CN1296714C - Method for determing sensitivity of limulus reagent - Google Patents
Method for determing sensitivity of limulus reagent Download PDFInfo
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- CN1296714C CN1296714C CNB031113982A CN03111398A CN1296714C CN 1296714 C CN1296714 C CN 1296714C CN B031113982 A CNB031113982 A CN B031113982A CN 03111398 A CN03111398 A CN 03111398A CN 1296714 C CN1296714 C CN 1296714C
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- 230000035945 sensitivity Effects 0.000 title claims abstract description 55
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 32
- 241000239218 Limulus Species 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 27
- 239000002158 endotoxin Substances 0.000 claims abstract description 74
- 238000011084 recovery Methods 0.000 claims abstract description 22
- 230000035484 reaction time Effects 0.000 claims abstract description 11
- 239000012480 LAL reagent Substances 0.000 claims abstract description 4
- 238000012360 testing method Methods 0.000 claims description 13
- 238000004737 colorimetric analysis Methods 0.000 claims description 3
- 238000012417 linear regression Methods 0.000 claims description 2
- 238000010606 normalization Methods 0.000 claims description 2
- 238000002834 transmittance Methods 0.000 claims description 2
- 238000004879 turbidimetry Methods 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 8
- 238000001514 detection method Methods 0.000 abstract description 8
- 238000002835 absorbance Methods 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 3
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- 241000239222 Tachypleus Species 0.000 description 43
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- 230000002346 endotoxic effect Effects 0.000 description 15
- 238000005259 measurement Methods 0.000 description 8
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- 229920006008 lipopolysaccharide Polymers 0.000 description 4
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- 238000007817 turbidimetric assay Methods 0.000 description 4
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Abstract
本发明涉及细菌内毒素的检测，具体地说是测定鲎试剂灵敏度的方法，其特征在于：1)采用3-6个浓度的标准内毒素，在内毒素测定仪上，对不同灵敏度的标准鲎试剂进行试验，将其反应时间的对数与内毒素浓度的对数做线性回归，得到相应的不同灵敏度的标准曲线，作为判断的依据；2)待测灵敏度的鲎试剂，与1～6个浓度的标准内毒素反应，在相应的反应时间或溶液的吸光度条件下，利用标准曲线分别计算内毒素浓度及内毒素的回收率，根据回收率在75-120%之内的标准曲线所对应的灵敏度，确定为待测鲎试剂的灵敏度。本发明的优点是快速、准确、应用范围广。The present invention relates to the detection of bacterial endotoxin, specifically is the method for measuring the sensitivity of Limulus reagent, it is characterized in that: 1) adopt the standard endotoxin of 3-6 concentration, on the endotoxin measuring instrument, to the standard limulus of different sensitivity The reagent is tested, and the logarithm of the reaction time and the logarithm of the endotoxin concentration are linearly regressed to obtain the corresponding standard curves of different sensitivities as the basis for judgment; The standard endotoxin reaction of the concentration, under the corresponding reaction time or the absorbance of the solution, use the standard curve to calculate the endotoxin concentration and the recovery rate of the endotoxin respectively, according to the standard curve corresponding to the recovery rate within 75-120% Sensitivity is determined as the sensitivity of the LAL reagent to be tested. The invention has the advantages of quickness, accuracy and wide application range.
Description
Technical field
The present invention relates to detection for bacterial endotoxin, specifically measure the method for sensitivity of the limulus reagent.
Background technology
Bacterial endotoxin (Bacterial Endotoxin) be gramnegative bacterium produce have various bioactive macromolecular substances, its main chemical compositions is lipopolysaccharides (LPS); Endotoxin is the major pollutants matter in the injection medicine (raw material etc.).
The detection for bacterial endotoxin method has rabbit test method(s), limulus test.Limulus test (LimulusAmebocyte Lysate, LAL, or Tachypleus Amebocyte Lysate, TAL) be the mechanism of utilizing tachypleus amebocyte lysate and endotoxin generation agglutinating reaction, with the endotoxic a kind of external detection method of the bacterial infection in qualitative or detection by quantitative medicine or the body blood.Adopt the peptide type compound that produces look or produce fluorescent, be used to analyze endotoxin, compound structure R1-A1-A2-A3-A4-B-R2, the R2 representative is produced look or is produced fluorescent group; Endotoxin activates the enzyme of LAL, and enzyme hydrolysis peptide type compound generates color [document 1. United States Patent (USP)s: name is called the Peptide-type substrates useful in the quantitative determination ofendotoxin. patent No. and is: US4510241]; Utilize the interaction of endotoxic lipopolysaccharides and polymyxins or octapeptide or similar cyclic peptide to measure endotoxin, marker enzyme is for further detecting [document 2. European patents: it is EP0265127 that name is called the Endotoxin assay. patent No.] on cyclic peptide or the lipopolysaccharides; Be used for tachypleus amebocyte lysate and measure the peptide type compound of usefulness, structure X-A1-A2-A3-B-R, A1-A2-A3 is an amino acid, B is an amide blend, R and tachypleus amebocyte lysate reaction discharge the back and produce amino, further form visible color [document 3. European patents: it is EP0228666 that name is called the Peptide substrates and method for thequantitative assay of endotoxin. patent No.]; A kind of reagent with the limulus blood amebocyte lysate, and mensuration contains endotoxic method in the serine protease sample [document 4. Chinese patents: name is called the reagent that is used for endotoxin measurement and uses this reagent to carry out method for measuring, and the patent No. is ZL94103286.8]; Be used for the reagent that contains LAL reagent and alkyl glucoside that the endotoxin specificity is differentiated, and use this reagent specificity to differentiate endotoxic method in the sample [document 5. Chinese patents: name is called and is used for the reagent that the endotoxin specificity is differentiated, the patent No. is ZL94117898.6]; A kind of horseshoe crab reagent bacterial endotoxin quick test box and using method, be by relevant apparatus such as the required tachypleus amebocyte lysate of bacterial endotoxins test, endotoxin, lysate, pipet droppers after special processing, divide cover to be packaged into kit, simplify experimental implementation [document 6. Chinese patents: name is called horseshoe crab reagent bacterial endotoxin quick test box and using method, and the patent No. is ZL95115774.4]; A kind of active directed production technology of tachypleus amebocyte lysate makes not vulnerable to pollution of tachypleus amebocyte lysate, and is highly sensitive, and self blank is long negative observing time, product stability height [document 7. Chinese patents: the patent No. is ZL95105652.2]; A kind ofly produce agglutinating reaction by tachypleus amebocyte lysate and bacterial endotoxin and realize that bacteriotoxin content in the water is carried out the instrument of determination limit [document 8. Chinese patents: name is called a kind of bacteria endotoxin detector, the patent No. is ZL95216488.4], it have simple in structure, with low cost, easy to operate, to detect result of determination reliable and meet plurality of advantages such as pharmacopeia regulation.
Utilize kinetic turbidimetric assay and dynamic colourimetry principle, can adopt instrument to come the endotoxic concentration C of quantitative measurement, promptly in the tachypleus amebocyte lysate course of reaction, observe the change color curve of its turbidity change curve or product color substance, a default absorbance OD value such as OD0.02 or flex point, the time of experiencing when beginning to rise to this preset value with response curve is as reaction time (T (OD0.02) or T50, second), the logarithm in this reaction time and the logarithm of endotoxin concns are linear, be that typical curve is LogT (OD0.02)=a+b LogC or Log T50=a+b LogC, by with typical curve relatively come endotoxin concns in the calculation sample.Relevant detection instrument commonly used both at home and abroad has U.S. LAL-5000 type, Japan and light Toxinometer ET-201 series, domestic Kingsoft river EDS-98, MB-80 series detection system and huge BET-32 series detection system.
Adopt limulus test, no matter be qualitative or the endotoxic content of quantitative measurement, the accuracy of sensitivity of the limulus reagent is vital; The assay method of conventional sensitivity of the limulus reagent is the method for limiting the quantity of: measure in advance respectively and formal mensuration, bacterial endotoxin working standard proportional diluted need be at least 4 concentration, each concentration is done 4 pipes, and used standard items are many, and length consuming time is prepared in test; The reaction temperature retention time is 60 ± 2 minutes, and the reaction time is long; Personal error appears in observations ocular estimate easily; The endotoxic recovery between 50-200%, wide ranges.
Summary of the invention
The objective of the invention is, a kind of method of measuring sensitivity of the limulus reagent is provided,, solved the method stability problem of measuring sensitivity of the limulus reagent to measure the sensitivity of tachypleus amebocyte lysate accurately and rapidly.
For achieving the above object, the technical solution used in the present invention is: the standard endotoxin solution that adopts a series of (3～6) variable concentrations, on endotoxin determinator, standard tachypleus amebocyte lysate to different sensitivity is tested respectively, the logarithm in its reaction time and the logarithm of endotoxin concns are done linear regression, obtain the typical curve of corresponding different sensitivity, as the foundation of judging; The tachypleus amebocyte lysate of sensitivity to be measured, standard endotoxin reaction with one or more (1～6) concentration, obtain the corresponding reaction time, utilize the endotoxin typical curve of different sensitivity tachypleus amebocyte lysate, calculate the endotoxin concns and the endotoxic recovery respectively, according to the pairing sensitivity of the typical curve of the recovery within 75-120%, determine the sensitivity of tachypleus amebocyte lysate to be measured again.
Described endotoxin determinator is to adopt kinetic turbidimetric assay or dynamic colorimetric determination to measure endotoxin, obtains the endotoxic typical curve of mensuration of corresponding different sensitivity tachypleus amebocyte lysate; Described kinetic turbidimetric assay is OD limit value method, transmittance limit value method or T50 normalization method; Standard sensitivity of the limulus reagent scope is preferably 0.01～0.5EU/ml; The range of sensitivity of unknown tachypleus amebocyte lysate is preferably in 0.01～0.5EU/ml.
The present invention has following advantage:
1. quick.The present invention adopts sizing technique to measure the sensitivity of tachypleus amebocyte lysate, owing to utilize the principle of its quantitative measurement, adopt the standard endotoxin of same concentration, on endotoxin determinator, respectively the standard tachypleus amebocyte lysate of different sensitivity is carried out revision test, obtain the average and the standard deviation in corresponding reaction time, adopt the endotoxin standard of same concentration can finish the mensuration of sensitivity of the limulus reagent then, weak point consuming time is prepared in test.In addition, on the endotoxin measurement instrument, adopt tachypleus amebocyte lysate directly to measure endotoxin, minimum test can obtain corresponding data in 10 minutes, was used for the calculating of sensitivity of the limulus reagent, and the reaction time is short.
2. accurate.The present invention can utilize existing endotoxin measurement instrument, realize accurate, the quantification mensuration of sensitivity of the limulus reagent, endotoxic recovery scope between 75-120%, the recovery scope of the 50-200% that requires much smaller than conventional method, the sensitivity of the tachypleus amebocyte lysate of calculating is accurate.Observations Instrument measuring absorbance method, the result is objective, accurate.
3. applied range.The present invention can be applicable to kinetic turbidimetric assay and dynamic colourimetry, is expected to be applied to the production process monitoring of scientific research, pharmaceutical production check, tachypleus amebocyte lysate and quality control etc., for measuring endotoxin in the medicine provides more science, objective method.
Description of drawings
Fig. 1 is the typical curve synoptic diagram of different sensitivity tachypleus amebocyte lysate.
Fig. 2 is that synoptic diagram is judged in the sensitivity of 0.06EU/ml tachypleus amebocyte lysate sample.
Fig. 3 is that synoptic diagram is judged in the sensitivity of 0.25EU/ml tachypleus amebocyte lysate sample.
Embodiment
1) is used for the typical curve of the different tachypleus amebocyte lysate of endotoxin measurement: based on typical curve LogT (OD0.02)=a+b LogC, employing standard endotoxin concns 0.22-22EU/ml, respectively to sensitivity be 0.5,0.25, the standard tachypleus amebocyte lysate of 0.06EU/ml tests, obtain corresponding standard curve and be (1) y=-0.6197x+3.1531; (2) y=-0.3725x+3.322; (3) y=-0.4129x+3.2038;
Based on typical curve Log (T50)=a+b LogC, adopt standard endotoxin concns 0.22-22EU/ml, be that the standard tachypleus amebocyte lysate of 0.01EU/ml is tested to sensitivity, obtain corresponding standard curve and be (4) y=-0.171x+2.89;
Based on typical curve Log (T50)=a+b LogC, adopt standard endotoxin concns 3.125,6.25,25EU/ml is that the standard tachypleus amebocyte lysate of 0.06EU/ml is tested to sensitivity, obtains corresponding standard curve and is (5) y=-0.377x+3.48;
2) sensitivity determination of tachypleus amebocyte lysate 1: the standard endotoxin of employing 4.4,2.2,0.88,0.44,0.22EU/ml, tachypleus amebocyte lysate 1 to be measured is tested, obtain T (OD0.02) value and be followed successively by 810,1200,1890,2110,2920 seconds.Adopt typical curve (1), (2), (3) to calculate the endotoxin concns and the endotoxic recovery respectively, the recovery is followed successively by (56%, 60%, 72%, 120%, 142%); (293%, 204%, 151%, 224%, 187%); (118%, 91%, 76%, 116%, 106%), the recovery are according to typical curve (3) result calculated, so the sensitivity of tachypleus amebocyte lysate 1 is 0.06EU/ml between 75-120%;
The sensitivity determination of tachypleus amebocyte lysate 2: the standard endotoxin of employing 4.4,2.2,0.88,0.44,0.22EU/ml, tachypleus amebocyte lysate 2 to be measured is tested, obtain T (OD0.02) value and be followed successively by 1150,1650,2240,2860,3600 seconds.Adopt typical curve (1), (2), (3) to calculate the endotoxin concns and the endotoxic recovery respectively, the recovery is followed successively by (32%, 36%, 55%, 74%, 102%); (114%, 87%, 95%, 99%, 107%); (50%, 42%, 50%, 56%, 64%), the recovery are according to typical curve (2) result calculated, so the sensitivity of tachypleus amebocyte lysate 2 is 0.25EU/ml between 75-120%.
1) is used for the typical curve of the different tachypleus amebocyte lysate of endotoxin measurement: adopt standard endotoxin concns 0.22-22EU/ml, to sensitivity is that the standard tachypleus amebocyte lysate of 0.125EU/ml is tested, based on typical curve LogT (OD0.02)=a+b LogC, obtain corresponding standard curve and be (6) y=-0.4204x+3.1932;
2) sensitivity determination of tachypleus amebocyte lysate 3: the standard endotoxin that adopts 4.4EU/ml, tachypleus amebocyte lysate 3 to be measured is tested, obtaining T (OD0.02) value is 820 seconds, adopt typical curve (1), (2), (4) to calculate the endotoxin concns and the endotoxic recovery respectively, the recovery is followed successively by 58%, 303%, 111%, the recovery is according to typical curve (6) result calculated, so the sensitivity of tachypleus amebocyte lysate 3 is 0.125EU/ml between 75-120%;
The sensitivity determination of tachypleus amebocyte lysate 3: the standard endotoxin that adopts 22EU/ml, tachypleus amebocyte lysate 3 to be measured is tested, obtaining T (OD0.02) value is 470 seconds, adopt typical curve (1), (2), (4) to calculate the endotoxin concns and the endotoxic recovery respectively, the recovery is followed successively by 27%, 253%, 79%, the recovery is according to typical curve (6) result calculated, so the sensitivity of tachypleus amebocyte lysate 3 is 0.125EU/ml between 75-120%.Instrument measuring was tested 470 seconds consuming time, about 8 minutes.
Comparative example 1.
The assay method of conventional sensitivity of the limulus reagent is the method for limiting the quantity of, need measure in advance and formal mensuration, need be with the bacterial endotoxin working standard with 2 times of proportional diluted, select to occur 4 border dilutions of positive and negative findings, each dilution is made 4 pipes, and 4 pipes of its maximum concentration should be all positive, 4 pipes of least concentration should be all negative, vibrate above-mentioned test tube mixing content gently, the sealing mouth of pipe, put in 37 ± 1 ℃ of water-baths, be incubated 60 ± 2 minutes observationss and calculate this batch sensitivity of the limulus reagent (λ) on request, the endotoxic recovery is between 50～200%; The standard endotoxin that is about to variable concentrations and tachypleus amebocyte lysate be 37 ℃ of insulation reaction 1 hour, gel whether occurs and judge, calculate sensitivity of the limulus reagent according to estimating.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0173021A2 (en) * | 1984-06-27 | 1986-03-05 | Wako Pure Chemical Industries, Ltd. | Process for measuring endotoxin |
| CN1140256A (en) * | 1995-10-06 | 1997-01-15 | 莫水晶 | Horseshoe crab reagent bacterial endotoxin quick test box and its use method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0173021A2 (en) * | 1984-06-27 | 1986-03-05 | Wako Pure Chemical Industries, Ltd. | Process for measuring endotoxin |
| CN1140256A (en) * | 1995-10-06 | 1997-01-15 | 莫水晶 | Horseshoe crab reagent bacterial endotoxin quick test box and its use method |
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