CN1093173C - Papain blood group diagnose reagent prepn. method - Google Patents
Papain blood group diagnose reagent prepn. method Download PDFInfo
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- CN1093173C CN1093173C CN99108817A CN99108817A CN1093173C CN 1093173 C CN1093173 C CN 1093173C CN 99108817 A CN99108817 A CN 99108817A CN 99108817 A CN99108817 A CN 99108817A CN 1093173 C CN1093173 C CN 1093173C
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Abstract
The present invention discloses a method for making blood groups and cross matching diagnostic reagents by latex of subtropical pawpaw. The technological steps comprise acid precipitation, alkali neutralization, chromatography and purification, elution, ultrafiltration and freeze drying. The blood preparation made by the method has the advantages of low cost, few impurities, high yield, high specific activity rate of enzyme, and meets requirements of medicine quality.
Description
The present invention relates to a kind of diagnostic reagent that is used for blood group and cross matching, be used for the external immunological experiments of blood group system such as Rh, Kidd, particularly prepare the method for blood group and cross matching diagnostic reagent with the natural rubber latex of subtropical crop pawpaw.
Papoid begins experimental technique as a kind of blood group immunology melon from nineteen forty-seven, its significant feature mechanism is that papoid is that a kind of natural plant contains sulfydryl (SH) endopeptidase, this enzyme have proteolytic enzyme and lipase activity, specificity is more widely arranged, animal and plant albumen, polypeptide, ester, acid amides etc. there is stronger hydrolysis ability, and possess the function that can repeat protolysate again plastein especially, for other enzymes can not be compared.Use the hydrolytic action of papoid erythrocyte membrane protein antigen is carried out restricted modification, blood group antigen such as Rh, Kidd are fully exposed, thereby realization antigen---antibody mediated immunity reaction, can be used for the blood donor, the phenotype diagnosis of Rh (D) the type blood group antigen of aspects such as blood recipient, pregnant person, hemolytic disease of newborn, the classification diagnosis of antibody.
ICSH (ICSH), International Society for the Study of Behavioural Development (ISBT), FDA (FDA) united in 1994 convened international 22 tame laboratories once to papoid the reaction on blood immunology work on a large scale, authenticated papoid to Rh (D) blood group system immuno agglutination reaction by summary, in best enzyme activity scope, under the condition of optimum manipulation method, it is sensitive laboratory diagnosis method, its result of experiment is better than the low ionic strength method, consistent side by side with antihuman globulin method diagnostic result, and expense is lower than above-mentioned two methods, and is easy to operate.The units concerned of international organization have unified the standardized content of papoid as blood grouping reagent for this reason, think to be necessary to produce the papoid preparation that a kind of special blood supply liquid immunological experiment uses, to guarantee the verity of experimental result.
At present external basic medical research developed country, amynologic diagnostic method to the Rh blood group system, make laws by medical administrative department, become the routine inspection project of medical institutions, Blood Center, China's " China's blood transfusion regulations for technical operation " one 97 editions, " national Clinical Laboratory working specification "-97 editions is with the at first method of papoid method as Rh blood group system immunology detection.The Ministry of Health also carries out the blood group immunological experiment to color enzyme process as front three hospital Blood Transfusion Department in the standard for acceptance of front three hospital, blood bank checks and accepts one of content.
General crowd's blood group is under the main typing of blood situation with ABO, most of people's whiles are also being deposited Rh blood group antigen or Rh blood group antibody, investigation in China before according to 1985, Rh antigen positive person accounts for 99.6% among the crowd of Han nationality, the Rh of ethnic minority antigen positive person accounts for 95%, the frequency that detects of the crowd of Han nationality imperfection antibody is 0.3-0.2%, and the recall rate of ethnic minority's imperfection antibody will have rising greatly.And medically conventional blood group experiment or cross matching experiment, can only diagnose the blood epidemic disease immunology agglutination reaction that is caused by thoroughness antibody, distinguishing abo blood group and intermiscibility thereof, can obtain diagnostic result and can only under 37 ℃ of conditions, experimentize by reagent such as papoids for the Rh blood group immuno agglutination reaction that is caused by imperfection antibody.
Because Rh blood group system and ABO blood group system have status of equal importance on clinical medicine, can cause the generation of malpractices such as blood recipient's death, lifelong deformity, stillborn foetus, hemolytic disease of newborn because of the immune response of Rh blood group system.This shows, use this nontoxic pure natural reagent of papoid, safe and reliable, experiment is accurate, significant.
Papaya (cayica papava) is a kind of torrid zone, subtropical plant, it has certain requirement to physical environment, weather, soil, the whole world mainly contains small part area plantations such as South East Asia, South America, China also has only several provinces and regions such as Guangdong and Guangxi Provinces, Hainan, Yunnan, Fujian, Guizhou, Taiwan to plant, but it is edible as fruit or vegetables that pawpaw plantation always mainly is a picking fruit, and the latex that also begins in recent years to utilize pawpaw fruit or cane to gather is made Chemicals and biological products.Main effective constituent in the pawpaw latex has: papoid, Disken, pawpaw peptase etc., hydrolysis ability is in various degree all arranged, but from pawpaw latex, extract blood group diagnosis papoid also in the exploratory stage, purify with special reagent after the external general employing pickling, separating impurity, but special reagent is still being maintained secrecy, domestic also have some units to produce as a trial, but its cost of manufacture height, purification difficulty are big, cost an arm and a leg, purposes is also failed more developing, so the papoid biochemical products still have many domestic blanks that are.
It is simple to the purpose of this invention is to provide a kind of leaching process, the yield height, and foreign matter content is few, and cost is low, can be used for the high purity of Rh blood group system diagnosis, the extracting method of the papoid of height ratio vigor.
The raw material sources that are used to extract papoid are in following several respects, and 1, the fresh latex of from bright pawpaw or cane, gathering and obtaining.2, the air-dry thing of pawpaw latex or dry thing.3, through the pawpaw latex product of other preliminary Physical Processing.
The present invention is used for the papoid of Blood Preparations owing to being used as pharmaceutical reagent, and the essential strictness of preparation process is controlled, and also should avoid other chemical substances to mix in the impurity while of removing inside and pollute and destroy its character, below the processing step of summary production:
One, Acid precipitation impurity elimination:
Because the natural latex of gathering back of pawpaw contains more impurity, and some unwanted chemical ingredientss are arranged and deposit, need be with these Impurity removals before refining, the method for removing is that the latex that receipts are adopted is back diluted with 1: 3 with deionized water, by filtering or centrifugal removal leaf and silt.Add thin liquids such as mineral acid example hydrochloric acid, sulfuric acid, phosphoric acid then, PH is transferred to 1-3, place, impurity in the latex is precipitated out under acidic conditions, in the Acid precipitation process, should under 4 ℃ of temperature, carries out during work, the limit adds sour limit and stirs evenly, acid is dispersed evenly in the latex, in order to avoid cause damage because of temperature variation influences enzyme activity too greatly, throw out is by filtering or centrifugal removing.
Two, alkali neutralization:
Through the filtrate of Acid precipitation, because solution is acid, neutralize, neutralizing agent uses mineral alkali, especially at NaOH, KOH, Ca (OH)
2In choose, solution is transferred to neutrality, about PH7, through semi-permeable membranes dialysis desalination.
Three, remove heavy metal:
The pawpaw latex of handling through acid ﹠ alkali liquid, also may exist the heavy metal element of trace, in order to obtain highly purified product, above-mentioned solution can be added reductive agent such as dithiothreitol (DTT) activates, and adding sequestrant such as EDFA (ethylenediamine tetraacetic acid (EDTA)), consumption is 1-2%, and heavy metals such as copper in the solution, manganese, iron, cadmium are removed, and separates by dialysis.But it is atomic that most of pawpaw planting sites contain heavy metal element, and visual generally speaking raw material is surveyed the inspection situation and this step that decides what to use.
Four, chromatography and purifying:
For the purity that improves papoid and the performance of reagent, need it is carried out chromatography and purifying, chromatography is to adopt the interpolation neutral salt to carry out, the above-mentioned pawpaw latex by volume that obtains is calculated, add the ammonium sulfate powder, make concentration reach 40-50%, placed 0.5-1 hour, allow the papoid crystalline deposit, centrifugal or press filtration separates.
Throw out is dissolved with small amount of deionized water, put in 0.01M acetic acid-sodium-acetate buffer, (this buffer solution ph 5.0), put into CMC-cellulose ion post of crossing through the equal abundant balance of damping fluid, enzyme solution approximates the ion column triplication, carry out ionic adsorption, allow positively charged papoid gene be combined on CMC-cellulose carrier, thereby obtain further chromatography purification.
Five, wash-out:
Elution process be by elutriant with papoid from above-mentioned CMC-cellulose carrier selectively desorb cement out, to obtain the best effective ingredient of target product, the eluent that the present invention uses always is 0.01M acetic acid, sodium-acetate buffer PH5.0 wash-out, hac buffer is added ion column with certain flow rate, collect protein peak, till the effluent liquid protein ingredient is minimum.
Six, ultra-filtration and separation concentrates:
The papoid that from elutriant, reclaims, can use ultrafiltration membrane treatment, molecular sieving technology by ultra-filtration membrane, particulate and moisture content in bacterium in the papoid behind the purifying, virus, the latex solid are removed, ultra-filtration process can also make the papoid behind the purifying be concentrated, shorten time of drying significantly, reduces manufacturing, cost and improve the quality of products.The present invention adopts the hollow fiber ultrafiltration membrane of molecular weight cut-off 10000 units, rejection can reach 93%, this ultra-filtration membrane mild condition, no phase change, be difficult for causing material damage, the macromolecular substance of papoid is retained down, and water and small molecular weight impurity can see through filter membrane, thereby reach the purpose that concentrates with purifying.
In ultra-filtration process, can adopt flocculation technique and ultra-filtration technique to combine, improve extract yield and shortening time.Flocculence be with the papoid liquid behind a certain amount of wash-out under constantly stirring, slowly add a certain amount of polysaccharide flocculant, add inorganic coagulant aids more successively and regulate PH, staticly settle after stirring, centrifuging is measured the transparence and the enzyme of supernatant liquor and is lived, described polyose flocculation agent is avirulent fc, addition 0.2g/L, PH5.0-5.5, inorganic flocculation coagulant aids is fc, dosage 0.2g/L, PH5.0-5.5.Ultrafiltration and concentration is that hollow fiber ultrafiltration membrane is cleaned with deionized water, and the above-mentioned clear liquid control certain pressure that will cross through flocculation treatment and flow cycling stream are crossed the ultrafiltration post and got final product up to the moisture content amount is minimum then.
Seven, lyophilize:
With the above-mentioned ultrafiltration and concentration liquid weighing bottling that makes, putting in the loft drier of freeze drier-20～-35 ℃ earlier into carries out quick-frozen and freezes, extract dry moisture content situation at vacuum pump then and under-20～-35 ℃, carry out sublimation drying, in bottle, add inert gas or nitrogen behind the dried powdered and preserve sealing, in order to avoid enzyme deactivation, then in preserving below 4 ℃.
The product of papoid is differentiated available ultraviolet method discriminating, and by relevant home or overseas standard test, the present invention adopts the Sodium phosphate dibasic method to do qualitative analysis, and water casein method and spectrophotometry are done quantitative analysis.Ratio vigor 〉=750 units/the mg of papoid.
The present invention compared with the prior art, its outstanding substantive distinguishing features and obvious improvement is:
1, raw material sources are extensive, can extract papain from pawpaw, and ground such as the Guangdong and Guangxi Provinces of China, Yunnan, Fujian all can be cultivated, and the conditional request of pawpaw cultivation is not high, and the plantation pawpaw not only can be gathered in the crops fruit and also can be gathered in the crops latex and make biotechnological formulation.
2, present Blood Preparations extraction cost height, China is populous, most of dependence on import of past, annual about 400,000,000 person-times of need carry out serological blood group diagnosis and cross matching experiment, the import fee that year has spent approximately reaches 600,000,000 yuan more than, the exploitation of this project will provide the blood group diagnostic products for China, reduce the foreign exchange expenditure.
3, the storage temperature of some blood system needs-20 ℃ at present, and papoid of the present invention is kept at 4 ℃ above freezing and gets final product, and does not need cryogenics and equipment, has reduced storage and running cost.
4, the pure natural reagent that makes with product of the present invention is nontoxic, safe and reliable, meets medical quality requirements fully.
5, papoid of the present invention, owing to can modify the antigen of erythrocyte membrane protein, strengthen blood group system antigen body immune responses such as Rh, Kidd, but also destroy the antigenicity of part blood group antigen, as blood groups such as M, N, Fya, Fyb, diagnose out correct result so can make blood group and cross matching detection single stage method and two step method, papoid can detect red corpuscle Rh (D) blood group system or change anti-E, anti-C, anti-Jka into, also can identify the feminine gender and the positive exactly, thus the generation of stopping malpractice.
6, method of the present invention is simple, and the manufacturing price of product is low, the yield height, and production process does not have waste water and gas to pollute.
Below be embodiments of the invention:
Embodiment one:
Get the fresh pawpaw latex of gathering, after pressing 1: 3 dilution proportion coarse filtration of whey slag with deionized water earlier, add hydrochloric acid 30% solution, transferring PH is 2, left standstill 1 hour, the centrifugal precipitation of removing, supernatant liquor adds the NaOH diluent and transfers about PH7, semi-permeable membranes dialysis desalination, get supernatant liquor and add dithiothreitol (DTT) and chela and contain each 1 ‰ consumption of agent EDTA, trace heavy metal is removed in dialysis, removes precipitation, filtrate slowly adds the ammonium sulfate powder, amount to be added reaches at 45% o'clock and leaves standstill, and allows precipitation separate out, and takes out precipitation and adds the triplication of deionized water by last ion column, solution acetate buffer solution (0.01M, PH5.0) fully balance CMC-cellulose column absorption of crossing is used 0.01M, PH5.0 acetate buffer solution wash-out, collect protein peak, make the outflow aqueous solution contain albumen and stop the most after a little while, behind ultrafiltration and concentration, trace is bottled with elutriant, add freeze drier and carry out freezing vacuum low temperature-30 ℃ sublimation drying, get finished product.
Embodiment two:
Get air-dry pawpaw latex, add earlier 5 times of water dissolution by weight, adding dilute sulphuric acid adjusting PH is 3, left standstill 2 hours, and the centrifugal precipitation of removing, clear liquid adds Ca (OH)
2Solution is transferred about PH7, with semi-permeable membranes dialysis desalination, get supernatant liquor add dithiothreitol (DTT) and sequestrant EDTA each 1.5 ‰, trace-metal salt is removed in dialysis, remove precipitation, filtrate adds ammonium sulfate, make when reaching strength of solution 40% and leave standstill, separate out precipitation, take out the precipitate with deionized water dilution, and to transfer PH with hac buffer be about 5, add polysaccharide flocculant fc-1 precipitation, use CMC-cellulose column absorption then, the acetic acid of using 0.01mph5.0 again is towards eluant solution, collect protein peak, contain to flowing out the aqueous solution that albumen is minimum to be stopped, with elutriant behind the hollow-fibre membrane ultrafiltration and concentration of 1000 units by volume or the weight bottling, get finished product with-25 ℃ vacuum and low temperature sublimation dryings, last in bottle, add nitrogen and preserve, can never degenerate in 3 years at 0-4 ℃ refrigerator.
Embodiment three:
Get the pawpaw fresh latex and add 3 times of dilutions of double distilled water, add 30% phosphoric acid, transferring PH is 1, leaves standstill half an hour, with contaminant filter, solution adds NaOH to be transferred about PH to 7, with semi-permeable membranes dialysis desalination, adds ammonium sulfate to solution then and reaches 50%, place and allowed its precipitation in 1 hour, with the double distilled water dilution, put to CMC-cellulose carrier absorption, with the flushing of PH5.0 hac buffer, collect protein peak, make the basic wash-out of papoid, add polysaccharide flocculant flocculation (also can flocculate) again, concentrate with the hollow cellulose membrane ultrafiltration, the back bottling, vacuum-sublimation drying under-35 ℃ of low temperature obtains finished product, transports blood station or hospital to and preserves in 4 ℃ of refrigerators.
Claims (5)
1. method of utilizing pawpaw latex to produce blood group diagnose reagent preparation, it is characterized in that its processing step is: (1), adds mineral acid contamination precipitation is separated towards rare or dissolving pawpaw latex with deionized water; (2) add sodium hydroxide or potassium hydroxide, calcium hydroxide neutralization, and with semi-permeable membranes dialysis desalination; (3) remove trace heavy metal; (4) chromatography and purifying; (5) wash-out of papoid; (6) use the ultra-filtration membrane ultrafiltration; (7) cryogenic vacuum sublimation drying; (8) adding inert gas or nitrogen-sealed preserves.
2. the method for utilizing pawpaw latex to produce blood group diagnose reagent preparation according to claim 1, it is characterized in that adding mineral acid, contamination precipitation to be separated be the PH that adds hydrochloric acid or sulfuric acid regulation solution be 1-3.
3. the method for utilizing pawpaw latex to produce blood group diagnose reagent preparation according to claim 1, it is characterized in that chromatography and purifying are to be added to the solution of removing behind the trace heavy metal with the ammonium sulfate powder to make the papoid crystalline deposit, transfer PH to 5 then, use the CMC-cellulose purification.
4. the method for utilizing pawpaw latex to produce blood group diagnose reagent preparation according to claim 1, the wash-out that it is characterized in that papoid is the papoid that is eluted in the CMC-cellulose purification with the hac buffer of PH5.
5. the method for utilizing pawpaw latex to produce blood group diagnose reagent preparation according to claim 1 is characterized in that the cryogenic vacuum sublimation drying is to carry out sublimation drying in-20～-35 ℃ under vacuum pump draws water part.
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| Application Number | Priority Date | Filing Date | Title |
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| CN99108817A CN1093173C (en) | 1999-06-19 | 1999-06-19 | Papain blood group diagnose reagent prepn. method |
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| CN99108817A CN1093173C (en) | 1999-06-19 | 1999-06-19 | Papain blood group diagnose reagent prepn. method |
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| CN1242429A CN1242429A (en) | 2000-01-26 |
| CN1093173C true CN1093173C (en) | 2002-10-23 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102286609B (en) * | 2011-06-22 | 2013-01-02 | 北京工商大学 | Method for detecting heavy metal pollution by using papain |
| JP7110360B2 (en) | 2017-10-09 | 2022-08-01 | テルモ ビーシーティー バイオテクノロジーズ，エルエルシー | Freeze-drying method |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| CN114181925A (en) * | 2020-09-14 | 2022-03-15 | 武汉禾元生物科技股份有限公司 | Industrial purification and freeze-drying method for recombinant proteinase K |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1088614A (en) * | 1992-12-24 | 1994-06-29 | 中国科学院昆明植物研究所 | Refined enzyme purification process for papain |
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- 1999-06-19 CN CN99108817A patent/CN1093173C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1088614A (en) * | 1992-12-24 | 1994-06-29 | 中国科学院昆明植物研究所 | Refined enzyme purification process for papain |
Non-Patent Citations (1)
| Title |
|---|
| 华东理工大学学报VOL.22NO.1 1996.2.1 用絮凝和超滤技术纯化木瓜蛋白酶 刘叶青等 * |
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