 3 F 2 1.21 7 B 4 1.68 (5) Anti-OmpC antibody producing hybridoma Cloning of 33 hybridomas producing antibodies showing high binding ability for Om and pC were cloned into BAL B /
c Gagn in a 966 microplate using mouse thymocytes as a single feeder layer (lx106/well)
on et, al (J, ImmunoJogic
Single-cell mayupulation (single cell mayupulation) according to the method
The cells were cloned using the cell manipulation method.

(6) Culture and purification of anti-OmpC antibody-producing hybridoma After cloning, hybridoma 5 was grown in culture.
x 10′ cells were administered with pristane 2 weeks before B
ALB/c mice were inoculated intraperitoneally.

After 10 to 14 days, ascitic fluid was collected from the abdominal cavity of the mouse and
The anti-OmpC monoclonal antibody was isolated by precipitation with % saturated ammonium sulfate. 10+ 000x for 15 minutes
g Dissolve the precipitate obtained by centrifugation in a small amount of 20mM) Lis-HCl buffer (pH 7, 8) containing 40mM NaCl, and use a dialysis tube to dilute the precipitate with 20mM NaCl.
) Dialysis was performed overnight at 4°C against Lis-HCl buffer (pH 7, 8). 10. Dispense the dialysis fluid for 15 minutes. OOOxg
The centrifuged supernatant was equilibrated with DEAE-HCl buffer (pH 7, 8) containing 20 mM NaCl.
After loading onto a cellulose (DE-52, Whatman Chemical Separate Container) column chromatography,
The IgG fraction was obtained by elution by continuously increasing the NaCl concentration from 20 mM to 400 mM.

(7) Antibody titer and reaction specificity of ascites Mouse ascites of cloned hybridoma was diluted with PBS.
The mixture was diluted 103 times with T and the reactivity to OmpC and its monomer, OmpF, and BSA and the antibody titer to OmpC were measured by the ELISA method described in section (4) (Table 2).

Table 2 2B11 2.0B 0.24 0.10 0.
04 5.52F10 1.9B 0.64 0
．． 13 0.0B 6284 1.55 2.3
2 0.22 0.10 5.52119 2.
27 1,20 0.08 0.06 6.53DB
2,19 0.19 0.07 0.06
4.53F2 1,60 2.08 0.03
0.06 67E4 1.74 2.17 0,
08 0.04 60CM-7H100,202,53
-0,10-OCM-7H10 is a monoclonal antibody against OmpC monomer. OmpC, OmpF
means trimer.

None of the eight monoclonal antibodies that react with the OmpC trimer reacted with the OmpF trimer SA. 2B11.3D8 and 8H1 did not react with the OmpC monomer but reacted with the OmpC trimer.

(8) Identification of the immunoglobulin class of the antibody 75 μl of the culture supernatant of the hybridoma was applied to a monoclonal antibody typing kit (Seikagaku Corporation) based on Ophthalony's double immunodiffusion method, and the mixture was heated to room temperature (23 ± 3°C). After incubation for 24 hours, the antigen-antibody precipitate was observed and the subclass was identified.

Antiserum includes mouse immunoglobulin T1, γ21,
Sheep polyclonal antibodies against rzb, T31α and μ (Miles) were used. Table 3 shows the subclasses of representative anti-OmpC monoclonal antibodies.

Table 3 We applied for deposit of hybridomas 2B11 and 2H9 as representatives of the hybridomas of the present invention to the Institute of Microbial Technology, Agency of Industrial Science and Technology, but the deposit was rejected.

Example 2 Preparation of antigen used for production of anti-OmpC monoclonal antibody Derivative of Escherichia coli Lcoli K-12 strain SM-131 (
OmpF-deficient strain; Matsuya+ma et a
l,, J, Bact.

158, 1041-1047.1984) in LB medium supplemented with 0.2 glucose (0.2% glucose, Bacto-
tryptone 1%, yeast extrac
t 0.5%, NaCj! 0.5%, pH 7.0) and harvested at the logarithmic growth phase.

The cells were treated as in Example 1 (1) to obtain a cell wall fraction and purified OmpC.

Example 3 Application of anti-OmpC monoclonal antibody to E. coli detection (1) Preparation of E. coli E. coli K-12 derivative JEIO11 and its LPS mutant strain NS-1 (TamakieL
, al., J., Bacteriol, 105.96
8-975.1971) as Heart Infusion.
The cells were cultured with shaking at 37° C. for 6 hours in a cellulose (HIB; Tasumi Seiyaku) medium using an L-shaped tube. This preculture solution was inoculated into a new HIB medium in a Sakaro flask, and after shaking culture at 37°C for 18 hours, centrifugation (4,000xg, 15 minutes)
Bacteria were collected by doing this. Washed twice with Hank's solution pH 7°4 containing 1% BSA (no phenol red),
Then, it was suspended in the same Hank's solution and used in subsequent experiments.

(2) Escherichia coli detection method Each anti-OmpC monoclonal antibody (mouse ascites) was diluted 10 times with Hank's solution PH7.4 with 1% BSA to 0.9 d of the bacterial suspension prepared in (1).
0.1 d was added and incubated at 37°C for 11 hours.

After washing three times with the same Hank's solution by centrifugation (3,000xg, 10 minutes), it was suspended in the same Hank's solution for 0.2 d, and then I! ! ■-Protein A (Amers
ham) was added and the ink was incubated at 37°C for 1 hour. Then, after washing three times, 1' of the antigen-antibody complex was removed.
The amount of I was measured using a γ-counter (Aroki KK).

Anti-OmpC monoclonal antibody and myeloma cell P3U
Table 4 shows the binding properties of I to H. coli JEloll and its LPS mutant strain N5-1 live bacterial hedron.

Table 4 2B11 60 652F10
0.3 92H4556 2H92041 3D8 9 55 3F2 0.2 27E4
0.1 418H1345 P3U1 (negative control) 0.1 0.30
CM-7H100,22 0m C rabbit blood' 47 60 anti-Om
Among the pC monoclonal antibodies, those that showed high binding to both Escherichia coli JEIOII and N5-1 live bacteria were
2B11.2H9 was the only one. On the other hand, monoclonal antibodies 2H4, 3D8.7E4 and 8H1 are N5-
It selectively bound only to viable bacteria at a high rate.

Since N5-1 is a mutant strain of Escherichia coli JEIOII that is partially deficient in bacterial surface polysaccharides, monoclonal antibody 2B11.2H9 is less susceptible to steric hindrance due to the presence of bacterial surface polysaccharides than other monoclonal antibodies. It was suggested that this antibody is suitable for the detection of Ligram-negative bacilli. Also, OmpCJIi
Monoclonal antibodies against T-bodies (e.g. OCM-7
H10) did not bind to these viable bacteria. Therefore, the binding ability of monoclonal antibody 2B11 to live Escherichia coli JEIOII bacteria was investigated by varying the amount of test bacteria. 2
Table 5 shows the binding rate when a 10-fold dilution of mouse ascites containing B11 was used.

Table 5 6 series = ljA2%4xlO
'CFU 34 x 10'
104'xlOb
234 x 10' 27
4xlO” 53 Escherichia coli JEI
OII 10' to 10' CFU or more of viable bacteria could be detected.

(3) Binding ability with Gram-negative bacilli other than E. coli Gram-negative bacilli were cultured by the method described in Example 3-(1) to prepare a bacterial suspension containing 750 Klett units. Thereafter, the binding ability to various bacterial cells was examined by the same method as in Example 3-(2). As an anti-Ompc monotalonal antibody,
Mouse ascites 2B11 diluted 10 times was used.

The results are shown in Table 6.

Table 6 Colonic atresia JEIOII 52% Citrobacter freundii CN346 55% Enterobacter cloacae CN7471 4% Salmonella typhimurium TL-18852% Shigella flexinelle 2a TL-15384% Klebsiella pneumoniae ATCC100313% S. marcescens X100 9% Proteus mirabilis CN2 425 3% Proteus bulcaris 0X-192% Pseudomonas aeruginosa PA103 3% Staphylococcus aureus 209P 8% (When P3U1 mouse ascites was used as a negative control instead of 2B11, the binding rate was 1% or less.) Anti- OmpC monoclonal antibody 2B11 selectively bound to E. coli JE1011 and Citrobacter freundii CN346 live bacteria.

(complete)

Claims (13)

[Claims] (1) Monoclonal antibody that reacts with E. coli outer membrane protein C trimer (2) The monoclonal antibody according to claim 1, wherein the monoclonal antibody is derived from a mouse. (3) The monoclonal antibody according to claim 1, wherein the monoclonal antibody is IgG. (4) The monoclonal according to claim 1, which reacts with E. coli outer membrane protein C trimer and does not react with E. coli outer membrane protein C monomer and E. coli outer membrane protein F trimer. antibody (5) The monoclonal antibody according to claim 1, which binds to living cells of E. coli and Citrobacter freundii. (6) Hybridoma cell line 2H9 or 2B1
1. The monoclonal antibody according to claim 1 produced by (7) Hybridoma cell lines producing monoclonal antibodies that react with E. coli outer membrane protein C trimer and their descendants (8) Hybridoma cell lines 2H9 and 2B11
The hybridoma cell line defined in claim 7 and its descendants named (9) Using a monoclonal antibody that specifically recognizes E. coli outer membrane protein C trimer, Gram-negative bacilli are detected by directly binding the living cells of the bacteria without heat treatment. A method for detecting Gram-negative bacilli characterized by (10) The detection method according to claim 9, wherein the Gram-negative bacillus is Escherichia coli or Citrobacter freundii (11) A composition containing as one of its components a monoclonal antibody that specifically recognizes E. coli outer membrane protein C trimer. (12) The composition according to claim 11 for the purpose of detecting Gram-negative bacilli. (13) E. coli outer membrane protein C, characterized by culturing a hybridoma cell line or its descendants that produce monoclonal antibodies that react with E. coli outer membrane protein C trimer.
Method for producing monoclonal antibodies that react with trimers