CN117467001A - Preparation method and application of clostridium perfringens antitoxin A serum - Google Patents
Preparation method and application of clostridium perfringens antitoxin A serum Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
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Abstract
The invention relates to the technical field of biological products for livestock, in particular to a preparation method and application of clostridium perfringens type A antitoxin serum. The preparation method of the clostridium perfringens type A antitoxin serum comprises the steps of performing basic immunity on a healthy animal at least twice by using a first immunogen, then performing booster immunity on the healthy animal at least twice by using a second immunogen, and harvesting the clostridium perfringens type A antitoxin serum; the first immunogen takes clostridium perfringens type A toxin as an immunogen component; the second immunogen takes clostridium perfringens A exotoxin as an immunogenic component. According to the invention, under the premise of ensuring safety, the exotoxin is used as a second immunogen instead of toxoid, so that the immunogenicity is ensured and improved, and the titer of the product is further improved.
Description
Technical Field
The invention relates to the technical field of biological products for livestock, in particular to a preparation method and application of clostridium perfringens type A antitoxin serum.
Background
Clostridium perfringens @Clostridium perfringens) Is one of main pathogenic bacteria causing various animal necrotic enteritis, enterotoxemia, human food poisoning and traumatic gas gangrene, and is also called clostridium welchiiC.welchii) Or clostridium perfringens, which is called clostridium perfringens, because it breaks down sugars in muscle and connective tissue, generates a large amount of gas, causes severe emphysema, and is able to form a capsule in the body. The bacterium is widely distributed in soil and animal digestive tracts, and traditionally, clostridium perfringens is divided into five toxin types of A (beta 1), B (alpha, beta, epsilon), C (alpha, beta), D (alpha, epsilon) and E (alpha, iota) by the difference of 4 main exotoxins (alpha, beta 0 and iota) produced by the bacterium. Clostridium perfringens type a can cause symptoms such as "sudden clostridium death", necrotic enteritis, limb injury, organ injury gangrene and the like of various animals, can also cause food poisoning of people, and is the most serious type of harm in clostridium perfringens.
The high immune serum is also called disease-resistant serum, and refers to animal serum containing high-titer specific antibodies. The animals are given repeated injections of specific pathogenic microorganisms or their metabolites to the appropriate animals to promote an immune response, and the serum contains a large amount of corresponding specific antibodies. The hyperimmune serum is mainly used for passive immunization, treatment and emergency prevention of diseases caused by corresponding pathogens. At present, the research on the hyperimmune serum of clostridium perfringens type A is less, and the problems of low serum titer, poor safety and the like exist.
In view of this, the present invention has been made.
Disclosure of Invention
In view of the technical problems in the background art, the invention provides a preparation method and application of clostridium perfringens type A antitoxin serum. The clostridium perfringens type A prepared by the preparation method provided by the invention has high serum-free titer and good safety, and can be used for emergency immunization of diseased animals.
Specifically, the technical scheme provided by the invention is as follows:
in a first aspect, the present invention provides a method for preparing clostridium perfringens type a antitoxin serum, wherein a healthy animal is subjected to at least two basic immunizations using a first immunogen, then the healthy animal is subjected to at least two booster immunizations using a second immunogen, and clostridium perfringens type a antitoxin serum is harvested; the first immunogen takes clostridium perfringens type A toxin as an immunogen component; the second immunogen takes clostridium perfringens A exotoxin as an immunogenic component.
Conventional methods of preparing antitoxin serum typically use toxoids for immunization. Toxoids are preparations which, after formaldehyde treatment, lose their toxicity and retain their immunogenicity and stimulate the body to produce protective immunity. The preparation of clostridium-containing bacillus product in the national livestock biological product code of the people's republic of China, 2000 edition, has the residual formaldehyde content of not more than 0.5 percent of formaldehyde solution (40 percent formaldehyde); the amount of formaldehyde solution (40% formaldehyde) in the other preparations should not exceed 0.2%. Clostridium perfringens type a is used as bacillus, and the addition amount of formaldehyde solution for inactivation is generally 0.5% -0.8%. The higher the formaldehyde concentration, the faster the detoxification, but the greater the loss of antigenicity. According to the invention, under the premise of ensuring safety, the exotoxin is used as a second immunogen instead of toxoid, so that the immunogenicity is ensured and improved, and the titer of the product is further improved.
In the present invention, preferably, the preparation method of clostridium perfringens type a exotoxin comprises: culturing clostridium perfringens CNM0708 strain a, and harvesting bacterial liquid; filtering clostridium perfringens A in the bacterial liquid to obtain clear liquid; concentrating the clear liquid by using hollow fibers to obtain clostridium perfringens exotoxin solution A. The clostridium perfringens type a CNM0708 strain is disclosed in the applicant's prior patent CN201711418333.6, and is preserved in the China Committee for culture Collection of microorganisms, with the preservation number of CGMCC No.14880.
The invention preferably uses a filter membrane of 0.22-0.45 μm to filter and sterilize clostridium perfringens type A in the bacterial liquid after the bacterial liquid is centrifuged for 20-30min at 6000-8000r/min, and clear liquid is obtained.
More preferably, the invention filters clostridium perfringens type a in the bacterial liquid by using a filter membrane with the diameter of 0.22 μm after centrifuging the bacterial liquid for 30min at 8000r/min to obtain clear liquid.
The invention preferably uses hollow fibers of 5-10KD for concentrating the clear liquid, the concentration degree is 10-15 times.
More preferably, the present invention uses 5KD hollow fibers to concentrate the supernatant to a degree of 10-fold concentration.
In the present invention, preferably, the clostridium perfringens type a toxin is prepared from the clostridium perfringens type a endotoxin solution by formaldehyde inactivation and detoxification.
According to the invention, formaldehyde solution is preferably used for inactivating and detoxication of the clostridium perfringens type A exotoxin solution to obtain clostridium perfringens type A toxin solution; the concentration of the formaldehyde solution is the original standard concentration (37-40% of formaldehyde content) of a commercial product, and the addition amount of the formaldehyde solution is 0.5-0.8 vol% of the clostridium perfringens exotoxin solution A.
More preferably, the invention uses formaldehyde solution (formaldehyde content is 37-40%) to inactivate and detoxify the clostridium perfringens type a exotoxin solution to obtain clostridium perfringens type a toxin solution; the addition amount of the formaldehyde solution is 0.5 vol% of the clostridium perfringens exotoxin solution A.
In the present invention, preferably, the first immunogen is formulated from clostridium perfringens type a toxin solution and aluminium hydroxide gel in a mass ratio of 4-5:1.
More preferably, the first immunogen is prepared from clostridium perfringens type a toxin solution and aluminum hydroxide gel according to a mass ratio of 4:1, has good immunogenicity, is favorable for the immunized animal to generate stronger adaptability to clostridium perfringens type a toxin in subsequent booster immunization, and generates higher serum titer of the antitoxin.
In the present invention, preferably, the healthy animal is 1.5-2.0kg healthy rabbit.
In the present invention, preferably, the number of times of the basic immunization is two, and the time interval between the two basic immunization is 14-28 days; the times of the boosting are two times, and the time interval between the two times of the boosting is 7-14 days; the time interval between the second basal immunization and the first booster immunization is 7-21 days.
More preferably, the number of basal immunizations is two, and the interval between the two basal immunizations is 21 days; the times of the boosting are two times, and the time interval between the two times of the boosting is 10 days; the time interval between the second basal immunization and the first boost immunization was 14 days. The preferred number and time intervals of immunization of the present invention help the immunized animal adapt to the immunogen and have a better response during subsequent immunization.
In a second aspect, the invention provides clostridium perfringens type a antitoxin serum, which is prepared by the preparation method of clostridium perfringens type a antitoxin serum, has the advantages of high titer, high safety and the like, and can be used for emergency immunization of diseased animals.
In a third aspect, the invention provides a method for preparing clostridium perfringens type a antitoxin serum or application of clostridium perfringens type a antitoxin serum in preparing a pharmaceutical preparation containing antitoxin serum. The clostridium perfringens type a antitoxin serum or the medicament may be used for immunization of a diseased animal.
The beneficial effects are that:
the invention provides a preparation method of clostridium perfringens type A antitoxin serum, which comprises the steps of performing basic immunity on a healthy animal at least twice by using a first immunogen, performing booster immunity on the healthy animal at least twice by using a second immunogen, and harvesting clostridium perfringens type A antitoxin serum; the first immunogen takes clostridium perfringens type A toxin as an immunogen component; the second immunogen takes clostridium perfringens A exotoxin as an immunogenic component. According to the invention, under the premise of ensuring safety, the exotoxin is used as a second immunogen instead of toxoid, so that the immunogenicity is ensured and improved, and the titer of the product is further improved. The invention performs antitoxin serum sterile test, safety test and serum titer determination according to the ' national biological product code for livestock of the people's republic of China ' 2000 edition. Serum titers were determined using KM mice weighing 16-20g, and the results showed that 30 mice minimum Mortem (MLD) toxins could be neutralized per 0.1ml of antitoxin serum. After 1.5-2.0kg healthy rabbits are challenged with 1 rabbit death amount of clostridium perfringens type A exotoxin, 1ml of clostridium perfringens type A antitoxin serum is injected intravenously at the edge of the ear, 5 rabbits are all alive, and the rabbits in the control group are all dead within 6 hours.
Detailed Description
The invention relates to a preparation method of clostridium perfringens type A antitoxin serum, and clostridium perfringens type A antitoxin serum prepared by the preparation method. Preparing exotoxin as a second immunogen by resuscitating and culturing clostridium perfringens strain A; adding formaldehyde solution according to the volume of 0.5% of the exotoxin solution for inactivation to obtain clostridium perfringens toxin solution A, and mixing according to the proportion of 4 parts of toxoid solution and 1 part of hydrogenated alumina gel to prepare toxoid inactivated vaccine as a first immunogen. 1.5-2.0kg healthy rabbits were subjected to two basic immunizations and two booster immunizations with the first immunogen and the second immunogen. Heart blood is collected 10 days after the last immunogen injection, serum is separated, and clostridium perfringens type a antitoxin serum is obtained. The clostridium perfringens type A hyperimmune serum prepared by the invention has high titer and good safety, and can be used for emergency immunization of diseased animals.
In one specific embodiment provided by the invention, the preparation method of the clostridium perfringens type a antitoxin serum comprises the following steps:
1) Inoculating clostridium perfringens strain A to a culture medium of the anaerobic meat liver soup, and standing and culturing at 36-37 ℃ for 12-16 hours to revive the strain; inoculating 2% -4% of a 4L anaerobic meat liver and stomach enzyme digestion soup culture medium, standing and culturing at 36-37 ℃ for 4-5 hours, and then harvesting bacterial liquid; the bacterial liquid is centrifugated for 30 minutes at 8000r/min, and then filtered by a 0.22 mu m filter membrane, and the centrifugated supernatant is concentrated by 10 times by 5KD hollow fiber to obtain clostridium perfringens type A exotoxin solution which is used as a second immunogen of clostridium perfringens type A antitoxin serum.
2) Adding formaldehyde solution (the formaldehyde concentration is 37-40%) with the volume ratio of 0.5% into clostridium perfringens type A exotoxin solution, inactivating and detoxifying at 37 ℃ for at least 2 times per day, and inactivating and detoxifying for 48-72 hours to obtain clostridium perfringens type A toxin solution. And preparing seedlings according to the ratio of the clostridium perfringens toxin solution A to the aluminum hydroxide gel of 4:1 to obtain the clostridium perfringens toxin inactivated vaccine A serving as a first immunogen.
3) Clostridium perfringens type a antitoxin serum preparations are divided into basic immunity and booster immunity. Performing basic immunization on 1.5-2.0kg healthy rabbits twice by using a first immunogen, and performing intramuscular injection of 1 ml/animal; two booster immunizations were then performed with the second immunogen and the ear margin was intravenously injected with 1ml of clostridium perfringens exotoxin solution a/v (concentrated toxin was diluted with physiological saline to 1 rabbit lethal dose per ml). Immunization procedures are shown in Table 3.
4) Heart blood is collected 10 days after the last immunogen injection, serum is separated, and clostridium perfringens type a antitoxin serum is obtained.
According to the technical scheme, an inactivated vaccine prepared from the clostridium perfringens toxin A and the hydrogenated alumina gel is used as a first immunogen for two basic immunizations. From rabbits not exposed to clostridium perfringens type a antigen, no antibodies were detected in the serum within days after the first injection, this delay period lasting for about one week, and when antibodies finally appeared in the serum, they peaked at 10-20d and declined and disappeared within weeks. The antibodies produced in this primary immune response are very few and the protection provided by it is relatively small. Thus, a second basic immunization was performed 21 days after the first injection, with the second immunization causing a delay period of no more than 2-3d for the antibody response, and the amount of antibody in the serum generally increased to a high level within two weeks, followed by a slow decline. The antibody amount in serum of immunized animals reaches a protection level 14 days after the second basic immunization, and the immune animals can completely resist the attack of 1 rabbit lethal dose of clostridium perfringens type A exotoxin. At this point, two booster immunizations were performed using the exotoxin as the second immunogen, the immune response being characterized by a shorter delay period and a higher level of antibody response.
The invention performs antitoxin serum sterile test, safety test and serum titer determination according to the ' national biological product code for livestock of the people's republic of China ' 2000 edition.
(1) Serum titers were determined using KM mice weighing 16-20g, and the results showed that 30 mice minimum Mortem (MLD) toxins could be neutralized per 0.1ml of antitoxin serum.
(2) After 1.5-2.0kg healthy rabbits are challenged with 1 rabbit death amount of clostridium perfringens type A exotoxin, 1ml of clostridium perfringens type A antitoxin serum is injected intravenously at the edge of the ear, 5 rabbits are all alive, and the rabbits in the control group are all dead within 6 hours.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention may be made without departing from the spirit and nature of the invention and are intended to be within the scope of the present invention. Unless otherwise indicated, all experimental materials, reagents, instruments and the like used in the examples of the present invention are commercially available; all technical measures in the examples of the present invention are conventional measures well known to those skilled in the art unless specifically indicated.
Example 1
The embodiment provides a preparation method of clostridium perfringens antitoxin A serum, which comprises the following steps:
(1) Immunogen preparation
Inoculating clostridium perfringens strain A (CNM 0708 strain, china Committee for culture Collection of microorganisms, with a collection number of CGMCC No.14880, which is disclosed in the prior patent CN201711418333.6 of the applicant) to an anaerobic meat liver broth culture medium, and performing stationary culture at 36-37 ℃ for 12-16 hours for strain recovery; inoculating 2% -4% of a 4L anaerobic meat liver and stomach enzyme digestion soup culture medium, standing and culturing at 36-37 ℃ for 4-5 hours, and then harvesting bacterial liquid; the bacterial liquid is centrifugated for 30 minutes at 8000r/min, and then filtered by a 0.22 mu m filter membrane, and the centrifugated supernatant is concentrated by 10 times by 5KD hollow fiber to obtain clostridium perfringens type A exotoxin solution which is used as a second immunogen of clostridium perfringens type A antitoxin serum. Adding 0.5% formaldehyde solution (the formaldehyde concentration is 37-40%, the formaldehyde solution is purchased from North Union Fine chemical development Co., tianjin, according with national standard GB/T685-2013) into the clostridium perfringens exotoxin solution A, inactivating and detoxication at 37 ℃, shaking for at least 2 times a day, and inactivating and detoxication for 48-72 hours to obtain the clostridium perfringens toxin solution A. And preparing seedlings according to the ratio of the clostridium perfringens toxin solution A to the aluminum hydroxide gel of 4:1 to obtain the clostridium perfringens toxin inactivated vaccine A serving as a first immunogen.
The anaerobic meat liver soup used above is prepared according to the composition and preparation method of China veterinary biological product regulations, and the composition is shown in the following table 1:
table 1: composition of liver soup for treating anaesthesia
The preparation method comprises the following steps:
1) Taking beef to remove fat and fascia, mincing with a meat mincer, mixing with liver blocks cut into about 100g, adding distilled water, stirring thoroughly, and cold soaking for 20-24 hr.
2) Boiling for 20-60 min, supplementing water, filtering with white cloth, removing meat residue, and taking out liver block.
3) Adding peptone and sodium chloride into the filtrate, heating to melt, adjusting pH to 7.8-8.0 with sodium hydroxide solution, and boiling for 10-20 min.
4) Filtering with filter paper or flannelette, adding glucose, stirring, and thawing.
5) The filtered liver block is washed, cut into small blocks, fully washed by distilled water and then packaged in test tubes or neutral glass bottles, the amount of which is about 1/10 of the expected packaged meat liver soup amount.
6) Packaging the filtrate into neutral container (such as test tube, and optionally adding appropriate amount of liquid paraffin), and sterilizing with steam at 116 deg.C under high pressure for 30-40 min.
The anaerobic meat liver and stomach enzyme digestion soup used above is prepared according to the composition and preparation method of China veterinary biological product regulations, and the composition is shown in the following table 2:
table 2: composition of anaerobic meat liver and stomach enzyme digestion soup
The preparation method comprises the following steps:
1) Adding hydrochloric acid and minced beef and liver into 65deg.C warm water, stirring, adding pepsin, stirring, and mixing at 56-58 deg.C.
2) Digestion is carried out at 53-55 ℃ for 22-24 hours, and stirring is carried out fully every hour for the first 10 hours.
3) Extracting supernatant, heating to 80deg.C, adding peptone, boiling, and adjusting pH to 7.6-7.8. Boiling for 10 min, filtering or precipitating to obtain supernatant, adding dextrin, dissolving, and packaging.
4) And autoclaving at 116℃for 40 minutes.
(2) Immunization program
Performing basic immunization on 1.5-2.0kg healthy rabbits twice by using a first immunogen, and performing intramuscular injection of 1 ml/animal; two booster immunizations were then performed with the second immunogen and the ear margin was intravenously injected with 1ml of clostridium perfringens exotoxin solution a/v (concentrated toxin was diluted with physiological saline to 1 rabbit lethal dose per ml). Immunization procedures were performed as in table 3.
TABLE 3 immunization program
(3) Extraction and inspection of antitoxin serum
Heart blood is collected 10 days after the last immunogen injection, serum is separated, and clostridium perfringens type a antitoxin serum is obtained. Antitoxin serum sterility test, safety test and serum titer determination are carried out according to the national institute of biological products for livestock, china, 2000 edition. Serum titers were determined using KM mice weighing 16-20g, and the results showed that 30 mice minimum Mortem (MLD) toxins could be neutralized per 0.1ml of antitoxin serum.
Example 2
This example describes the determination of virulence of clostridium perfringens type a exotoxins in example 1, as follows:
(1) Minimum Lethal Dose (MLD) of mice
The clostridium perfringens type a exotoxins described above were diluted to the desired titer with 1% peptone water according to the dilution route shown in table 4 below, 16-20g KM mice were used, each titer toxin was injected into the mice by tail vein at a dose of 0.2 ml/x 5, and the minimum lethal dose of 0.003ml was determined by observation for 24 hours.
Table 4: toxin dilution route
(2) Minimal mortality of rabbit (MLD)
The clostridium perfringens type a exotoxins described above were diluted to the desired titer with 1% peptone water according to the dilution route shown in table 5 below, 1.5-2.0kg healthy rabbits were intravenously injected at the ear margin of each titer toxin liquid at a dose of 1.0 ml/x 5 animals, and the minimum lethal dose of the rabbits was determined to be 0.12ml (40 mice MLD) by observation for 24 hours.
Table 5: toxin dilution route
Example 3
In this example, the antitoxic performance of clostridium perfringens antitoxin a serum obtained in example 1 was tested by using a rabbit emergency immunization test, and the test is specifically as follows:
(1) The test process comprises the following steps:
rabbit emergency immunization test was performed using clostridium perfringens type a antitoxin serum prepared in example 1 of the present invention. Taking 10 healthy rabbits of 1.5-2.0kg, and dividing the rabbits into a test group and a control group, wherein each group comprises 5 rabbits. Both rabbits were given an ear margin intravenous injection of 1 ml/dose of clostridium perfringens exotoxin solution a (concentrated toxin was diluted with physiological saline to a lethal dose per ml of 1 rabbit), and the test group was given an intravenous injection of 1ml of antitoxin serum after challenge, and observed for 24 hours.
(2) Results:
all rabbits in the test group 5 survived, and all rabbits in the control group died within 6 hours.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
- A method for preparing clostridium perfringens type a antitoxin serum, characterized in that a first immunogen is used for at least two basic immunizations on a healthy animal, then a second immunogen is used for at least two booster immunizations on the healthy animal, and clostridium perfringens type a antitoxin serum is obtained; the first immunogen takes clostridium perfringens type A toxin as an immunogen component; the second immunogen takes clostridium perfringens A exotoxin as an immunogenic component.
- 2. The method for preparing clostridium perfringens type a antitoxin serum according to claim 1 wherein said method for preparing clostridium perfringens type a exotoxin comprises: culturing clostridium perfringens CNM0708 strain a, and harvesting bacterial liquid; filtering clostridium perfringens A in the bacterial liquid to obtain clear liquid; concentrating the clear liquid by using hollow fibers to obtain clostridium perfringens exotoxin solution A.
- 3. The method for preparing clostridium perfringens type a antitoxin serum according to claim 2 wherein after the bacterial liquid is centrifugated for 20-30min at 6000-8000r/min, the clostridium perfringens type a in the bacterial liquid is filtered out by using a 0.22-0.45 μm filter membrane to obtain clear liquid; and/or concentrating the clear liquid by using 5-10KD hollow fibers, wherein the concentration degree is 10-15 times of concentration.
- 4. A method for preparing clostridium perfringens type a antitoxin serum according to claim 3 wherein said clostridium perfringens type a toxin is prepared from said clostridium perfringens type a exotoxin solution by formaldehyde inactivation and detoxification.
- 5. The method for preparing clostridium perfringens type a antitoxin serum according to claim 4 wherein said clostridium perfringens type a exotoxin solution is inactivated and detoxified with formaldehyde solution to obtain clostridium perfringens type a toxin solution; the addition amount of the formaldehyde solution is 0.5-0.8 vol% of the clostridium perfringens exotoxin solution A.
- 6. The method for preparing clostridium perfringens type a antitoxin serum according to claim 5 wherein said first immunogen is prepared from clostridium perfringens type a toxin solution and aluminum hydroxide gel in a mass ratio of 4-5:1.
- 7. The method for preparing clostridium perfringens type a antitoxin serum according to any one of claims 1-6 wherein said healthy animal is 1.5-2.0kg healthy rabbit.
- 8. The method for preparing clostridium perfringens type a antitoxin serum according to claim 7 wherein said basic immunization is performed twice at a time interval of 14-28 days; the times of the boosting are two times, and the time interval between the two times of the boosting is 7-14 days; the time interval between the second basal immunization and the first booster immunization is 7-21 days.
- 9.A clostridium perfringens antitoxin serum obtainable by a process according to any one of claims 1 to 8.
- 10. A method of preparing clostridium perfringens type a antitoxin serum according to any one of claims 1-8 or the use of clostridium perfringens type a antitoxin serum according to claim 9 for the preparation of pharmaceutical formulations comprising antitoxin serum.
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| CN104829712A (en) * | 2015-04-23 | 2015-08-12 | 山东农业大学 | C and D type C.perfringens antitoxin serum and preparation method thereof |
| CN108892725A (en) * | 2018-04-18 | 2018-11-27 | 山东农业大学 | A kind of preparation method of E type C.perfringens antitoxic serum |
| CN109954135A (en) * | 2017-12-25 | 2019-07-02 | 金宇保灵生物药品有限公司 | Ox A type C.perfringens inactivates toxoid vaccine and preparation method thereof |
| CN111500482A (en) * | 2019-01-31 | 2020-08-07 | 内蒙古金宇保灵生物技术研究院有限公司 | Sheep A-type clostridium perfringens strain, inactivated vaccine thereof and vaccine preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104829712A (en) * | 2015-04-23 | 2015-08-12 | 山东农业大学 | C and D type C.perfringens antitoxin serum and preparation method thereof |
| CN109954135A (en) * | 2017-12-25 | 2019-07-02 | 金宇保灵生物药品有限公司 | Ox A type C.perfringens inactivates toxoid vaccine and preparation method thereof |
| CN108892725A (en) * | 2018-04-18 | 2018-11-27 | 山东农业大学 | A kind of preparation method of E type C.perfringens antitoxic serum |
| CN111500482A (en) * | 2019-01-31 | 2020-08-07 | 内蒙古金宇保灵生物技术研究院有限公司 | Sheep A-type clostridium perfringens strain, inactivated vaccine thereof and vaccine preparation method |
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