JP2000290298A - Monoclonal antibody which reacts with adenovirus and detection of adenovirus therewith - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a monoclonal antibody which highly sensitively reacts not only with serum type adenoviruses, especially prototype adenoviruses, but also with clinically separated strains. SOLUTION: This monoclonal antibody reacts with adenoviruses of serium types 1, 3, 4, 5, 6, 7, 8, 11, 19, and 37, especially commonly exhibits high reactivity with all the adenovirus of serium types 3, 4, 8, 19, and 37, and does not react with viruses except the adenoviruses. The monoclonal antibody is preferably produced from a hybridoma which is obtained by fusing a mouse myeloma cell with the splenic cell of a mouse immunized with the mixture of the 3, 4, 8, 19 and 37 type adenoviruses.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody which reacts with high sensitivity to a wide range of serotypes of adenovirus, and which can be used for the examination and diagnosis of adenovirus. It belongs to the disease inspection and diagnosis technology.

[0002]

BACKGROUND ART Adenovirus is known as a pathogen such as acute fever pharyngitis, pharyngeal conjunctivitis, acute respiratory tract inflammation, viral pneumonia, acute follicular conjunctivitis, epidemic keratoconjunctivitis and the like. Currently, 47 serotypes are known as human adenoviruses, but in the future, clinical strains will be mutated and the number of serotypes may increase. When an adenovirus infects a human, it presents various clinical symptoms and has few specific medical conditions, so it is difficult to prove adenovirus infection from the clinical symptoms. In addition, adenovirus is highly infectious, and it is necessary to prove virus infection at an early stage in order to prevent outbreaks. At present, the detection and identification of adenovirus is performed using the isolation culture-neutralization method or PCR-RF.
Although the LP method is used, the isolation culture-neutralization method requires a long period of two weeks before determination, and the PCR-RFLP method does not prove that the pathogen retained infectivity. Each has its own problems. In addition, immunochromatography using an anti-adenovirus antibody and a method using EIA have been developed as a quick and simple method for detecting adenovirus, but the positive rate in ophthalmic areas where the amount of sample that can be collected is small is low. There is a need for a more sensitive rapid diagnostic method, which is 60% or less, or an anti-adenovirus monoclonal antibody that can be used for the method. Representative prior art relating to a monoclonal antibody against adenovirus and an adenovirus detection method using the same include those described in the following literature. <1> Application of a Solid-Phase Immunofluoromet
ric Assay to the Selection of Monoclonal Antibody
Specific for the Adenovirus Group-Reactive Hexon A
ntigen, Arch-Virol. 1984; 80, 1-10, <2> Comparison of Time-Resolved Fluoroimmunoassa
y with Monoclonal Capture-Biotinylated Detector En
zyme Immunoassay for Adenovirus Antigen Detection,
J-Clinical Microbiology, 1987 Sept, 1662-1667, <3> Characterization of adenovirus hexons by the
ir epitope composition, Arch-Virol. 1996; 141: 1891-1
907, <4> Rapid diagnosis of adenovirus pharygoconjunct
ival fever; use of a monoclonal antibody-based ELIS
A test during an outbreak, J-Virol-Methods, 1989,
Jun; 24 (3): 307-12, <5> Adenovirus Hexon Monoclonal Antibody That Is
Group Specific and Potentially Useful as a Diagnos
tic Reagent, J-Clinical Microbiology, 1983Feb,
360-364,

[0003]

At present, a large number of monoclonal antibodies against adenovirus have been produced as described in the above-mentioned prior art documents, but none of them has sufficient sensitivity for detecting adenovirus, and many serotypes of adenovirus have been produced. There is a need for monoclonal antibodies that react with high sensitivity not only to viruses, especially prototype adenoviruses, but also to clinical isolates. The present inventors have conducted research to respond to those requests.

[0004]

Means for Solving the Problems The present inventors have conducted various studies to produce monoclonal antibodies having high reactivity to a wide range of serotypes of adenovirus.
The present invention is based on the finding that monoclonal antibodies obtained by immunizing mice by mixing five types of adenovirus antigens of types 3, 4, 8, 19 and 37 react with a wide range of serotype adenoviruses. Was completed.

That is, the present invention reacts with adenovirus serotypes 1, 3, 4, 5, 6, 7, 8, 11, 19 and 37, and reacts with adenovirus types 3, 4, 8, 19 and 37. The present invention also relates to a monoclonal antibody that reacts with adenovirus, characterized in that it exhibits high reactivity but does not react with viruses other than adenovirus. , A monoclonal antibody produced by a hybridoma obtained by fusing mouse spleen cells and mouse myeloma cells immunized with the mixture of types 19 and 37, wherein the monoclonal antibody is adenovirus 3, 4 , 8, 19 and 3
The present invention relates to a monoclonal antibody characterized by having an absorbance OD _{490 nm} of 0.40 or more when the reactivity to type 7 is measured by ELISA, and a method for detecting adenovirus using the monoclonal antibody. .

[0006]

The present invention will be described in detail below with reference to examples.の Preparation of anti-adenovirus monoclonal antibody (1) Animal immunization and preparation of antibody-producing cells Adenovirus clinical strain, Ad3x, Ad4x, Ad8x, Ad19x, Ad3
Each of 5 types of 7x was cultured using HEp-2 cells, and those having cytopathic effect (CPE) +++ were cultured at 60 ° C for 2 hours.
The cells inactivated by the time treatment are used as immunizing antigens (hereinafter, inactivated antigens) and intraperitoneally administered to 3- to 10-week-old BALB / C mice (♀) together with Freund's complete adjuvant. Thereafter, intraperitoneal administration of the inactivated antigen together with incomplete Freund's adjuvant is repeated 2 to 5 times every two weeks. When performing cell fusion, the immunized mouse was subjected to fusion
Four days before, the inactivated antigen is intraperitoneally administered for final immunization. On the day of cell fusion, the spleen is removed and spleen cells are prepared. In other words, spleen is Iscove's Modified Dulbecco's Medium
(IMDM), shred in mesh,
After centrifugation at 000 rpm for 5 minutes, the supernatant is discarded, and washing with IMDM is repeated three times to provide splenocytes for fusion. (2) Preparation of myeloma cells A mouse-derived NS-1 (ATCC name; P3 / NS1 / 1 / Ag4-1) cell line was cultured in a normal medium to obtain 2 × 10 ⁷ or more cells upon cell fusion. The parent strain was used for fusion. (3) Preparation of hybridoma The spleen cells and myeloma cells obtained in (1) and (2) were
Mix so as to have a ratio of 5 to 10: 1.
After centrifugation for 5 minutes, discard the supernatant, disintegrate the precipitated cell group well, add 1 ml of 50% polyethylene glycol (PEG 1000-5000) at 37 ° C. with stirring for 1 minute, and further add 1-2 ml of IMDM. Add several times for a total volume of 20ml
Add until After centrifugation at 1000 rpm x 5 minutes,
Discard the supernatant and prepare the BALB prepared as feeder cells.
/ c mouse thymocytes (1 × 10 ⁸ ) were added and loosened gently, and HAT (sodium hypoxanthine, aminopterin, thymidine) medium /
200 ml of [IMDM, fetal calf serum (FCS) 20%] is added, and the cells are gently suspended by suction and blowing out with a female pipette. This suspension is dispensed 100 μl at a time into a 96-well culture microplate and cultured at 37 ° C. for 24 hours in a 5% CO ₂ incubator. Thereafter, when wells with a yellowish color appear in the culture solution, approximately every two days,
Discard 100 μl of culture supernatant and add 100 μl of fresh HAT medium. While repeating this operation for 10 to 14 days, 5
Incubate at 37 ° C. in a% CO ₂ incubator. During the medium exchange during this time, the resulting culture supernatant is shown below.
Subjected to ELISA, adenovirus 3, 4, 8, 19 and 3
Wells producing antibodies reactive with any of the seven types are selected.

{Circle around (7)} Screening method Inactivated and purified virus of Ad3x, Ad4x, Ad8x, Ad19x, Ad37x
A 2 μg / ml / PBS mixed solution was dispensed into a 96-well microtiter plate at 50 μl / well and left standing at room temperature for 1 hour or more or at 4 ° C. overnight for sensitization.
Wash three times with BS and block with 1% BSA / PBS. To this antigen-sensitized plate, 50 μl / well of a sample solution suspected of containing an antibody is dispensed, and reacted at room temperature for 1 hour or more or at 4 ° C. overnight. After the reaction, PB containing 0.05% Tween20
Washed three times with S and peroxidase-labeled anti-mouse IgG
Diluted with PBS to the appropriate concentration, dispensed 50 μl / well,
Incubate for 1 hour at room temperature, and again contain PBS containing 0.05% Tween20.
Wash 3 times. Orthophenylenediamine, a substrate of peroxidase, was dispensed at 100 μl / well for 5 minutes to 3 minutes.
After the color was developed for 0 minutes, the reaction was stopped by adding 100 μl / well of 2N-H ₂ SO ₄ , and the OD value at 490 nm was measured with a spectrophotometer. . EL
Positive in ISA, ie adenovirus 3x, 4
x, 8x, 19x, is a well producing antibodies that react with any of the 37x type, and, for the wells where hybridomas have been grown in colonies, cloned by limiting dilution, stably Adenovirus 3x,
Those with strong antibody titers for all 4x, 8x, 19x, and 37x types were selected as monoclonal antibodies reactive to a wide range of serotypes of adenoivirus. In the case of the selected medium, the medium is gradually converted to an HT (hypoxanthine sodium, thymidine) medium, and then to a normal medium. In this way, three (C9, C14, C27)
One kind of monoclonal antibody (C41) was obtained from the cell fusion of three kinds of monoclonal antibodies and three spleens. The obtained monoclonal antibody was deposited with the National Institute of Bioscience and Biotechnology,
9: FERM P-17354, C14: FERM P-
17355, C27: FERM P-17356, C41:
Deposited as FERM P-17357.

(1) Mass production of monoclonal antibody pristane (2,6,10,14-tetramethylpentadecane)
5 to 8 weeks old mice (BALB / c, ♀) which were intraperitoneally administered with 5 ml and bred for 2 to 4 weeks, the anti-adenovirus monoclonal antibody-producing hybridoma cells obtained in (3) were obtained for 2 to 1 times.
0 × 10 ⁶ cells / animal are administered intraperitoneally. Hybridomas develop ascites tumors in 10 to 21 days, so ascites is collected from the mice, centrifuged (3000 rpm × 5 minutes) to remove solids, and the supernatant is passed through a Protein A column. The IgG fraction was collected and used as a purified monoclonal antibody IgG fraction, and the IgG concentration was determined by measuring absorbance at 280 nm.

(4) Reactivity of C9, C14, C27 and C41 The reactivity of C9, C14, C27 and C41 thus obtained to adenovirus was examined by the following two methods. Method 1 ELISA method 1 Six types of adenovirus antigens (Ad3x, Ad4x, Ad8x, Ad19,
An operation of binding a clinical isolate of Ad37x and a prototype Ad37p) to a 96-well microtiter plate was performed. All the adenovirus antigens used here were purified by cesium centrifugation and obtained from the Ophthalmology Department of the Yokohama City University School of Medicine. All of them had been inactivated by heating at 60 ° C. for 2 hours, and the hexon protein concentration was determined by the BCA method. The names of each clinical isolate are as follows. Ad3x = TC21309, Ad4x = Sapporo 95-0187, Ad8x = 19751, Ad19x
= V92-8266, Ad37x = TC-20850-1. Each of these adenoviruses was added in PBS at 2 μg / ml.
And dispensed into a 96-well microtiter plate (manufactured by PVC, Sumitomo Bakelite, MS7196F) at 50 μl / well. After sensitizing at room temperature (22 ° C to 26 ° C) for 1 hour,
Wash three times with PBS containing 0.05% Tween 20, and wash with 1% BSA / P
Block with BS at 37 ° C for 4 hours. Each antibody of C9, C14, C27 and C41 was added to this antigen-sensitized plate with
50 μl / well of a solution adjusted to a concentration of 0.1 μg / ml is dispensed, and reacted at room temperature (22 ° C. to 26 ° C.) for 1 hour. After the reaction,
The plate was washed three times with PBS containing 05% Tween 20 and peroxidase-labeled anti-mouse IgG ((H + L) (MB330 CODE330) was added to PB.
Dilute 2000 times with S, dispense 50 μl / well,
(22 ° C. to 26 ° C.) The reaction is carried out for 1 hour, followed by washing three times again with PBS containing 0.05% Tween 20. Tablets of orthophenylenediamine (opd), a substrate for peroxidase (opd 15 mg, Na ₂
(Consisting of 91.3 mg of SO _2, 6.8 mg of citric acid, 16.9 mg of Na ₂ CO ₃ )
Was dissolved in 12 ml of 2M-Tris-citrate buffer having a pH of 5.2 containing 0.015% H ₂ O ₂ , dispensed at 100 μl / well, and allowed to develop color at room temperature (22 ° C. to 26 ° C.) for 30 minutes. 2N-H ₂
The reaction was stopped by adding 100 μl / well of SO _{4, and} 490 nm-65 using a plate reader (Molecular Devices Vmax).
The OD value at 0 nm was measured. As a control of anti-adenovirus monoclonal antibody, <Chemicon MAB8052
> Was used. MAB8052 is a monoclonal antibody produced by a hybridoma obtained by immunization with Ad3 type,
Reacts with adenovirus of the serotype of the species. The clone name is
<20/11>. (References: <1>, <2> among descriptions in the prior art) Regarding the obtained antibodies, three tests were performed for each, and the average value and standard deviation were calculated. Tables 1 to 4 show the obtained results. The resulting values are for OD 490-650 nm. In addition,
The background value when only PBS was reacted with the antigen-binding plate instead of the antibody was less than 10 mOD and was ignored.

[0010]

[Table 1]

[0011]

[Table 2]

[0012]

[Table 3]

[0013]

[Table 4]

From the above results, all of the obtained monoclonal antibodies C9, C14, C27, and C41 have the same or higher reactivity to adenovirus as MAB8052, and under the measurement conditions carried out by the present inventors, Ad37x It was found that the MAB8052 had an OD value of less than 0.3 for each type, whereas the OD value of each antibody was 0.4 or more. Also, Ad37
For the p-type, MAB8052 has an OD value of less than 0.4, whereas for all antibodies, the OD value is 0.6 or more. For the Ad4x type, the OD value of MAB8052 is less than 0.45. In contrast, all antibodies have an OD value of 0.5 or more, and for the Ad8x type, MAB8052 has an OD value of less than 0.5, whereas all antibodies have an OD value of 0.7 or more. Value, MAB for Ad19x type
All antibodies showed an OD value of 0.9 or more, while 8052 had an OD value of less than 0.8. Therefore, C9, C14, C
All four types of acquired antibodies, 27 and C41, show higher reactivity against adenovirus than MAB8052, and have the lowest OD value.
It became clear that the absorbance of Ad40x was 0.40 OD or more. These OD values were measured under the above measurement conditions. Essentially, 50 μl of the antibody solution was reacted with the antigen-binding plate, and the color was developed with peroxidase-labeled anti-mouse IgG and its substrate, orthophenylenediamine. Can be said to have been measured by the ELISA method.

Method 2 Fluorescent Antibody Method The following experiment was performed to confirm the reactivity with non-inactivated adenovirus. P for C9, C14, C27, C41
After dilution with BS, 10 adenovirus serotypes (1p, 3
Each of the antigens p, 4p, 5p, 6p, 7p, 8p, 11p, 19p, and 37p) is spread on a slide glass, and incubated at 37 ° C for 60 minutes in a humid box. FITC-labeled anti-mouse IgG was added, incubated at 37 ° C. for 60 minutes, washed, and then inspected with a fluorescence microscope. Those with fluorescence are considered positive. Each antibody was diluted with PBS to a final concentration below which Ad3p clearly shows fluorescence, and reacted with 10 adenovirus serotypes (C9: 12.0 μg / ml, C1).
4: 5.3 μg / ml, C27: 5.3 μg / ml, C41: 12.2 μg / ml). As a result, in any of the antibodies of the above concentrations C9, C14, C27 and C41, Ad1p, 3p, 4p, 5p, 6p, 7p, 8p, 11p, 1p, 1p
Reactivity was observed with all 10 antigens of 9p and 37p.
Further, in order to confirm whether C9, C14, C27, and C41 cross-react with viruses and bacteria other than adenovirus, reactions with the viruses and bacteria shown in Table 5 were performed using the above-described fluorescent antibody method. . When the antibody titer was less than 1, that is, when no reactivity was observed at the following antibody stock concentrations, it was considered negative [C9 (770 μg / ml) C14 (680 μg / ml) C27 (6
70 μg / ml) C41 (780 μg / ml)].

[0016]

[Table 5]

From these results, it was confirmed that C9, C14, C27, and C41 had no reactivity with the above viruses and bacteria other than adenovirus. From the above results, the monoclonal antibodies C9, C14, C27, and C41 produced by the obtained hybridomas all reacted with all the adenoviruses used in Examples and reacted with viruses and bacteria other than adenoviruses. Did not.

[0018]

The monoclonal antibodies of the present invention, specifically, the monoclonal antibodies designated as C9, C14, C27 and C41 are all monoclonal antibodies having specific reactivity to adenovirus, and are commercially available adenovirus. Reactivity with adenovirus is higher than that of monoclonal antibody MAB8052.
It shows an absorbance higher than OD _{490 nm} . Further, the monoclonal antibody of the present invention is a method for detecting a wide range of serotypes of adenovirus using the monoclonal antibody (EIA method,
RIA method, fluorescent antibody method, immunochromatography method, immunoelectrophoresis method, and other modifications and applications thereof), and high sensitivity can be obtained.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Norihiko Ito 21-17 Nagatasannodai, Minami-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Shigeaki Ono 141-10 Tomiokanishi, Kanazawa-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Aoki Kouki 4F0Term 4B064 AG27 CA10 CA20 CC24 CE12 DA15 4B065 AA90X AA95Y AB04 BA08 BD15 CA25 CA46 4H045 AA11 AA30 DA76 EA53 FA72 GA15 GA26

Claims (4)

[Claims] 1. Adenovirus serotypes 1, 3, 4, 5,
Reacts with types 6, 7, 8, 11, 19 and 37 and shows high reactivity with adenovirus types 3, 4, 8, 19 and 37, but does not react with viruses other than adenovirus. A monoclonal antibody that reacts with adenovirus. 2. Adenovirus 3, 4, 8, 19 and 3
The adenovirus-responsive monoclonal antibody according to claim 1, wherein the monoclonal antibody is produced by a hybridoma obtained by fusing mouse spleen cells and mouse myeloma cells immunized with the type 7 mixture. 3. Adenovirus 3, 4, 8, 19 and 3
The monoclonal antibody reactive with adenovirus according to claim 1, wherein the absorbance OD _490nm when the reactivity to type 7 is measured by ELISA is 0.40 or more. 4. A method for detecting an adenovirus, comprising using the monoclonal antibody reactive to the adenovirus according to claim 1.