JPH0315396A - Monoclonal antibody against pathogenic virus of fishes and its production - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody having specificity for pathogenic viruses of fishes by fusing an immunized cell of a mammal immunized with a specific pathogenic viral antigen of fishes to a myelomatous cell and culturing the resultant hybridoma. CONSTITUTION:An immunized cell of a mammal is obtained by using one kind of pathogenic viral antigen for fishes selected from the group of infectious hematopoietic organ necrotic viruses(IHNV), infectious pancreatic necrotic viruses(IPNV) and halibut Rhabdoviruses(HRV). The resultant immunized cell is then fused to a myelomatous cell to provide a hybridoma, which is subsequently cultured. As a result, one kind of monoclonal antibody selected from monoclonal antibodies IgG and IgM against the infectious hematopoietic organ necrotic viruses, monoclonal antibodies IgG and IgM against the infectious pancreatic necrotic viruses and monoclonal antibodies IgG and IgM against the halibut Rhabdoviruses is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a monoclonal antibody against a fish pathogenic virus and a method for producing the same.

[Problems to be solved by conventional technology and inventions] From the perspective of securing animal protein resources, fish and shellfish farming has become popular all over the world in recent years, and in Japan, the fish farming business is rapidly increasing year by year. It is developing into

However, although managing healthy fish and preventing infectious diseases are unresolved issues in fish farming operations, in practice it is difficult to manage the health of fish. Among various fish epidemics, viral infections in particular spread extremely quickly compared to other bacterial epidemics, and there are no effective treatments, so once a viral disease occurs,
Often suffers great damage. Therefore, it is strongly desired to establish an effective epidemic prevention method for viral diseases.

Given this current situation, temperature control and chemotherapy have been used as epidemic prevention methods for viral infections. Preventive immunization method. Methods for sterilizing eggs have been studied. However, with the exception of egg sterilization, no epidemic prevention effects have been reported, and the reality is that there is no practical epidemic prevention method. Particularly in the case of viral diseases, early detection of the pathogen before the outbreak of an epidemic is the top priority. Currently, the detection and diagnosis of fish virus diseases involves examination of the appearance of diseased fish, virus isolation, physical and chemical properties of the isolated virus, antiviral serum neutralization test, etc., but these tests require a long time and are not simple. There is a need for the development of methods for diagnosing and treating viral diseases.

[Means to solve the problem]

As a result of repeated investigations into the use of monoclonal antibodies as diagnostic and therapeutic agents for fish viral diseases, the present inventors discovered that a specific monoclonal antibody was suitable for this purpose. The present invention was completed by establishing a method for producing the monoclonal antibody using a hybridoma obtained by fusing mouse lymphocytes with mouse myeloma cells. That is, the present invention provides the following (7), ({) and (
(c) Monoclonal antibodies with specificity for one fish pathogenic virus selected from the group (7) Monoclonal antibodies against infectious hematopoietic necrosis virus (IHNV) IgG and IgM (b) Infectious pancreatic necrosis virus ( monoclonal antibodies IgG and IgM against Hiramapudovirus (IPNV); (7) above, which corresponds to the above antigen by culturing a hybridoma obtained by fusing myeloma cells with the immune cells of a mammal immunized with one selected fish pathogenic virus antigen.
A monoclonal antibody (hereinafter sometimes abbreviated as fish pathogenic virus antibody or simply monoclonal antibody) having specificity for one type of fish pathogenic virus selected from the group of , C4) and (c) is produced, and the monoclonal antibody is The present invention provides a method for producing a monoclonal antibody, which comprises collecting a monoclonal antibody.

Next, the method for producing the fish pathogenic virus antibody of the present invention will be described in detail below.

The fish pathogenic virus antibody of the present invention is obtained from immune cells of a mammal that has been immunized with one fish pathogenic virus selected from the group of infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, and hiramerapdovirus as an immunizing antigen. This can be obtained by culturing a hybridoma obtained by fusion of a myeloma cell and a myeloma cell to produce antibodies to the fish pathogenic virus, and collecting the antibodies to the fish pathogenic virus. The above-mentioned mammalian immune cells are prepared by a conventional method using one type of fish pathogenic virus selected from the group of infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, and hiramelapdovirus as an immunizing antigen. Prepared by immunizing mammals. Here, the fish pathogenic virus used as the immunizing antigen can be one isolated by a conventional method from a culture solution of cells positive for the fish pathogenic virus. Isolation of the above-mentioned fish pathogenic virus is carried out by a conventional centrifugation method, and this is further carried out by a conventional centrifugation method. It is preferable to purify according to the density gradient ultracentrifugal partition method etc. In addition, the mammal to be immunized with the fish pathogenic virus is not particularly limited, but is preferably selected in consideration of compatibility with the myeloma cells used for cell fusion.
Generally, mice, rats, etc. are used. The immunization method is also carried out by a general method, for example, the fish pathogenic virus is diluted to an appropriate concentration with a normal buffer solution, made into a suspension with Freund's auxiliary solution, etc., and administered to the animal by subcutaneous injection. A booster immunization is performed 2 to 5 weeks after the initial immunization, and the total dose is about 120 μg protein amount of fish pathogen virus obtained from 5001 dl of culture fluid of fish pathogen virus positive cells cultured at 2 to 3 x 10 cells/mouse. It is preferable to make it so that As the immune cells, it is preferable to use lymphocytes extracted from splenic cells extracted about 3 days after the final immunization. In addition, various known cell lines, such as P3 (P3/X63-Ag8 )
[Nature, i-mail, 495-497 (1975)]
, P3-LI1[Current↑opics i
n Microbiology and Im+
*unology+81,1~ma(197B)], N
S-1 [Eur. J. Is+munol. ．． 6, 5
11-519 (1976)], MPC-11 (Call
．． 8, 405”415 (1976)], SP2/0
[Nature, 276 269-270 (197
8)], FO [J. Immunol. Meth. ．． 3
5.1-21 (1980)], X63.6.55.3.
[J. Immuno1. 123 1548-15
50 (1979)], S194 [J. Exp. Med
．． , 148 313-323 (1978)] and R210 in rats [Nature. 277131
~133 (1979), etc. The above-mentioned fusion reaction between immune cells and myeloma cells can be carried out using basically known methods such as Oi and Herzenberg.
Selected Methods g)
Cejlular Ia+muno1ogy+35
1-371, W. I{. Freeman & G.
o. , TLSA Publishing (1980)]. More specifically, the fusion reaction is carried out in a conventional nutrient medium, for example in the presence of a fusion promoter. Here, as the fusion promoter, commonly used ones such as polyethylene glycol (PEG), Sendai virus (HVJ), etc. are used, and if desired, auxiliary agents such as dimethyl sulfoxide are added to increase the fusion efficiency. You can also.

The ratio of immune cells to myeloma cells used is the same as in normal methods, for example, the ratio of immune cells to myeloma cells may be about 1 to 10 times. As the medium for the above-mentioned fusion, for example, RPM as used for the proliferation of the above-mentioned osteoma cell line.
I-1640 medium, HEM medium. Various other usual media used for this type of cell culture can be used, and it is usually advisable to exclude serum supplements such as fetal calf serum (FCS). Fusion is carried out by thoroughly mixing a predetermined amount of the above immune cells and myeloma cells in the above medium, and adding PE warmed to about 37°C in advance.
This is done by adding G solution, for example, one with an average molecular weight of about 1000 to 6000, to a normal medium at a concentration of about 30 to 60 w/v2 and mixing. Thereafter, the desired fused cells (hybridoma) are formed by repeating the operations of sequentially adding an appropriate medium, centrifuging, and removing the supernatant.

The resulting desired hybridomas are isolated by culturing them in a conventional selection medium, such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Culture in the HAT medium may be carried out for a sufficient period of time, usually from several days to several weeks, to kill cells other than the target hybridoma (unfused cells, etc.). The hybridoma thus obtained is searched for a strain producing the antibody of interest and single cloned according to the usual limiting dilution method. The above-mentioned search for the desired hybridoma can be performed, for example, by indirect immunofluorescence. Enzyme antibody assay (EIA), neutralization reaction method.
Sedimentation reaction method. Complement fixation reaction method, agglutination reaction method. It is carried out by various methods generally used for antibody detection, such as the Ouchterlony method and the RIA method ["Hybridoma method and monoclonal antibody Jθ Shu R&D Planning, published, pp. 30-53, March 5, 1980]".

The hybridoma producing the monoclonal antibody of the present invention obtained as described above can be subcultured in a conventional medium and can be easily stored for a long period of time in liquid nitrogen. All hybridomas shown in the examples below are maintained in a state where they can be distributed to the Department of Cell Control Engineering, Faculty of Agriculture, Kyushu University.

The monoclonal antibody of the present invention can be produced from the specific hybridoma described above by culturing the hybridoma according to a conventional method and separating the desired antibody from the culture supernatant. A method is adopted in which the antibody is administered to a certain type of chewing milk, allowed to grow, and the desired antibody is isolated from the ascites. The former method is successful in obtaining high purity, and the latter method is suitable for mass production. For the former method, for example, E-RDF medium supplemented with 10% fetal bovine serum (Hironori Murakami et al., Nippon No Kagaku Shi. 58, 57) is used at a concentration of 2 x 10' cells/1.
Monoclonal antibodies having specificity for the target fish pathogenic virus can be produced by suspending the above hybridoma in a medium such as 5 (1984) ") and culturing it in a 5% C08 incubator at 35-38°C. The present invention will now be explained in more detail with reference to Examples.
The present invention is not limited to this. Reference Example 1 Preparation of monoclonal antibody-producing hybridoma against infectious hematopoietic necrosis virus (IHNV) IHNV purified by the 11 density gradient ultracentrifugation method was given to 5-week-old male BALB/C mice for protein concentration. 40
, c/g in Freund's complete adjuvant (Difco
(manufactured by the company) and was inoculated intraperitoneally. Thereafter, the mice were immunized with the same amount of virus every two weeks, and the mice were dissected when the anti-IHNV blood antibody titer had risen sufficiently. 2X10'' lymphocytes aseptically removed from the spleen and 2XIO' mouse myeloma cells P3-X63- in logarithmic growth phase.
Ag. 8. U1 (hereinafter referred to as P3-U1, purchased from Dainippon Pharmaceutical), and polyethylene glycol
Molecular weight 1000. (manufactured by Wako Pure Chemical Industries, Ltd.). Next, 15% fetal calf serum (hereinafter referred to as FCS) was added to the fused cells. -RDF medium (Kyokuto Seiyaku ■), 1
B2ng/d aminopterin. 13.6μg/xi!
The cells were cultured for 14 days at 37°C in a 5% C08 incubator using a medium supplemented with hypoxanthine and 3.9 μg/yd thidine (hereinafter referred to as HAT medium). Take 50 ul of culture supernatant of hybridomas grown in HAT medium and add purified IHNV (500 n
g protein/di) was dispensed onto a 966 microplate coated with 966 microplates, and hybridomas producing anti-IHNV-specific monoclonal antibodies were screened by ELISA. The obtained positive hybridoma was cloned twice by the limiting dilution method.

Reference Example 2 Production of hybridoma producing monoclonal antibodies against infectious pancreatic necrosis virus (IPNV)! IPNV purified by It1 density gradient ultracentrifugation was administered to 5-week-old male BALB/C mice at a protein level of 40 u.
g with Freund's complete adjuvant (manufactured by Difco)
It was inoculated intraperitoneally. Thereafter, the mice were immunized with the same amount of virus every two weeks, and the mice were dissected when the anti-IPNV blood antibody titer had increased sufficiently. 2XlO' lymphocytes aseptically removed from the spleen and 2X in logarithmic growth phase.
10' mouse myeloma cells P3-11 were mixed and fused using polyethylene glycol (molecule i1100G, manufactured by Wako Pure Chemical Industries, Ltd.). Next, the fused cells were transferred to 15% FCS-supplemented E-RDF medium (
Using HAT medium (manufactured by Kyokuto Seiyaku ■) at 37°C and 5% C
HAT cultured for 14 days in an Ot incubator.
The culture supernatant of hybridomas grown in the medium was
ux was taken and dispensed into a 966 microplate coated with purified IPNV (500 ng protein/d), and hybridomas producing anti-IPNV-specific monoclonal antibodies were screened by ELISA. The obtained positive hybridoma was cloned twice by the limiting dilution method.

Reference Example 3 Production of a hybridoma producing a monoclonal antibody against the pathogenic rhapdovirus (HRV) of flounder! II1! HRV purified by density gradient ultracentrifugation was administered to 5-week-old male BALB/C mice at a protein level of 40.
tlg was inoculated intraperitoneally with Freund's complete adjuvant (manufactured by Difco). Thereafter, the mice were immunized with the same amount of virus every two weeks, and the mice were dissected when the anti-10RV blood antibody titer had risen sufficiently. 2XIO” lymphocytes aseptically removed from the spleen and 2 in logarithmic growth phase.
X10' mouse myeloma cells P3-111 were mixed and fused using polyethylene glycol (molecular weight 1000, manufactured by Wako Pure Chemical Industries, Ltd.). Next, the fused cells were transferred to 15% FCS-supplemented E-RDP medium (
(manufactured by Kyokuto Seiyaku) using HAT medium at 37°C for 5 minutes.
% CO in an incubator for 14 days. H.A.
The culture supernatant of hybridoma grown in T medium was
Aliquots of 0 al were taken and dispensed into 966 microplates coated with purified IIRV (500 ng protein/M1), and hybridomas producing anti-+1 RV-specific monoclonal antibodies were screened by the FiLISA method. The positive hybridomas obtained were cloned twice by limiting dilution. Example 1 Anti-IHNV monoclonal antibody (specificity test by LISA method) Anti-111NV monoclonal antibody-producing hybridoma HG-59-9.HM-2.1 in E-RDF medium supplemented with 10% fetal bovine serum
1M-3 and HM-11 were added to 2XlOS cells/
The cells were suspended at 37°C and cultured in a 5% CO2 incubator, followed by centrifugation to collect the culture supernatant. Purified IHNV. IPNV and HRV antigens were sequentially lh from 5ttg protein/1 to 0.04 μg protein/d.
It was serially diluted and coated on a 966 microplate (manufactured by Nunc), and its specificity was examined by HLISA. In addition, the monoclonal antibody Ig produced by HG-59-9
For the G specificity test, 25 μg protein/1 viral antigen was diluted in 8 serial dilutions. As a result, HG-
The monoclonal antibody IgG produced by 59-9 (shown in Figure 1) has a concentration of 25 μg protein/II! IH with a concentration of l
It clearly reacted not only with NV antigen but also at a concentration of 0.39 μg protein/1, and with other viral antigens IPNV and I.
It did not react with IRV antigen. In addition, IgM-producing hybridoma IIM-2 (shown in Figure 2), H
The monoclonal antibodies produced by M-3 (shown in Figure 3) and Ii-11 (shown in Figure 4) reacted to the same extent not only with IHNV antigen but also with HRV antigen belonging to the same rhabdovirus. This means that the monoclonal antibodies produced by these hybridomas can be used against not only IHNV but also H
This is thought to be because it recognizes the same antigenic determinant that IIV also has, and provides important knowledge not only in the detection of viral antigens but also in the classification of fish viruses.

(b) Monoclonal antibody subclass}IG-59-
9. HM-2, }IM-3. The subclass of the monoclonal antibody produced by HM-11 was determined by the BLISA method using a typing kit (manufactured by Tago). As a result, the subclass of IgG produced by HG-59-9 was IgGl, and the light chain was κ chain. Also, H.M.
-2, HM-3. All of the 15M light chains produced by HM-11 were κ chains. (C) Viral proteins recognized by monoclonal antibodies. The purified virus was mixed with a 4% acrylamide concentration gel.
sDs~polyacrylamide ξ gel electrophoresis was performed using a 2% acrylamide ξ-ad separation gel. Electrophoresis is performed using Le
ong et al. (Developoq. Biol. Stand
ard. ．． 49+43 (1981)) (7) method, 25mA 1? It lasted about 40 minutes. After electrophoresis, the viral proteins on the gel were analyzed by Tosbin et al. (Proc.
Natl. Acad. Sci. U. S. A. ．．
16+ 4350 (1979)), and the nitrocellulose was blocked in PBS supplemented with 0.2% gelatin and reacted with a monoclonal antibody. Protein A-Gold and Sensitizer (Yiyun Prot Assay Kit, Bio
-Rad) to detect the virus protein recognized by the monoclonal antibody. , In addition, Pre-stained SOS-PA was used as a molecular weight marker.
GE standards (manufactured by Bio-Rad) were used. As a result, the monoclonal antibodies produced by HG-59-9 and HM-2 both recognized the N protein among the five proteins (L, G, N.M1.l'l2) of IHNV (shown in Figure 5). ), but the monoclonal antibody produced by HM-2 is related to HRV, which also belongs to the rhabdovirus.
(a)
This confirmed the results obtained using the ELISA method.

Example 2 Anti-IPNV monoclonal antibody (a) Specificity test by ELISA method Anti-IP
NV monoclonal antibody producing hybridoma 4PG-4
and 4PM-7 were recovered by the method described in Example 1, and purified 111NV, IPN was diluted in 18 steps to the concentrations mentioned above.
When reacting with V and HRV and measuring its specificity by ELISA, it was found that IgG-producing hybridoma 4PG-
The monoclonal antibody produced by No. 4 (shown in FIG. 7) reacted only with the IPNV antigen and did not react with the other two virus IHNV and HRV antigens, confirming its specificity.

In addition, the monoclonal antibodies produced by IgM-producing hybridoma 4PM-7 (shown in Figure 8) were similarly found to have strong specificity, confirming that these monoclonal antibodies can be used to detect IPNV, which belongs to Birnavirus. It was done. (b) Monoclonal antibody subclass 4PG-4
The subclass of monoclonal antibodies produced by 4PG-7 was investigated in the same manner as in (b) of Example 1, and the subclass of IgG produced by 4PG-4 was IgG2.
b, and the light chains of 4PG-4 and 4PG-7 are both κ
It was a chain. Example 3 Anti-HRV monoclonal antibody (a) [! Specificity test using LISA method Anti-HRV monoclonal antibody producing hybridoma +1RG-25^
, HRG-128, HRG-188, HRM-3
A and HIIM-4A were cultured by the method described in Example 1, and the monoclonal antibodies produced by them were purified I.
HNV, IPNV. It was reacted with HRV antigen by the method of Example 1, and its specificity was investigated. As a result, IgG-producing hybridoma I {RG-25A
(The monoclonal antibodies produced by aL IIRG-12B (shown in Figure 10) and IIRG-18B (shown in Figure 11) shown in Figure 9 react only with HRV antigens, confirming their specificity. On the other hand, monoclonal antibodies produced by IgM-producing hybridomas IIRM-3A (shown in Figure 12) and HRM-4A (shown in Figure 13) also react specifically with HRV antigens, and the host We found that the present invention could also be useful for virological research between the same rhabdoviruses, HRV isolated from different flounder diseased fish and the salmonid fish pathogenic virus IHNV. (B) Monoclonal antibody class and subclass} 112G-25A. HRG-128 and III
The subclass of the monoclonal antibody IgG produced by G-18B was investigated in the same manner as in Example 1 (b), and all were found to be IgG1. Furthermore, all of the light chains of 1 gM of the monoclonal antibodies produced by these antibodies and HRM-3^ and HRM-4A were κ chains.

(C) Viral proteins recognized by monoclonal antibodies When the viral proteins recognized by the monoclonal antibodies produced by HRG-25A and HRM-4A were investigated in the same manner as in (C) of Example 1, HRG-25^ and Both monoclonal antibodies produced by IIRM-4A recognized the intermediate protein among the five proteins of HRV (shown in Figure 14). [Effects of the Invention] The fish pathogenic virus monoclonal antibody of the present invention is effective against infectious hematopoietic necrosis virus (I), which mainly infects salmonids.
It reacts only with HNV), infectious pancreatic necrosis virus (IPNV), and hirame rhabdovirus (HRV).
These antibodies are specific antibodies that do not react with cells that host these viruses. Only 500ng of these antibodies
When reacted with protein/d virus specimen, ELIS
It can be easily detected by method A.

Therefore, it is promising as a future fish virus diagnostic agent to replace the identification of fish pathogenic viruses and the diagnosis of viral diseases, which have so far relied on antiserum polyclonal antibodies.

Furthermore, by combining these monoclonal antibodies with antiviral agents, it can be used to treat viral diseases that have been considered difficult to use with straw cell therapy, and can be developed as a therapeutic agent. Furthermore, this time, the virus-specific monoclonal antibodies are not only IgG (IgM has also been produced, so monoclonal antibodies that recognize different antigenic determinants can be used to bind and react with virus antigens using the sandwich method, which was not possible until now). It has become possible to detect viruses with higher sensitivity than the ELISA method.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the specificity of monoclonal antibody tgc produced by hybridoma HG-59-9 to IIINV antigen. FIG. 2 is a diagram showing the specificity of monoclonal antibody 1gH produced by hybridoma IIM-2 to IHNV antigen. FIG. 3 is a diagram showing the specificity of 1 gM of the monoclonal antibody produced by hybridoma II-3 to the IHNV antigen. FIG. 4 is a diagram showing the specificity of monoclonal antibody 1gH produced by hybridoma HM-11 to IHNV antigen. Figure 5 shows hybridomas HG~59-9 and IIM-
FIG. 2 is a diagram showing the IHNV proteins recognized by the monoclonal antibody produced by No. 2. FIG. 6 is a diagram showing the HRV protein recognized by the monoclonal antibody produced by hybridoma HH-2. FIG. 7 is a diagram showing the specificity of monoclonal antibody IgG produced by hybridoma 4PG-4 to IPNV antigen. FIG. 8 is a diagram showing the specificity of monoclonal antibody 1gH produced by hybridoma 4PM-7 to IPNV antigen. FIG. 9 is a diagram showing the specificity of monoclonal antibody IgG produced by hybridoma HRG-25A to HRV antigen. FIG. 10 is a diagram showing the specificity of monoclonal antibody IgG produced by hybridoma HRG-12B to HRV antigen. FIG. 11 is a diagram showing the specificity of monoclonal antibody IgG produced by hybridoma HRG-188 for HRV antigen. FIG. 12 is a diagram showing the specificity of 1 gM of the monoclonal antibody produced by hybridoma HRM-3A to the HRV antigen. FIG. 13 is a diagram showing the specificity of 1 gM of the monoclonal antibody produced by hybridoma IIRM-4A to the NRV antigen. Figure 14 shows hybridomas HRG-25A and IIR.
Recognition by the monoclonal antibody produced by M-4A
FIG. 2 is a diagram showing the protein of RV.

Claims (1)

[Claims] (1) A monoclonal antibody having specificity for one kind of fish pathogenic virus selected from the following groups (a), (b) and (c). (a) Monoclonal antibodies IgG and IgM against infectious hematopoietic necrosis virus (IHNV) (b) Monoclonal antibodies IgG and IgM against infectious pancreatic necrosis virus (IPNV) (c) Monoclonal antibodies IgG and IgM against hirame rhabdovirus (HRV) and IgM (2) fusion of myeloma cells with mammalian immune cells immunized with one fish pathogenic virus antigen selected from the group of infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, and flounder rhabdovirus. The hybridoma obtained by A method for producing a monoclonal antibody, which comprises collecting the antibody. (a) Monoclonal antibodies IgG and IgM against infectious hematopoietic necrosis virus (IHNV) (b) Monoclonal antibodies IgG and IgM against infectious pancreatic necrosis virus (IPNV) (c) Monoclonal antibodies IgG and IgM against hirame rhabdovirus (HRV) and IgM