CN104524569A - Infectious bronchitis egg yolk antibody oral solution and preparation method thereof - Google Patents
Infectious bronchitis egg yolk antibody oral solution and preparation method thereof Download PDFInfo
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- CN104524569A CN104524569A CN201410762568.7A CN201410762568A CN104524569A CN 104524569 A CN104524569 A CN 104524569A CN 201410762568 A CN201410762568 A CN 201410762568A CN 104524569 A CN104524569 A CN 104524569A
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Abstract
The invention discloses an infectious bronchitis egg yolk antibody oral solution and a preparation method thereof. According to a formula, the infectious bronchitis egg yolk antibody oral solution comprises the following raw materials in percentage by volume: 95-105 of a whole-egg water solution containing an egg yolk antibody, 0.2-0.3 of octanoic acid, 0.2-0.3 of formaldehyde, 0.01-0.02 of merthiolate and 800-1000 units of mycillin in every milliliter of the oral solution. Whole eggs with high immunoglobulin are adopted during preparation, so that the clear egg yolk antibody oral solution is obtained. The egg yolk antibody oral solution prepared by using the preparation method is not only low in loss, high in immune active substance content, high in biological activity and free of clinical side effects in the production process, but also contains a great number of various proteins in egg white, so that the adverse effects on the antibody can be reduced during oral administration.
Description
Technical field
The invention belongs to birds biological product technical field, relate to a kind of infectious bronchitis yolk antibody oral liquid and preparation method thereof.
Background technology
Infectious bronchitis of chicken is the acute high degree in contact respiratory infectious disease of one of the chicken that infectious bronchitis virus causes, and belongs to coronaviridae coronavirus genus.This virus is not strong to environmental resistance, irritated to common disinfectant, has certain resistance to low temperature.Infectious bronchitis virus has very strong variability, has isolated more than 30 serotype in the world at present.In these strains, majority can make trachea produce specific lesions, and some strain can cause nephropathy and Genital canal lesion.Mainly through air borne, also the propagation such as feedstuff, drinking-water, bedding and padding can be passed through.Face and examine feature and be dyspnea, send rale, cough, mouth breathing, sneeze.Primary disease is widely current in all over the world, is the important epidemic disease of poultry husbandry.
A kind of Mitochondrion IgG that yolk antibody IgY is, IgY is functionally equivalent to mammal IgG, but different in structure, has the space conformation of typical immunoglobulin.Yolk antibody has lot of superiority.(1) due to the gap of birds and mammal phylogenetics, birds are more suitable for the specific antibody of producing resisting mammal antigen.Yolk antibody can not excite complement system, does not react with the antibody of resisting mammal antigen, and not with the Fc receptors bind of mammal and antibacterial, this is significant in immunologic diagnosis.(2) yolk antibody has bioactive immunoglobulin as one, is storing, produces, processes, ingests and ensureing in digestion process that its stability is very crucial.Multitest shows, IgY has good stability, acidproof, alkaline-resisting, heat-resisting.Yolk antibody is more easily preserved, and deposits 6 months antagonist activity and have no significant effect under 4 DEG C of conditions under depositing 5 years or room temperature condition.(3) yolk antibody amount is large, and cost is low, is convenient to large-scale production.Antibody from yolk concentration energy long term maintenance is being equivalent to even exceed in the level of Serum antibody concentrations.Generally, yolk antibody does not have toxic and side effects to animal, not only avoids the shortcoming that serum is expensive, yield poorly, the side effect that can serum avoided to cause over the course for the treatment of by yolk antibody treatment Animal diseases.Therefore, the application that yolk antibody is diagnosed at diseases of bird and livestock, treats and prevented gets more and more.The preparation of current yolk antibody and production technology are ripe, the production that can guarantee both quality and quantity.
Summary of the invention
The invention provides a kind of infectious bronchitis yolk antibody oral liquid and preparation method thereof, solve the infectious bronchitis yolk antibody inconvenient problem with use of injection.
The object of the invention is to be realized by following form:
A kind of infectious bronchitis yolk antibody oral liquid, comprises the following raw material calculated according to volume ratio: containing the shell egg aqueous solution 95-105 of yolk antibody, sad 0.2-0.3, formaldehyde 0.2-0.3, thimerosal 0.01-0.02; Also comprise the mycillin of 800-1000 units per ml oral liquid.
Preparation process is:
1) inactivated vaccine for avian infectious bronchitis is adopted to carry out reinforced immunological to chicken: adopt three immune programme for children, every minor tick 10 days, every each vaccine consumption of chicken is 2ml, when antibody titer reaches 1:(128-256) time, carry out high-immunity egg collection;
2) 0.1-0.2% bromo geramine is used to carry out disinfection to high-immunity egg;
3) by 2) in the egg pulp of high-immunity egg carry out emulsifying, emulsification times 7-10min, then add pH be 4.2-4.3, temperature is the hcl acidifying water of 4-6 DEG C, the addition of hcl acidifying water is 5.5-6.5 times of egg pulp volume, after mixing, be cooled to 3-5 DEG C, leave standstill 18-24 hour;
4) get 3) in the supernatant, be warmed up to 25-28 DEG C, it is sad to add, stir 1-2h, leave standstill 10-12h, filter; Wherein sad volume: supernatant volume is (0.2-0.3): (95-105);
5) add formaldehyde, thimerosal, mycillin, make the shell egg aqueous solution of yolk antibody: sad: formaldehyde: thimerosal
Volume ratio be (95-105): (0.2-0.3): (0.2-0.3): (0.01-0.02), the content of mycillin is 800-1000 units per ml oral liquid;
6) subpackage, preserves.
Filtered by the filter membrane of 1.0mm, 0.45mm, 0.22mm successively when step 4) is filtered.
The present invention is by clarifying yolk antibody oral liquid to obtaining after the extraction of high-immunity egg shell egg, low temperature acidification, sad process.Shell egg uses in the extraction of yolk antibody by the present invention, can directly orally use clinically, containing a large amount of different kinds of proteins in Ovum Gallus domesticus album, the value of high-immunity egg can be made full use of, improve immunity and the stress ability of animal body, bioavailability is high, has the impregnable advantage of biological activity.
Detailed description of the invention
Embodiment 1:
The formula of infectious bronchitis yolk antibody oral liquid comprises following raw material: by shell egg aqueous solution 95 L, sad 0.2 L, formaldehyde 0.3 L, thimerosal 0.02 L containing yolk antibody, and the mycillin of 1000 units per ml oral liquids.
Its preparation method comprises the following steps:
1) adopt commercially available inactivated vaccine for avian infectious bronchitis (H120 strain) to carry out reinforced immunological, every each vaccine consumption of chicken is 2ml, and every minor tick 10 days, carries out three immune programme for children, when antibody titer reaches 1:256, starts to collect high-immunity egg.
2) high-immunity egg will be collected arrange, 0.2% bromo geramine sterilization, for subsequent use.
3) water dilution method acidification: by 2) step high-immunity egg shell egg squeeze into carry out in Pasteur's tank body fully emulsified, emulsification times 7min, then the egg pulp after emulsifying is added the hcl acidifying water that pH is 4.2, temperature is 4 DEG C, hcl acidifying water addition is 6 times of egg pulp volume, mixing, place in 4 DEG C of environment and staticly settle for 18 hours, get supernatant; Wherein, hcl acidifying water in water, adds hydrochloric acid make its pH value reach 4.2.
4) sad process; By the supernatant of step 3), the shell egg aqueous solution namely containing yolk antibody weighs, and be warmed up to 25 DEG C, it is sad to add, and fully mixes 1h, then leaves standstill 10h; Wherein, by volume, the supernatant: sad=95:0.2;
5) step 4) gained clear liquor is used successively to the membrane filtration of 1.0mm, 0.45mm, 0.22mm, leave and take filtrate;
6) to step 5) gained filtrate adds formaldehyde, thimerosal, mycillin; By volume, containing the shell egg shell egg aqueous solution of yolk antibody: sad: formaldehyde: thimerosal=95:0.2:0.3:0.02; The content of mycillin in this oral liquid is 1000 units per ml;
7) subpackage, preserves.
For determining yolk antibody oral liquid placement effect duration at normal temperatures, do following test: this product be kept in 37 DEG C of incubators, preserve 24 months, 1 week, 2 weeks respectively, January, February, April, August, December, after 16 months, 20 months, 24 months, by agar diffusion (AGP) method, the yolk antibody measuring each point in time sampling product is tired change, the results are shown in Table 1.
Table 1. room temperature places the change that oral liquid different time sections is tired
result shows:yolk antibody can deposit 16 months under the preservation of room temperature.
Quality standard:
[character] this product is weak yellow liquid.
[steriling test] is undertaken by " Chinese veterinary pharmacopoeia ", asepsis growth.
[mycoplasma inspection] is undertaken by " Chinese veterinary pharmacopoeia ", grows without mycoplasma.
[exogenous virus inspection] is undertaken by " Chinese veterinary pharmacopoeia ", conforms with the regulations.
[safety verification] with 7 Japanese instar chickling 5, each oral 5 plumage parts, observe 2 weeks, all should be good for and live.
[efficacy test]
1) protection detects: with 7 Japanese instar chickling 30, wherein 10, often only oral 2 plumage part yolk antibodies, and another 20 contrast isolated rearings.24h, get 10 rear virus liquids of chickling intramuscular injection inoculation IBV dilution in test group and matched group, record 96h death condition, the protective rate of test group is more than 95%, and counteracting toxic substances matched group mortality rate is more than 92%, and chickling is not any change blank group.
2) healing power measures: with 7 Japanese instar chickling 120, make negative control for 40, the virus liquid of 80 eye dripping inoculation IBV dilution, and after infection 12h, wherein 40, often only oral this product 2 plumage part, every day 1 time, continuous 3 days, another 40 were done positive control, isolated rearing.72h after treatment, record each group of death condition, counteracting toxic substances positive controls mortality rate is more than 91%, and treatment group survival rate is more than 93%, and healthy negative control group 40 is not only any change.
Embodiment 2:
The formula of infectious bronchitis yolk antibody oral liquid comprises following raw material: by shell egg aqueous solution 105 L, sad 0.2 L, formaldehyde 0.2 L, thimerosal 0.01 L containing yolk antibody, and the mycillin of 800 units per ml oral liquids.
Its preparation method comprises the following steps:
1) adopt commercially available inactivated vaccine for avian infectious bronchitis (H120 strain) to carry out reinforced immunological, every each vaccine consumption of chicken is 2ml, and every minor tick 10 days, carries out three immune programme for children, when antibody titer reaches 1:128, starts to collect high-immunity egg.
2) high-immunity egg will be collected arrange, 0.1% bromo geramine sterilization, for subsequent use.
3) water dilution method acidification: by 2) step high-immunity egg shell egg squeeze into carry out in Pasteur's tank body fully emulsified, emulsification times 10min, then the egg pulp after emulsifying is added the hcl acidifying water that pH is 4.3, temperature is 6 DEG C, hcl acidifying water addition is 5.5 times of egg pulp volume, mixing, place in 3 DEG C of environment and staticly settle for 24 hours, get supernatant; Wherein, hcl acidifying water in water, adds hydrochloric acid make its pH value reach 4.3.
4) sad process; By the supernatant of step 3), the shell egg aqueous solution namely containing yolk antibody weighs, and be warmed up to 32 DEG C, it is sad to add, and fully mixes 2h, then leaves standstill 12h; Wherein, by volume, the supernatant: sad=105:0.2;
5) step 4) gained clear liquor is used successively to the membrane filtration of 1.0mm, 0.45mm, 0.22mm, leave and take filtrate;
6) to step 5) gained filtrate adds formaldehyde, thimerosal, mycillin; By volume, containing the shell egg shell egg aqueous solution of yolk antibody: sad: formaldehyde: thimerosal=105:0.2:0.2:0.01; The content of mycillin in this oral liquid is 800 units per ml;
7) subpackage, preserves.
For determining yolk antibody oral liquid placement effect duration at normal temperatures, do following test: this product be kept in 37 DEG C of incubators, preserve 24 months, 1 week, 2 weeks respectively, January, February, April, August, December, after 16 months, 20 months, 24 months, by agar diffusion (AGP) method, the yolk antibody measuring each point in time sampling product is tired change, the results are shown in Table 1.The results are shown in Table 2.
Table 2. room temperature places the change that injection different time sections is tired
result shows:yolk antibody can deposit 16 months under the preservation of room temperature.
Quality standard:
[character] this product is weak yellow liquid.
[steriling test] is undertaken by " Chinese veterinary pharmacopoeia ", asepsis growth.
[mycoplasma inspection] is undertaken by " Chinese veterinary pharmacopoeia ", grows without mycoplasma.
[exogenous virus inspection] is undertaken by " Chinese veterinary pharmacopoeia ", conforms with the regulations.
[safety verification] with 7 Japanese instar chickling 5, each oral 5 plumage parts, observe 2 weeks, all should be good for and live.
[efficacy test]
1) protection detects: with 7 Japanese instar chickling 30, wherein 10, often only oral 2 plumage part yolk antibodies, and another 20 contrast isolated rearings.24h, get 10 rear virus liquids of chickling intramuscular injection inoculation IBV dilution in test group and matched group, record 96h death condition, the protective rate of test group is more than 95%, and counteracting toxic substances matched group mortality rate is more than 92%, and chickling is not any change blank group.
2) healing power measures: with 7 Japanese instar chickling 120, make negative control for 40, the virus liquid of 80 eye dripping inoculation IBV dilution, and after infection 12h, wherein 40, often only oral this product 2 plumage part, every day 1 time, continuous 3 days, another 40 were done positive control, isolated rearing.72h after treatment, record each group of death condition, counteracting toxic substances positive controls mortality rate is more than 85%, and treatment group survival rate is more than 92%, and healthy negative control group 40 is not only any change.
Embodiment 3:
The formula of infectious bronchitis yolk antibody oral liquid comprises following raw material: by shell egg aqueous solution 100L, the sad 0.3L, formaldehyde 0.3 L, thimerosal 0.02 L that contain yolk antibody, and the mycillin of 1000 units per ml oral liquids.
Its preparation method comprises the following steps:
1) adopt commercially available inactivated vaccine for avian infectious bronchitis (H120 strain) to carry out reinforced immunological, every each vaccine consumption of chicken is 2ml, and every minor tick 10 days, carries out three immune programme for children, when antibody titer reaches 1:256, starts to collect high-immunity egg.
2) high-immunity egg will be collected arrange, 0.2% bromo geramine sterilization, for subsequent use.
3) water dilution method acidification: by 2) step high-immunity egg shell egg squeeze into carry out in Pasteur's tank body fully emulsified, emulsification times 8min, then the egg pulp after emulsifying is added the hcl acidifying water that pH is 4.3, temperature is 5 DEG C, hcl acidifying water addition is 6.5 times of egg pulp volume, mixing, place in 5 DEG C of environment and staticly settle for 20 hours, get supernatant; Wherein, hcl acidifying water in water, adds hydrochloric acid make its pH value reach 4.3.
4) sad process; By the supernatant of step 3), the shell egg aqueous solution namely containing yolk antibody weighs, and be warmed up to 30 DEG C, it is sad to add, and fully mixes 2h, then leaves standstill 11h; Wherein, by volume, the supernatant: sad=100:0.3;
5) step 4) gained clear liquor is used successively to the membrane filtration of 1.0mm, 0.45mm, 0.22mm, leave and take filtrate;
6) to step 5) gained filtrate adds formaldehyde, thimerosal, mycillin; By volume, containing the shell egg shell egg aqueous solution of yolk antibody: sad: formaldehyde: thimerosal=100:0.3:0.3:0.02; The content of mycillin in this oral liquid is 1000 units per ml;
7) subpackage, preserves.
With 7 Japanese instar chickling 100, first group 50 this product of feeding groups, only penetrate liquid product group (Liaoning Yikang, 201305012) as commercially available yolk antibody liquid for second group 50, isolated rearing, the virus liquid of eye dripping inoculation IBV dilution, when showing clinical symptoms, starts treatment, first group of every only oral this product 2ml, every day 1 time, continuous 3 days, second group of commercially available prod 2 ml, every day 1 time, continuous 3 days.72h after treatment, records each group of morbidity and treatment situation.Table 3.
Table 3 experimental conditions
Embodiment 4:
A kind of formula of infectious bronchitis yolk antibody oral liquid comprises following raw material: calculate according to volume ratio, by containing the shell egg aqueous solution 98 of yolk antibody, the mycillin of sad 0.3, formaldehyde 0.2, thimerosal 0.01,1000 units per ml.
Its preparation method comprises the following steps:
1) adopt commercially available inactivated vaccine for avian infectious bronchitis (H120 strain) to carry out reinforced immunological, every each vaccine consumption of chicken is 2ml, and every minor tick 10 days, carries out three immune programme for children, when antibody titer reaches 1:128, starts to collect high-immunity egg.
2) high-immunity egg will be collected arrange, 0.1% bromo geramine sterilization, for subsequent use.
3) water dilution method acidification: by 2) step high-immunity egg shell egg squeeze into carry out in Pasteur's tank body fully emulsified, emulsification times 9min, then the egg pulp after emulsifying is added the hcl acidifying water that pH is 4.3, temperature is 6 DEG C, hcl acidifying water addition is 5.5 times of egg pulp volume, mixing, place in 5 DEG C of environment and staticly settle for 22 hours, get supernatant; Wherein, hcl acidifying water in water, adds hydrochloric acid make its pH value reach 4.3.
4) sad process; By the supernatant of step 3), the shell egg aqueous solution namely containing yolk antibody weighs, and be warmed up to 28 DEG C, it is sad to add, and fully mixes 2h, then leaves standstill 12h; Wherein, by volume, the supernatant: sad=98:0.3;
5) step 4) gained clear liquor is used successively to the membrane filtration of 1.0mm, 0.45mm, 0.22mm, leave and take filtrate;
6) to step 5) gained filtrate adds formaldehyde, thimerosal, mycillin; By volume, containing the shell egg shell egg aqueous solution of yolk antibody: sad: formaldehyde: thimerosal=98:0.3:0.2:0.01; The content of mycillin in this oral liquid is 1000 units per ml;
7) subpackage, preserves.
Embodiment 5:
The formula of infectious bronchitis yolk antibody oral liquid comprises following raw material: by shell egg aqueous solution 102 L, sad 0.2 L, formaldehyde 0.3 L, thimerosal 0.01 L containing yolk antibody, and the mycillin of 800 units per ml oral liquids.
Its preparation method comprises the following steps:
1) adopt commercially available inactivated vaccine for avian infectious bronchitis (H120 strain) to carry out reinforced immunological, every each vaccine consumption of chicken is 2ml, and every minor tick 10 days, carries out three immune programme for children, when antibody titer reaches 1:256, starts to collect high-immunity egg.
2) high-immunity egg will be collected arrange, 0.2% bromo geramine sterilization, for subsequent use.
3) water dilution method acidification: by 2) step high-immunity egg shell egg squeeze into carry out in Pasteur's tank body fully emulsified, emulsification times 10min, then the egg pulp after emulsifying is added the hcl acidifying water that pH is 4.2, temperature is 4 DEG C, hcl acidifying water addition is 6 times of egg pulp volume, mixing, place in 5 DEG C of environment and staticly settle for 24 hours, get supernatant; Wherein, hcl acidifying water in water, adds hydrochloric acid make its pH value reach 4.2.
4) sad process; By the supernatant of step 3), the shell egg aqueous solution namely containing yolk antibody weighs, and be warmed up to 30 DEG C, it is sad to add, and fully mixes 2h, then leaves standstill 12h; Wherein, by volume, the supernatant: sad=102:0.2;
5) step 4) gained clear liquor is used successively to the membrane filtration of 1.0mm, 0.45mm, 0.22mm, leave and take filtrate;
6) to step 5) gained filtrate adds formaldehyde, thimerosal, mycillin; By volume, containing the shell egg shell egg aqueous solution of yolk antibody: sad: formaldehyde: thimerosal=102:0.2:0.3:0.01; The content of mycillin in this oral liquid is 800 units per ml;
7) subpackage, preserves.
Claims (3)
1. an infectious bronchitis yolk antibody oral liquid, is characterized in that: comprise the following raw material calculated according to volume ratio: containing the shell egg aqueous solution 95-105 of yolk antibody, sad 0.2-0.3, formaldehyde 0.2-0.3, thimerosal 0.01-0.02; Also comprise the mycillin of 800-1000 units per ml oral liquid.
2. a preparation method for infectious bronchitis yolk antibody oral liquid as claimed in claim 1, is characterized in that concrete steps are:
1) inactivated vaccine for avian infectious bronchitis is adopted to carry out reinforced immunological to chicken: adopt three immune programme for children, every minor tick 10 days, every each vaccine consumption of chicken is 2ml, when antibody titer reaches 1:(128-256) time, carry out high-immunity egg collection;
2) 0.1-0.2% bromo geramine is used to carry out disinfection to high-immunity egg;
3) by 2) in the egg pulp of high-immunity egg carry out emulsifying, emulsification times 7-10min, then add pH be 4.2-4.3, temperature is the hcl acidifying water of 4-6 DEG C, the addition of hcl acidifying water is 5.5-6.5 times of egg pulp volume, after mixing, be cooled to 3-5 DEG C, leave standstill 18-24 hour;
4) get 3) in the supernatant, be warmed up to 25-28 DEG C, it is sad to add, stir 1-2h, leave standstill 10-12h, filter; Wherein sad volume: supernatant volume is (0.2-0.3): (95-105);
5) formaldehyde, thimerosal, mycillin is added, make the shell egg aqueous solution of yolk antibody: sad: formaldehyde: the volume ratio of thimerosal is (95-105): (0.2-0.3): (0.2-0.3): (0.01-0.02), and the content of mycillin is 800-1000 units per ml oral liquid;
6) subpackage, preserves.
3. the preparation method of infectious bronchitis yolk antibody oral liquid according to claim 2, is characterized in that: filtered by the filter membrane of 1.0mm, 0.45mm, 0.22mm successively when step 4) is filtered.
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Non-Patent Citations (1)
| Title |
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| 张小莺 等: "《卵黄抗体技术》", 31 May 2011 * |
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Application publication date: 20150422 |
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