CN1283663C - Antithemolytic streptococcus, staphylococcus and candida albicans lgG antibody and preparation thereof - Google Patents
Antithemolytic streptococcus, staphylococcus and candida albicans lgG antibody and preparation thereof Download PDFInfo
- Publication number
- CN1283663C CN1283663C CN 200510031150 CN200510031150A CN1283663C CN 1283663 C CN1283663 C CN 1283663C CN 200510031150 CN200510031150 CN 200510031150 CN 200510031150 A CN200510031150 A CN 200510031150A CN 1283663 C CN1283663 C CN 1283663C
- Authority
- CN
- China
- Prior art keywords
- staphylococcus
- candida albicans
- immunogen
- igy antibody
- hemolytic streptococcus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The present invention discloses a specific chicken IgG antibody resisting hemolytic streptococcus, staphylococcus and candida albicans, and a preparation method thereof. In the method, the thallus and the bacterium fragments of hemolytic streptococcus, staphylococcus and candida albicans are prepared into Freund's complete adjuvant immunogens and Freund's incomplete adjuvant immunogens; egg laying hens are immunized in a subcutaneous multipoint mode; chicken IgG antibodies are coarsely extracted from egg yolk of the immunized hens by a water dilution method; the ammonium sulfate salting out method is used for purifying the chicken IgG antibodies for obtaining chicken IgG antibodies resisting hemolytic streptococcus, staphylococcus and candida albicans. The products such as spraying agents, aerosols, nose dropping liquid, buccal tablets, etc., prepared by the specific chicken IgG antibody of the present invention resisting hemolytic streptococcus, staphylococcus and candida albicans can be used for treating upper respiratory tract infection inflammation such as acute or chronic rhinitis; chronic rhinitis, nasosinusitis, tonsillitis, pharyngitis, laryngitis, bronchitis, etc. caused by streptococcus, staphylococcus and candida albicans.
Description
Technical field
The present invention relates to a kind of antibody and preparation method thereof, particularly a kind of anti-hemolysis suis, staphylococcus and candida albicans IgY antibody and preparation method.
Background technology
Hemolytic streptococcus has the many different kinds and the bacterial strain of type, and they go back the common antigen of various degrees and different quantities except specific separately antigen or title identification antigen is arranged; Staphylococcus also is so, and the existence of common antigen is arranged between the different kinds and the bacterial strain of type; Exist common antigen between the candidiasis bacterial strain of kind that Candida albicans is different and type.Around this principle, we use several strain Hemolytic streptococcuss, staphylococcus and Candida albicans immunity bird inlay, the chicken IgY antibody of specificity anti-hemolysis suis, staphylococcus and Candida albicans that this chicken produces just can be not only and the bacterial isolates generation specific antigens-antibody response of immunity, and can with the common antigen that has of not of the same race and type, that is: the bacterial strain of common antigenic determinant produces antigen-antibody response.When chicken IgY antibody and bacterium surface antigen generation antigen-antibody response, just may change the bacterium surface structure, suppress bacterium some functionally activies, bacteria growing inhibiting, minimizing and inhibition bacterium pair cell stick, promote phagocytic cells such as segmented cell and scavenger cell engulfing and remove to bacterium.Moreover, use specificity chicken IgY antibody preventing dental caries and treatment infant rotavirus diarrhoea (Randomized clinically, placebo-controlled, clinical trial ofhyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea.J PediatrGastroenterol Nutr.2001 Jan; 32 (1): 19-25), prevention gallbladder cystic fibrosis patient charrin's disease (Oraladministration of specific yolk antibodies (IgY) may prevent Pseudomonas aeruginosa infections inpatients with cystic fibrosis:A phase I feasibility study.Pediatr Pulmonol.2003 Jun; 35 (6): 433-40), alleviate the microbial gastritis of helicobacter pylorus (Effect of dietary anti-Helicobacter pylori-urease immunoglobulin Y onHelicobacter pylori infection.Aliment Pharmacol Ther.2004 Jul; 20 Suppl 1:185-92), in addition the protection monkey by intracellular toxin B infringement (Retention of specific yolk IgY in the human oral cavity.BioDrugs.2002; 16 (6): 433-7).
Specificity anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody are different with the antibiotic action principle, it combines with bacterium surface corresponding antigens generation specificity, change this antigenic state, sealed antigenic function and the activity that influences bacterium; Covered bacterium surface and tissue, cell bonded acceptor, stoped bacterial adhesion, repelled the existence of bacterium at inflammation part in tissue, cell; Changed bacterium surface structure and electric field, promoted phagocytic cells such as segmented cell and scavenger cell to the engulfing and removes of bacterium, the while can not be brought out the generation of Resistant strain again fully.Therefore current antibiotic abuse and widely Resistant strain deposit under the living situation, more demonstrate the importance and the feasibility of specificity anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody preparation.Action principle according to above-mentioned specificity chicken IgY antibody, specificity anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody preparation can be treated all effectively by Hemolytic streptococcus, staphylococcus and Candida albicans, and have bacterium and a fungus-caused upper respiratory tract infection inflammation that common antigenic determinant is arranged with these three kinds of bacterium, as: anxious, chronic rhinitis, sinusitis paranasal sinusitis, tonsillitis, pharyngitis, laryngitis, bronchitis etc.
Summary of the invention
The purpose of this invention is to provide a specific specificity anti-hemolysis suis, staphylococcus and candida albicans IgY antibody and preparation method thereof, and with this Antybody therapy by Hemolytic streptococcus and staphylococcus, and the upper respiratory tract infection inflammation that causes of Candida albicans, as: anxious, chronic rhinitis, sinusitis paranasal sinusitis, tonsillitis, pharyngitis, laryngitis, bronchitis etc.
Purpose of the present invention can take following method to realize:
A kind of anti-hemolysis suis, staphylococcus and candida albicans IgY antibody is characterized in that it is obtained by following method:
(1) immunogen preparing:
Get 50～70 parts of morbific Hemolytic streptococcus and Hemolytic streptococcus bacterial chips, 20～40 parts of staphylococcus and staphylococcus bacterial chips, Candida albicans and Candida albicans bacterial chip are mixed into bacterium liquid for 10～20 parts;
The bacterium liquid of equivalent volumes is mixed with the Fu Shi Freund's complete adjuvant of equivalent volumes, and stirring and emulsifying is made water-in-oil Fu Shi Freund's complete adjuvant Hemolytic streptococcus, staphylococcus and Candida albicans immunogen;
The bacterium liquid of equivalent volumes is mixed with the freund 's incomplete adjuvant of equivalent volumes, and stirring and emulsifying is made water-in-oil freund 's incomplete adjuvant Hemolytic streptococcus, staphylococcus and Candida albicans immunogen;
In every milliliter of freunds complete adjuvant immunogen and the freunds incomplete adjuvant immunogen, all contain bacterium 2,100,000,000～2,700,000,000;
(2) immunity:
Give bird inlay with the subcutaneous and muscle multi-point injection of freunds complete adjuvant immunogen, every bird inlay injection 1ml immunogen, use the subcutaneous and muscle multi-point injection of freunds incomplete adjuvant immunogen 1ml to give bird inlay then week about, after using freunds incomplete adjuvant immunogen booster immunization secondary, freunds incomplete adjuvant immunogen 1ml is subcutaneous to give bird inlay booster immunization secondary with the muscle multi-point injection every re-using in three weeks, front and back immune five times altogether;
The immunity back was collected the egg of immune hen on the 30th day for the first time.Anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody that the egg of the 30th day to the 300th day the immune hen in immune for the first time back all has height to tire;
(3) preparation of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract:
Collect the egg of the immune hen of immunity after 30 days, the egg surface disinfects in alcohol, get yolk, remove membrane of yolk and get egg yolk liquid, ratio adding distil water with egg yolk liquid and distilled water 1: 7 by volume～9, fully stir, and with 1N HCl adjusting potential of hydrogen to pH5.0～5.2, under 4 ℃ of temperature, left standstill 10 hours;
With yolk diluent high speed centrifugation 30 minutes, get supernatant, it is 5～8 times of the hollow fiber membrane ultrafiltration device ultrafiltration and concentration of 100kDa that yolk diluent supernatant is placed molecular weight cut-off, adding 0.02%～2.0% chlorhexidine gluconate then fully stirs, and use 1N NaOH to regulate potential of hydrogen, and under 4 ℃ of temperature, left standstill 10 hours to pH7.0～7.5; After this high speed centrifugation is 15 minutes, gets supernatant, and the weeding of grease metallic substance with the sterilization filtering with microporous membrane degerming of supernatant through 0.45 μ～0.22 μ, promptly gets anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract.
Hemolytic streptococcus in the described immunogen preparing and Hemolytic streptococcus bacterial chip are beta hemolytic streptococcus and bacterial chip, and described staphylococcus and staphylococcus bacterial chip are streptococcus aureus and bacterial chip.
The bacterium in the described immunogen preparing and the ratio of bacterial chip are 50～80 parts on bacteriums, 20～50 parts of bacterial chips.
Bird inlay in the described immunity comprises the native chicken of family that raising under the conventional environment, cleaning ambient are raised down and the SPF environment is raised down, white hair Gallus Domesticus, the blue brown chicken in sea, Lai Hangji, Luo Man chicken, star chicken.
The preparation method of anti-hemolysis suis, staphylococcus and candida albicans IgY antibody comprises the steps:
With the anti-hemolysis suis after the weeding of grease metallic substance, staphylococcus and Candida albicans chicken IgY antibody crude extract add the saturation ratio of sulfuric acid amine to 55%, fully stir, room temperature left standstill 4～12 hours, high speed centrifugation is 10 minutes then, get precipitation, to precipitate and use distilled water diluting to the original volume amount, the saturation ratio that adds sulfuric acid amine to 33% again, fully stir, room temperature left standstill 4～12 hours, and high speed centrifugation is 10 minutes then, get precipitation, with the dialysis tubing of packing into behind the dissolved in distilled water, use the distill water dialysis desalination, promptly get the anti-hemolysis suis, staphylococcus and the thick pure product of Candida albicans chicken IgY antibody.
Also comprise the steps: to add pharmaceutically acceptable additives and make sprays, aerosol, nasal drops and the lozenge that contains anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody.
Press the purity of anti-hemolysis suis, staphylococcus and the Candida albicans chicken IgY antibody crude extract of method extraction of the present invention, measure through irreducibility SDS-PAGE, the purity of chicken IgY antibody reaches (〉=30%) more than 30%; And the purity of anti-hemolysis suis, staphylococcus and the thick pure product of Candida albicans chicken IgY antibody that process method of the present invention is extracted is measured through irreducibility SDS-PAGE, and the purity of chicken IgY antibody reaches (〉=85%) more than 85%.
Anti-hemolysis suis by method extraction of the present invention, the biological value of staphylococcus and Candida albicans chicken IgY antibody crude extract, measure through ELISA (enzyme-linked immunosorbent assay) method, specificity anti-hemolysis suis, the chicken IgY antibodies Hemolytic streptococcus of staphylococcus and Candida albicans, the biological value of staphylococcus and Candida albicans hybrid antigen reaches the specificity anti-hemolysis suis in every milligram of albumen, tiring of staphylococcus and Candida albicans chicken IgY antibody more than or equal to 1: 512, promptly tire 〉=1: 512/mg albumen perhaps claims in the crude extract specificity anti-hemolysis suis in every milligram of albumen, the ratio of the chicken IgY antibody of staphylococcus and Candida albicans is lived to 〉=1: 512; And the anti-hemolysis suis of extracting by method of the present invention, the biologic activity of staphylococcus and the thick pure product of Candida albicans chicken IgY antibody, that is: tire, measure through ELISA (enzyme-linked immunosorbent assay) method, specificity anti-hemolysis suis, the chicken IgY antibodies Hemolytic streptococcus of staphylococcus and Candida albicans, the biological value of staphylococcus and Candida albicans hybrid antigen reaches the specificity anti-hemolysis suis in every milligram of albumen, tiring of staphylococcus and Candida albicans chicken IgY antibody more than or equal to 1: 2048, promptly tire 〉=1: 2048/mg albumen perhaps claims in the thick pure product specificity anti-hemolysis suis in every milligram of albumen, the ratio of the chicken IgY antibody of staphylococcus and Candida albicans is lived to 〉=1: 2048.
Specificity anti-hemolysis suis, staphylococcus and the Candida albicans chicken IgY antibody part of producing by method of the present invention impose on nasal cavity, oral cavity, pharynx, larynx and bronchial mucosa, both there be not the limitation and the side effect of systemic administration, have again that local application is rapid-action, dosage is few, good effect, do not absorb, do not destroy normal microflora in nasal cavity, oral cavity, pharynx, larynx, the segmental bronchus ecotope, do not cause effect such as local flora imbalance, also have no side effect, more do not have the local problems such as easily producing resistance of using of antibiotic.Chicken IgY antibody is the activating complement system not, does not produce non-specific binding with Rheumatoid factors, polyclonal, fixing staphylococcal protein A not, and not with Protein G, and human cell FC receptors bind, thereby be difficult for taking place transformation reactions.Anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody extract do not produce acute toxic reaction and chronic toxicity reaction, belong to actual nontoxic level; It is negative that anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody extract three cause test, belongs to no teratogenesis, carcinogenic, mutagenic goods.
Embodiment
Describe implementation of the present invention in detail below in conjunction with embodiment.
Embodiment 1:
Preparation 500 gram anti-hemolysis suis, staphylococcus and the Candida albicans chicken IgY antibody crude extract technological process of production:
(1) immunogenic preparation:
Come from the isolating Hemolytic streptococcus in acute tonsillitis patient tonsilla inflammation position, staphylococcus and Candida albicans, the menses basic medium adds the blood agar and the tryptone broth culture of 10% fresh sheep blood, and the Hemolytic streptococcus of sabouraud's agar separation and Culture and amplification cultivation, staphylococcus, Candida albicans, confirm as Hemolytic streptococcus, staphylococcus and Candida albicans through evaluation.With above-mentioned thalline through the phenylic acid inactivation treatment.In addition,, the bacterium thalline is pulverized be cracked into fragment tiny and/or molecule bacterium thalline ultrasonication 30 minutes with the ultrasonic grinding machine.
Hemolytic streptococcus and Hemolytic streptococcus fragment, staphylococcus and staphylococcus fragment, Candida albicans and Candida albicans fragment all mix by 60: 30: 10 quantity parts.Wherein the ratio of bacterium and bacterial chip is 70 parts on a bacterium, 30 parts of bacterial chips.
The bacterium liquid of 200ml is mixed with the Fu Shi Freund's complete adjuvant of 200ml, use the high speed agitator stirring and emulsifying, make the water-in-oil freunds complete adjuvant immunogen.The preparation of freunds incomplete adjuvant immunogen then is: the bacterium liquid of 200ml is mixed with the freund 's incomplete adjuvant of 200ml, use the high speed agitator stirring and emulsifying, make the water-in-oil freunds incomplete adjuvant immunogen.Contain bacterium 2,100,000,000 in every milliliter of immunogen;
(2) immune bird inlay: use syringe with freunds complete adjuvant immunogen subcutaneous and tens of points of muscle inject extra large blue brown bird inlay to 27 weeks, every injection 1ml immunogen, freunds incomplete adjuvant immunogen 1ml is subcutaneous to give bird inlay with the muscle multi-point injection every using in a week then, be total to secondary, after this freunds incomplete adjuvant immunogen 1ml is subcutaneous to give bird inlay booster immunization secondary altogether with the muscle multi-point injection every re-using in three weeks, front and back immune five times altogether, immune hen adds up to 300;
(3) preparation of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract: collect the egg of 60～80 days the immune hen in immunity back for the first time, the alcohol of use 70% carries out the egg surface sterilization, get yolk, remove membrane of yolk and get egg yolk liquid, with egg yolk liquid and 1: 9 by volume ratio adding distil water of distilled water, fully stir, and use 1N HCl to regulate potential of hydrogen, placed 4 ℃ of refrigerator-freezers 10 hours to pH5.0;
Then with yolk diluent high speed centrifugation 10000rpm, 30 minutes, get supernatant, supernatant is placed 8 times of the hollow fiber membrane ultrafiltration device ultrafiltration and concentration of molecular weight cut-off 100kDa, adding 1.0% chlorhexidine gluconate then fully stirs, and use 1NNaOH to regulate potential of hydrogen to pH7.0～7.5, and place 4 ℃ 10 hours, after this high speed centrifugation 10000rpm, 15 minutes, get supernatant, the weeding of grease metallic substance, with the sterilization filtering with microporous membrane degerming of supernatant, promptly get the anti-hemolysis suis through 0.45 μ～0.22 μ, staphylococcus and Candida albicans chicken IgY antibody crude extract; Anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract are measured through ELISA (enzyme-linked immunosorbent assay) method, tiring of chicken IgY antibody in the crude extract in every milligram of albumen is 1: 1024/mg albumen claims perhaps in the crude extract that the ratio work of the chicken IgY antibody in every milligram of albumen is 1: 1024.In chicken IgY antibody crude extract, the purity of chicken IgY antibody is determined as 32.1% through irreducibility SDS-PAGE.
Embodiment 2:
Preparation 500 gram anti-hemolysis suis, staphylococcus and the thick pure product technological process of production of Candida albicans chicken IgY antibody:
(1) immunogenic preparation:
Come from the isolating beta hemolytic streptococcus in acute tonsillitis patient tonsilla inflammation position, streptococcus aureus and Candida albicans, the menses basic medium adds the blood agar and the tryptone broth culture of 10% fresh sheep blood, and the beta hemolytic streptococcus of sabouraud's agar separation and Culture and amplification cultivation, streptococcus aureus, Candida albicans, confirm as beta hemolytic streptococcus, streptococcus aureus and Candida albicans through evaluation.With above-mentioned thalline through the phenylic acid inactivation treatment.In addition,, the bacterium thalline is pulverized be cracked into fragment tiny and/or molecule bacterium thalline ultrasonication 30 minutes with the ultrasonic grinding machine.
Beta hemolytic streptococcus and beta hemolytic streptococcus fragment, streptococcus aureus and streptococcus aureus fragment, Candida albicans and Candida albicans fragment all mix by 55: 30: 15 quantity parts.Wherein the ratio of bacterium and bacterial chip is 60 parts on a bacterium, 40 parts of bacterial chips.
The bacterium liquid of 500ml is mixed with the Fu Shi Freund's complete adjuvant of 500ml, use the high speed agitator stirring and emulsifying, make the water-in-oil freunds complete adjuvant immunogen.The preparation of freunds incomplete adjuvant immunogen then is: the bacterium liquid of 500ml is mixed with the freund 's incomplete adjuvant of 500ml, use the high speed agitator stirring and emulsifying, make the water-in-oil freunds incomplete adjuvant immunogen.Contain bacterium 2,100,000,000 in every milliliter of immunogen;
(2) immune bird inlay: use syringe with freunds complete adjuvant immunogen subcutaneous and tens of points of muscle inject extra large blue brown bird inlay to 27 weeks, every injection 1ml immunogen, use the bird inlay that freunds incomplete adjuvant immunogen 1ml is subcutaneous and the muscle multi-point injection is given every a week then, be total to secondary, after this freunds incomplete adjuvant immunogen 1ml is subcutaneous to give bird inlay booster immunization secondary altogether with the muscle multi-point injection every re-using in three weeks, front and back immune five times altogether, immune hen adds up to 800;
(3) preparation of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract: collect the egg of 60～80 days the immune hen in immunity back for the first time, the alcohol of use 70% carries out the egg surface sterilization, get yolk, remove membrane of yolk and get egg yolk liquid, with egg yolk liquid and 1: 9 by volume ratio adding distil water of distilled water, fully stir, and use 1N HCl to regulate potential of hydrogen, placed 4 ℃ of refrigerator-freezers 10 hours to pH5.0;
Then with yolk diluent high speed centrifugation 10000rpm, 30 minutes, get supernatant, supernatant is placed 8 times of the hollow fiber membrane ultrafiltration device ultrafiltration and concentration of molecular weight cut-off 100kDa, adding 1.0% chlorhexidine gluconate then fully stirs, and use 1NNaOH to regulate potential of hydrogen to pH7.0～7.5, and place 4 ℃ 10 hours, after this high speed centrifugation 10000rpm, 15 minutes, get supernatant, the weeding of grease metallic substance promptly gets anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract;
With the anti-hemolysis suis after the weeding of grease metallic substance, staphylococcus and Candida albicans chicken IgY antibody crude extract add the saturation ratio of sulfuric acid amine to 55%, fully stir, room temperature left standstill 4 hours, high speed centrifugation 10000rpm is 10 minutes then, get precipitation, to precipitate and use distilled water diluting to the original volume amount, the saturation ratio that adds sulfuric acid amine to 33% again, fully stir, room temperature left standstill 4 hours, and high speed centrifugation 10000rpm is 10 minutes then, get precipitation, with the dialysis tubing of packing into after the distilled water diluting dissolving, use the distill water dialysis desalination, promptly get the anti-hemolysis suis, staphylococcus and the thick pure product of Candida albicans chicken IgY antibody; Anti-hemolysis suis, staphylococcus and the thick pure product of Candida albicans chicken IgY antibody are measured through the ELISA method, tiring of every milligram of proteic specificity chicken IgY antibody is 1 in anti-hemolysis suis, staphylococcus and the thick pure product of Candida albicans chicken IgY antibody: 2048/mg albumen claims perhaps in anti-hemolysis suis, staphylococcus and the thick pure product of Candida albicans chicken IgY antibody that the ratio work of the specificity chicken IgY antibody in every milligram of albumen is 1: 2048.In the thick pure product of chicken IgY antibody, the purity of chicken IgY antibody is determined as 91.6% through irreducibility SDS-PAGE.
Embodiment 3:
Oral spray, the tracheae aerosol of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody:
Anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract 0.20%
Mentha camphor 0.12%
Bright orange essence 0.35%
Apple flavour 0.20%
Pandan paste 0.20%
Sweetener 0.20%
Distilled water is supplemented to 100ml
Earlier, add mentioned component then successively, at last distilled water be supplemented to 100ml with the stoste of 95% dissolve with ethanol mentha camphor to 12%, through the sterilization filtering with microporous membrane degerming of 0.22 μ, sterile filling go into disinfectant sprays and aerosol jar in.
Embodiment 4:
The nasal drop of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody:
Anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract 0.30%
Bright orange essence 0.20%
Sweetener 0.10%
Distilled water is supplemented to 100ml
60ml distilled water is added in the beaker, add mentioned component more successively, at last distilled water is supplemented to 100ml, through the sterilization filtering with microporous membrane degerming of 0.22 μ, sterile filling is gone in the bottle of disinfectant nasal drop.
Embodiment 5:
The lozenge of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody:
Anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody extract 1.0%
Mentha camphor 0.20%
Bright orange essence 0.50%
Apple flavour 0.30%
Pandan paste 0.40%
Carboxymethyl cellulose 0.15%
Magnesium Stearate 0.10%
Sorbyl alcohol is supplemented to 100g
Above-mentioned lozenge medicine low temperature is granulated, be pressed into difform buccal tablet then, every 0.5g.
Embodiment 6:
Oral spray, the tracheae aerosol of anti-hemolysis suis, staphylococcus and the thick pure product of Candida albicans chicken IgY antibody:
The thick pure product 0.05% of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody
Mentha camphor 0.12%
Bright orange essence 0.35%
Apple flavour 0.20%
Pandan paste 0.20%
Sweetener 0.20%
Distilled water is supplemented to 100ml
Earlier, add mentioned component then successively, at last distilled water be supplemented to 100ml with the stoste of 95% dissolve with ethanol mentha camphor to 12%, through the sterilization filtering with microporous membrane degerming of 0.22 μ, sterile filling go into disinfectant sprays and aerosol jar in.
Experimental example 1:
Hemolytic streptococcus, staphylococcus and three kinds of bacteriums of Candida albicans and fungi immunogen are by the blue brown bird inlay in method immunity sea of the present invention, get the egg of 60～80 days the immune hen in immunity back for the first time, and prepare the crude extract of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody by method of the present invention, use the ELISA method and measure the different antigenic biological values (seeing Table 1) of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibodies:
Tiring of table 1 anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract
| Immunogen | Detect antigen | Chicken IgY antibodies is tired |
| Hemolytic streptococcus, staphylococcus and Candida albicans thalline+bacterial chip | Hemolytic streptococcus | 1: 512/mg albumen |
| Staphylococcus | 1: 256/mg albumen | |
| Candida albicans | 1: 128/mg albumen | |
| Chain, Portugal, white three kinds of thalline hybrid antigens | 1: 1024/mg albumen |
Can be clear that according to above-mentioned experimental result: (1). Hemolytic streptococcus, staphylococcus and three kinds of bacterium thalline of Candida albicans+bacterial chip immunogen immune bird inlay can produce the specificity chicken IgY antibody that resists three kinds of bacteriums and fungi respectively; (2). every kind of dosage difference (that is: ratio difference) that bacterium is used during according to immune bird inlay, tiring of the chicken of the specificity separately IgY antibody that is produced is also inequality, and tiring of the Production by Bacteria live chickens IgY antibody that immunizing dose is big is also high, otherwise then low; (3). the hybrid antigen of Hemolytic streptococcus, staphylococcus and Candida albicans chicken IgY antibody and Hemolytic streptococcus, staphylococcus and three kinds of bacterium of Candida albicans can both react, and total tiring of specificity chicken IgY antibody also increased its certain additive effect of tiring; (4). by above-mentioned experiment as seen, a kind of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody can be treated by above-mentioned three kinds of microbial urgency, chronic rhinitis, sinusitis paranasal sinusitis, tonsillitis, pharyngitis, laryngitis, bronchitis etc.; (5). simultaneously also can be with a kind of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY Antybody therapy by any bacterium or fungus-caused urgency in above-mentioned three kinds of bacterium, chronic rhinitis, sinusitis paranasal sinusitis, tonsillitis, pharyngitis, laryngitis, bronchitis etc.; (6). can also expand to thus by the bacterium and the fungus-caused urgency of common antigenic determinant being arranged with above-mentioned three kinds of bacterium, the chronic upper respiratory tract infection disease, as: anxious, chronic rhinitis, sinusitis paranasal sinusitis, tonsillitis, pharyngitis, laryngitis, bronchitis etc. can be used anti-hemolysis suis of the present invention, staphylococcus and Candida albicans chicken IgY Antybody therapy.
Experimental example 2:
Hemolytic streptococcus, staphylococcus and three kinds of bacterial immunities of Candida albicans are former in the blue brown bird inlay in method immunity sea of the present invention, get the egg of 60～80 days the immune hen in immunity back for the first time, and prepare the thick pure product of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody by method of the present invention, use the ELISA method and measure the different antigenic biological values (seeing Table 2) of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibodies:
Tiring of table 2 anti-hemolysis suis, staphylococcus and the Candida albicans chicken thick pure product of IgY antibody
| Immunogen | Detect antigen | Chicken IgY antibodies is tired |
| Hemolytic streptococcus, staphylococcus and Candida albicans thalline+bacterial chip | Hemolytic streptococcus | 〉=1: 1024/mg albumen |
| Staphylococcus | 1: 512/mg albumen | |
| Candida albicans | 1: 256/mg albumen | |
| Chain, Portugal, white three kinds of thalline hybrid antigens | 〉=1: 2048/mg albumen |
Can be clear that according to above-mentioned experimental result: (1). prepare the anti-hemolysis suis by method of the present invention, the thick pure product of staphylococcus and Candida albicans chicken IgY antibody are than preparing the anti-hemolysis suis by method of the present invention in the experimental example 1, the specificity chicken IgY antibodies Hemolytic streptococcus of the crude extract of staphylococcus and Candida albicans chicken IgY antibody, three kinds of bacterium hybrid antigens of staphylococcus and Candida albicans and Hemolytic streptococcus, it is high that three kinds of bacteriums of staphylococcus and Candida albicans and the antigenic separately biological value of fungi are all wanted, and is to increase in proportion substantially; (2). Hemolytic streptococcus, staphylococcus and the former immune bird inlay of three kinds of bacterium mixed immunities of Candida albicans can produce the specificity chicken IgY antibody that resists three kinds of bacteriums and fungi respectively; (3). every kind of dosage difference (that is: ratio difference) that bacterium is used during according to immune bird inlay, tiring of the chicken of the specificity separately IgY antibody that is produced is also inequality, and tiring of the Production by Bacteria live chickens IgY antibody that immunizing dose is big is also high, otherwise then low; (4). the hybrid antigen of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody and Hemolytic streptococcus, staphylococcus and three kinds of bacterium of Candida albicans can both react, and total tiring of specificity chicken IgY antibody also increased its certain additive effect of tiring; (5). by above-mentioned experiment as seen, a kind of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody can be treated by above-mentioned three kinds of microbial urgency, chronic rhinitis, sinusitis paranasal sinusitis, tonsillitis, pharyngitis, laryngitis, bronchitis etc.; (6). simultaneously also can be with a kind of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY Antybody therapy by any bacterium or fungus-caused urgency in above-mentioned three kinds of bacterium, chronic rhinitis, sinusitis paranasal sinusitis, tonsillitis, pharyngitis, laryngitis, bronchitis etc.; (7). can also expand to thus by the bacterium and the fungus-caused urgency of common antigenic determinant being arranged with above-mentioned three kinds of bacterium, the chronic upper respiratory tract infection disease, as: anxious, chronic rhinitis, sinusitis paranasal sinusitis, tonsillitis, pharyngitis, laryngitis, bronchitis etc. can be used anti-hemolysis suis of the present invention, staphylococcus and Candida albicans chicken IgY Antybody therapy.
Experimental example 3:
Compare the immunne response of Different Chicken kind and produce the anti-hemolysis suis; the ability of staphylococcus and Candida albicans chicken IgY antibody: with the described specific specificity anti-hemolysis suis of claim 1; the preparation method of staphylococcus and Candida albicans chicken IgY antibody crude extract; immune respectively mass-producing raise in cages sea blue brown bird inlay and Lay Hangzhoupro bird inlay; and the native chicken of putting in a suitable place to breed of laying eggs; adopt the egg of 60～80 days the immune hen in immunity back for the first time; and by the described method extraction of claim 1 anti-hemolysis suis; the crude extract of staphylococcus and Candida albicans chicken IgY antibody; use the ELISA method and measure the anti-hemolysis suis; staphylococcus and the antigenic biological value of Candida albicans chicken IgY antibodies the results are shown in Table 3:
The bird inlay of table 3 Different Chicken kind is to same antigenic immunne response ability and specificity chicken IgY antibody titer
| The chicken kind | Immunogen | Detect antigen | Chicken IgY antibodies is tired |
| The blue brown chicken in sea | Hemolytic streptococcus, staphylococcus and Candida albicans thalline+bacterial chip | Hemolytic streptococcus, staphylococcus and Candida albicans hybrid antigen | 1: 1024/mg albumen |
| Lay Hangzhoupro chicken | 1: 1024/mg albumen | ||
| The soil chicken | 1: 512～1024/mg albumen |
Can be clear that according to above-mentioned experimental result: under identical immune condition, the anti-hemolysis suis of the Different Chicken kind of preparing by method of the present invention, the crude extract of staphylococcus and Candida albicans chicken IgY antibody, its specificity anti-hemolysis suis, the biological value basically identical of staphylococcus and Candida albicans chicken IgY antibody, wherein have only the anti-hemolysis suis of native chicken in different batch preparations, in the crude extract of staphylococcus and Candida albicans chicken IgY antibody, specificity anti-hemolysis suis, the biological value of staphylococcus and Candida albicans chicken IgY antibody changes slightly, but difference is not remarkable.
Experimental example 4:
The Hemolytic streptococcus of preparing by method of the present invention, the thalline of staphylococcus and Candida albicans and Hemolytic streptococcus, two kinds of blue brown bird inlays in immunogen immune sea such as the thalline+bacterial chip of staphylococcus and Candida albicans, and prepare the anti-hemolysis suis respectively, staphylococcus and Candida albicans thalline chicken IgY antibody crude extract and anti-hemolysis suis, the thalline of staphylococcus and Candida albicans+bacterial chip chicken IgY antibody crude extract, use the ELISA method and measure their anti-hemolysis suis, the hybrid antigen of staphylococcus and Candida albicans in conjunction with biological value, the results are shown in Table 4:
The blue brown hen specific IgY in table 4 different immunogen inductive sea is in conjunction with the biological value of three kinds of bacteriums
| The chicken kind | Immunogen | Detect antigen | Chicken IgY antibody | Antibodies is tired |
| The blue brown laying hen in sea | Hemolytic streptococcus, staphylococcus and Candida albicans thalline | Hemolytic streptococcus, staphylococcus and Candida albicans hybrid antigen | Anti-hemolysis suis, staphylococcus and Candida albicans somatic antibody | 1: 512～1024/mg albumen |
| Hemolytic streptococcus, staphylococcus and Candida albicans thalline+bacterial chip | Anti-hemolysis suis, staphylococcus and Candida albicans thalline+bacterial chip antibody | 1: 1024/mg albumen |
Can be clear that according to above-mentioned experimental result: under identical immune condition, prepare Hemolytic streptococcus by method of the present invention, staphylococcus and Candida albicans thalline and Hemolytic streptococcus, the blue brown laying hen in two kinds of different immunogen immune sea such as staphylococcus and Candida albicans thalline+bacterial chip, and prepare the anti-hemolysis suis of the egg of 60～80 days immune hens in immunity back for the first time, staphylococcus and Candida albicans thalline chicken IgY antibody crude extract and anti-hemolysis suis, staphylococcus and Candida albicans thalline+two kinds of chicken IgY antibody such as bacterial chip chicken IgY antibody crude extract.These two kinds of chicken IgY antibody react with Hemolytic streptococcus, staphylococcus and Candida albicans O antigen respectively, these two kinds of chicken IgY antibody biology are in conjunction with tiring certain difference as a result, wherein the biology of the chicken IgY antibody of anti-hemolysis suis, staphylococcus and Candida albicans thalline+bacterial chip is higher and more stable in conjunction with tiring, the biology of the chicken IgY antibody of anti-hemolysis suis, staphylococcus and Candida albicans thalline is in conjunction with variant between tiring then batch, from 1: 512/mg albumen-1: 1024/mg albumen.These presentation of results Hemolytic streptococcuss, staphylococcus and the immunogenic effect of Candida albicans thalline+bacterial chip are better than simple Hemolytic streptococcus, staphylococcus and the immunogenic effect of Candida albicans thalline.
Experimental example 5:
The immune effect of Hemolytic streptococcus, staphylococcus and Candida albicans thalline+tens of somes muscle of bacterial chip immunogen and subcutaneous injection immunity and simple several somes intramuscular injection or simple several somes subcutaneous injection immunity relatively the results are shown in Table 5:
The biological value of the specific IgY antibody that the blue brown hen in the different immunization ways inductive of table 5 sea produces relatively
| Immunization ways | Immunogen | Detect antigen | Chicken IgY antibody | Antibodies is tired |
| Tens of muscle and subcutaneous injection | Hemolytic streptococcus, staphylococcus and Candida albicans thalline+bacterial chip | Hemolytic streptococcus, staphylococcus and Candida albicans hybrid antigen | Anti-hemolysis suis, staphylococcus and Candida albicans thalline+bacterial chip antibody | 1: 1024/mg albumen |
| Several somes intramuscular injection | 1: 256～512/mg albumen | |||
| Several somes subcutaneous injections | 1: 512/mg albumen |
Can be clear that according to above-mentioned experimental result: the identical immunogen of pressing method preparation of the present invention is with the different blue brown bird inlays in the immunization ways identical sea of immunity, and the anti-hemolysis suis of preparing the egg of 60～80 days immune hens in immune for the first time back by method of the present invention, the crude extract of staphylococcus and Candida albicans chicken IgY antibody, its anti-hemolysis suis, the biological value that mode produced that the biological value of staphylococcus and Candida albicans chicken IgY antibody will be counted tens of somes muscle of the present invention and subcutaneous injection immunity is the highest, and therefore immunization method of the present invention is best.
Experimental example 6:
The comparison of the time length of the high biological value specificity chicken IgY antibody that Hemolytic streptococcus, staphylococcus and Candida albicans thalline+tens of somes muscle of bacterial chip immunogen and subcutaneous injection immunity and simple several somes intramuscular injection or simple several somes subcutaneous injection immunity are produced the results are shown in Table 6:
The time ratio of the high specific IgY antibody of tiring of the blue brown hen generation in the different immunization ways inductive of table 6 sea
| Immunization ways | Immunogen | Detect antigen | Chicken IgY antibody | Time | Antibodies is tired |
| Tens of muscle and subcutaneous injection | Hemolytic streptococcus, staphylococcus and Candida albicans thalline+bacterial chip | Hemolytic streptococcus, staphylococcus and Candida albicans hybrid antigen | Anti-hemolysis suis, staphylococcus and Candida albicans thalline+bacterial chip antibody | The 280th～300 day egg IgY antibody after the first immunisation | 1: 512/mg albumen |
| Several somes intramuscular injection | 1: 128～256/mg albumen | ||||
| Several somes subcutaneous injections | 1: 128～256/mg albumen |
Can be clear that according to above-mentioned experimental result: with the different blue brown bird inlays in the immunization ways identical sea of immunity, and prepare the crude extract of anti-hemolysis suis, staphylococcus and the Candida albicans chicken IgY antibody of the egg of 280～300 days immune hens in immunity back for the first time by method of the present invention by the identical immunogen of method of the present invention preparation.Reach 9～10 months anti-hemolysis suis by ELISA method mensuration, the variation of tiring of staphylococcus and Candida albicans chicken IgY antibody, found that the method for tens of somes muscle and subcutaneous injection immunity, they are specificity anti-hemolysis suis after 9～10 months, staphylococcus and Candida albicans chicken IgY antibody titer descend not obvious, and the hen of simple several somes intramuscular injection or simple several somes subcutaneous injection immunity produces specificity anti-hemolysis suis, the ability of staphylococcus and Candida albicans chicken IgY antibody obviously descends, and specificity chicken IgY antibody titer also obviously descends.This further specifies, aspect the time length that the specificity anti-hemolysis suis that the mode of tens of somes muscle of the present invention and subcutaneous injection immunity is tired at height, staphylococcus and Candida albicans chicken IgY antibody produce significant advantage is arranged, effect is significantly better than other two kinds of methods.
Experimental example 7:
The effect of the method for the method of five immunizations of the present invention and traditional three immunizations relatively the results are shown in Table 7:
The comparison that the blue brown hen in the different immune time inductive of table 7 sea produces high specific IgY antibody ability of tiring
| Immunization ways | Immunogen | Detect antigen | Time | Chicken IgY antibodies is tired | Time | Chicken IgY antibodies is tired |
| Five immunizations of the present invention | Hemolytic streptococcus, staphylococcus, Candida albicans thalline+bacterial chip | Hemolytic streptococcus, staphylococcus and Candida albicans hybrid antigen | The 60th～80 day egg IgY antibody after the first immunisation | 1: 1024/mg albumen | The 280th～300 day chicken IgY antibody after the first immunisation | 1: 512/mg albumen |
| Three times traditional immunizations | 1: 512～1024/mg albumen | 1: 128～256/mg albumen |
Can be clear that according to above-mentioned experimental result: by the extra large blue brown bird inlay of the extra large blue brown bird inlay of five immunization method immunity of the present invention and traditional three immunization method immunity relatively its specificity anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY production of antibodies ability, and by method of the present invention prepare the first time immune after 60～80 days anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract and 280～300 days anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract.Measure the biological value of the two kinds of inductive specificity chicken IgY of immunization method institute antibody of different time by the ELISA method, the result shows that the biological value of the specificity chicken IgY antibody that two kinds of immunization methods produce changes and is little that five immunization methods of the present invention are better than three times traditional immunization methods slightly in time of the 60th～80 day after the first immunisation; But after reaching time of 280～300 days, five immunization methods of the present invention are well more a lot of than three times traditional immunization methods, and the biological value of its specificity anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody has very significant difference by comparison.Five immunization methods of the present invention immune hen to produce time of high specificity anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody of tiring longer.
Experimental example 8:
Three kinds of different proportion of composing with fungi of bacterium such as Hemolytic streptococcus, staphylococcus and Candida albicans the results are shown in Table 8 to the influence that specificity anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody produce:
The comparison that the immunogen of the different ratio of componentss of three kinds of bacterium of table 8 induces the blue brown hen in sea to produce specificity chicken IgY antibody
| The ratio of chain, Portugal, bacterium such as white | Immunogen | Detect antigen | Detect antibody | Antibodies is tired |
| 6∶3∶1 | Hemolytic streptococcus, staphylococcus, Candida albicans thalline+bacterial chip | Hemolytic streptococcus, staphylococcus and Candida albicans hybrid antigen | Anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody | 1: 1024/mg albumen |
| 5.5∶3∶1.5 | 1: 1024/mg albumen | |||
| 5∶4∶1 | 1: 1024/mg albumen | |||
| 5∶3∶2 | 1: 1024/mg albumen | |||
| 7∶2∶1 | 1: 1024/mg albumen |
Can be clear that according to above-mentioned experimental result: though press the identical immunogen Hemolytic streptococcus of method preparation of the present invention, staphylococcus is different with the fungi proportion of composing with three kinds of bacteriums such as Candida albicanss, but immunity is laid eggs behind the extra large blue brown hen, and the anti-hemolysis suis of preparing the egg of 60～80 days immune hens in immune for the first time back by method of the present invention, the crude extract of staphylococcus and Candida albicans chicken IgY antibody, its anti-hemolysis suis, the biological value of the chicken IgY antibody of staphylococcus and Candida albicans hybrid antigen is consistent substantially.Though three kinds of bacteriums such as this explanation Hemolytic streptococcus, staphylococcus and Candida albicans are different with the fungi proportion of composing, the biological value that produces total anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody is constant.
Experimental example 9:
Three kinds of different proportion of composing with fungi of bacterium such as Hemolytic streptococcus, staphylococcus and Candida albicans are to difference anti-hemolysis suis, and the influence that the chicken IgY antibody of staphylococcus and anti-candida albicans produces the results are shown in Table 9:
The influence of the specificity chicken IgY antibody of different three kinds of different bacterium of ratio of components antagonism of three kinds of bacterium of table 9 and fungi
| The ratio of chain, Portugal, bacterium such as white | Immunogen | Detect antigen | Detect antibody | Chicken IgY antibodies is tired |
| 6∶3∶1 | Hemolytic streptococcus, staphylococcus, Candida albicans thalline+bacterial chip | Hemolytic streptococcus, staphylococcus and Candida albicans hybrid antigen | Anti-hemolysis suis chicken IgY antibody | 1: 512/mg albumen |
| Staphylococcus chicken IgY antibody | 1: 256/mg albumen | |||
| Anti-candida albicans chicken IgY antibody | 1: 128/mg albumen | |||
| 5∶4∶1 | Anti-hemolysis suis chicken IgY antibody | 1: 512/mg albumen | ||
| Staphylococcus chicken IgY antibody | 1: 256～512/mg albumen | |||
| Anti-candida albicans chicken IgY antibody | 1: 128/mg albumen | |||
| 5∶3∶2 | Anti-hemolysis suis chicken IgY antibody | 1: 512/mg albumen | ||
| Staphylococcus chicken IgY antibody anti-candida albicans chicken IgY antibody | 1: 256/mg albumen | |||
| 1: 128～256/mg albumen | ||||
| 7∶2∶1 | Anti-hemolysis suis chicken IgY antibody | 1: 512～1024/mg albumen | ||
| Staphylococcus chicken IgY antibody | 1: 128～256/mg albumen | |||
| Anti-candida albicans chicken IgY antibody | 1: 128/mg albumen |
Can be clear that according to above-mentioned experimental result: Hemolytic streptococcus, the different proportion of composing with fungi of three kinds of bacteriums such as staphylococcus and Candida albicans prepare immunogen by method of the present invention respectively, immunity lay eggs the sea blue brown hen, and the anti-hemolysis suis of preparing the egg of 60～80 days immune hens in immune for the first time back by method of the present invention, the crude extract of staphylococcus and Candida albicans chicken IgY antibody, the streptococcic antibody titer of its anti-hemolysis, antistaphylococcic antibody titer, and the increase and decrease of essentially identical antibody titer takes place respectively in the anti-candida albicans antibody titer with the increase and decrease of different bacteriums and fungi immunizing dose.The variation of these antibody titers is is all effectively increasing and decreasing in the chicken IgY antibody activity scope, though three kinds of bacteriums such as this explanation Hemolytic streptococcus, staphylococcus and Candida albicans are different with the fungi proportion of composing, they induce chicken to produce the biological value of chicken IgY antibody of anti-hemolysis suis, staphylococcus and Candida albicans still in useful range.
Experimental example 10:
Anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody are engulfed streptococcus aureus to neutrophil leucocyte influence: method of the present invention is prepared anti-hemolysis suis, staphylococcus and the Candida albicans chicken IgY antibody crude extract and streptococcus aureus or deactivation streptococcus aureus and people's ear-lobe blood or the venous blood of living of the egg of 60～80 days immune hens in immunity back for the first time, at 37 ℃, 5%CO
2, to hatch jointly in the incubator of saturated humidity 30 minutes, the result has promoted people's ear-lobe blood and venous blood neutrophil leucocyte to engulf streptococcus aureus alive and deactivation streptococcus aureus.The phagocytic rate of neutrophil leucocyte increases along with the rising of IgY concentration, sees Table 10, table 11, and table 12:
The effect that table 10 specificity chicken IgY antibody is engulfed the deactivation streptococcus aureus to ear-lobe blood neutrophil leucocyte
| The IgY final concentration | Neutrophil leucocyte number (individual) | Engulf the cell count (individual) of bacterium | Engulf total plate count (individual) | Cytophagy rate (%) | Phagocytic index |
| 3.0mg/ml 1.5mg/ml 0.0mg/ml | 200.00±0.00 200.00±0.00 200.00±0.00 | 162.58±2.08 148.39±0.97 122.99±1.43 | 394.84±25.61 401.30±31.54 277.01±21.26 | 81.33±5.17 74.20±2.98 61.60±4.06 | 1.97±0.22 2.02±0.30 1.40±0.22 |
Annotate: 1, cytophagy rate (%): 1 group: 3 groups of t=0.009; 1 group: 2 groups of t=0.048; 2 groups: 3 groups of t=0.026.
2, phagocytic index: 1 group: 3 groups of t=0.008; 1 group: 2 groups of t=0.760; 2 groups: 3 groups of t=0.057.
Table 11 specificity chicken IgY antibody is engulfed the streptococcus aureus of deactivation to the venous blood neutrophil leucocyte effect
| The IgY final concentration | Neutrophil leucocyte number (individual) | Engulf the cell count (individual) of bacterium | Engulf total plate count (individual) | Cytophagy rate (%) | Phagocytic index |
| 3.0mg/ml 1.5mg/ml 0.0mg/ml | 200.00±0.00 200.00±0.00 200.00±0.00 | 163.25±3.40 154.50±6.03 128.50±4.43 | 438.25±24.46 396.25±30.35 283.25±13.52 | 81.63±1.70 77.25±3.01 63.13±2.78 | 2.19±0.12 1.98±0.15 1.42±0.07 |
Annotate: 1, cytophagy rate (%): 1 group: 3 groups of t=0.003; 1 group: 2 groups of t=0.021; 2 groups: 3 groups of t=0.016.
2, phagocytic index: 1 group: 3 groups of t=0.0004; 1 group: 2 groups of t=0.070; 2 groups: 3 groups of t=0.007.
Table 12 specificity chicken IgY antibody is engulfed streptococcus aureus alive to the venous blood neutrophil leucocyte effect
| The IgY final concentration | Neutrophil leucocyte number (individual) | Engulf the cell count (individual) of bacterium | Engulf total plate count (individual) | Cytophagy rate (%) | Phagocytic index |
| 3.0mg/ml 1.5mg/ml 0.0mg/ml | 200.00±0.00 200.00±0.00 200.00±0.00 | 165.25±3.40 149.75±2.06 134.00±3.56 | 461.75±27.94 441.75±34.22 351.50±20.40 | 82.63±0.00 74.88±0.01 67.00±0.02 | 2.31±0.01 2.21±0.17 1.76±0.10 |
Annotate: 1, cytophagy rate (%): 1 group: 3 groups of t=0.002; 1 group: 2 groups of t=0.001; 2 groups: 3 groups of t=0.011.
2, phagocytic index: 1 group: 3 groups of t=0.0134; 1 group: 2 groups of t=0.187; 2 groups: 3 groups of t=0.037.
Can be clear that according to above-mentioned experimental result: prepare for the first time anti-hemolysis suis, staphylococcus and the Candida albicans chicken IgY antibody crude extract of the egg of 60～80 days immune hens in immunity back by method of the present invention, it can promote human neutrophil to strengthen to engulf alive and streptococcus aureus deactivation.Neutrophil leucocyte is engulfed streptococcus aureus alive and is engulfed indifference between the streptococcus aureus of deactivation.This opsonophagocytosis promoter action may be with to have changed bacterium surface structure and electrical effect relevant.This has illustrated anti-hemolysis suis, staphylococcus and the Candida albicans chicken IgY antibody crude extract of preparing by method of the present invention, and its treatment is by the mechanism of action of bacterium such as Hemolytic streptococcus, staphylococcus and Candida albicans and fungoid inflammation.
Claims (5)
1, a kind of anti-hemolysis suis, staphylococcus and candida albicans IgY antibody is characterized in that it is obtained by following method:
(1) immunogen preparing:
Get 50～70 parts of morbific Hemolytic streptococcus and Hemolytic streptococcus bacterial chips, 20～40 parts of staphylococcus and staphylococcus bacterial chips, Candida albicans and staphylococcus bacterial chip are mixed into bacterium liquid for 10～20 parts;
The bacterium liquid of equivalent volumes is mixed with the Fu Shi Freund's complete adjuvant of equivalent volumes, and stirring and emulsifying is made water-in-oil Fu Shi Freund's complete adjuvant Hemolytic streptococcus, staphylococcus and Candida albicans immunogen;
The bacterium liquid of equivalent volumes is mixed with the freund 's incomplete adjuvant of equivalent volumes, and stirring and emulsifying is made water-in-oil freund 's incomplete adjuvant Hemolytic streptococcus, staphylococcus and white beads immunogen;
In every milliliter of freunds complete adjuvant immunogen and the freunds incomplete adjuvant immunogen, all contain bacterium 2,100,000,000～2,700,000,000;
(2) immunity:
Give bird inlay with the subcutaneous and muscle multi-point injection of freunds complete adjuvant immunogen, every bird inlay injection 1ml immunogen, use the subcutaneous and muscle multi-point injection of freunds incomplete adjuvant immunogen 1ml to give bird inlay then week about, after using freunds incomplete adjuvant immunogen booster immunization secondary, freunds incomplete adjuvant immunogen 1ml is subcutaneous to give bird inlay booster immunization secondary with the muscle multi-point injection every re-using in three weeks, front and back immune five times altogether;
(3) preparation of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract:
Collect the egg of the immune hen of immunity after 30 days, the egg surface disinfects in alcohol, get yolk, remove membrane of yolk and get egg yolk liquid, ratio adding distil water with egg yolk liquid and distilled water 1: 7 by volume～9, fully stir, and with 1N HCl adjusting potential of hydrogen to pH5.0～5.2, under 4 ℃ of temperature, left standstill 10 hours;
With yolk diluent high speed centrifugation 30 minutes, get supernatant, it is 5～8 times of the hollow fiber membrane ultrafiltration device ultrafiltration and concentration of 100kDa that yolk diluent supernatant is placed molecular weight cut-off, adding 0.02%～2.0% chlorhexidine gluconate then fully stirs, and use 1N NaOH to regulate potential of hydrogen, and under 4 ℃ of temperature, left standstill 10 hours to pH7.0～7.5; After this high speed centrifugation is 15 minutes, gets supernatant, and the weeding of grease metallic substance with the sterilization filtering with microporous membrane degerming of supernatant through 0.45 μ～0.22 μ, promptly gets anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract.
2, anti-hemolysis suis according to claim 1, staphylococcus and candida albicans IgY antibody, it is characterized in that Hemolytic streptococcus and Hemolytic streptococcus bacterial chip in the described immunogen preparing are beta hemolytic streptococcus and bacterial chip, described staphylococcus and staphylococcus bacterial chip are streptococcus aureus and bacterial chip.
3, anti-hemolysis suis according to claim 1, staphylococcus and candida albicans IgY antibody is characterized in that the bird inlay in the described immunity comprises the native chicken of family that raising under the conventional environment, cleaning ambient are raised down and the SPF environment is raised down, white hair Gallus Domesticus, the blue brown chicken in sea, Lai Hangji, Luo Man chicken, star chicken.
4, the preparation method of anti-hemolysis suis as claimed in claim 1, staphylococcus and candida albicans IgY antibody is characterized in that comprising the steps:
(1) immunogen preparing:
Get 50～70 parts of morbific Hemolytic streptococcus and Hemolytic streptococcus bacterial chips, 20～40 parts of staphylococcus and staphylococcus bacterial chips, Candida albicans and staphylococcus bacterial chip are mixed into bacterium liquid for 10～20 parts;
The bacterium liquid of equivalent volumes is mixed with the Fu Shi Freund's complete adjuvant of equivalent volumes, and stirring and emulsifying is made water-in-oil Fu Shi Freund's complete adjuvant Hemolytic streptococcus, staphylococcus and Candida albicans immunogen;
The bacterium liquid of equivalent volumes is mixed with the freund 's incomplete adjuvant of equivalent volumes, and stirring and emulsifying is made water-in-oil freund 's incomplete adjuvant Hemolytic streptococcus, staphylococcus and white beads immunogen;
In every milliliter of freunds complete adjuvant immunogen and the freunds incomplete adjuvant immunogen, all contain bacterium 2,100,000,000～2,700,000,000;
(2) immunity:
Give bird inlay with the subcutaneous and muscle multi-point injection of freunds complete adjuvant immunogen, every bird inlay injection 1ml immunogen, use the subcutaneous and muscle multi-point injection of freunds incomplete adjuvant immunogen 1ml to give bird inlay then week about, after using freunds incomplete adjuvant immunogen booster immunization secondary, freunds incomplete adjuvant immunogen 1ml is subcutaneous to give bird inlay booster immunization secondary with the muscle multi-point injection every re-using in three weeks, front and back immune five times altogether;
(3) preparation of anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract:
Collect the egg of the immune hen of immunity after 30 days, the egg surface disinfects in alcohol, get yolk, remove membrane of yolk and get egg yolk liquid, ratio adding distil water with egg yolk liquid and distilled water 1: 7 by volume～9, fully stir, and with 1N HCl adjusting potential of hydrogen to pH5.0～5.2, under 4 ℃ of temperature, left standstill 10 hours;
With yolk diluent high speed centrifugation 30 minutes, get supernatant, it is 5～8 times of the hollow fiber membrane ultrafiltration device ultrafiltration and concentration of 100kDa that yolk diluent supernatant is placed molecular weight cut-off, adding 0.02%～2.0% chlorhexidine gluconate then fully stirs, and use 1N NaOH to regulate potential of hydrogen, and under 4 ℃ of temperature, left standstill 10 hours to pH7.0～7.5; After this high speed centrifugation is 15 minutes, gets supernatant, and the weeding of grease metallic substance with the sterilization filtering with microporous membrane degerming of supernatant through 0.45 μ～0.22 μ, promptly gets anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody crude extract;
(4) with the anti-hemolysis suis after the weeding of grease metallic substance, staphylococcus and Candida albicans chicken IgY antibody crude extract add the saturation ratio of sulfuric acid amine to 55%, fully stir, room temperature left standstill 4～12 hours, high speed centrifugation is 10 minutes then, get precipitation, to precipitate and use distilled water diluting to the original volume amount, the saturation ratio that adds sulfuric acid amine to 33% again, fully stir, room temperature left standstill 4～12 hours, and high speed centrifugation is 10 minutes then, get precipitation, with the dialysis tubing of packing into behind the dissolved in distilled water, use the distill water dialysis desalination, promptly get the anti-hemolysis suis, staphylococcus and the thick pure product of Candida albicans chicken IgY antibody.
5, the preparation method of anti-hemolysis suis according to claim 4, staphylococcus and candida albicans IgY antibody is characterized in that also comprising the steps: to add pharmaceutically acceptable additives makes sprays, aerosol, nasal drops and the lozenge that contains anti-hemolysis suis, staphylococcus and Candida albicans chicken IgY antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510031150 CN1283663C (en) | 2005-01-12 | 2005-01-12 | Antithemolytic streptococcus, staphylococcus and candida albicans lgG antibody and preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510031150 CN1283663C (en) | 2005-01-12 | 2005-01-12 | Antithemolytic streptococcus, staphylococcus and candida albicans lgG antibody and preparation thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1680445A CN1680445A (en) | 2005-10-12 |
| CN1283663C true CN1283663C (en) | 2006-11-08 |
Family
ID=35067233
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510031150 Active CN1283663C (en) | 2005-01-12 | 2005-01-12 | Antithemolytic streptococcus, staphylococcus and candida albicans lgG antibody and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1283663C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110680917A (en) * | 2019-09-30 | 2020-01-14 | 南京道大药业有限公司 | Preparation method and application of anti-propionibacterium acnes and staphylococcus composite IgY antigen |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955544A (en) * | 2010-06-10 | 2011-01-26 | 东北师范大学 | Candida albicans secreted aspartic proteinase (Sap2) coding epitope sequence-synthesizing and bacteriophage shell protein-bonding hybrid protein and use thereof |
| MY166794A (en) | 2011-08-19 | 2018-07-23 | Ostrich Pharma Kk | Antibody and antibody-containing composition |
| CN103520719B (en) * | 2013-10-24 | 2016-11-23 | 邹潮 | A kind of buccal antibody product and preparation method thereof |
| CN103877561A (en) * | 2014-03-31 | 2014-06-25 | 陈光健 | Pharmaceutical composition containing small-molecular active peptides |
| CN106749650B (en) * | 2016-12-19 | 2021-06-25 | 姚善龙 | Preparation method and application of composite IgY antibody for resisting cold virus, streptococcus pneumoniae and staphylococcus |
| CN108623680A (en) * | 2017-03-17 | 2018-10-09 | 蒙永祥 | Compound antibody extract and its preparation method and application |
| CN108992669B (en) * | 2018-08-27 | 2022-03-22 | 广州汇高生物科技有限公司 | Composite yolk antibody composition for preventing and treating respiratory tract infection, aerosol inhalation solution, preparation process and application |
| CN112957464A (en) * | 2021-02-24 | 2021-06-15 | 江秀坤 | Application of chicken egg yolk immunoglobulin in preparing throat-moistening health-care product |
-
2005
- 2005-01-12 CN CN 200510031150 patent/CN1283663C/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110680917A (en) * | 2019-09-30 | 2020-01-14 | 南京道大药业有限公司 | Preparation method and application of anti-propionibacterium acnes and staphylococcus composite IgY antigen |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1680445A (en) | 2005-10-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1283663C (en) | Antithemolytic streptococcus, staphylococcus and candida albicans lgG antibody and preparation thereof | |
| CN1226829A (en) | Method for treating gastrointestinal damage | |
| CN102908615B (en) | Novel haemophilus parasuis disease trivalent inactivated vaccine and preparation method thereof | |
| CN1062605C (en) | Escherichia coli vaccine | |
| CN109666609A (en) | A kind of Rhodococcus ruber fermentation process and its application as adjuvant in animal vaccine | |
| CN102977208A (en) | Preparation method, application and medicine composition and preparation of specific egg yolk immunoglobulin (IgY) and acinetobacter baumannii, as well as preparation and kit | |
| CN1206243C (en) | Yolk antibody and antigen of pig's infective enterogastritis virus and its preparing process | |
| CN1038559C (en) | Vaccine against E. coli septicaemia in poultry | |
| CN1454901A (en) | Gynaecological anti-infective specificity IgY and its combined preparation | |
| CN1218962C (en) | Somatostatin yolk antibody and its preparing process | |
| CN101074260A (en) | Production of hygrophilous monospermous bacterium main-protective antigen univalent and multivalent vitelline antibody and use in aquatic animal | |
| CN1178674C (en) | Bacteriostatic compositions for salmonellae | |
| KR100267746B1 (en) | Oral immunological preparation containing egg-yolk antibodies, for prevention and treatment of porcine diarrhea caused by enterotoxigenic escherichia coli | |
| CN1292794C (en) | Preparation of immuno-stimulation composition for hemolycin in monad | |
| US20120009198A1 (en) | Compositions and Methods for Modulating Immune Response | |
| CN113845588A (en) | Preparation method and application of yolk antibody for resisting porcine rotavirus | |
| KR102077701B1 (en) | Vaccin composition containing novel inactivated Salmonella Gallinarum whole cells for preventing or treating Fowl Typhoid | |
| CN109223716B (en) | Quadruple yolk antibody soluble powder for resisting porcine epidemic diarrhea, swine fever, pseudorabies and transmissible gastroenteritis and preparation method thereof | |
| RU2470663C1 (en) | Swine salmonellosis vaccine, method for preparing, method for preventing swine salmonellosis | |
| CN1384119A (en) | Composite yolk antibody for resisting fowl's viral blight and its prepn and application | |
| DK2376113T3 (en) | PREPARATIONS AND DOSAGE REGIMES WHICH INCLUDES A CLOSTRIDIUM VACCINE AND levamisole | |
| CN111793137A (en) | Hp tetravalent antigen and preparation method and application thereof | |
| CN106749650B (en) | Preparation method and application of composite IgY antibody for resisting cold virus, streptococcus pneumoniae and staphylococcus | |
| US20120308579A1 (en) | Method of making and using a composition for delivering viral immunogen immunoglobulin inhibitor to the nasal pharyngeal membrane | |
| CN101058607A (en) | Anti-helicobacter pylori thallus, cell toxin egg yolk antibody product, preparation and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee |
Owner name: JIANGXI 3L MEDICAL PRODUCTS GROUP CO., LTD. Free format text: FORMER NAME: 3L MEDICAL PRODUCTS GROUP CO., LTD., JIANGXI |
|
| CP01 | Change in the name or title of a patent holder |
Address after: 330029 No. 599 torch street, hi tech Zone, Jiangxi, Nanchang Patentee after: Jiangxi 3L Medical Products Group Co., Ltd. Address before: 330029 No. 599 torch street, hi tech Zone, Jiangxi, Nanchang Patentee before: Jiangxi 3L Medical Product Group Co., Ltd. |