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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- amebocyte lysate
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Abstract
The present invention relates to a method for detecting bacterial endotoxin, more specifically to a method for detecting the sensitivity of limulus reagents. The present invention is characterized in that 1, standard endotoxins with 3 to 6 concentrations are adopted to detect the standard limulus reagents with different sensitivity on an endotoxin detecting machine, the logarithm of reaction time and the logarithm of the endotoxin concentration are calculated via linear regression to obtain corresponding standard curves with different sensitivity used as judgment reference; 2, the limulus reagents with sensitivity to be detected can be reacted with the standard endotoxins with 1 to 6 concentrations; under the conditions of corresponding reaction time or light absorbance, the endotoxin concentration and endotoxin recovery rate are respectively calculated via the standard curves; the sensitivity of the limulus reagents to be detected are determined according to the sensitivity corresponding to the standard curves with the recovery rate of 75 to 120%. The present invention has the advantages of high speed, exact detection 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.
Claims (4)
1. measure the method for sensitivity of the limulus reagent, it is characterized in that:
1) the standard endotoxin of 3-6 concentration of employing on endotoxin determinator, is tested the standard tachypleus amebocyte lysate, and the logarithm in its reaction time and the logarithm of endotoxin concns are done linear regression, obtains corresponding standard curve, as the foundation of judging;
Wherein: the range of sensitivity of standard tachypleus amebocyte lysate is at 0.01～0.5EU/ml;
2) tachypleus amebocyte lysate of sensitivity to be measured, standard endotoxin reaction with 1～6 concentration, obtain the corresponding reaction time, utilize typical curve to calculate the endotoxin concns and the endotoxic recovery respectively, according to the pairing sensitivity of the typical curve of the recovery within 75-120%, be defined as the sensitivity of tachypleus amebocyte lysate to be measured.
2. according to the sensitivity of method of the described mensuration tachypleus amebocyte lysate of claim 1, it is characterized in that: described endotoxin determinator is to adopt kinetic turbidimetric assay or dynamic colorimetric determination to measure endotoxin, is measured endotoxic typical curve accordingly.
3. according to the sensitivity of method of the described mensuration tachypleus amebocyte lysate of claim 2, it is characterized in that: described kinetic turbidimetric assay is OD limit value method, transmittance limit value method or T50 normalization method.
4. according to the sensitivity of method of the described mensuration tachypleus amebocyte lysate of claim 1, it is characterized in that: the range of sensitivity of tachypleus amebocyte lysate to be measured is at 0.01～0.5EU/ml.
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Citations (2)
| 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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Patent Citations (2)
| 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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