CN1054204C - Active directional production process for limulus reagent - Google Patents
Active directional production process for limulus reagent Download PDFInfo
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- CN1054204C CN1054204C CN 95105652 CN95105652A CN1054204C CN 1054204 C CN1054204 C CN 1054204C CN 95105652 CN95105652 CN 95105652 CN 95105652 A CN95105652 A CN 95105652A CN 1054204 C CN1054204 C CN 1054204C
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- amebocyte lysate
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- tachypleus amebocyte
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Abstract
The present invention relates to an active directional production process for limulus reagents. An anticoagulant, a washing agent, a disintegrant, a sensitizer and a sensitive protective agent are centrifugally separated twice so that the walls of limulus blood cells are difficult to become a block by denaturalization and are easy to disintegrate for enhancing the activity of solidifying protease. A prepared limulus reagent has the advantages of difficult pollution, high sensitivity, long self blank negative observation time and high product stability. The present invention protects limulus resources, avoids product overstock and waste due to blind production, and enhances enterprise economic benefit.
Description
The invention belongs to a kind of active directed production technology of tachypleus amebocyte lysate.
At present, the production technology of tachypleus amebocyte lysate has of all kinds, as publishing in February, 1979, the 35th page of " Fujian medicine " magazine " preparation of east limulus blood cell lysate and sensitivity determination thereof ", the sensitivity of the limulus reagent of production is low, at 10ng-0.1ng/ml, tachypleus amebocyte lysate self is short blank negative observing time, only be 2-4 hour, of poor quality, product stability is poor.
The object of the invention is to provide a kind of active directed production technology of tachypleus amebocyte lysate, and the tachypleus amebocyte lysate of production is used to detect harmful bacterial endotoxin, and is highly sensitive, and quality is good.Product is active directed, production is economized in raw materials, and protect the king crab raw material resources better.
The present invention with anti-coagulants, suspending washing agent, crack agent, sensitizer, handle to quick protective agent, separate by two times centrifugal, product lot quantity productive capacity is strong, every batch of 0.1ml loading amount reaches 22000, its production technology is as follows:
1, new fresh and alive king crab is cleaned, with 75% alcohol sterilization, the ostium blood sampling adds anti-coagulants, makes king crab blood at 10 ℃.
2, under 750-800 rev/min speed with the centrifuging of king crab blood, abandon blue serum, the white cell 1: 4 by volume (v/v) of remainder is added the suspending washing agent, after the processing, 1: 5 by volume (v/v) adds cell and cracks agent, to haemocyte is cracked fully, deposit acquisition cell Dissolve things inside at 0 ℃.
3, the adding volume ratio is 1: 0.6 CHCl
32-5 ℃ of following jolting 10 minutes, centrifuging under 2590 rev/mins-3000 rev/mins speed, the collecting cell Dissolve things inside is a tachypleus amebocyte lysate stoste; add sensitizer 7% (g/v); preliminary examination adds to quick protective agent again, makes (1-3)-β Glucn (fungi polysaccharide) of every ml soln 100ng get final product packing; in subzero 45 ℃ of-55 ℃ of freeze-drying; 35 ℃ with lower sealing, quality inspection, lettering finished product.
The normal method for making that adopts several reagent of production technology: 1. anti-coagulants is: working concentration is that NaOH or the HCI solution of 0.03M heated 40 minutes, remove the bacterial endotoxin in the solvent, transfer pH value to neutral, add pre-assigned aseptic auxiliary material solution dilution, make and contain theophylline concentration 0.025M in the anti-coagulants solution, NaC10.5M, transferring pH value with tuis-HCl is 7.6, add the decolouring of 0.6% (g/v) active carbon adsorption, heat suction filtration packing in 15 minutes again 120 ℃ of autoclave sterilizations 60 minutes, anti-coagulants and king crab blood volume ratio are 1: 4.5 (v/v).
2. the suspending washing agent is to heat 40 minutes with 0.025MNaOH or HCl solution, destroy contaminated bacteria in the solvent, accent pH value neutrality, adding concentration is the NaCl solution of 0.5M, transferring pH value with tris-HCl is 7.6, adds the decolouring of 0.6% (g/v) active carbon adsorption, the suction filtration packing, standby at the 120C autoclaving, washing agent is 1: 4 (v/v) with the ratio of limulus blood cell surplus amount.
3. cracking agent is with 0.1M concentration NaOH solution heating 40 minutes, transfers pH value neutrality, transfers PH7.6 with 0.03Mtris-HCl solution, adds the decolouring of 0.6% (g/v) active carbon adsorption, the suction filtration packing, and autoclave sterilization is standby.Crack the limulus blood cell volume ratio 1: 8 (v/v) that agent and centrifuging go out.
4. sensitizer heated 40 minutes with the NaOH or the HCl solution of 0.15M concentration, transferred pH value neutrality, and being used to dilute NaCl solution is 0.5M, CaCl
2Solution is 0.31M, MgCl
2Solution is 0.41M (g/v), adds acticarbon 0.6% adsorption bleaching that heat suction filtration packing in 15 minutes, autoclaving is standby.Sensitizer and tachypleus amebocyte lysate stoste volume ratio 7% (g/v).
5. the active protective agent of regulating, (claiming to quick protective agent again).Heated 40 minutes with 0.05-0.1MNaOH or HCl solution; accent pH value neutrality; add 1-3-β Glucn (fungi polysaccharide); making concentration is 100ng/ml; as tachypleus amebocyte lysate active matter protective agent; this reagent is mainly used in and adds in the reagent stoste, makes the usefulness of the controlled experiment of tachypleus amebocyte lysate susceptibility, and has improved the single-minded gel reaction of tachypleus amebocyte lysate and bacterial endotoxin.
The concentration of each reagent of the present invention is suitable, can not cause cell membrane atrophy sex change to be piece, can not cause cracking difficulty and the activity that suppresses to solidify white enzyme of dawn; Can not cause limulus blood cell to shift to an earlier date molten account yet and break.And, solvent is carried out apyrogeneity handle, prevent that tachypleus amebocyte lysate is contaminated, improve solvent purity.Therefore; the present invention prepares tachypleus amebocyte lysate yield quality height; 0.05-0.006ng/ml is brought up in product sensitivity, improves more than 200 times than the 10ng-0.1ng/ml of relevant technologies, and self reaches 12-24 hour blank positive observing time; high 6 times than interrelated data report; product is deposited the effect phase and was extended to more than 2 years from 1 year, and product quality reaches the U.S. FDA quality standard, realizes active directed production; save king crab blood raw material, protected the king crab resource effectively.Avoided simultaneously blindly producing causing product overloading and waste, improved the business economic benefit.
Raw material of the present invention is mainly derived from Tachypleus tridentatus, and male and female are not limit, and for guaranteeing king crab blood quality, after king crab breaks away from the water surface before blood sampling, can not surpass two days later, and guarantee that individuality is not injured.Before the production, all produce the necessary aseptic process of container, to the preparation of various auxiliary reagents, at first solvent are carried out pyrogen and remove processing in advance, adopt dilution method to prepare then, to guarantee all ingredients quality.Blood sampling, the king crab that at first will live is rinsed well with tap water, after the thorough disinfection, takes over blood sampling (unsuitable too small in order to avoid stop up) with suitably big small pinhead, and blue blood flows in the receiving bottle naturally, and centrifuging first is 750-800 rev/min, in order to avoid cell rupture.Isolated first haemocyte makes the cell suspendible so generally add with suspending washing agent jog, so that return the usefulness of bottle because amount is less.Washing times should be decided on the amount retained in the inherent bottle of haemocyte.Before the freeze-drying,, must add CHCl for improving product sensitivity
3By volume ratio 1: 0.6% (g/v) makes the coagulogen in the cell Dissolve things inside precipitate reject, should do preliminary examination earlier and guarantee product quality before packing.
Claims (6)
1, a kind of active directed production technology of tachypleus amebocyte lysate with anti-coagulants, washing agent, crack agent, sensitizer, to quick protective agent king crab blood handled, is separated by two times centrifugal, it is characterized in that production technology is as follows:
A, new fresh and alive king crab is clean, the ethanol disinfection with 75%, the ostium blood sampling, adding anti-coagulants concentration is that 0.025M theophylline liquor capacity ratio is 1: 4.5, at 10 ℃ of system king crab blood;
B, under 750-800 rev/min speed with the centrifuging of king crab blood, abandon blue serum, with white cell adding in 1: the 4 by volume suspending detergent concentration of remainder is the NaCl solution of 0.5M, after the processing, the cell that adds the tris-HCl solution of 0.03M cracks agent, to haemocyte is cracked fully, under 0 ℃, deposit and obtain the cell Dissolve things inside:
C, adding volume ratio are 1: 0.6 CHCl
3, 2-5 ℃ of following jolting, centrifuging under 2500 rev/mins speed, the collecting cell Dissolve things inside is a tachypleus amebocyte lysate stoste, it is 0.5M that the adding sensitizer makes NaCl concentration, CaCl
20.31M mixed liquor 7% (g/v), and preliminary examination is added to quick protective agent again; every milliliter contains (1-3)-β-Glucn (fungi polysaccharide) 100rg concentration and gets final product packing; improve tachypleus amebocyte lysate and bacterial endotoxin specific reaction, in subzero 45 ℃ of-55 ℃ of freeze-drying, at 35 ℃ with the lower sealing manufactured goods.
2, according to the active directed production technology of the described tachypleus amebocyte lysate of claim 1, it is characterized in that anti-coagulants: be to use the NaOH of 0.025M or HCl solution to heat 40 minutes to adding, remove the bacterial endotoxin in the solvent, transfer pH value to neutral, add pre-assigned aseptic auxiliary material solution dilution, make in every 100ml solution and contain theophylline 0.025M, NaCl0.5M, transferring pH value with tris-HCl is 7.6-7.8, add the decolouring of 0.6% (g/v) active carbon adsorption, heat suction filtration packing in 15 minutes again 120 ℃ of autoclave sterilizations 60 minutes, anti-coagulants and king crab blood volume ratio are 1: 4.5.
3, according to the active directed production technology of the described tachypleus amebocyte lysate of claim 1, it is waited to levy and is: the suspending washing agent of adding is to heat 40 minutes with 0.025M NOH or HCl solution, destroy contaminated bacteria in the solvent, accent pH value neutrality, it is 0.5M concentration that adding NaCl solution makes every milliliter of content, transferring pH value with tris-HCl is 7.6-7.8, add the decolouring of 0.6% (g/v) active carbon adsorption, the suction filtration packing, standby at 120 ℃ of autoclavings, washing agent is 1: 4 (v/v) with the ratio of limulus blood cell surplus amount.
4, according to the active directed production technology of the described tachypleus amebocyte lysate of claim 1, it is characterized in that: the agent that cracks of adding is to heat 40 minutes with 0.08M NOH or HCl solution, accent pH value neutrality, transferring pH value with 0.03Mtris-HCl solution is 7.6-7.8, add the decolouring of 0.6% (g/v) active carbon adsorption, the suction filtration packing, autoclave sterilization is standby, cracks the limulus blood cell volume ratio 1: 8 that agent and centrifuging go out.
5, according to the active directed production technology of the described tachypleus amebocyte lysate of claim 1, it is characterized in that: the sensitizer of adding heated 40 minutes with 0.15MNOH or HCl solution, transferred pH value neutrality, and adding NaCl solution is 0.5M, CaCl
2Solution is 0.31M, MgCl
2Solution is to add carbon 0.6% (g/v) adsorption bleaching in the solution of 0.41M, heats suction filtration packing in 15 minutes, and autoclaving is standby, sensitizer and tachypleus amebocyte lysate stoste volume ratio 7%.
6, according to the active directed production technology of the described tachypleus amebocyte lysate of claim 1; it is characterized in that: the activity of adding is regulated protective agent; claim again to quick protective agent; be to heat 40 minutes with 0.05-0.1M NOH or HCl solution; accent pH value neutrality; adding (1-3)-β-Glucn (fungi polysaccharide) concentration is the activity protecting agent of 100ng/ml, as the sensitivity adjusting reagent of tachypleus amebocyte lysate, to realize active directed production.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 95105652 CN1054204C (en) | 1995-06-09 | 1995-06-09 | Active directional production process for limulus reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 95105652 CN1054204C (en) | 1995-06-09 | 1995-06-09 | Active directional production process for limulus reagent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1144337A CN1144337A (en) | 1997-03-05 |
| CN1054204C true CN1054204C (en) | 2000-07-05 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 95105652 Expired - Fee Related CN1054204C (en) | 1995-06-09 | 1995-06-09 | Active directional production process for limulus reagent |
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| Country | Link |
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| CN (1) | CN1054204C (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1328584C (en) * | 2003-11-29 | 2007-07-25 | 丁友玲 | Process for preparing anti-interference horseshoe crab agent |
| CN102866255B (en) * | 2012-09-13 | 2014-12-24 | 莫水晶 | Kit for detecting fungus (1-3)-beta-D glucan in human body fluid and application method of kit |
| CN102901726B (en) * | 2012-09-14 | 2014-08-13 | 莫水晶 | Preparation and application of detection kit for blood bacterial endotoxin |
| CN111449005A (en) * | 2020-05-25 | 2020-07-28 | 北部湾大学 | Sustainable blood sampling method based on artificial culture of Tachypleus tridentatus |
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1995
- 1995-06-09 CN CN 95105652 patent/CN1054204C/en not_active Expired - Fee Related
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| Publication number | Publication date |
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| CN1144337A (en) | 1997-03-05 |
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Owner name: ZHANJIANG BOKANG MARINE BIOLOGICAL CO., LTD. Free format text: FORMER OWNER: MO SHUIJING Effective date: 20070216 |
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Effective date of registration: 20070216 Address after: 524005 Guangdong Province, Zhanjiang City Road No. 188 Mazhang District zuiun Patentee after: Zhanjiang Bokang ocean Biotechnology Co. Ltd. Address before: 524005 marine biological products factory, Haibin Road, Zhanjiang Development Zone, Guangdong 40, China Patentee before: Mo Shuijing |
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Granted publication date: 20000705 Termination date: 20110609 |