CN103341163A - Double-effect horse antiserum used for rabies and tetanus and preparation method thereof - Google Patents
Double-effect horse antiserum used for rabies and tetanus and preparation method thereof Download PDFInfo
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- CN103341163A CN103341163A CN2013102759498A CN201310275949A CN103341163A CN 103341163 A CN103341163 A CN 103341163A CN 2013102759498 A CN2013102759498 A CN 2013102759498A CN 201310275949 A CN201310275949 A CN 201310275949A CN 103341163 A CN103341163 A CN 103341163A
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Abstract
The invention discloses a double-effect horse antiserum used for rabies and tetanus. The double-effect horse antiserum is prepared by a method comprising the following steps of: performing injection immunotherapy to a healthy horse through a rabies virus inactivation antigen and a tetanus toxoid antigen and bringing antigen serum into existence; sampling blood; selecting blood plasma, wherein the blood plasma per 1ml contains 100 to 20,000 international units of rabies antiserum based on titers, and 1,100 to 20,000 international units of antitetanic serum based on titers; diluting to 2 to 4 times through injection water; purifying and refining; and then adding sodium chloride and thiomersal, thus obtaining the double-effect horse antiserum. The double-effect horse antiserum is used for preventing a patient with an exposed wound caused by biting or scratching by a rabid dog or other rabid animals from rabies and tetanus; the double-effect horse antiserum contains double-effect antibodies against rabies and tetanus, so that the patient can be prevented from rabies and tetanus after being injected once; and the double-effect horse antiserum is simple and convenient to use, and has high social benefit and economic benefit.
Description
Technical field
The invention belongs to field of biological pharmacy, be specifically related to a kind of rabies, tetanus economic benefits and social benefits horse anteserum and preparation method thereof.
Background technology
Rabies are a kind of disease of natural focus; be all people beast property suffered from diseases altogether of susceptible of all homoiothermic animals of being caused by rabies virus; in case death rate of the onset is 100%; in time carry out the injection of Rab/Ser or immunoglobulin; and the timely inoculation of rabies vaccine, be unique protective measure.It is very severe that the rabies of China are prevented and treated situation.
When the people was subjected to wound, clostridium tetani can be invaded human body through wound, discharged a kind of height toxic protein---tetanopasmin, caused nervous symptoms limb spasm paralysis (locality tetanus) and whole body tic (holotetanus).
The people by dog bite after, need prevent rabies and tetanus simultaneously.Existing method generally is to inject tetanus immune globulin and rabies immune globulin respectively, because injection volume is bigger, entry needle is inferior more, and expense is very high, and the patient is difficult to accept.Another is the injection horse anteserum, horse anteserum is with behind the antigen immune horses, horses produce the efficient antibody of resisting pathogenic microbes, the serum that will contain efficient antibody separates, purification is prepared from, the rabies antiserum that unit price is arranged in the market, and tetanus antiserum, but since its all be the antibody protein of horse extract is heterologous protein eventually, heavy dose of injection produces anaphylaxis inevitably, there is not the listing of tetanus and antilyssic economic benefits and social benefits serum at present, having only application number is that 200910088039.2 Chinese invention patent discloses a kind of rabies, tetanus double-titer human immunoglobulin, owing to be to obtain after the normal population immunity, limited so originate, price is high, obtains the very big difficulty of existence on a large scale.
Summary of the invention
The objective of the invention is to remedy the deficiency of prior art and product usefulness, provide a kind of rabies of can preventing of human can prevent tetanic rabies, tetanus economic benefits and social benefits horse anteserum and preparation method thereof simultaneously again.
Technical scheme is as follows:
A kind of rabies, tetanus economic benefits and social benefits horse anteserum, it is with rabies virus inactivation antigen and tetanus toxoid antigen healthy horses to be carried out injecting immune and make it to produce antibody serum, blood sampling is also chosen every 1ml and is contained rabies antiserum 100 ~ 20000 ius of tiring, contain the tire blood plasma of 1100 ~ 20000 ius of antitetanic serum, carry out purification refine and add sodium chloride with after 2 ~ 4 times of the water for injection dilutions, thimerosal is prepared from, described rabies, every 1ml contains rabies antiserum and tires and be no less than 200 ius in the tetanus economic benefits and social benefits horse anteserum, contain antitetanic serum and tire and be not less than 2000 ius, F (ab ')
2Purity be that the content of 60 ~ 95%, IgG is 1 ~ 10%, Protein content is 10 ~ 150g/L, the content of sodium chloride is 7.5 ~ 9.5g/L, the content of ammonium sulfate is 0.2 ~ 1.0g/L, the content of antiseptic thimerosal is 0.07 ~ 0.10g/L.
A kind of rabies, tetanus economic benefits and social benefits method for preparing horse anti blood serum may further comprise the steps:
1) selects healthy horses, at 0,14 day injection rabies virus inactivation antigen, carried out the initial immunity injection of tetanus toxoid antigen and the 3rd pin injection of rabies virus inactivation antigen then in 21 days, the last booster immunization injection of carrying out rabies virus inactivation antigen and tetanus toxoid antigen in 45 and 60 days simultaneously, rabies virus inactivation antigen and the tetanus toxoid antigen of per injection are 5ml
Tiring of described rabies virus inactivation antigen is 15 ~ 50IU/ml, and its adjuvant is the incomplete freund adjuvant of water-in-oil type that liquid paraffin and lanoline are made into,
Tiring of described tetanus toxoid antigen is 100 ~ 500Lf/ml, and its adjuvant is the incomplete freund adjuvant of water-in-oil type that liquid paraffin and lanoline are made into;
2) after immunity is finished the 15th day gather blood plasma, choose every 1ml and contain rabies antiserum 100 ~ 20000 ius of tiring, contain the tire blood plasma of 1100 ~ 20000 ius of antitetanic serum, with 2 ~ 4 times of water for injection dilutions;
3) blood plasma after the dilution is purified refining, namely.
Wherein, the blood plasma after the dilution is to adopt following method purification refine:
1) blood plasma after will diluting is adjusted pH value to 3.1 ~ 3.7, and adding pepsin to pepsic whole content then is 3 ~ 12u/g, digests to add ammonium sulfate precipitation, filtration after 60 ~ 120 minutes, gets precipitation;
2) will precipitate with water for injection and dissolve fully, add 0.8 ~ 1.2% Alumen by weight, adjust pH value to 7.7 ~ 7.9, adsorb after 1 hour and filter, remove Alumen, the supernatant ultrafiltration and concentration, adding water for injection in the concentrated solution, to adjust protein content be 10 ~ 150g/L, and adding sodium chloride, thimerosal, to make the concentration of sodium chloride be that the concentration of 7.5 ~ 9.5g/L, thimerosal is 0.07 ~ 0.1g/L, adjust pH to 6.0 ~ 7.0;
3) with step 2) solution that obtains is with 0.20 micron filter membrane aseptic filtration, and the pressure of aseptic filtration is 0.01 ~ 0.15MPa.
Horse anteserum and people source immunoglobulin have the difference of essence, and Chinese Pharmacopoeia is classified as inhomogeneous product fully with it.This product is not simple two kinds of sero-fast mixing, but utilize the animal body immunoreation, an antibody molecule that utilizes different antibodies molecular locus difference and produce carries the antibody of two kinds of antipathogen effects simultaneously, cooperates immune programme for children and dosage to obtain immune serum through being prepared from.In addition, horses belong to biology not of the same race with respect to the people, the antigen amount of immunity and the use of adjuvant are all inequality, and antibody protein must carry out more complicated preparation after obtaining, purify, but because the source is simple, large-scale acquisition and preparation easily, so it is cheap, its unit tires than human normal immunoglobulin's height simultaneously, and use amount is few, and injection volume is little in the use, entry needle lacks, if the economic benefits and social benefits antiserum will further reduce the use amount of half, can maximum reduce heterologous protein to the anaphylaxis of human body, thereby have people source globulin and the at present independent irreplaceable superiority of cause of disease antiserum clinically.
Rabies of the present invention, tetanus economic benefits and social benefits horse anteserum only produce precipitation line with the serum of anti-horse, are positive with horse IgG antibody response.Described rabies, tetanus economic benefits and social benefits horse anteserum are used for being carried out rabies and tetanus prevention by the patient after mad dog or other crazy animal bites or the exposure of scratching.Owing to contain rabies, tetanus economic benefits and social benefits antibody simultaneously, patient's shot can prevent rabies and tetanus, and is easy to use, has bigger social benefit and economic benefit.
The specific embodiment
Embodiment 1
A kind of rabies, tetanus economic benefits and social benefits horse anteserum, it is prepared from by the following method:
1, select suitable 4 ~ 12 years old 10 of healthy horses, at 0,14 day injection rabies virus inactivation antigen 5ml, tiring of its antigen was 25IU/ml respectively, and its adjuvant uses liquid paraffin and lanoline to be made into the incomplete freund adjuvant of " Water-In-Oil "; Carried out first the exempting from of tetanus toxoid antigen then injected in the 21st day, tiring of antigen is 200Lf/ml, its adjuvant uses liquid paraffin and lanoline to be made into the incomplete freund adjuvant of " Water-In-Oil ", carries out the 3rd pin injection of rabies virus inactivation antigen simultaneously, and every kind of antigen is respectively 5ml; Carried out the injection booster immunization of rabies virus inactivation antigen and tetanus toxoid antigen after finishing simultaneously in 45,60 days, every kind of antigen is respectively 5ml.Set up detailed horse immune inoculation record.
2, the collection of blood plasma
Carry out horses and gather plasma sample and carry out the detection of serum antibody titer, the examination blood tetanus antibody 1100 ~ 20000IU/ml that tires, the horses of rabies virus antibodies 100 ~ 20000IU/ml can be taken a blood sample.The horses that do not reach the collection standard for serum titer carry out booster immunization, make it reach the collection standard, and the horses of finishing for collection carry out booster immunization simultaneously in order to carrying out sampled plasma next time.Every dry goods is gathered blood plasma 5000 ± 500ml.The blood plasma of gathering contains rabies, tetanus economic benefits and social benefits antibody, and freezing is preserved.Dilute 3 times with water for injection before using.
3, blood plasma purification refine
1) blood plasma after will diluting is adjusted pH value to 3.4, and adding pepsin to pepsic whole content then is 8u/g, digest to add ammonium sulfate (restraining ammonium sulfate by per 100 gram Digestive systems addings 15) after 90 minutes and saltout, filter (filter press), gets precipitation;
2) will precipitate with water for injection and dissolve fully, add 1% Alumen by weight, adjust pH value to 7.8, adsorb after 1 hour and filter (add kieselguhr and help filter), remove Alumen, the supernatant ultrafiltration and concentration (is concentrated into 1/10 of volume earlier, after 6 times of water for injection dilutions, ultrafiltration and concentration is to 1/10 of volume again), adding water for injection in the concentrated solution, to adjust protein content be 50g/L, and adding sodium chloride, thimerosal, to make the concentration of sodium chloride be that the concentration of 8.5g/L, thimerosal is 0.08g/L, adjust pH to 6.5;
3) with step 2) solution that obtains is with 0.20 micron filter membrane aseptic filtration, and the pressure of aseptic filtration is 0.01 ~ 0.15MPa.Aseptic filtration should finish in ultrafiltration and concentration and carry out in back 2 hours.
Antitoxin stock solution behind the purification should be put 2 ~ 8 ℃ and be kept in Dark Place 1 ~ 12 month as stable phase.
Rabies of the present invention, tetanus economic benefits and social benefits horse anteserum, its dosage form is liquid dosage form, packing is as the criterion with the rabies NAT, and its specification has 400IU/2.0ml/ bottle, 1000IU/5.0ml/ bottle, and including tetanic antibody titer is 500 ~ 2000IU/ml.
The raw blood plasma that the present invention gathers is answered cryopreservation, and storage life is no more than 3 years.
Rabies of the present invention, tetanus economic benefits and social benefits antiserum are used for being prevented to take place rabies and tetanus by mad dog or other crazy animal bites or the exposure patient that scratches, its use amount is calculated with the rabies NAT, can take the circumstances into consideration to increase to 80 ~ 100IU) body weight by the 40IU/kg(especially severe and calculate, gradation injection in 1 ~ 2 day.
The quality testing result:
Embodiment 2
1) select with 110 of batch healthy mices, laboratory was fed 3 days, treated that white mice conforms.
2) will carry out 37 ℃ of neutralizations of mixing with rabies virus, tetanolysin respectively by the product of embodiment gained.
3) with the rabies virus antiserum (Wuhan Biological Products Inst. that buys, lot number: 20120504) with 37 ℃ of neutralizations of rabies virus mixing, tetanus antiserum (Wuhan Biological Products Inst., lot number: 20120607) carry out 37 ℃ of neutralizations of mixing with tetanolysin.
4) the according to the form below experiment method carries out grouping experiment:
5) observe 14 days continuously, the mouse death of all falling ill of injection rabies virus, tetanic symptom and dead all appears in the mouse of injection tetanus vibrio, the mouse and the mouse that does not carry out any medicine injection of every group 20 injection neutralization back mixed liquors, none only occurs dead, and normal growth appears in body weight, prolong and observed one month, the mouse of not finding injection neutralization back mixed liquor occurs any unusual, every group of 20 mouse of in Wuhan Biological Products Inst.'s commodity serum that contrast injection is bought and back mixed liquor, none only occurs dead, and normal growth appears in body weight, prolong and observed one month, do not find that the mouse of injection neutralization back mixed liquor occurs any unusual.
Conclusion: application rabies, tetanus economic benefits and social benefits horse anteserum can prevent rabies and tetanic generation, have the same preventive effect of monospecific antiserum, have better curative effect.
Claims (3)
1. rabies, tetanus economic benefits and social benefits horse anteserum, it is characterized in that: it is with rabies virus inactivation antigen and tetanus toxoid antigen healthy horses to be carried out injecting immune and make it to produce antibody serum, blood sampling is also chosen every 1ml and is contained rabies antiserum 100 ~ 20000 ius of tiring, contain the tire blood plasma of 1100 ~ 20000 ius of antitetanic serum, purification refine is prepared from carrying out after 2 ~ 4 times of the water for injection dilutions, described rabies, every 1ml contains rabies antiserum and tires and be no less than 200 ius in the tetanus economic benefits and social benefits horse anteserum, contain antitetanic serum and tire and be not less than 2000 ius, F (ab ')
2The purity of fragment is not less than 60%.
2. rabies, tetanus economic benefits and social benefits method for preparing horse anti blood serum is characterized in that may further comprise the steps:
1) selects healthy horses, at 0,14 day injection rabies virus inactivation antigen, carried out the initial immunity injection of tetanus toxoid antigen and the 3rd pin injection of rabies virus inactivation antigen then in 21 days, the last booster immunization injection of carrying out rabies virus inactivation antigen and tetanus toxoid antigen in 45 and 60 days simultaneously, rabies virus inactivation antigen and the tetanus toxoid antigen of per injection are 5ml
Tiring of described rabies virus inactivation antigen is 15 ~ 50IU/ml, and its adjuvant is the incomplete freund adjuvant of water-in-oil type that liquid paraffin and lanoline are made into,
Tiring of described tetanus toxoid antigen is 100 ~ 500Lf/ml, and its adjuvant is the incomplete freund adjuvant of water-in-oil type that liquid paraffin and lanoline are made into;
2) after immunity is finished the 15th day gather blood plasma, choose every 1ml and contain rabies antiserum 100 ~ 20000 ius of tiring, contain the tire blood plasma of 1100 ~ 20000 ius of antitetanic serum, with 2 ~ 4 times of water for injection dilutions;
3) the blood plasma purification refine after the dilution, namely.
3. rabies according to claim 2, tetanus economic benefits and social benefits method for preparing horse anti blood serum, it is characterized in that: the method for described purification refine may further comprise the steps:
1) blood plasma after will diluting is adjusted pH value to 3.1 ~ 3.7, and adding pepsin to pepsic whole content then is 3 ~ 12u/g, digests to add ammonium sulfate precipitation, filtration after 60 ~ 120 minutes, gets precipitation;
2) will precipitate with water for injection and dissolve fully, add 0.8 ~ 1.2% Alumen by weight, adjust pH value to 7.7 ~ 7.9, adsorb after 1 hour and filter, remove Alumen, the supernatant ultrafiltration and concentration, adding water for injection in the concentrated solution, to adjust protein content be 10 ~ 150g/L, and adding sodium chloride, thimerosal, to make the concentration of sodium chloride be that the concentration of 7.5 ~ 9.5g/L, thimerosal is 0.07 ~ 0.1g/L, adjust pH to 6.0 ~ 7.0;
3) with step 2) solution that obtains is with 0.20 micron filter membrane aseptic filtration, and the pressure of aseptic filtration is 0.01 ~ 0.15MPa.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107096026A (en) * | 2017-03-08 | 2017-08-29 | 苏文全 | The preparation method of one species-specific proteins composition |
| CN114470184A (en) * | 2021-12-30 | 2022-05-13 | 孙嘉玮 | Combined vaccine for tetanus, canine distemper and rabies for livestock |
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| CN1947728A (en) * | 2005-10-11 | 2007-04-18 | 上海赛伦生物技术有限公司 | Method for preparing horse anti blood serum |
| CN101502651A (en) * | 2009-03-16 | 2009-08-12 | 山西康宝生物制品股份有限公司 | Hydrophobia, tetanus double-titer human immunoglobulin and preparation method thereof |
| CN101591393A (en) * | 2009-06-30 | 2009-12-02 | 同路生物制药有限公司 | A kind of rabies, tetanus double-titer human immunoglobulin and preparation method thereof with and application in the pharmacy thing |
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- 2013-07-03 CN CN201310275949.8A patent/CN103341163B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1947728A (en) * | 2005-10-11 | 2007-04-18 | 上海赛伦生物技术有限公司 | Method for preparing horse anti blood serum |
| CN101502651A (en) * | 2009-03-16 | 2009-08-12 | 山西康宝生物制品股份有限公司 | Hydrophobia, tetanus double-titer human immunoglobulin and preparation method thereof |
| CN101591393A (en) * | 2009-06-30 | 2009-12-02 | 同路生物制药有限公司 | A kind of rabies, tetanus double-titer human immunoglobulin and preparation method thereof with and application in the pharmacy thing |
Non-Patent Citations (1)
| Title |
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| 国家药典委员会: "《中华人民共和国药典》", 31 January 2010 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107096026A (en) * | 2017-03-08 | 2017-08-29 | 苏文全 | The preparation method of one species-specific proteins composition |
| CN114470184A (en) * | 2021-12-30 | 2022-05-13 | 孙嘉玮 | Combined vaccine for tetanus, canine distemper and rabies for livestock |
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| CN103341163B (en) | 2016-02-10 |
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