CN101732732B - Bovine type tuberculin standard substance and preparation method thereof - Google Patents
Bovine type tuberculin standard substance and preparation method thereof Download PDFInfo
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Abstract
The invention relates to bovine type tuberculin standard substance and a preparation method thereof. The bovine type tuberculin standard substance is prepared by the following steps of: preparing bovine type tuberculin through processes of culturing, inactivating, filtering, purifying, and the like by utilizing mycobacteriumbovis separated in China; measuring the contents of protein, nucleic acid and polysaccharide in the bovine type tuberculin; and preparing the bovine type tuberculin standard substance by combining biological test measurement.
Description
Technical field
The present invention relates to bovine type tuberculin standard substance and preparation method thereof, belong to standard substance technology and veterinary biologics technical field.
Background technology
Bovine tuberculosis is a kind of chronic bacillary disease to the animal and human that is caused by Mycobacterium bovis, and this disease is worldwide distribution, also is the main infectious disease of China cattle, can propagate to the mankind, makes the people suffer from tuberculosis, threatens bigger to human beings'health.Because China carries out elimination system to the positive cattle of bovine tuberculosis, the prapes diagnostic techniques is particularly important, and the bovine tuberculin diagnostic techniques is unique diagnostic method that OIE (OIE) is recommended.The demarcation of tiring of purification bovine tuberculosis rhzomorph product is the skin allergic reaction method with Cavia porcellus, as contrast, through the comparison of dermoreaction area, confirms iu (IU) content of bovine tuberculin product with statistical method with standard substance.By the specified reference laboratory of OIE (OIE) international standard substance is provided, also is referred to as the base standard article, various countries' laboratory is traced to the source with international standard substance and is prepared the national standard article of country separately, is used for the demarcation of tuberculin product.Yet the prescription of standard substance is high, and method for preparing and check or scaling method are strict, about preparation procedure and scaling method are not seen relevant report so far.
Summary of the invention:
The objective of the invention is to set up method that a kind of utilization prepares bovine type tuberculin standard substance by the cow mycobacteria of isolated in China to prepare qualified bovine type tuberculin standard substance.
The present invention realizes through technology path once; Promptly be through utilizing cow mycobacteria to prepare the bovine tuberculosis rhzomorph through cultivation, deactivation, filtration, purification and freeze-dry process process by isolated in China, and through to wherein albumen, nucleic acid and measurement of the polysaccharide content and combine the dermal sensitivity test of Cavia porcellus and cattle to measure to prepare bovine type tuberculin standard substance.
Detailed description of the present invention:
One, the preparation of bovine type tuberculin standard substance
1. culture medium
The logical synthetic medium of Soviet Union (veterinary biologics Rule Committee of the Ministry of Agriculture. People's Republic of China's veterinary biologics rules; Version in 2000; Chemical Industry Press; 2000) composition comprises as follows, asparagine, citric acid, magnesium sulfate, dipotassium hydrogen phosphate, ferric ammonium citrate, glycerol etc., and every kind of composition is analytical pure.
2. strain
Mycobacterium bovis C68001 and C68002 strain that the present invention is used are provided by China Veterinery Drug Inspection Office.
(1) strain properties
1) the strain form is observed under optical microscope, and thalline is elongated, directly or slightly curved bacillus, is about 1.5～4 microns, and is wide 0.2～0.6 micron, Gram-positive, and acid-fast stain is positive.
2) cultural character is cultivated more than 20 days at Pei Shi (Petragnani) solid medium, and the single bacterium colony of growth is faint yellow graininess, and is comparatively dry.Well-grown in the logical culture medium of Soviet Union is cultivated 20 days culture medium top layers and more rich Mycoderma should be occurred, and liquid should be limpid, and growth is pure, and incubation time was looked growing state general 2～3 months.
3) virulence is to tame rabbit ear vein injection viable bacteria 6～10mg, and rabbit can cause death.
(2) production prepares with seed
1) after the first order seed preparation broke a seal freeze-drying lactobacillus, inoculation coulee culture medium (seeing appendix 1) was put in 37 ℃ of greenhouses and is cultivated about about two weeks, and development growth is yellow particle shape bacterium colony or lawn, is first order seed.
2) the secondary seed preparation hooks up first order seed lawn or bacterium colony with the platinum shovel, evenly coats on several Zhi Xiaochuan medium slant, puts in 37 ℃ of greenhouses and cultivates about 10 days; Hook up lawn with the platinum shovel from the coulee medium slant, at the logical abundant porphyrize of synthetic medium (seeing appendix 2) inboard wall of test tube of Soviet Union, test tube then tilts; Contact thalline gently with culture medium; It is floated on the culture medium liquid level, slowly move in 37 ℃ of greenhouses and cultivated 7～15, then form thin Mycoderma in media surface.Choosing a truffles film with the platinum ring is transplanted in the logical synthetic medium bottle of Soviet Union; In 37 ℃ of greenhouses, cultivated 7～15, can form the rubber-like Mycoderma at the culture bottle liquid surface, Mycoderma is transplanted in the logical synthetic medium bottle of Soviet Union again; In 37 ℃ of greenhouses, cultivated 7～15; On the pin liquid surface, form little have gauffer, Mycoderma that elasticity is stronger, this Mycoderma promptly can be used as mass-produced seed, through perusal; Liquid must be limpid, and pure, thin white, the no living contaminants of Mycoderma is as secondary seed.
3. the preparation of bovine tuberculosis rhzomorph
(1) inoculation picking Mycoderma is put on the liquid level of bottle slowly, holds a bottle person and has filled in bottle stopper, replaces gently.
After inoculation finishes, tackle vaccinated medium bottle and check, find to have the seed sinking or the person of scattering to inoculate by bottle.Inoculation carefully moves in the greenhouse after finishing, and puts down gently on culturing rack.
(2) be incubated at and cultivate 2～3 months Mycodermas under the room temperature and should grow thicklyer, should stop to cultivate.
(3) deactivation is cultivated and is finished, and culture is through 121 ℃ of 30min inactivation treatment.
(4) mixing culture that coarse filtration at first respectively gets the corresponding lot numbers of two bacterial strains, identical bottle number mixes the back and adds the layer of copper yarn with double gauze and filter and remove Mycoderma.
(5) fine filtering is in seitz filter (model: 2000ml production firm: add 8 filter plate (models: first III the safe Bioisystech Co., Ltd in the northern part of the country in Beijing); The manufacturer: the source, Dalian is along the Filters company limited); Opening the air compressor power supply makes purified water fully through behind the filter mixed bacteria liquid being sucked storage tank; Make it to pass through fully filter again, be bovine tuberculosis rhzomorph filtrating.
(6) purification joins 40% trichloroacetic acid solution (seeing appendix) of 1 times of volume in the tuberculin filtrate of 9 times of volumes slowly, and the limit edged stirs, and behind the mixing, leaves standstill 14～24h at 2～8 ℃, makes albumen precipitation.Then carefully with supernatant sucking-off, collecting precipitation.With 1% trichloroacetic acid solution (seeing appendix) with the precipitate mixing that suspends again, centrifugation again (4000r/min, 2～8 ℃, 30min) remove supernatant, so repeat 3 times.After the last centrifugal end, abandon supernatant and take by weighing wet albumen net weight.
(7) heavily dissolve filtration precipitate is added a small amount of 1mol/L sodium hydroxide solution (seeing appendix) earlier, make it dissolving.Calculate the volume that dilutes back rhzomorph cumulative volume and needs pH 7.4 phosphate buffers (seeing appendix) according to wet albumen net weight and suitable dilution multiple.After the dilution, transfer pH to 7.4, filter the material standed for that is bovine type tuberculin standard substance with the sterilization Seitz filter.
4. the preparation of appendix culture medium and reagent
(1) coulee culture medium
1) composition is seen table 1
The composition of table 1 coulee culture medium
4 of new fresh hen eggs
2% malachite green oxalate aqueous solution 6ml
Saline solution 100ml
Annotate: saline solution prescription, method for making
Monosodium glutamate (containing Sodium Glutamate 80%) 1g
Potassium dihydrogen phosphate (acidity) 1g
Glycerol 6ml
Pre-cooling water for injection 94ml
With the above component water-bath dissolving after-filtration packing of heating, 121 ℃ of sterilizations 30 minutes, subsequent use.
2) method for making
Fresh hen egg is soaped brush, rinse well, will clean egg and immerse in 75% ethanol several minutes with tap water.With sterile gauze egg surface ethanol is dried.Remove egg one end housing with aseptic nipper,, and make a call to an aperture with this end down, egg liquid is flowed into have in the wide mouthed bottle of bead, rock and make the yellow and white mix homogeneously in the upper end.With shaking up in saline solution and the 2% malachite green oxalate 6ml impouring egg liquid, filter the packing test tube through two-layer gauze sterilization funnel.Test tube is put into the inclined-plane in serum coagulator, 85-90 ℃ of sterilization 1 hour, second day can repeat once, takes out that inspection is qualified does not promptly put 2-8 ℃ of refrigerator and preserve subsequent use through having.
(2) the logical synthetic medium of Soviet Union
1) composition is seen table 2
The composition of the logical synthetic medium of table 2 Soviet Union
Asparagine 4g
Citric acid 2g
Aqueous magnesium sulfate 0.5g
Dipotassium hydrogen phosphate 0.5g
Ferric ammonium citrate 0.05g
Glycerol 60ml
Pre-cooling water for injection 940ml
2) method for making
Solid constituent in the prescription is added in a small amount of pre-cooling water for injection.Slowly heating makes all material dissolving.Go into Yu Shui and glycerol.With sodium hydroxide solution adjustment pH to 7.0.Heated and boiled filters the back packing.Sterilized 30 minutes in 40 minutes or 121 ℃ with 116 ℃ of sterilizations.
(3) trichloroacetic acid solution (40% and 1%)
40% trichloroacetic acid solution: the 400g trichloroacetic acid is dissolved in the 1000ml pre-cooling water for injection gets final product;
1% trichloroacetic acid solution: 1 part of 40% trichloroacetic acid solution and 9 parts of pre-cooling water for injection mix homogeneously are got final product.
(4) 1mol/L sodium hydroxide solution
40g sodium hydroxide (analytical pure) is added pre-cooling water for injection to get final product to 1000ml.
(5) 1mol/L hydrochloric acid solution
With 83.3ml concentrated hydrochloric acid (mass percentage concentration: 36-38%; Molar concentration: about 12mol/L) and 917.7ml pre-cooling water for injection mix homogeneously get final product.
(6) pH7.4 phosphate buffer
First liquid: the 1/30mol/L disodium hydrogen phosphate, get the 11.9g disodium hydrogen phosphate and be dissolved in the 1000ml pre-cooling water for injection and get final product;
Second liquid: the 1/30mol/L potassium dihydrogen phosphate, get the 4.5g potassium dihydrogen phosphate and be dissolved in the 1000ml pre-cooling water for injection and get final product.
With 81 parts of first liquid and 19 parts of second liquid mix homogeneously, transfer pH to 7.4, sterilized 30 minutes for 121 ℃.
Two, the evaluation of bovine tuberculosis rhzomorph (material standed for)
1. steriling test
Undertaken by " People's Republic of China's veterinary biologics rules " (version in 2000) prescriptive procedure.
2. determining the protein quantity
The purification bovine tuberculosis rhzomorph semi-finished product that the steriling test that takes a morsel is qualified adopt Kjeldahl, and the concrete operations step is referring to attaching " cattle type purification tuberculin national standard article protein content determination standard operating instructions " according to the determining the protein quantity result,
With pH 7.4 phosphate buffers (phenol content is 0.5% (W/V)) purification bovine tuberculosis rhzomorph semi-finished product are diluted to every 1m1 and contain 1.8mg protein.
3. purity testing (Physicochemical and Biological Studies on Various Preparations ofTuberculin Purified Protein Derivative APPLIED MICROBIOLOGY; March; 1965; Copyright
1965 American Society for Microbiology; Vol.13, No.2; Pharmacopoeia of People's Republic of China 2005 three ones " bacillus calmette-guerin vaccine purified protein derivative ").
(1) nucleic acid content is measured
These article of getting 2～3ml adopts ultraviolet visible spectrophotometry, measures absorbance in wavelength 260nm place, presses E
1cm 1%=200 calculate nucleic acid content.
(2) determination of polysaccharide
With normal saline solution dilution anhydrous grape saccharide, preparation 0～100 μ g/ml, glucose standard solution; Sulphuric acid 225ml is joined in the 75ml physiological sodium chloride solution, and other takes by weighing anthrone 0.6g, joins in the 10ml ethanol; Above-mentioned solution is mixed, be configured to the anthrone mixed liquor.Accurately measure 1.0ml respectively, variable concentrations standard glucose solution, and these article add 4.0ml anthrone mixed liquor, are mixed, and measure absorbance with wavelength 620nm place, the calculating polyoses content after putting boiling water bath 20min.
(3) criterion
Every 1mg protein contains polysaccharide and the nucleic acid total amount should not be higher than 0.1mg.
(4) material standed for selects protein content suitable; The material standed for (every 1mg protein contains polysaccharide and the nucleic acid total amount should not be higher than 0.1mg) that nucleic acid and polysaccharide total amount end exceed standard is used for quantitative packing, lyophilisation; With the quantitative packing of packing instrument of error≤1%, by suitable freeze-drying curve lyophilization.
Three, the check of bovine type tuberculin standard substance
1. the uniformity test sample thief is 15, with the tuberculin protein content of every ampoule of Kjeldahl mensuration, and meansigma methods, standard deviation, the coefficient of variation, the standard deviation and the coefficient of variation answer≤5%.
2. stability test is measured with hot accelerated stability test, checks under hot conditions the time that sample is placed.And with these data as foundation, be that the next group stability test does the basis.
3. titration (for the first time)
(1) pilot study
1) Cavia porcellus sensitization is selected 10～12 of albino guinea-pigs about body weight 400g; (earlier the bacillus tuberculosis bovis culture is scraped, weighs, grinds at femoribus internus deep intramuscular injection bacillus tuberculosis bovis sensitinogen respectively; Add the dilution of an amount of normal saline, 121 ℃ of deactivations 30 minutes, the reuse incomplete Freund is processed oil emulsion; Make every 1ml contain bacillus tuberculosis bovis 8～10mg, 80 ℃ of water-baths were sterilized 2 hours after the packing.Steriling test qualified rearmounted 2～8 ℃ subsequent use) 0.5ml.
2) Cavia porcellus is surveyed quick sensitization after 5 weeks, and every sensitized guinea pig buttocks is taken out a fritter by hair (about 3cm
2, avoid injecting sensitinogen one side), cattle type purification tuberculin international standard substance or national standard article are diluted to every 1ml in the 2nd and contain tuberculin 32.5IU, in epilation place intradermal injection 0.1ml, injection back 48 h observation, injection site red swelling of the skin area reaches 1cm
2Above person can be used for titration.
(2) formal mensuration
Testing sample is diluted to 4 kinds of variable concentrations that every 1ml contains tuberculin albumen (be equivalent to 32 by 1mg, 500IU calculates) 25,50,100 and 200IU; (biological standard calibrating institute of Britain country, down together) also does same dilution with international standard substance.Select 8 qualified Cavia porcelluss of sensitization, breast abdominal part two sides are removed (about 3 * 9cm by alopecia areata,trichomadesis in the proxima luce (prox. luc) of injecting
2); Adopt samsara to change point mode and inject this 8 kinds of rhzomorph diluents successively in epilation place of every Cavia porcellus; Every some intradermal injection 0.1ml; Inject back 24 hours with each injection point red swelling of the skin area of vernier caliper measurement, calculate each gradient of testing sample and each dilution factor of international standard substance and cause red swelling of the skin area (be judged to be the master with 24 hours, can do judgement for the second time if necessary in 42 hours) on one's body 8 Cavia porcelluss.(OIE's work, translate at veterinary office of the Ministry of Agriculture/Chinese world animal health and epidemiology center. terrestrial animal diagnostic test and vaccine handbook [S] .2007:399-410.)
4. safety verification
With normal saline purification bovine tuberculosis rhzomorph is diluted to every 1ml 32500IU.With 2 of the Cavia porcelluss of body weight 350～450g, every lumbar injection tuberculin 1ml observed 10.
5. specificity check
(1) checks with cattle
With 20 of healthy susceptible cattle, be divided into two groups, 10 every group.
First group of seized tuberculin of left side of neck intradermal injection, right side injection international standard substance.
Second group of left side of neck intradermal injection international standard substance, seized tuberculin is injected on the right side.
The every 1ml of each intradermal injection contains the rhzomorph 0.1ml of 32500IU, injects judgement in back 72 hours, and indivedual cattle are had various reactions when causing, and can do injection for the second time at once at different parts.Observe seized tuberculin and international standard substance cattle is had or not non-specific inflammatory reaction.
(2) check with Cavia porcellus
Carry out the specificity check with the Cavia porcellus of body weight 350～450g, its method is basic identical with " titration (for the first time) ", but rhzomorph do not dilute, and every 1ml contains 32500IU, intradermal injection 0.1ml.Inject judgement in back 48 hours, observation has or not any inflammatory reaction.
6. the general statistical value with each result of cooperation unit of confirming of definite value (cooperation is demarcated) national standard article potency unit is represented, generally needs to carry out through several experienced laboratory cooperations.Participant should adopt unified design, unified method and unified record format, and calibration result must be through statistical procedures (calibration result need be obtained independently effective result at least 5 times).
(1) data statistics is demarcated the data of being measured through cooperation, adopts one one of veterinary drug allusion quotation of the People's Republic of China (PRC) (version in 2005), the quantitative response parallel line assay method in the bioassay statistic law.(metering technology normalized JJG1006-94 " primary standard material " State Bureau of Technical Supervision of country of the People's Republic of China (PRC); One one of in 2005 version of People's Republic of China's veterinary drug allusion quotation)
(2) iu is confirmed through calculating the iu content of 1mg tuberculin after the data statistics of cooperation calibration result, with this determined value as the national standard article.
Positive effect of the present invention:
The present invention relates to a kind of bovine type tuberculin standard substance and preparation method thereof.Bovine type tuberculin standard substance involved in the present invention is through utilizing the cow mycobacteria by isolated in China to prepare the bovine tuberculosis rhzomorph through technical processs such as cultivation, deactivation, filtration, purification, and through to wherein albumen, nucleic acid and measurement of the polysaccharide content and combine biological test to measure to prepare bovine type tuberculin standard substance.Method of the present invention makes standard substance prepare science, rigorous, perfect more, makes each step scientific and precise according to the standard substance preparation procedure, demarcate through cooperation, and data statistics, the definite value that guarantees to tire is accurate.
The preparation of 1 N of type purification of embodiment tuberculin national standard material
1. culture medium
The logical synthetic medium of Soviet Union (veterinary biologics Rule Committee of the Ministry of Agriculture. People's Republic of China's veterinary biologics rules; Version in 2000; Chemical Industry Press; 2000) composition comprises as follows, asparagine 4g, citric acid 2g, magnesium sulfate 0.5g, dipotassium hydrogen phosphate 0.5g, ferric ammonium citrate 0.05g, glycerol 60ml, and every kind of composition is analytical pure.Adding distil water 940ml prepares 50000ml altogether.
2. strain
Mycobacterium bovis C68001 and C68002 strain (China Veterinery Drug Inspection Office provides).
(1) production prepares with seed
1) first order seed preparation
After inoculation broke a seal freeze-drying lactobacillus, inoculation coulee culture medium was put in 37 ℃ of greenhouses and is cultivated about about two weeks, and development growth is yellow particle shape bacterium colony or lawn, is first order seed.
2) secondary seed preparation
Hook up first order seed lawn or bacterium colony with the platinum shovel, evenly coat on several Zhi Xiaochuan medium slant, put in 37 ℃ of greenhouses and cultivated about 10 days, can physically well develop.This culture is as the liquid domestication.
For make the tuberculosis strain that is grown on the solid medium can liquid medium within growth and breeding, adapting to the needs that tuberculin is produced, should in growth early stage with bacterial classification inoculation on fluid medium, carry out seed domestication work.Hook up lawn with the platinum shovel from the coulee medium slant; At the logical abundant porphyrize of synthetic medium inboard wall of test tube of Soviet Union; The test tube that tilts then contacts thalline gently with culture medium, and it is floated on the culture medium liquid level; Slowly move in 37 ℃ of greenhouses and cultivated 7～15, then form thin Mycoderma in media surface.Choosing a truffles film with the platinum ring is transplanted in the logical synthetic medium bottle of Soviet Union; In 37 ℃ of greenhouses, cultivated 7～15, can form the rubber-like Mycoderma at the culture bottle liquid surface, Mycoderma is transplanted in the logical synthetic medium bottle of Soviet Union again; In 37 ℃ of greenhouses, cultivated 7～15; On the pin liquid surface, form little have gauffer, Mycoderma that elasticity is stronger, this Mycoderma promptly can be used as mass-produced seed, is secondary seed.
3) secondary seed is identified
Perusal, liquid must be limpid, pure, thin white, the no living contaminants of Mycoderma.The culture not limpid for liquid discards.
3. rhzomorph manufacturing
1) inoculation picking Mycoderma is put on the liquid level of bottle slowly, holds a bottle person and has filled in bottle stopper, replaces gently.
After inoculation finishes, tackle vaccinated medium bottle and check, find to have the seed sinking or the person of scattering to inoculate by bottle.Inoculation carefully moves in the greenhouse after finishing, and puts down gently on culturing rack.
2) must make regular check on temperature, humidity and have or not varied bacteria growing between the cultivation culture period.The polluter is timely, and autoclaving discards.
3) deactivation was cultivated 2～3 months, and the Mycoderma growth is thicker, stopped to cultivate, and culture is put 121 ℃ of deactivation 30min.
4) mixing coarse filtration at first adds the layer of copper yarn with double gauze and the culture bottle culture of two the identical bottle of the same lot number of bacterial strain numbers is mixed to filter removes Mycoderma.
5) fine filtering adds 8 first 3 filter plates in filter, opens the air compressor power supply and makes purified water pass through filter fully.Mixed bacteria liquid is sucked storage tank, make it to pass through fully filter again, be tuberculin filtrate.About 40000ml.
6) purification joins 40% trichloroacetic acid solution (seeing appendix) of 1 times of volume in the tuberculin filtrate of 9 times of volumes slowly, and the limit edged stirs, and behind the mixing, leaves standstill 14-24 hour at 2～8 ℃, makes albumen precipitation.Then carefully with supernatant sucking-off, collecting precipitation.With the precipitate mixing that suspends again, (4000r/min, 30min) removes supernatant, 3 times like this by 2～8 ℃ in centrifugation again with 1% trichloroacetic acid solution (seeing appendix).After the last centrifugal end, abandon supernatant and weigh, and deduct centrifuge tube weight and obtain wet albumen net weight, approximately about 70g.
Heavily dissolve and filter.Precipitate is added (about 7ml) 1mol/L sodium hydroxide solution (seeing appendix) on a small quantity earlier, make it dissolving.Calculate the volume that dilutes back rhzomorph cumulative volume and needs pH7.4 phosphate buffer (seeing appendix) according to wet albumen net weight and suitable dilution multiple.Measuring a small amount of pH7.4 phosphate buffer earlier dilutes rhzomorph concentrated solution in the centrifuge tube; And be transferred to the 3000ml bottle with graduated cylinder; PH 7.4 phosphate buffers of reuse residual volume carry out 2 washings and successively are transferred to the 3000ml bottle centrifuge tube and graduated cylinder, and this is single batch purification bovine tuberculosis rhzomorph stock solution.Purification bovine tuberculosis rhzomorph stock solution is suitably diluted, transfer pH to 7.4, filter with the sterilization Seitz filter and be material standed for.
The calibrating of 4 material standed fors
(1) steriling test
Carry out for 448 pages by " People's Republic of China's veterinary biologics rules " (version in 2000) appendix.
(2) determining the protein quantity
The purification bovine tuberculosis rhzomorph semi-finished product that the steriling test that takes a morsel is qualified adopt Kjeldahl (44 pages of appendix of " People's Republic of China's veterinary drug allusion quotation " version in 2000) to measure every 1ml total nitrogen, calculate protein content.The concrete operations step referring to; Attach " cattle type purification tuberculin national standard article protein content determination standard operating instructions " according to the determining the protein quantity result; With pH7.4 phosphate buffer (phenol content is 0.5% (W/V)) purification bovine tuberculosis rhzomorph semi-finished product are diluted to every 1ml and contain 58,500IU/1.8mg.
(3) the every 1mg protein of purity testing contains polysaccharide and the nucleic acid total amount should not be higher than 0.1mg.
1) nucleic acid content is measured
These article of getting 2～3ml adopts ultraviolet visible spectrophotometry, measures absorbance in wavelength 260nm place, presses E
1cm 1%=200 calculate nucleic acid content, and measuring the result is 0.002mg/mg protein.
2) determination of polysaccharide
With physiological sodium chloride dilution anhydrous grape saccharide, preparation 0～100ug/ml, glucose standard solution; Sulphuric acid 225ml is joined in the 75ml physiological sodium chloride solution, and other takes by weighing anthrone 0.6g, joins in the 10ml ethanol; Above-mentioned solution is mixed, be configured to the anthrone mixed liquor.Accurately measure 1.0ml respectively, variable concentrations standard glucose solution, and these article add 4.0ml anthrone mixed liquor, are mixed, and put boiling water bath and measure absorbance with wavelength 620nm place after 20 minutes, and calculating polyoses content is 0.072mg/1mg/mg protein.
5. material standed for selects protein content suitable, and nucleic acid and polysaccharide total amount do not exceed the material standed for lyophilizing (every 1mg protein contains polysaccharide and the nucleic acid total amount should not be higher than 0.1mg) of standard.Nucleic acid and polyoses content amount to 0.072mg/1mg protein.
6. quantitatively packing, lyophilisation with the quantitative packing of packing instrument of error≤1%, are pressed suitable freeze-drying curve lyophilization.1163 ampoules of 1200ml tuberculin packing, every branch loading amount 1ml is about residue 30ml.
The product inspection of 2 Ns of type purifications of embodiment tuberculin national standard material
1. the uniformity test sample thief is 15, measures every ampoule tuberculin protein content result with Kjeldahl and sees table 3.
Uniformity result after the lyophilizing of table 3 cattle type purification tuberculin
15 ampoule meansigma methods X are 100.2130%, and standard deviation S is 4.7855%, and coefficient of variation CV is 4.8%.
2. stability test international standard substance regulation storage life is 8 years; We quicken preservation effect under 50 ℃ of conditions of anti-aging test inspection tuberculin albumen with heat; Sample is placed 50 ℃ to be saved to through efficacy determinations and can to reach the qualification determination standard in 21 days in 7,14,21 and 28 days; Ratio was 0.1109 in the 28th day, judged that defective (the qualification determination standard is: 0.9-1.1), see table 4.
Table 4 stability test (anti-aging test) result
3. the Preliminary Determination of tiring
(1) Cavia porcellus sensitization
Select albino guinea-pig 10-12 about body weight 400g; (earlier the bacillus tuberculosis bovis culture is scraped, weighs, grinds at femoribus internus deep intramuscular injection bacillus tuberculosis bovis sensitinogen respectively; Add the dilution of an amount of normal saline, 121 ℃ of deactivations 30 minutes, the reuse incomplete Freund is processed oil emulsion; Make every 1ml contain bacillus tuberculosis bovis 8～10mg, 80 ℃ of water-baths were sterilized 2 hours after the packing.Steriling test qualified rearmounted 2～8 ℃ subsequent use) 0.5ml.
(2) Cavia porcellus is surveyed quick
Sensitization takes out a fritter by hair (about 3cm with every sensitized guinea pig buttocks after 5 weeks
2, avoid injecting sensitinogen one side), cattle type purification tuberculin international standard substance or national standard article are diluted to every 1ml in the 2nd and contain tuberculin 32.5IU, in epilation place intradermal injection 0.1ml, injection back 48 h observation, injection site red swelling of the skin area reaches 1cm
2Above person can be used for titration, and every Cavia porcellus all reaches 1cm
2More than.
Testing sample is diluted to 4 kinds of variable concentrations that every 1ml contains tuberculin albumen (be equivalent to 32 by 1mg, 500IU calculates) 25,50,100 and 200IU; International standard substance is also done same dilution.Select 8 qualified Cavia porcelluss of sensitization, breast abdominal part two sides are removed (about 3 * 9cm by alopecia areata,trichomadesis in the proxima luce (prox. luc) of injecting
2); Adopt samsara to change point mode and inject this 8 kinds of rhzomorph diluents successively in epilation place of every Cavia porcellus; Every some intradermal injection 0.1ml; Inject back 24 hours with each injection point red swelling of the skin area of vernier caliper measurement, calculate each gradient of testing sample and each dilution factor of international standard substance and cause the red swelling of the skin area on one's body 8 Cavia porcelluss.Dilution factor average response area and international standard substance average response area ratio through calculating each sample to be measured are: 25IU ratio 0.07 50IU ratio 0.068 100IU ratio 0.064 and 200IU ratio 0.0114.
4. safety verification
With normal saline purification bovine tuberculosis rhzomorph is diluted to every 1ml 32500IU.With 2 of the Cavia porcelluss of body weight 350-450g, every lumbar injection tuberculin 1ml observed 10.The result is all strong to live.
5. specificity check
(1) checks with cattle
With 20 of healthy susceptible cattle, be divided into two groups, 10 every group.
First group of seized tuberculin of left side of neck intradermal injection, right side injection international standard substance.
Second group of left side of neck intradermal injection international standard substance, seized tuberculin is injected on the right side.
The every 1ml of each intradermal injection contains the rhzomorph 0.1ml of 32500IU, injects judgement in back 72 hours.Measure the result and all do not have irritated reaction.
(2) check with Cavia porcellus
Cavia porcellus with body weight 350-450g carries out the specificity check, and every 1ml contains 32500IU rhzomorph, intradermal injection 0.1ml.Inject judgement in back 48 hours, the result does not all have any inflammatory reaction.
6, definite value (cooperation is demarcated) is used the titration method, and the general statistical value with each result of cooperation unit of confirming of national standard article potency unit is represented, generally needs to carry out through several experienced laboratory cooperations.Participant should adopt unified design, unified method and unified record format, and calibration result must be through statistical procedures (calibration result need be obtained independently effective result at least 5 times).We at first draw up a plan for, and are demarcated by units such as biological pharmaceutical factory, Heilungkiang, biological pharmaceutical factory, Jilin, biological pharmaceutical factory, Chengdu, middle prison institutes, and per unit is carried out independent trials 2 times.
(1) data statistics is demarcated the data measured through cooperation, adopt quantitative response parallel line assay method in the bioassay statistic law (Chinese veterinary drug allusion quotation committee. version in 2005 of People's Republic of China's veterinary drug allusion quotation. Chinese agriculture publishing house, 2006)
(2) the iu content that calculates the 1mg tuberculin after the iu specified data is added up is 32322IU, with this determined value as the national standard article.
Claims (1)
1. method for preparing bovine type tuberculin standard substance is characterized in that preparation process is following:
(1) culture medium
Be stated from the logical synthetic medium of Soviet Union in " veterinary biologics Rule Committee of the Ministry of Agriculture, People's Republic of China's veterinary biologics rules, version in 2000; Chemical Industry Press, 2000 ", composition comprises as follows; Asparagine 4g, citric acid 2g, magnesium sulfate 0.5g, dipotassium hydrogen phosphate 0.5g, ferric ammonium citrate 0.05g, glycerol 60ml; Every kind of composition is analytical pure, and adding distil water 940ml prepares 50000ml altogether;
(2) strain
Mycobacterium bovis C68001 that China Veterinery Drug Inspection Office provides and C68002 strain;
Production prepares with seed
1) first order seed preparation
After inoculation broke a seal freeze-drying lactobacillus, inoculation coulee culture medium was put in 37 ℃ of greenhouses and was cultivated for two weeks, and development growth is yellow particle shape bacterium colony or lawn, is first order seed;
2) secondary seed preparation
Hook up first order seed lawn or bacterium colony with the platinum shovel, evenly coat on several Zhi Xiaochuan medium slant, put in 37 ℃ of greenhouses and cultivated 10 days, can physically well develop, this culture is as the liquid domestication;
For make the tuberculosis strain that is grown on the solid medium can liquid medium within growth and breeding, adapting to the needs that tuberculin is produced, should in growth early stage with bacterial classification inoculation on fluid medium, carry out seed domestication work; Hook up lawn with the platinum shovel from the coulee medium slant; At the logical abundant porphyrize of synthetic medium inboard wall of test tube of Soviet Union; The test tube that tilts then contacts thalline gently with culture medium, and it is floated on the culture medium liquid level; Slowly move in 37 ℃ of greenhouses and cultivated 7～15, then form thin Mycoderma in media surface; Choosing a truffles film with the platinum ring is transplanted in the logical synthetic medium bottle of Soviet Union; In 37 ℃ of greenhouses, cultivated 7～15, can form the rubber-like Mycoderma at the culture bottle liquid surface, Mycoderma is transplanted in the logical synthetic medium bottle of Soviet Union again; In 37 ℃ of greenhouses, cultivated 7～15; On the pin liquid surface, form little have gauffer, Mycoderma that elasticity is stronger, this Mycoderma promptly can be used as mass-produced seed, is secondary seed;
3) secondary seed is identified
Perusal, liquid must be limpid, pure, thin white, the no living contaminants of Mycoderma, the culture not limpid for liquid discards;
(3) rhzomorph manufacturing
1) inoculation picking Mycoderma is put on the liquid level of bottle slowly, holds a bottle person and has filled in bottle stopper, replace gently,
After inoculation finishes, tackle vaccinated medium bottle and check by bottle, find to have the seed sinking or the person of scattering to inoculate, inoculation carefully moves in the greenhouse after finishing, and puts down gently on culturing rack;
2) must make regular check on temperature, humidity and have or not varied bacteria growing between the cultivation culture period, the polluter is timely, and autoclaving discards;
3) deactivation was cultivated 2～3 months, and the Mycoderma growth is thicker, stopped to cultivate, and culture is put 121 ℃ of deactivation 30min;
4) mixing coarse filtration at first adds the layer of copper yarn with double gauze and the culture bottle culture of two the identical bottle of the same lot number of bacterial strain numbers is mixed to filter removes Mycoderma;
5) fine filtering adds 8 first 3 filter plates in filter, opens the air compressor power supply and makes purified water pass through filter fully; Mixed bacteria liquid is sucked storage tank, make it to pass through fully filter again, be tuberculin filtrate, 40000ml;
6) purification joins 40% trichloroacetic acid solution of 1 times of volume in the appendix in the tuberculin filtrate of 9 times of volumes slowly, and the limit edged stirs, and behind the mixing, leaves standstill 14-24 hour at 2～8 ℃, makes albumen precipitation; Then carefully with supernatant sucking-off, collecting precipitation; With the precipitate mixing that suspends again, again through 4000r/min, 2～8 ℃, supernatant is removed in 30min centrifugation with 1% trichloroacetic acid solution in the appendix, 3 times like this, after the last centrifugal end, abandons supernatant and weighs, and deduct centrifuge tube weight and obtain wet albumen net weight, 70g;
Heavily dissolve filtration precipitate is added the about 7ml of 1mol/L sodium hydroxide solution in the appendix earlier, make it dissolving; Calculate the volume that dilutes pH7.4 phosphate buffer in back rhzomorph cumulative volume and the needs appendix according to wet albumen net weight and suitable dilution multiple; Measuring a small amount of pH7.4 phosphate buffer earlier dilutes rhzomorph concentrated solution in the centrifuge tube; And be transferred to the 3000ml bottle with graduated cylinder; PH 7.4 phosphate buffers of reuse residual volume carry out 2 washings and successively are transferred to the 3000ml bottle centrifuge tube and graduated cylinder, and this is single batch purification bovine tuberculosis rhzomorph stock solution; Purification bovine tuberculosis rhzomorph stock solution is suitably diluted, transfer pH to 7.4, filter with the sterilization Seitz filter and be material standed for;
(4) material standed for calibrating
1) steriling test
Carry out for 448 pages by version " People's Republic of China's veterinary biologics rules " appendix in 2000;
2) determining the protein quantity
The purification bovine tuberculosis rhzomorph semi-finished product that the steriling test that takes a morsel is qualified; Adopt by the Kjeldahl of 44 pages of records of " People's Republic of China's veterinary drug allusion quotation " appendix of version in 2000 and measure every 1ml total nitrogen; Calculate protein content; The concrete operations step referring to; Attach " cattle type purification tuberculin national standard article protein content determination standard operating instructions " according to the determining the protein quantity result, using phenol content is that 0.5% (W/V) pH7.4 phosphate buffer is diluted to every 1ml with purification bovine tuberculosis rhzomorph semi-finished product and contains 58,500 IU/1.8mg;
3) the every 1mg protein of purity testing contains polysaccharide and the nucleic acid total amount should not be higher than 0.1mg;
1. nucleic acid content is measured
These article of getting 2～3ml adopts ultraviolet visible spectrophotometry, measures absorbance in wavelength 260nm place, presses
2. determination of polysaccharide
With physiological sodium chloride dilution anhydrous grape saccharide, preparation 0～100ug/ml, glucose standard solution; Sulphuric acid 225ml is joined in the 75ml physiological sodium chloride solution, and other takes by weighing anthrone 0.6g, joins in the 10ml ethanol; Above-mentioned solution is mixed, be configured to the anthrone mixed liquor; Accurately measure 1.0ml respectively, variable concentrations standard glucose solution, and these article add 4.0ml anthrone mixed liquor, are mixed, and put boiling water bath and measure absorbance with wavelength 620nm place after 20 minutes, and calculating polyoses content is 0.072mg/1mg/mg protein;
(5) material standed for selects protein content suitable, and nucleic acid and polysaccharide total amount do not exceed the material standed for lyophilizing of standard, and every 1mg protein contains polysaccharide and the nucleic acid total amount should not be higher than 0.1mg, and nucleic acid and polyoses content amount to 0.072mg/1mg protein;
(6) quantitatively packing, lyophilisation, with the quantitative packing of packing instrument of error≤1%, by suitable freeze-drying curve lyophilization, 1163 ampoules of 1200ml tuberculin packing, every branch loading amount 1ml, residue 30ml;
The preparation of appendix culture medium and reagent
(1) coulee culture medium
1) composition
2) method for making
Fresh hen egg is soaped brush, rinse well, will clean egg and immerse in 75% ethanol several minutes with tap water; With sterile gauze egg surface ethanol is dried, removed egg one end housing with aseptic nipper, down this end; And make a call to an aperture in the upper end, and egg liquid is flowed into have in the wide mouthed bottle of bead, rock and make the yellow and white mix homogeneously; With shaking up in saline solution and the 2% malachite green oxalate 6ml impouring egg liquid, filter the packing test tube through two-layer gauze sterilization funnel, test tube is put into the inclined-plane in serum coagulator; 85-90 ℃ of sterilization 1 hour, second day can repeat once, takes out that inspection is qualified does not promptly put 2-8 ℃ of refrigerator and preserve subsequent use through having;
(2) the logical synthetic medium of Soviet Union
1) composition
2) method for making
Solid constituent in the prescription is added in a small amount of pre-cooling water for injection; Slowly heating makes all material dissolving; Go into Yu Shui and glycerol, with sodium hydroxide solution adjustment pH to 7.0; Heated and boiled filters the back packing, sterilizes 30 minutes in 40 minutes or 121 ℃ with 116 ℃ of sterilizations;
(3) 40% and 1% trichloroacetic acid solution
40% trichloroacetic acid solution: the 400g trichloroacetic acid is dissolved in the 1000ml pre-cooling water for injection gets final product,
1% trichloroacetic acid solution: 1 part of 40% trichloroacetic acid solution and 9 parts of pre-cooling water for injection mix homogeneously are got final product;
(4) 1mol/L sodium hydroxide solution
Analytical pure 40g sodium hydroxide is added pre-cooling water for injection to get final product to 1000ml;
(5) 1mol/L hydrochloric acid solution
With mass percentage concentration: 36%-38%; The 83.3ml concentrated hydrochloric acid of molar concentration: 12mol/L and 917.7ml pre-cooling water for injection mix homogeneously get final product;
(6) pH7.4 phosphate buffer
First liquid: 1/30 mol/L disodium hydrogen phosphate is got the 11.9g disodium hydrogen phosphate and is dissolved in the 1000ml pre-cooling water for injection and gets final product;
Second liquid: 1/30 mol/L potassium dihydrogen phosphate is got the 4.5g potassium dihydrogen phosphate and is dissolved in the 1000ml pre-cooling water for injection and gets final product;
With 81 parts of first liquid and 19 parts of second liquid mix homogeneously, transfer pH to 7.4, sterilized 30 minutes for 121 ℃.
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| CN105606556B (en) * | 2016-01-28 | 2018-09-21 | 广西壮族自治区中医药研究院 | The ultraviolet spectrophotometry of polyoses content in fokien angiopteris rhizome medicinal material |
| CN109395099B (en) * | 2018-12-13 | 2020-02-21 | 北京祥瑞生物制品有限公司 | Method for improving stability of tuberculin BCG-PPD skin test diagnostic reagent |
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| CN1715422A (en) * | 2004-06-30 | 2006-01-04 | 中国农业科学院哈尔滨兽医研究所 | Novel Mycobacterium bovis and the method for quick identification of mycobacterium tuberculosis and application |
| CN1810838A (en) * | 2005-01-28 | 2006-08-02 | 中国农业科学院哈尔滨兽医研究所 | Recombinant antigen protein for diagnosing ox tuberculosis and its prepn process |
| CN1858238A (en) * | 2006-03-22 | 2006-11-08 | 李洪敏 | Quick tubercle myco-bacillus culture medium |
| CN1888076A (en) * | 2005-06-29 | 2007-01-03 | 李建林 | Fast identification method for ox tubercle bacillus and its drug sensitive test kit |
| CN101294958A (en) * | 2008-05-13 | 2008-10-29 | 中国兽医药品监察所 | Dot-ELISA fast detecting reagent kit for bovine tuberculosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1715422A (en) * | 2004-06-30 | 2006-01-04 | 中国农业科学院哈尔滨兽医研究所 | Novel Mycobacterium bovis and the method for quick identification of mycobacterium tuberculosis and application |
| CN1810838A (en) * | 2005-01-28 | 2006-08-02 | 中国农业科学院哈尔滨兽医研究所 | Recombinant antigen protein for diagnosing ox tuberculosis and its prepn process |
| CN1888076A (en) * | 2005-06-29 | 2007-01-03 | 李建林 | Fast identification method for ox tubercle bacillus and its drug sensitive test kit |
| CN1858238A (en) * | 2006-03-22 | 2006-11-08 | 李洪敏 | Quick tubercle myco-bacillus culture medium |
| CN101294958A (en) * | 2008-05-13 | 2008-10-29 | 中国兽医药品监察所 | Dot-ELISA fast detecting reagent kit for bovine tuberculosis |
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