CN101502651A - Hydrophobia, tetanus double-titer human immunoglobulin and preparation method thereof - Google Patents
Hydrophobia, tetanus double-titer human immunoglobulin and preparation method thereof Download PDFInfo
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- CN101502651A CN101502651A CNA2009100739578A CN200910073957A CN101502651A CN 101502651 A CN101502651 A CN 101502651A CN A2009100739578 A CNA2009100739578 A CN A2009100739578A CN 200910073957 A CN200910073957 A CN 200910073957A CN 101502651 A CN101502651 A CN 101502651A
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Abstract
The invention relates to a rabies and tetanus double valence human normal immunoglobulin, the rabies valence of antibody is not less than 100IU/mL, the tetanus valence of antibody is not less than 60IU/mL, the content of protein is not more than 175g/L, the immunoglobulin purity is not lower than 92% of the total protein content. The preparation method of source plasma of the protein is to select plasma supplier to be inoculated with rabies vaccine and then with adsorbed tetanus vaccine at least 1-5 days later to obtain the source plasma with rabies valence of antibody being not less than 6IU/mL and the tetanus valence of antibody being not less than 5IU/mL and ensure that the rabies valence of antibody of the source plasma is not less than 10IU/m and the tetanus valence of antibody is not less than 8IU/mL after mixture. The immunoglobulin of the invention can be used to immunize patients suffering exposed wounds caused by biting and scratching of a mad dog or other mad animals.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of biological immune goods, particularly relate to and a kind ofly can prevent human rabies and tetanic economic benefits and social benefits valency immunoglobulin and preparation method thereof simultaneously.
Background technology
Rabies are that a kind of pathogen is the elasticity virus of ribonucleic acid type.World Health Organization's statistics, there is rubies epidemiology the countries and regions of the whole world more than 2/3rds, and China and south east asia are in rabies district occurred frequently, the world, and its case accounts for more than 70% of the whole world.Rabies are in case morbidity still can't be cured at present, and mortality rate almost reaches 100%.According to the mad dog immunization scheme that WHO recommends, the resurrectionist is injected Human Rabies Immunoglobulin (RIg), intramuscular injection rabies vaccine then is to guarantee resurrectionist's immune effect.It is present unique effective prevention method that this method is known as by World Health Organization (WHO).
Tetanus is a kind of acute specific infection disease of serious threat human life health, is caused by clostridium tetani intrusion human body wound, growth and breeding, generation toxin.Clostridium tetani and toxin thereof can not be invaded normal skin and mucosa, so after tetanus all occurs in wound.All open injuries all have the tetanic possibility of generation.Human body tetanus immune globulin (TIg) is to have the tire blood plasma of tetanus antibody of height through tetanus toxoid (TD) immunity again after Hepatitis B virus vaccine inoculation, separate purification through cold ethanol method fractional precipitation, pressure filtration, and, be used to prevent and treat tetanus through viral inactivation treatment.
World Health Organization's requirement, to the sick rabic while of lyssodexis active prevention, also antitetanus in advance.The rabies human immunoglobulin and the tetanus human normal immunoglobulin of the clinical use of China are single tiring at present, need multiple injection during use, clinical use inconvenience, and expense height.Prepare and a kind ofly can prevent the rabies economic benefits and social benefits valency immunoglobulin of antitetanus generation in advance again simultaneously, will have bigger social benefit and economic benefit.
Summary of the invention
The objective of the invention is to remedy the deficiency of prior art and product usefulness, provide a kind of and can prevent the rabies economic benefits and social benefits valency immune globulin Pseudobulbus Bletillae (Rhizoma Bletillae) preparation method of antitetanus generation in advance again simultaneously.
Rabies provided by the invention, tetanus double-titer human immunoglobulin contain rabies antibody and the tetanus antibody that height is tired, and its immunoglobulin purity is not less than 92% of total protein content; Rabies antibody is tired and is not less than 100IU/ml, and tetanus antibody is tired and is not less than 60IU/mL; Protein content is not more than 175g/L.
Rabies provided by the invention, tetanus double-titer human immunoglobulin only produce precipitation line with anti-people's serum, all do not produce precipitation line with the serum of anti-horse, anti-cattle, anti-pig, anti-sheep.
Rabies provided by the invention, tetanus double-titer human immunoglobulin and normal human serum relatively, main precipitation line should be IgG, and its IgG monomer and dimeric content sum are not less than 92%.
Rabies, tetanus double-titer human immunoglobulin are made conventional liquid type in the present invention or freeze-dried formulation uses.
The preparation method of rabies of the present invention, tetanus double-titer human immunoglobulin is with the cold ethanol Protein Separation method fractional precipitation of economic benefits and social benefits valency raw blood plasma through routine, extract immunoglobulin components, make immunoglobulin liquid or freeze-dried formulation through the operation of necessity such as pressure filtration, centrifugal, viral inactivation treatment.The key of its preparation method is the preparation of raw blood plasma, and concrete grammar is:
1) selection is healthy, and the blood supply slurry person who meets the requirement of blood products raw blood plasma regulation inoculates rabies vaccine, uses adsorbed Tetanus Vaccine at least after 15 days again and carries out immunity inoculation;
2) with the rabies antibody and the tetanus antibody of solid-phase enzyme-linked immune method (ELISA) detection by quantitative raw blood plasma, the rabies antibody of single blood plasma is tired and is not less than 6IU/mL, tetanus antibody is tired and is not less than 5IU/mL, other meets " blood products raw blood plasma rules " requirement, gathers to be rabies, tetanus double-titer human immunoglobulin raw blood plasma;
3) mix rabies, the tetanus double-titer human immunoglobulin raw blood plasma of gathering, the rabies antibody of blood plasma is tired and is not less than 10IU/mL after mixing, and tetanus antibody is tired and is not less than 8IU/mL.
The raw blood plasma that the present invention collects answers cryogenic freezing to store, and its storage life is no more than 2 years.As the mixed material blood plasma of preparation rabies, tetanus double-titer human immunoglobulin raw material, every batch of need are mixed by the raw blood plasma that is no less than 100 blood supply slurry persons.
Rabies of the present invention, tetanus double-titer human immunoglobulin can be used for by mad dog or other crazy animal bites or scratch exposing patient's immunity.Its use amount is tired in rabies antibody, presses the 20IU/kg body weight and calculates a shot.When injecting accumulated dose greater than 10mL as the required immunoglobulin of individuality, can gradation injection in 1～2 day.Can carry out the injection of rabies vaccine subsequently.
Rabies provided by the invention, a tetanus double-titer human immunoglobulin shot can prevent rabies and tetanus, and be easy to use, has bigger social benefit and economic benefit.The raw blood plasma preparation method of immunoglobulin of the present invention is simple, and the immunoglobulin preparation cost is low.
The specific embodiment
Embodiment 1
1, blood supply slurry person's immunity:
Select healthyly, meet the blood supply slurry person that blood products raw blood plasma regulation requires, inoculated 2,2,1,1,2 doses of rabies vaccine at 0,3,7,14,28 day respectively.Inoculation rabies vaccine after 30 days is used adsorbed Tetanus Vaccine again, connects to be not less than 0.5mL according to 0,14,28 day dosage and to carry out immunity inoculation, and sets up detailed immune record.
2, blood supply slurry person's plasma screening collection:
Select rabies antibody and tetanus antibody in solid-phase enzyme-linked immune method (ELISA) difference detection by quantitative blood supply slurry person's serum or the blood plasma, rabies antibody is tired and is not less than 6IU/mL, tetanus antibody is tired and is not less than 5IU/mL simultaneously, protein content (biuret method) is not less than 55g/L, alanine aminotransferase (reitman-frankel method) is not higher than 25 units, the hbs antigen feminine gender, the syphilis feminine gender, the HIV-1/HIV-2 negative antibody, the blood plasma that HCV negative antibody etc. meets national requirements is privilege blood plasma, by blood products raw blood plasma rules requirement collection is rabies, tetanus double-titer immunoglobulin raw blood plasma, cryogenic freezing stores, and storage life is no more than 2 years.
3, the preparation of rabies, tetanus double-titer human immunoglobulin:
(1) 100 above blood supply slurry persons' blood plasma is melted mixes, mix back blood plasma rabies antibody and tire and be not less than 10IU/mL, tetanus antibody and tire that to be not less than 8IU/mL be acceptable material blood plasma;
(2) the aseptic blood plasma after the separation, centrifugal removal cryoprecipitate under 0～4 ℃;
(3) blood plasma after the removal cryoprecipitate is regulated pH value to 6.8～7.2 with acetate buffer solution, and adding 95% ethanol to reactant liquor ultimate density is 8～10%, extremely-1～-3 ℃ of reacting liquid temperature, centrifugal must the precipitation;
(4) adding 95% ethanol to reactant liquor ultimate density in supernatant is 19～21%, regulates pH value to 6.5～6.8 with acetate buffer solution, and reacting liquid temperature separates to such an extent that precipitate to-4～-5 ℃;
(5) with the precipitation of step (4) lysate stirring and dissolving with 15～20 times of precipitation capacities;
(6) adding 95% ethanol to reactant liquor ultimate density in supernatant is 14～17%, regulates pH value to 5.0～5.2 with acetate buffer solution, and reacting liquid temperature separates to such an extent that precipitate to-4～-5 ℃;
(7) adding 95% ethanol to reactant liquor ultimate density in supernatant is 24～26%, regulates pH value to 7.0～7.2 with the sodium bicarbonate buffer, and reacting liquid temperature separates to such an extent that precipitate to-5～-7 ℃;
(8) with the precipitation of step (7) the lysate stirring and dissolving with 10 times of precipitation capacities, regulating pH value is 7.0～7.2, temperature to 0～2 ℃, and protein concentration is 1.5～2.5%, centrifugal must the precipitation;
(9) precipitation with step (8) is stirred to dissolving with lysate, through ultrafiltration, dealcoholysis, desalination, concentrated, obtains containing rabies, the tetanus double-titer human immunoglobulin stock solution that height is tired;
(10) pH value of adjusting above-mentioned stock solution is 3.8～4.2, and protein concentration 5% is incubated through 24～25 ℃ and to be put 21 days and the Geminivirus inactivation treatment of nano-film filtration;
(11) stock solution of adjustment deactivation, make protein concentration be no more than 17.5%, rabies antibody is tired and is not less than 100IU/mL, and tetanus is tired and is not less than 60IU/mL, pH value is 6.6～7.2, and adds a certain proportion of glycine and an amount of maltose is made semi-finished product as protective agent;
(12) be packed as finished product, assay approval.
4, calibrating:
(1) activity
Rabies antibody is tired and is not less than 100IU/mL, and tetanus is tired and is not less than 60IU/mL.
(2) differentiate
Immune double diffusion method: only produce precipitation line, do not produce precipitation line with the serum of anti-horse, anti-cattle, anti-pig, anti-sheep with anti-people's serum.
Immunoelectrophoresis: compare with normal human serum, main precipitation line should be IgG.
(3) protein content
Be not higher than 175g/L.
(4) purity
Immunoglobulin content is not less than 92% of total protein.
(5) molecular size distribution
IgG monomer and dimer content sum are not less than 92%.
(6) outward appearance
Should be colourless or faint yellow supernatant liquid, can be with opalescence, muddiness should not occur.
(7) heat stability
Insulation was used the visible foreign matters testing fixture, perusal gel-freeization or floccule after 4 hours in 57 ℃ ± 0.5 ℃ water-bath.
(8) pH value
With physiological sodium chloride solution the test sample protein content is diluted to 10g/L, pH value is 6.6～7.2.
(9) other meets human normal immunoglobulin's requirement.
Embodiment 2
The rabies, the tetanus double-titer human immunoglobulin that use embodiment 1 to prepare carry out immunity to the patient, and wherein dog hinders 100 person-portions, and damaged by rats is hindered 20 person-portions, and cat hinders 20 person-portions, pig 20 person-portions of biting.Observed 4 years continuously, do not see morbidity and death.
Claims (10)
1, rabies, tetanus double-titer human immunoglobulin, contain rabies antibody and tetanus antibody that height is tired, its immunoglobulin purity is not less than 92% of total protein, tiring of rabies antibody is not less than 100IU/mL, tetanus antibody is tired and is not less than 60IU/mL simultaneously, and protein content is not more than 175g/L.
2, rabies according to claim 1, tetanus double-titer human immunoglobulin is characterized in that described immunoglobulin only produces precipitation line with the AHS, do not produce precipitation line with anti-horse, cattle, pig, sheep blood serum.
3, rabies according to claim 1, tetanus double-titer human immunoglobulin is characterized in that described immunoglobulin and normal human serum relatively, and main precipitation line is IgG.
4, rabies according to claim 1, tetanus double-titer human immunoglobulin is characterized in that the IgG monomer of described immunoglobulin and dimer content sum are not less than 92%.
5, rabies according to claim 1, tetanus double-titer human immunoglobulin, the dosage form that it is characterized in that described immunoglobulin is liquid type or freeze-dried formulation.
6, the preparation method of the described rabies of claim 1, tetanus double-titer human immunoglobulin is characterized in that the preparation method of its raw blood plasma is:
1) selection is healthy, and the blood supply slurry person who meets the requirement of blood products raw blood plasma regulation inoculates rabies vaccine, uses adsorbed Tetanus Vaccine at least after 15 days again and carries out immunity inoculation;
2) rabies antibody of detection by quantitative raw blood plasma and tetanus antibody, single blood plasma rabies antibody is tired and is not less than 6IU/mL, tetanus antibody is tired and is not less than 5IU/mL, other meets " blood products raw blood plasma rules " requirement, gathers to be rabies, tetanus double-titer human immunoglobulin raw blood plasma;
3) mix rabies, the tetanus double-titer human immunoglobulin raw blood plasma of gathering, the rabies antibody of blood plasma is tired and is not less than 10IU/mL after mixing, and tetanus antibody is tired and is not less than 8IU/mL.
7, the preparation method of rabies according to claim 6, tetanus double-titer human immunoglobulin is characterized in that every batch mixed material blood plasma is mixed by the raw blood plasma that is no less than 100 blood supply slurry persons.
8, the described rabies of claim 1, tetanus double-titer human immunoglobulin are by mad dog or other crazy animal bites or scratch and expose application in patient's immunity.
9, rabies according to claim 8, tetanus double-titer human immunoglobulin are by mad dog or other crazy animal bites or scratch and expose application in patient's immunity, the use amount that it is characterized in that described immunoglobulin is tired in rabies antibody, presses the 20IU/kg body weight and calculates.
10, rabies according to claim 9, tetanus double-titer human immunoglobulin are by mad dog or other crazy animal bites or scratch and expose application in patient's immunity, when it is characterized in that individual immunity globulin injection accumulated dose greater than 10mL, gradation injection in 1～2 day.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2009100739578A CN101502651A (en) | 2009-03-16 | 2009-03-16 | Hydrophobia, tetanus double-titer human immunoglobulin and preparation method thereof |
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| CNA2009100739578A CN101502651A (en) | 2009-03-16 | 2009-03-16 | Hydrophobia, tetanus double-titer human immunoglobulin and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103341163A (en) * | 2013-07-03 | 2013-10-09 | 孙文 | Double-effect horse antiserum used for rabies and tetanus and preparation method thereof |
| CN105770890A (en) * | 2016-03-08 | 2016-07-20 | 苏文全 | Specific protein composition and application method thereof |
| CN107096026A (en) * | 2017-03-08 | 2017-08-29 | 苏文全 | The preparation method of one species-specific proteins composition |
| CN108918880A (en) * | 2018-04-26 | 2018-11-30 | 武汉生命科技股份有限公司 | Tetanus immune globulin blood sample screening reagent box, preparation method and application method |
-
2009
- 2009-03-16 CN CNA2009100739578A patent/CN101502651A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103341163A (en) * | 2013-07-03 | 2013-10-09 | 孙文 | Double-effect horse antiserum used for rabies and tetanus and preparation method thereof |
| CN105770890A (en) * | 2016-03-08 | 2016-07-20 | 苏文全 | Specific protein composition and application method thereof |
| WO2017152744A1 (en) * | 2016-03-08 | 2017-09-14 | 苏文全 | Specific protein composition and mehtod for using same |
| CN107096026A (en) * | 2017-03-08 | 2017-08-29 | 苏文全 | The preparation method of one species-specific proteins composition |
| CN108918880A (en) * | 2018-04-26 | 2018-11-30 | 武汉生命科技股份有限公司 | Tetanus immune globulin blood sample screening reagent box, preparation method and application method |
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Open date: 20090812 |