CN1557490A - Microorganism infection resisting immune globulin - Google Patents

Abstract

The present invention relates to immune globulin preparation for resisting microbe infection, and is especially the preparation process and application of immune globulin preparation prepared with milk for resisting microbe infection. The present invention features that monovalent or multivalent antigen vaccine with proper antigen or antigen combination is used to inoculate selected health dairy cow repeatedly to make it enter to and maintain in high-sensitivity immune state while the dairy cow serum is monitored and the bioactivity of immune globulin in milk is tested, and the collected milk is separated and purified with ultrafiltering membrane to prepare high-purity immune globulin preparation for resisting microbe infection.

Description

The immunoglobulin of anti-microbial infection

Affiliated technical field

The present invention relates to a kind of preparation method and application thereof of immunoglobulin preparation of anti-microbial infection, relate in particular to a kind of employing vaccine virus immunization milk cattle, bring out its high sensitiveness, with the method and the application thereof of the immunoglobulin preparation of its milk produced anti-microbial infection.

The invention technical background

The abundant immunoglobulin A of content is all arranged on normal human's gastrointestinal tract and the respiratory mucosa, resist the multiple invasive organism of invading human body, when immune function of human body weakens or lack, white A content of body cavities mucosal immunity ball dawn can significantly reduce, thereby make pathogenic microorganism invade human body, cause the generation and the development of all kinds of infectious disease.

Immunologic function is not grown as yet and is finished in the infant health, and mainly the immunoglobulin by breast milk comes killing microorganisms, so all kinds of infection take place the baby of not breast fed easily.

Low or the immunologic function of immune function of human body lacks also can cause serious infection, and especially acquired immune deficiency disease patient (AIDS patient) can very easily suffer from various infectious disease because of immunodeficiency.

As everyone knows, contain a large amount of immunoglobulins in the cattle colostrums, can resist various infected by microbes, but the cattle colostrums source is less, easily collecting is not easy to form large batch of production scale.

Multinomial experimentation is verified, by the immunity inoculation milk cattle, can bring out milk cattle and produce specific antibody to resist each quasi-microorganism, and the technology that United States Patent (USP) (4,324,784) is announced can be prevented and treated dental caries with the specific antibody that Streptococcus mutans inoculation milk cattle produces.

Further discover, adopt complex antigen sensitization milk cattle, bring out milk cattle and enter high responsive immune state, the effectively multiple microorganism of antagonism of the antibody capable that produces in its excretory milk.

Technical problem to be solved by this invention is, selects suitable antigen inoculation milk cattle, brings out the high responsive immune state of milk cattle, and from its excretory milk the immunoglobulin of preparation anti-microbial infection, be used for prevention and treat all kinds of infected by microbes.

Summary of the invention

The present invention solves the problems of the technologies described above the technical scheme that is adopted, it is characterized in that, select the combination of suitable antigen or antigen, preparation unit price or multivalence complex antigen vaccine, the healthy milk cattle of immunity inoculation through selecting, monitoring inoculation milk cattle serum has entered high responsive immune state to determine it, repeat to append the vaccine of inoculation sufficient dosage to keep its high responsive immune state, the biological activity of immunoglobulin in the test milk, from the milk of collecting, by adopting supermicro filtration membrane to separate the method for purifying, prepare the immunoglobulin of high-purity anti-microbial infection.

Technical characterictic of the present invention is, independent antigen or complex antigen can be used as the vaccine of preparation inoculation milk cattle, antigenic type can be microorganism or other materials such as antibacterial, virus, sporinite, spirillum, enters high responsive immune state as long as this antigen can cause the milk cattle of inoculation.Optimum antigenic type is an antibacterial, preferably adopts the killed bacterial vaccine of polyvalent antigen form.

Technical characterictic of the present invention is; the immunity inoculation object of selecting can be milk cattle or milk goat; preferably healthy milk cattle; vaccination ways can be subcutaneous injection, intradermal injection, intravenous injection, peritoneal injection, intramammary injection, oral, coloclysis, intramuscular injection etc.; as long as this inoculation form can make inoculation animal sensitization; best vaccination ways is intramuscular injection; if intramuscular injection can not cause high responsive immune state; can adopt the method simultaneous inoculation of intravenous injection or intramammary injection simultaneously, enter high responsive immune state with the provocative inoculation animal.It is enough that the dosage of inoculation is wanted, and dosage range is from 1, times, and 10, sup.6cells is to 1.times.10.sup.8cells.

Technical characterictic of the present invention is whether monitor the inoculation milk cattle to the antigen sensitivity, and whether enter high responsive immune state.There are a lot of known methods can detect this sensitivity, comprising specific antibody titre in cutaneous sensibility test, the mensuration serum, the best way is to test the antibody agglutination that milk cattle inoculation front and back serum produces the polyvalent antigen combination vaccine respectively, if the specific antibody agglutination after inoculation, occurs, then confirm to have entered high responsive immune state.

Technical characterictic of the present invention is, after the high responsive immune state of milk cattle is brought out in the first immunisation inoculation, should repeat to append vaccinal injection, to keep this high responsive immune state of having brought out, duplicate injection should be kept enough dosage, at least should equal dosage of inoculation first, but escalated dose in case of necessity, but be no more than 150% of predose, the time of repeated inoculation and number of times depend on antigenic selection and combination, normally per two all duplicate injections once, but general duplicate injection 4～6 times, but serum antibody agglutination of per at least in the meantime two weeks monitoring, if the immunologic tolerance phenomenon appears in the inoculation milk cattle, the milk that then should stop using this milk cattle is as the feedstock production immunoglobulin.

Technical characterictic of the present invention is, preparing white method of high purity immune ball dawn is, the milk of collecting is adopted the centrifugation defat, adjust milk PH to 4.5～4.9 with sour agent, preferably PH4.7～4.8 are heated 30 ℃～50 ℃, best 40 ℃, 20 minutes～40 minutes heat time heating time, best 30 minutes, can be cooled to 4 ℃ after standing over night, use filter cloth filter, the direct spray drying in white back of agglutinative cheese dawn of nylon filter cloth filter filtering preferably, or adding alkaline agent, adjustment is spray dried to dry powder behind PH to 6.8～7.0, and is perhaps directly white with the centrifuge centrifugation cheese dawn after heating, centrifugation time 15 minutes～45 minutes, best 30 minutes, 3000～6000 rev/mins of centrifugal speeds, best 5000 rev/mins, after the centrifugation removal cheese dawn is white, the supernatant molecular weight is 100,000～120, the supermicro filtration membrane separating and filtering of 000D, concentrate is concentrated into original volume 10～30% o'clock, lyophilization or spray drying prepare dry powder, and temperature can not be above 140 °F during spray drying.

Technical characterictic of the present invention is, in order to keep the biological activity of immunoglobulin, all technology all needs strict control temperature, when milk raw material carried out pasteurization, temperature can not surpass 140 °F, and the time can not be above 15 seconds, in whole process of preparation, all will note controlling temperature, when spray drying, temperature can not be above 140 °F.

Technical characterictic of the present invention is that in the immunoglobulin dry powder of the present invention's preparation, immunoglobulin content is about 8%, wherein immunoglobulin (IgG) is a Main Ingredients and Appearance, biologically active can adopt multiple known method to measure, and antibody agglutination is a kind of simple effective method.

Technical characterictic of the present invention is, the immunoglobulin of the present invention's preparation can be prepared into the various ways preparation and use, can directly use dry powder formulations, or add solution and be mixed with oral liquid and use, perhaps be mixed with into tablet with the other drug adjuvant, capsule, powder, electuary, granule, oil preparation, Emulsion etc. are taken, can also be prepared into food as food additive uses, as milk, milk powder, instant coffee, instant tea, cookies, cake, ice fine jade pouring etc., can also be prepared into chewing gum, the buccal tablet form, with microorganism in the antagonism oral cavity, or be prepared into aerosol with antagonism throat or respiratory tract microorganism, can also be prepared into water-soluble solution and ultrasonic atomizing device common application with the microorganism of antagonism upper respiratory tract.

Technical characterictic of the present invention is, the immunoglobulin preparation of the present invention's preparation is applicable to infant, is particularly useful for not adopting breast-fed babies to prevent and treats the gastrointestinal tract infected by microbes; Be applicable to have a delicate constitution, crowd lower for the resistance, immunologic hypofunction is used for prevention and treatment gastrointestinal tract infected by microbes, respiratory tract infected by microbes, oral cavity, bottleneck throat infected by microbes; Be applicable to that the patient that immunologic hypofunction or immunologic function lack is used for preventing and treating all kinds of infected by microbes, especially AIDS patient's gastrointestinal infection; Also be applicable to all kinds of cavity organ infections that the patient that takes adrenal cortex hormones drug is for a long time taken place.

Technical characterictic of the present invention is, the immunoglobulin preparation of the inventive method preparation is used to prevent and the dosage for the treatment of all kinds of infected by microbes is 0.1～20g/ days, scope is 1～10g/ days preferably, preferably 2～5g/ days, depend on infection type and patient's states, take nontoxic pair of effect for a long time, can use jointly with any other medicine or nutritional labeling.

The invention has the beneficial effects as follows that the high purity immune ball dawn of preparation can effectively be resisted all kinds of pathogenic microorganism in vain, take nontoxic pair of effect for a long time, raw material sources are abundant, and preparation process is simple, popularizes easily.

The specific embodiment

The invention will be further described below in conjunction with embodiment:

Embodiment 1

Immunity inoculation and antibody titer detect

Select escherichia coli (Escherichia Coli for use, American Type Culture StrainNo.13076), cultivated 48 hours under 37 ℃ of temperature with the standard bacteria culture media, 60 ℃ of two hours deactivation antibacterials, centrifugal collection antibacterial, with DDW flushing five times, being diluted to ultimate density with normal saline is 4.times.10.sup.8cells, select healthy milk cattle, at intragluteal injection 5ml, inject once weekly then, continuously around the injection, inject altogether 5 times, the 5th week not, collect inoculation milk cattle milk, adopt little sticking collection program determination Chinese People's Anti-Japanese Military and Political College enterobacteria antibody, with the milk of the milk cattle before the inoculation not in contrast, table 1 monitors its result.

Table 1: milk Chinese People's Anti-Japanese Military and Political College enterobacteria titre contrast before and after the immunity inoculation

After the preceding immunity inoculation of immunity inoculation

0 5,000

Synopsis 1 result shows that Chinese People's Anti-Japanese Military and Political College's enterobacteria antibody titer significantly raises after immunity inoculation, proves that immunity inoculation brought out high immune sensitiveness.

Example 2

Preparation composite bacterial vaccine

(American Type Culture Collection) obtains following strain from U.S. spawn culture center: and staphylococcus aureus (staphylococcus aureus, ATCC#.11631), staphylococcus epidermidis (staphylococcusepidermidis.155), staphylococcus pyogenes 1 type (streptococcus pyogenes.Typel.8671), 3 types (Type3.10389), 5 types (type 5.12347), 8 types (Type 8.12349), 12 types (type 12.11434), 14 types (type 14.12972), 18 types (type 18.12357), 22 types (type 22.10403), aerobacteria (Aerobacteraerogenes.884), escherichia coli (Escherichia Coli.11126), enteritis (sramana) bacillus (Salmonellaenteritidis 13076), bacillus pyocyaneus (Pseudomonas aeruginosa 7700), pneumobacillus (KlebsiellaPneumoniae.9590), mouse typhus (sramana) bacillus (Salmonella typhimurium.13311), hemorrhage bacillus (Haemophilus typhimurium.9333), proteus vulgaris (Proteus vulgaris 13315), shigella (Shigella dysenteriae.13313), Diplococcus pneumoniae (Diplococcus pneumoniae.6303), Streptococcus mutans (Strep.sanguinis 10556), streptococcus micros (Strep.Mitis.6249), go out Streptococcus sanguis (Strep.Sanguinis.10556), streptococcus salivarius (Strep.Salivarius.13419), hypogalactia streptococcus (Strep.Agalactiae.13813).Every kind of microbionation according to the rules, adopt suitable culture fluid, under 37 ℃ of constant temperature, cultivated 48 hours, whether observe cultivation normal, if antibacterial is well-grown in culture medium, with centrifuge separation and Culture liquid, antibacterial is washed five times with physiological saline solution, with 60 ℃ of temperature deactivations 2 hours, kill all antibacterials, from the antibacterial of deactivation, gather sample and continue in culture medium, to cultivate (cultivating 2 hours down for 37 ℃), confirm no bacteria living, the antibacterial lyophilization of collecting becomes the antibacterial powder, above-mentioned every kind of powder takes by weighing 1g, with 500ml normal saline suspendible, and dilution repeatedly, until being mixed with the solution that concentration is 2.times.10.sup.8 bacterial cells/ml, (on 660nm, showing 1.0 ODUs).

Embodiment 3

With combination vaccine immunity inoculation milk cattle

Select healthy milk cattle, composite bacterial vaccine virus immunization milk cattle with embodiment 1 preparation, adopt the intramuscular injection form, at intragluteal injection 5ml vaccine, append injection once every two all identical dosage, repeat altogether to append five times, injecting back 5 days for the first time, gather the milk of inoculation milk cattle, adopt the antibody titer of measured by radioimmunoassay complex antigen, and with immunity inoculation not before the milk gathered compare, find that the antibody titer of complex antigen increases Radix Achyranthis Bidentatae after the immunity inoculation, prove that inoculating milk cattle is in high responsive immune state.

Embodiment 4

The preparation immunoglobulin

30 liters in the milk of collection immunity inoculation milk cattle, the centrifugation defat, add 1N HCL and transfer PH to 4.7, heat to 40 ℃, continue 30 minutes, white with the nylon cloth filtration removal cheese dawn, collect 20 liters of caseic pair of product serums of preparation, directly with the vacuum pump input molecular weight being housed is 10, the staggered tangent plane fluid ultrafiltration apparatus (TFF) of 000D supermicro filtration membrane, input is pressed and is 3Bar, and the output pressure is 2Bar, abandoning supernatant, the concentrated solution of collecting directly is delivered to molecular weight is housed is 100, the above-mentioned same equipment of 000D supermicro filtration membrane, the collection concentrate directly carries out lyophilization and becomes powder, obtains high purity immune ball dawn white spirit powder.

Embodiment 5

The preparation immunoglobulin

Collect the milk of immune milk cattle, defat, acid adjustment, heating, the filtering cheese dawn is white, collects serum, carries out pasteurization (temperature is 140 time remainings 15 seconds), serum directly is pumped to staggered tangent plane fluid ultrafiltration apparatus (TFF) (U.S. Pall company product), selecting the molecular weight of supermicro filtration membrane is 120,000, is concentrated into former volume 20%, collect concentrate directly with 140 temperature spray drying, obtain highly purified immunoglobulin dry powder.

Embodiment 6

Measure the biological activity of immunoglobulin

Prepare antigen with escherichia coli, glue the collection reaction with the immunoglobulin for preparing among this antigen and the embodiment 4, in the test reaction, immunoglobulin combines with antigenic specificity, and titre reaches 3400, proves the immunoglobulin biologically active.

Embodiment 7

Clinical verification immunoglobulin curative effect

Select 8 of the diarrhoea patients of hypoimmunity at random, take the immunoglobulin of embodiment 4 preparations, taking dose 5g for each person every day played 7 patients number of times of suffering from diarrhoea in second day and obviously reduces, and played diarrhoea on the 4th day to stop, and did not have other any pair of reaction.

Claims (10)

1. the present invention relates to a kind of preparation method and application thereof of immunoglobulin preparation of anti-microbial infection, relate in particular to a kind of employing vaccine virus immunization milk cattle, bring out its high sensitiveness, method and application thereof with the immunoglobulin preparation of its milk produced anti-microbial infection, it is characterized in that, select the combination of suitable antigen or antigen, preparation unit price or multivalence complex antigen vaccine, the healthy milk cattle of immunity inoculation through selecting, monitoring inoculation milk cattle serum has entered high responsive immune state to determine it, repeat to append the vaccine of inoculation sufficient dosage to keep its high responsive immune state, the biological activity of immunoglobulin in the test milk, from the milk of collecting, separate the method for purifying by adopting supermicro filtration membrane, the immunoglobulin of preparation high-purity anti-microbial infection.

In accordance with the method for claim 1 in, technical characterictic of the present invention is, independent antigen or complex antigen can be used as the vaccine of preparation inoculation milk cattle, antigenic type can be microorganism or other materials such as antibacterial, virus, sporinite, spirillum, as long as can causing the milk cattle of inoculation, this antigen enters high responsive immune state, optimum antigenic type is an antibacterial, preferably adopts the killed bacterial vaccine of polyvalent antigen form.

In accordance with the method for claim 1 in; technical characterictic of the present invention is; the immunity inoculation object of selecting can be milk cattle or milk goat; preferably healthy milk cattle; vaccination ways can be a subcutaneous injection; intradermal injection; intravenous injection; peritoneal injection; intramammary injection; oral; coloclysis; intramuscular injection etc.; as long as this inoculation form can make inoculation animal sensitization; best vaccination ways is intramuscular injection; if intramuscular injection can not cause high responsive immune state; can adopt the method simultaneous inoculation of intravenous injection or intramammary injection simultaneously; enter high responsive immune state with the provocative inoculation animal; it is enough that the dosage of inoculation is wanted, and dosage range is from 1, times; 10, sup.6 cells is to 1.times.10.sup.8cells.

In accordance with the method for claim 1 in, technical characterictic of the present invention is, whether should monitor the inoculation milk cattle to the antigen sensitivity, and whether entered high responsive immune state, there are a lot of known methods can detect this sensitivity, comprising specific antibody titre in cutaneous sensibility test, the mensuration serum, the best way is to test the antibody agglutination that milk cattle inoculation front and back serum produces the polyvalent antigen combination vaccine respectively, if the specific antibody agglutination after inoculation, occurs, then confirm to have entered high responsive immune state.

In accordance with the method for claim 1 in, technical characterictic of the present invention is, after the high responsive immune state of milk cattle is brought out in the first immunisation inoculation, should repeat to append vaccinal injection, to keep this high responsive immune state of having brought out, duplicate injection should be kept enough dosage, at least should equal dosage of inoculation first, but escalated dose in case of necessity, but be no more than 150% of predose, the time of repeated inoculation and number of times depend on antigenic selection and combination, normally per two all duplicate injections once, but general duplicate injection 4～6 times, but serum antibody agglutination of per at least in the meantime two weeks monitoring, if the immunologic tolerance phenomenon appears in the inoculation milk cattle, the milk that then should stop using this milk cattle is as the feedstock production immunoglobulin.

In accordance with the method for claim 1 in, technical characterictic of the present invention is, preparing white method of high purity immune ball dawn is, the milk of collecting is adopted the centrifugation defat, adjust milk PH to 4.5～4.9 with sour agent, PH4.7～4.8 preferably, heat 30 ℃～50 ℃, best 40 ℃, 20 minutes～40 minutes heat time heating time, best 30 minutes, standing over night after can being cooled to 4 ℃, use filter cloth filter, the direct spray drying in white back of agglutinative cheese dawn of nylon filter cloth filter filtering preferably, or add alkaline agent, be spray dried to dry powder after adjusting PH to 6.8～7.0, perhaps directly white after heating with the centrifuge centrifugation cheese dawn, centrifugation time 15 minutes～45 minutes, best 30 minutes, 3000～6000 rev/mins of centrifugal speeds, best 5000 rev/mins, after the centrifugation removal cheese dawn was white, the supernatant molecular weight was 100,000～120, the supermicro filtration membrane separating and filtering of 000D, concentrate is concentrated into original volume 10～30% o'clock, and lyophilization or spray drying prepare dry powder, and temperature can not be above 140 °F during spray drying.

In accordance with the method for claim 1 in, technical characterictic of the present invention is, in order to keep the biological activity of immunoglobulin, all technology all needs strict control temperature, when milk raw material carries out pasteurization, temperature can not be above 140 °F, time can not surpass 15 seconds, in whole process of preparation, all will note controlling temperature, when spray drying, temperature can not be above 140 °F.

In accordance with the method for claim 1 in, technical characterictic of the present invention is, the immunoglobulin of the present invention's preparation can be prepared into the various ways preparation and use, can directly use dry powder formulations, or add solution and be mixed with oral liquid and use, perhaps be mixed with into tablet with the other drug adjuvant, capsule, powder, electuary, granule, oil preparation, Emulsion etc. are taken, can also be prepared into food as food additive uses, as milk, milk powder, instant coffee, instant tea, cookies, cake, ice fine jade pouring etc., can also be prepared into chewing gum, the buccal tablet form, with microorganism in the antagonism oral cavity, or be prepared into aerosol with antagonism throat or respiratory tract microorganism, can also be prepared into water-soluble solution and ultrasonic atomizing device common application with the microorganism of antagonism upper respiratory tract.

In accordance with the method for claim 1 in, technical characterictic of the present invention is, the immunoglobulin preparation of the present invention's preparation is applicable to infant, is particularly useful for not adopting breast-fed babies to prevent and treats the gastrointestinal tract infected by microbes; Be applicable to have a delicate constitution, crowd lower for the resistance, immunologic hypofunction is used for prevention and treatment gastrointestinal tract infected by microbes, respiratory tract infected by microbes, oral cavity, bottleneck throat infected by microbes; Be applicable to that the patient that immunologic hypofunction or immunologic function lack is used for preventing and treating all kinds of infected by microbes, especially AIDS patient's gastrointestinal infection; Also be applicable to all kinds of cavity organ infections that the patient that takes adrenal cortex hormones drug is for a long time taken place.

In accordance with the method for claim 1 in, technical characterictic of the present invention is, the immunoglobulin preparation of the inventive method preparation is used to prevent and the dosage for the treatment of all kinds of infected by microbes is 0.1～20g/ days, scope is 1～10g/ days preferably, preferably 2～5g/ days, depend on infection type and patient's states, take nontoxic pair of effect for a long time, can use jointly with any other medicine or nutritional labeling.