CA2039168A1 - Neutralizing monoclonal antibody to infectious pancreatic necrosis virus - Google Patents
Neutralizing monoclonal antibody to infectious pancreatic necrosis virusInfo
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- CA2039168A1 CA2039168A1 CA 2039168 CA2039168A CA2039168A1 CA 2039168 A1 CA2039168 A1 CA 2039168A1 CA 2039168 CA2039168 CA 2039168 CA 2039168 A CA2039168 A CA 2039168A CA 2039168 A1 CA2039168 A1 CA 2039168A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
ABSTRACT
The present invention relates to a monoclonal antibody to infectious pancreatic necrosis virus, which is produced by hybridoma 4PG-3N, and a method for remedying and/or preventing the infectious pancreatic necrosis in salmonids by using this monoclonal antibody.
The present invention relates to a monoclonal antibody to infectious pancreatic necrosis virus, which is produced by hybridoma 4PG-3N, and a method for remedying and/or preventing the infectious pancreatic necrosis in salmonids by using this monoclonal antibody.
Description
2~3~16~
SPECIFIC~TION
NEUTRALIZINC. MONOCLONAI, ANTIBODY TO INFECTIOUS
PANCREATIC NECROSIS VIRUS
Technical Field The present invention relates to a neutralizing monoclo-nal antibody to infectious pancreatic necrosis virus and a use thereof.
Background Art In order to secure animal protein resources, multifica-tion and raising of fishes and shellfishes have recently been vigorously carried out throughout the world, and also in our country, the raising enterprises have rapidly developed year after year.
With this development, disease damages of fishes tend to increase year after year. Among various fish diseases, virus diseases spread at much higher propagation speeds than other bacterial diseases and there is not an effective agent for the remedy of virus diseases. Accordingly, once a virus disease breaks out, a great deal of damage is often done.
Under this background, as the means of disinfection of virus diseases, there have been investigated the control of the water temperature, the chemotherapy using an antibiotic substance or the like, the immunological prevention and the disinfection of eggs. However, no preventing effect has been reported except the effect by the disinfection of eggs, 203~1~8 and there is no practically disinfection method.
Accordingly, establishment of effective methods for the prevention and remedy of virus diseases is eagerly desired.
Under this background, Caswell-Reno et al. report a neutralizing monoclonal antibody to infectious pancreatic necrosis virus [P. Caswell-Reno et al., J. Gen. Virol (1986), 67, 2193-2205].
This neutralizing monoclonal antibody to infectious pancreatic necrosis virus is not an antibody for use in the remedy of infectious virus diseases, and since the specificity is somewhat high, the monoclonal antibody has no neutralizing effect to a certain kind of an infectious pancreatic necrosis virus.
Disclosure of the Invention The inventors made investigations with a view to utiliz-ing a monoclonal antibody having a neutrailizing effect to infectious pancreatic necrosis virus as a therapeutic agent for the infectious pancreatic necrosis (IPN) in fishes, especially salmonids, and as the result, it was found that a specific monoclonal antibody answers this purpose.
Furthermore, the inventors completed the methods for the remedy and prevention of the infectious pancreatic necrosis in salmonids by using this specific monoclonal antibody.
Thus, the present invention has now been completed.
More specifically, in accordance with the present invention, there are provided a neutralizing monoclonal 2~3~
antibody (often referred to as "anti-IPNV antibody"
hereinafter) to infectious pancreatic necrosis virus (often referred to as "IPNV" hereinafter), which is produced by hybridoma 4PG-3N, and a method for the remedy and prevention of the infectious pancreatic necrosis in salmonids, which comprises using this monoclonal antibody.
Brief Description of the Drawing Fig. 1 is a diagram illustrating the specificity of the monoclonal antibody of the present invention produced by hybridoma 4PG-3N to IPNV.
Best Mode for Carrying Out the Invention The present invention provides an anti-IPNV antibody and a method for the remedy and prevention of IPNV in salmonids. A general process for the preparation of the anti-IPNV antibody of the present invention will now be described in detail.
The immunocyte as one parent cell to be used for the cell fusion can be prepared by immunizing a mammal according to a conver.tional method using IPNV as the immune antigen.
The kind of IPNV as the immune antigen is not particularly critical, and IPNV separated from an IPNV positive cell according to a conventional method can be used. The separation of IPNV is accomplished according to usual means such as centrifugal separation, and if necessary, purifica-tion is carried out by the density gradient ultracentrifugal separation using sucrose or the like. The mammal to be ~39:~6~
immunized by IPNV is not particularly critical, and an ap-propriate mammal is selected in view of the compatibility with the myeloma cell to be used for the cell fusion. In general, a mouse, a rat and the like are used.
The immunization can be carried out according to a conventional method. For example, IPNV is diluted to an appropriate concentration, a suspension is prepared from the dilution together with Freund's adjuvant or the like, and the suspensiorl is administered to a mammal by hypodermic injection. Incidentally, it is preferred that after 3 to 5 weeks from the initial immunization, booster immunization be carried out so that the protein amount of IPNV obtained from about 500 ml of a culture liquid of IPNV positive cells cultured at a total administration amount of 2 x 105 to 3 x 105 cells/ml is 120 ~g per mammal.
A known myeloma cell of a mammal can be used as the other parent cell to be fused with -the so-obtained immunized cell (lymphocyte). For example, there can be mentioned P3(P3/X63-Ag8)[Nature, 256, 495 to 497 (1975)3, P3-Ul[Current Topics in Microbiology and Immunology, 81, 1 to 7 (1978)], NS-l[Eur. J. Immunol., 6, 511 to 519 (1976)], MPC-ll[Cell, 8, 405 to 415 (1976)], SP2/O[Nature, 276, 269 to 270 (1978)], FO[J. Immunol. Meth., 35, 1 to 21 (1980)], X63.6.55.3.[J.
Immunol., 123, 1548 to 1550 (1979)] and S1~4[J. Exp. Med., 148, 313 to 323 (1978)] and R210[Nature, 277, 131 to 133 (1979] in rats.
2~3~1~8 The fusion reaction between the lymphocyte and the myeloma cell is, in principle, performed according to a known method, for example, the method of Oi and Herzenberg [Selected Methods in Cellular Immunology, 351 to 371, published by W.H. Freeman & Co., U.S.A. (1980)]. More specifically, this fusion reaction is carried out in an ordinary nutrient culture medium in the presence of a fusion promotor. As the fusion promotor, there can be used polyethylene glycol (PEG) and Sendai virus (HVJ). In this case, the fusion efficiency can be increased by adding an adjuvant such as dimethylsulfoxide.
The ratio between the lymphocyte and the myeloma cell can be the same as adopted in the conventional method. For example, the lymphocyte is used in an amount about 1 to 10 times higher than the amount of the myeloma cell.
RPMI-1640 medium and MEM medium conventionally used for the propagation of myeloma cells can be used as the medium for the fusion reaction, and it is generally preferred that a serum adjuvant such as fetal calf serum (FCS) should be removed. The fusion reaction is accomplished by suffi-ciently fusing predetermined amounts of the lymphocyte and the myeloma cell in the above-mentioned medium, and mixing a solution of PEG having an average molecular weight of 1000 to 6000 heated in advance at about 37C with the medium so that the concentration is about 30 to about 60 w/v~. Then, operations of adding an appropriate medium to 2~3g~6~
the mixture, carrying out the centrifugal separation and removing the supernatant are repeated to obtain a desired fused cell (hybridoma).
The separation of the formed hybridoma is accomplished by carrying out the culturing in a ordinary selecting medium, for example, HAT medium (a medium containing hypoxanthine, aminopterin and thymidine). Namely, the culturing is conducted for a time sufficien-t for cells other than the indented hybrldoma (that is, unfused cells) to die, generally for several days to several weeks, whereby the hybridoma is separated.
The separated hybridoma is subjected to the retrieval for an intended antibody-producing strain and the mono-cloning according to an ordinary limiting dilution method.
The retrieval for the intended hybridoma can be carreid out according to various methods conventionally adopted for detection of antibodies, for example, the indirect immuno-fluorescence method, the enzyme-antibody method, the neutralization reaction method, the sedimentation reaction method, the complement fixation method, the agglutination method, the Ouchterlony method and the RIA method (see "Hybridoma Methods and Monoclonal Antibodies", pages 30 to 53, published by R & D Planning on March 5, 1982).
The hybridoma producing the neutralizing monoclonal antibody to IPNV, is obtained in the above-mentioned manner.
It was found that hybridoma 4PG-3N obtained by using ATCC
2~3~1~8 VR299 strain as the IPNV strain with the neutralizing activity being as the indicator has excellent characteristics.
As the method for preparing the anti-IPNV antibody of the present invention from the so-obtained specific hybridoma, there can be mentioned a method in which the hybridoma is cultured according to conventional procedures and the desired antibody is separated from the supernatant of the culture medium, and the method in which the hybridoma is admini-stered to a mammal compatible with the hybridoma to effect propagation and the desired antibody is separated from the abdominal dropsy. The former method is suitable for obtain-ing a product having a high purity, and the latter method is suitable for the mass production.
When the obtained anti-IPNV antibody is used for the remedy and prevention of IPNV in salmonids, the hypodermic injection method and the immersion method are adopted as the administration method.
In case of hypodermic injection, the anti-IPNV antibody is dissolved in a buffer solution (having a pH value of 6.5 to 7.5) such as a phosphate buffer solution at a con-centration of about 100 ~g/ml and the solution is inoculated into salmonids in an amount of 1 to 5 ml/kg.
The immersion method is mainly used for fingerlings.
For example, about 1000 of one-to-two-months-old fingerlings were immersed in about 50 Q of an anti-IPNV antibody solution (the anti-IPNV antibody concentration is 100 ~g/ml) for about .
2 ~ 6 ~
three hours per day and this immersion is conducted for 1 week to effect inoculation. In this case, it is preferred that the anti-IPNV antibody solution be one formed by dis-solving the anti-IPNV antibody in water for breeding of fingerings.
By administering the antl-IPNV antibody in the above-mentioned manner, there can be attained a sufficient effect for the remedy and prevention of IPN in salmonids.
Examples The present invention will now be described in detail with reference to the following experiment and examples.
Experiment The process for the preparation of the neutralizing monoclonal antibody to the infectious pancreatic necrosis virus will now be described.
At first, 40 ~g as the protein of the virus purified by the sucrose density gradient ultracentrifugal separation method was inoculated together with Freund's complete adjuvant (supplied by Difco) into the abdominal cavity of a 5-week-old male mouse of the BALB/c line, and the mouse was immunized with the same amount of the virus (ATCC VR299 strain) at intervals of 2 weeks without using the adjuvant.
When the anti-IPNV antibody value in blood was sufficiently increased, the mouse was dissected. Then, 2 x 108 of lymphocytes obtained from the spleen were mixed with 2 x 107 of mouse myeloma cells P3 x 63 - Ag.8.UI (purchased from 2~3~8 Dai-Nippon Seiyaku) in the logarithmic propagation phase and the fusion reaction was carried out by using polyethylene glycol (having a molecular weight of 1000 and supplied by Wako Junyaku).
The fused cells were cultured at 37C for 14 days in a 5% C2 incubator by using a medium (referred to as "HAT
medium") formed by adding 182 ng/ml of aminopterin, 13.6 ~g/ml of hypoxanthine and 3.9 ~g of thymidine to a 15~ fetal calf serum-added E-RDF medium (supplied by Kyokuto Seiyaku).
The culture supernatant of the hybridoma propagated in the HAT medium was collected 50 ~1 each and mixed with 50 ~1 of the infectious pancreatic virus (ATCC VR299 strain) injected in advance in a 96-well microplate at 100 PFV/well, and reaction was carried out at 15C for 1 hour. After the reaction, 3 x 10 per ml of chnook salmon cells CHSE-214 was injected 100 ~1 each and the culturing was conducted at 15C for 1 week.
After the culturing, the cells were fixed with 10~
formalin and stained with 0.1~ Crystal Violet, and whether or not the cytopathic effect was attained was checked.
The supernatant of the hybridoma where the cytopathic, effect by IPNV was not observed was screened as positive neutralizing activity to IPNV.
The obtained neutralizing monoclonal antibody producing hybridoma was cloned twice by the limiting dilution method.
The obtained hybridoma 4PG-3N was deposited at g _ 203~ 8 Fermentation Research Institute, Agency of Industrial Science and Technology Japan on September 1, 1989 (Deposi-tion No. FERM P-10984), and the deposition was changed to International Deposition on August 16, 1990 (Deposition No.
FE~M BP-3068).
Example 1 The hybridoma 4PG-3N (FERM BP-3068) producing the neutralizing monoclonal antibody to IPNV was suspended to a 10~ fetal calf serum-added E-RDF medium at a concentration of 2 x 105 cells/ml, and the culturing was conducted at 37C in a 5% CO2 incubator for 2 days and the supernatant of the culture fluid was recovered by the centrifugal separation.
(a) Class and subclass of monoclonal antibody produced by 4PG-3N
The class and subclass of the monoclonal antibody produced by 4PG-3N, prepared according to the above-mentioned method, were determined by the ELISA method using a typing kit (supplied by Tago). As the result, the class was IgG
and the subclass was IgG2b, and the light chain was k chain. The protein of IPNV recognized by this monoclonal antibody was VP2 protein (molecular weight was 51 Kd).
(b) Neutralizing activity to salmonid viruses Not only the infectious pancreatic necrosis virus but also infectious hematopoietic nectrosis virus (IHNV) and Oncorhychus mas_ virus (OMV) were tested as salmonids 2 0 ~ 8 viruses, and the neutralizing ac-tivity of the monoclonal antibody produced by 4PG-3N was examined. The above-mentioned supernatant of the culture fluid was reacted with the same amount of a virus-cultured fluid at 15C for 1 hour, and the obtained results were compared with the results of the control where a 10% fetal calf serum-added E-RDF medium was used and the degree of the neutralization of the virus was calculated according to the plaque reduc-tion method. The obtained results are shown in Table 1.
As is apparent from the results shown in the table, the infectivity of 6.5 x 107 PFU/ml observed in the control was reduced to 5.1 x 105 PFU/ml when themonoclonal antibody was reacted at a concentration of 1.3 ~g/ml, and the neutralization activity was confirmed. However, IHNV and OMV were not neutralized by the monoclonal antibody.
Table 1 Virus infectivity Virus antibody _1 Control *2 (log value) *3 IPNV 5.1 x 105 6.5 x 107 2.1 IHNV 1.8 x 105 2.4 x 105 0.1 OMV 3.5 x 104 3.5 x 104 0.0 Note *1: reaction between monoclonal antibody produced by 4PG-3N
and virus-cultured fluid *2: reaction between 10~ fetal calf serum-added R-RDF medium used for culturing 4PG-3N and virus-cultured fluid *3: logarithmic value of difference between virus infectivity obtained by reaction with control and virus infectivity obtained by reaction with monoclonal antibody 2 0 3 ~ 3L 6 8 (c) Reaction specificity to IPNV
The reaction specificity to purified IPNV, IHNV and Hirame Rhabdovirus (HRV) was examined according to the ELISA method. A 96-well microplate (supplied by Nunc) was coated with purified IPNV, IHNV or HRV diluted to 1/2 in sequence from 5 ~g to 0.3 ~g of protein/ml, and the reac-tivity of the monoclonal antibody was examined. The obtained results are shown in Fig. l. It was confirmed that the monoclonal antibody IgG produced by 4PG-3N reacted specifically only with IPNV but did not react with IHNV and HRV at all, and the monoclonal antibody IgG had a neutraliz-ing activity to IPNV and was an antibody specific to IPNV.
The neutralizing monoclonal antibody to IPNV according to the present invention neutralized IPNV with an activity of 2.1 calculated as the logarithmic value at a concentra-tion of 1.3 ~g of protein/ml. When the reactivity with IPNV was examined according to the ELISA method, since the monoclonal antibody reacted specifically with the virus even at a concentration of 1 ~g of protein per ml, it was confirmed that the monoclonal antibody had a neutralizing activity to IPNV and was a specific antibody to IPNV.
(d) Neutralizing activity to various strains of IPNV
The neutralizing activity was examined by the pla~ue reduction method with respect to two IPNV strains isolated in Japan (Towada and Nichiro), one IPNV strain isolated in Europe (Bonnamy) and three IPNV strains isolated in U.S.A.
203~
(VR299, Buhl and West Buxton). A sufficient neutralizing activity was obtained at an effective concentration (100 ~g/ml) for each strain.
Example 2 The IPNV antibody was dissolved in a phosphate buffer solution (having a pH value of 7.2) to give a concentration of 100 ~g/ml, and the solution was filled in ampoules having a volume of 5 ml to obtain hypodermic injections.
Industrial Applicability The neutralizing monoclonal antibody to IPNV according to the present invention can be utilizing as a medicine for the remedy of the infectious pancreatic necrosis, which has been considered to be difficult to cure, and moreover, attainment of an effect of disinfecting IPNV adhering to eggs and spermatozoa of IPNV-carrying salmon parent fishes is expected and the monoclonal antibody will be utilized for preventing the infection with IPNV in salmonids in the future.
SPECIFIC~TION
NEUTRALIZINC. MONOCLONAI, ANTIBODY TO INFECTIOUS
PANCREATIC NECROSIS VIRUS
Technical Field The present invention relates to a neutralizing monoclo-nal antibody to infectious pancreatic necrosis virus and a use thereof.
Background Art In order to secure animal protein resources, multifica-tion and raising of fishes and shellfishes have recently been vigorously carried out throughout the world, and also in our country, the raising enterprises have rapidly developed year after year.
With this development, disease damages of fishes tend to increase year after year. Among various fish diseases, virus diseases spread at much higher propagation speeds than other bacterial diseases and there is not an effective agent for the remedy of virus diseases. Accordingly, once a virus disease breaks out, a great deal of damage is often done.
Under this background, as the means of disinfection of virus diseases, there have been investigated the control of the water temperature, the chemotherapy using an antibiotic substance or the like, the immunological prevention and the disinfection of eggs. However, no preventing effect has been reported except the effect by the disinfection of eggs, 203~1~8 and there is no practically disinfection method.
Accordingly, establishment of effective methods for the prevention and remedy of virus diseases is eagerly desired.
Under this background, Caswell-Reno et al. report a neutralizing monoclonal antibody to infectious pancreatic necrosis virus [P. Caswell-Reno et al., J. Gen. Virol (1986), 67, 2193-2205].
This neutralizing monoclonal antibody to infectious pancreatic necrosis virus is not an antibody for use in the remedy of infectious virus diseases, and since the specificity is somewhat high, the monoclonal antibody has no neutralizing effect to a certain kind of an infectious pancreatic necrosis virus.
Disclosure of the Invention The inventors made investigations with a view to utiliz-ing a monoclonal antibody having a neutrailizing effect to infectious pancreatic necrosis virus as a therapeutic agent for the infectious pancreatic necrosis (IPN) in fishes, especially salmonids, and as the result, it was found that a specific monoclonal antibody answers this purpose.
Furthermore, the inventors completed the methods for the remedy and prevention of the infectious pancreatic necrosis in salmonids by using this specific monoclonal antibody.
Thus, the present invention has now been completed.
More specifically, in accordance with the present invention, there are provided a neutralizing monoclonal 2~3~
antibody (often referred to as "anti-IPNV antibody"
hereinafter) to infectious pancreatic necrosis virus (often referred to as "IPNV" hereinafter), which is produced by hybridoma 4PG-3N, and a method for the remedy and prevention of the infectious pancreatic necrosis in salmonids, which comprises using this monoclonal antibody.
Brief Description of the Drawing Fig. 1 is a diagram illustrating the specificity of the monoclonal antibody of the present invention produced by hybridoma 4PG-3N to IPNV.
Best Mode for Carrying Out the Invention The present invention provides an anti-IPNV antibody and a method for the remedy and prevention of IPNV in salmonids. A general process for the preparation of the anti-IPNV antibody of the present invention will now be described in detail.
The immunocyte as one parent cell to be used for the cell fusion can be prepared by immunizing a mammal according to a conver.tional method using IPNV as the immune antigen.
The kind of IPNV as the immune antigen is not particularly critical, and IPNV separated from an IPNV positive cell according to a conventional method can be used. The separation of IPNV is accomplished according to usual means such as centrifugal separation, and if necessary, purifica-tion is carried out by the density gradient ultracentrifugal separation using sucrose or the like. The mammal to be ~39:~6~
immunized by IPNV is not particularly critical, and an ap-propriate mammal is selected in view of the compatibility with the myeloma cell to be used for the cell fusion. In general, a mouse, a rat and the like are used.
The immunization can be carried out according to a conventional method. For example, IPNV is diluted to an appropriate concentration, a suspension is prepared from the dilution together with Freund's adjuvant or the like, and the suspensiorl is administered to a mammal by hypodermic injection. Incidentally, it is preferred that after 3 to 5 weeks from the initial immunization, booster immunization be carried out so that the protein amount of IPNV obtained from about 500 ml of a culture liquid of IPNV positive cells cultured at a total administration amount of 2 x 105 to 3 x 105 cells/ml is 120 ~g per mammal.
A known myeloma cell of a mammal can be used as the other parent cell to be fused with -the so-obtained immunized cell (lymphocyte). For example, there can be mentioned P3(P3/X63-Ag8)[Nature, 256, 495 to 497 (1975)3, P3-Ul[Current Topics in Microbiology and Immunology, 81, 1 to 7 (1978)], NS-l[Eur. J. Immunol., 6, 511 to 519 (1976)], MPC-ll[Cell, 8, 405 to 415 (1976)], SP2/O[Nature, 276, 269 to 270 (1978)], FO[J. Immunol. Meth., 35, 1 to 21 (1980)], X63.6.55.3.[J.
Immunol., 123, 1548 to 1550 (1979)] and S1~4[J. Exp. Med., 148, 313 to 323 (1978)] and R210[Nature, 277, 131 to 133 (1979] in rats.
2~3~1~8 The fusion reaction between the lymphocyte and the myeloma cell is, in principle, performed according to a known method, for example, the method of Oi and Herzenberg [Selected Methods in Cellular Immunology, 351 to 371, published by W.H. Freeman & Co., U.S.A. (1980)]. More specifically, this fusion reaction is carried out in an ordinary nutrient culture medium in the presence of a fusion promotor. As the fusion promotor, there can be used polyethylene glycol (PEG) and Sendai virus (HVJ). In this case, the fusion efficiency can be increased by adding an adjuvant such as dimethylsulfoxide.
The ratio between the lymphocyte and the myeloma cell can be the same as adopted in the conventional method. For example, the lymphocyte is used in an amount about 1 to 10 times higher than the amount of the myeloma cell.
RPMI-1640 medium and MEM medium conventionally used for the propagation of myeloma cells can be used as the medium for the fusion reaction, and it is generally preferred that a serum adjuvant such as fetal calf serum (FCS) should be removed. The fusion reaction is accomplished by suffi-ciently fusing predetermined amounts of the lymphocyte and the myeloma cell in the above-mentioned medium, and mixing a solution of PEG having an average molecular weight of 1000 to 6000 heated in advance at about 37C with the medium so that the concentration is about 30 to about 60 w/v~. Then, operations of adding an appropriate medium to 2~3g~6~
the mixture, carrying out the centrifugal separation and removing the supernatant are repeated to obtain a desired fused cell (hybridoma).
The separation of the formed hybridoma is accomplished by carrying out the culturing in a ordinary selecting medium, for example, HAT medium (a medium containing hypoxanthine, aminopterin and thymidine). Namely, the culturing is conducted for a time sufficien-t for cells other than the indented hybrldoma (that is, unfused cells) to die, generally for several days to several weeks, whereby the hybridoma is separated.
The separated hybridoma is subjected to the retrieval for an intended antibody-producing strain and the mono-cloning according to an ordinary limiting dilution method.
The retrieval for the intended hybridoma can be carreid out according to various methods conventionally adopted for detection of antibodies, for example, the indirect immuno-fluorescence method, the enzyme-antibody method, the neutralization reaction method, the sedimentation reaction method, the complement fixation method, the agglutination method, the Ouchterlony method and the RIA method (see "Hybridoma Methods and Monoclonal Antibodies", pages 30 to 53, published by R & D Planning on March 5, 1982).
The hybridoma producing the neutralizing monoclonal antibody to IPNV, is obtained in the above-mentioned manner.
It was found that hybridoma 4PG-3N obtained by using ATCC
2~3~1~8 VR299 strain as the IPNV strain with the neutralizing activity being as the indicator has excellent characteristics.
As the method for preparing the anti-IPNV antibody of the present invention from the so-obtained specific hybridoma, there can be mentioned a method in which the hybridoma is cultured according to conventional procedures and the desired antibody is separated from the supernatant of the culture medium, and the method in which the hybridoma is admini-stered to a mammal compatible with the hybridoma to effect propagation and the desired antibody is separated from the abdominal dropsy. The former method is suitable for obtain-ing a product having a high purity, and the latter method is suitable for the mass production.
When the obtained anti-IPNV antibody is used for the remedy and prevention of IPNV in salmonids, the hypodermic injection method and the immersion method are adopted as the administration method.
In case of hypodermic injection, the anti-IPNV antibody is dissolved in a buffer solution (having a pH value of 6.5 to 7.5) such as a phosphate buffer solution at a con-centration of about 100 ~g/ml and the solution is inoculated into salmonids in an amount of 1 to 5 ml/kg.
The immersion method is mainly used for fingerlings.
For example, about 1000 of one-to-two-months-old fingerlings were immersed in about 50 Q of an anti-IPNV antibody solution (the anti-IPNV antibody concentration is 100 ~g/ml) for about .
2 ~ 6 ~
three hours per day and this immersion is conducted for 1 week to effect inoculation. In this case, it is preferred that the anti-IPNV antibody solution be one formed by dis-solving the anti-IPNV antibody in water for breeding of fingerings.
By administering the antl-IPNV antibody in the above-mentioned manner, there can be attained a sufficient effect for the remedy and prevention of IPN in salmonids.
Examples The present invention will now be described in detail with reference to the following experiment and examples.
Experiment The process for the preparation of the neutralizing monoclonal antibody to the infectious pancreatic necrosis virus will now be described.
At first, 40 ~g as the protein of the virus purified by the sucrose density gradient ultracentrifugal separation method was inoculated together with Freund's complete adjuvant (supplied by Difco) into the abdominal cavity of a 5-week-old male mouse of the BALB/c line, and the mouse was immunized with the same amount of the virus (ATCC VR299 strain) at intervals of 2 weeks without using the adjuvant.
When the anti-IPNV antibody value in blood was sufficiently increased, the mouse was dissected. Then, 2 x 108 of lymphocytes obtained from the spleen were mixed with 2 x 107 of mouse myeloma cells P3 x 63 - Ag.8.UI (purchased from 2~3~8 Dai-Nippon Seiyaku) in the logarithmic propagation phase and the fusion reaction was carried out by using polyethylene glycol (having a molecular weight of 1000 and supplied by Wako Junyaku).
The fused cells were cultured at 37C for 14 days in a 5% C2 incubator by using a medium (referred to as "HAT
medium") formed by adding 182 ng/ml of aminopterin, 13.6 ~g/ml of hypoxanthine and 3.9 ~g of thymidine to a 15~ fetal calf serum-added E-RDF medium (supplied by Kyokuto Seiyaku).
The culture supernatant of the hybridoma propagated in the HAT medium was collected 50 ~1 each and mixed with 50 ~1 of the infectious pancreatic virus (ATCC VR299 strain) injected in advance in a 96-well microplate at 100 PFV/well, and reaction was carried out at 15C for 1 hour. After the reaction, 3 x 10 per ml of chnook salmon cells CHSE-214 was injected 100 ~1 each and the culturing was conducted at 15C for 1 week.
After the culturing, the cells were fixed with 10~
formalin and stained with 0.1~ Crystal Violet, and whether or not the cytopathic effect was attained was checked.
The supernatant of the hybridoma where the cytopathic, effect by IPNV was not observed was screened as positive neutralizing activity to IPNV.
The obtained neutralizing monoclonal antibody producing hybridoma was cloned twice by the limiting dilution method.
The obtained hybridoma 4PG-3N was deposited at g _ 203~ 8 Fermentation Research Institute, Agency of Industrial Science and Technology Japan on September 1, 1989 (Deposi-tion No. FERM P-10984), and the deposition was changed to International Deposition on August 16, 1990 (Deposition No.
FE~M BP-3068).
Example 1 The hybridoma 4PG-3N (FERM BP-3068) producing the neutralizing monoclonal antibody to IPNV was suspended to a 10~ fetal calf serum-added E-RDF medium at a concentration of 2 x 105 cells/ml, and the culturing was conducted at 37C in a 5% CO2 incubator for 2 days and the supernatant of the culture fluid was recovered by the centrifugal separation.
(a) Class and subclass of monoclonal antibody produced by 4PG-3N
The class and subclass of the monoclonal antibody produced by 4PG-3N, prepared according to the above-mentioned method, were determined by the ELISA method using a typing kit (supplied by Tago). As the result, the class was IgG
and the subclass was IgG2b, and the light chain was k chain. The protein of IPNV recognized by this monoclonal antibody was VP2 protein (molecular weight was 51 Kd).
(b) Neutralizing activity to salmonid viruses Not only the infectious pancreatic necrosis virus but also infectious hematopoietic nectrosis virus (IHNV) and Oncorhychus mas_ virus (OMV) were tested as salmonids 2 0 ~ 8 viruses, and the neutralizing ac-tivity of the monoclonal antibody produced by 4PG-3N was examined. The above-mentioned supernatant of the culture fluid was reacted with the same amount of a virus-cultured fluid at 15C for 1 hour, and the obtained results were compared with the results of the control where a 10% fetal calf serum-added E-RDF medium was used and the degree of the neutralization of the virus was calculated according to the plaque reduc-tion method. The obtained results are shown in Table 1.
As is apparent from the results shown in the table, the infectivity of 6.5 x 107 PFU/ml observed in the control was reduced to 5.1 x 105 PFU/ml when themonoclonal antibody was reacted at a concentration of 1.3 ~g/ml, and the neutralization activity was confirmed. However, IHNV and OMV were not neutralized by the monoclonal antibody.
Table 1 Virus infectivity Virus antibody _1 Control *2 (log value) *3 IPNV 5.1 x 105 6.5 x 107 2.1 IHNV 1.8 x 105 2.4 x 105 0.1 OMV 3.5 x 104 3.5 x 104 0.0 Note *1: reaction between monoclonal antibody produced by 4PG-3N
and virus-cultured fluid *2: reaction between 10~ fetal calf serum-added R-RDF medium used for culturing 4PG-3N and virus-cultured fluid *3: logarithmic value of difference between virus infectivity obtained by reaction with control and virus infectivity obtained by reaction with monoclonal antibody 2 0 3 ~ 3L 6 8 (c) Reaction specificity to IPNV
The reaction specificity to purified IPNV, IHNV and Hirame Rhabdovirus (HRV) was examined according to the ELISA method. A 96-well microplate (supplied by Nunc) was coated with purified IPNV, IHNV or HRV diluted to 1/2 in sequence from 5 ~g to 0.3 ~g of protein/ml, and the reac-tivity of the monoclonal antibody was examined. The obtained results are shown in Fig. l. It was confirmed that the monoclonal antibody IgG produced by 4PG-3N reacted specifically only with IPNV but did not react with IHNV and HRV at all, and the monoclonal antibody IgG had a neutraliz-ing activity to IPNV and was an antibody specific to IPNV.
The neutralizing monoclonal antibody to IPNV according to the present invention neutralized IPNV with an activity of 2.1 calculated as the logarithmic value at a concentra-tion of 1.3 ~g of protein/ml. When the reactivity with IPNV was examined according to the ELISA method, since the monoclonal antibody reacted specifically with the virus even at a concentration of 1 ~g of protein per ml, it was confirmed that the monoclonal antibody had a neutralizing activity to IPNV and was a specific antibody to IPNV.
(d) Neutralizing activity to various strains of IPNV
The neutralizing activity was examined by the pla~ue reduction method with respect to two IPNV strains isolated in Japan (Towada and Nichiro), one IPNV strain isolated in Europe (Bonnamy) and three IPNV strains isolated in U.S.A.
203~
(VR299, Buhl and West Buxton). A sufficient neutralizing activity was obtained at an effective concentration (100 ~g/ml) for each strain.
Example 2 The IPNV antibody was dissolved in a phosphate buffer solution (having a pH value of 7.2) to give a concentration of 100 ~g/ml, and the solution was filled in ampoules having a volume of 5 ml to obtain hypodermic injections.
Industrial Applicability The neutralizing monoclonal antibody to IPNV according to the present invention can be utilizing as a medicine for the remedy of the infectious pancreatic necrosis, which has been considered to be difficult to cure, and moreover, attainment of an effect of disinfecting IPNV adhering to eggs and spermatozoa of IPNV-carrying salmon parent fishes is expected and the monoclonal antibody will be utilized for preventing the infection with IPNV in salmonids in the future.
Claims (2)
1. A neutralizing monoclonal antibody to infectious pancreatic necrosis virus, which is produced by hybridoma 4PG-3N.
2. A method for the remedy and/or prevention of the infectious pancreatic necrosis in salmonids, which comprises using a neutralizing monoclonal antibody to infectious pancreatic necrosis virus, which is produced by hybridoma 4PG-3N.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JPHEI1-227582 | 1989-09-04 | ||
| JP22758289 | 1989-09-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2039168A1 true CA2039168A1 (en) | 1991-03-05 |
Family
ID=16863176
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2039168 Abandoned CA2039168A1 (en) | 1989-09-04 | 1990-08-31 | Neutralizing monoclonal antibody to infectious pancreatic necrosis virus |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA2039168A1 (en) |
| WO (1) | WO1991003570A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015104551A1 (en) * | 2014-01-08 | 2015-07-16 | Aqua Gen As | Treating susceptibility |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994004565A2 (en) * | 1992-08-26 | 1994-03-03 | Proteus Molecular Design Limited | Ipnv vaccine |
-
1990
- 1990-08-31 WO PCT/JP1990/001117 patent/WO1991003570A1/en active Application Filing
- 1990-08-31 CA CA 2039168 patent/CA2039168A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015104551A1 (en) * | 2014-01-08 | 2015-07-16 | Aqua Gen As | Treating susceptibility |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1991003570A1 (en) | 1991-03-21 |
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