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Method for determining sensitivity of limulus reagent

A sensitive, Limulus reagent technology, applied in the field of bacterial endotoxin detection, can solve the problems of prone to human error, long test preparation time, long reaction time, etc., and achieve objective results, short test preparation time, and short reaction time. Effect

Inactive Publication Date: 2007-01-24
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

[0005] Whether it is qualitative or quantitative determination of the content of endotoxin by the limulus test, the accuracy of the sensitivity of the limulus reagent is crucial; the conventional method of measuring the sensitivity of the limulus reagent is the limited method: the pre-determination and the formal determination are carried out respectively. The bacterial endotoxin working standard is diluted to at least 4 concentrations, and 4 tubes are made for each concentration. There are many standards used, and the test preparation takes a long time; the reaction incubation time is 60±2 minutes, and the reaction time is long; the observation results are visually observed method, prone to human error; the recovery rate of endotoxin is between 50-200%, with a wide range

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  • Method for determining sensitivity of limulus reagent

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Embodiment 1

[0017] 1) Standard curves of different Limulus reagents used for endotoxin determination: based on the standard curve LogT(OD0.02)=a+b LogC, the standard endotoxin concentration is 0.22-22EU / ml, and the sensitivity is 0.5, 0.25, 0.06 respectively The standard limulus reagent of EU / ml is tested, and the corresponding standard curve obtained is (1) y=-0.6197x+3.1531; (2) y=-0.3725x+3.322; (3) y=-0.4129x+3.2038;

[0018] Based on the standard curve Log(T50)=a+b LogC, using the standard endotoxin concentration of 0.22-22EU / ml, the standard limulus reagent with a sensitivity of 0.01EU / ml is tested, and the corresponding standard curve is obtained as (4)y=- 0.171x+2.89;

[0019] Based on the standard curve Log(T50)=a+b LogC, using standard endotoxin concentrations of 3.125, 6.25, and 25EU / ml, the standard limulus reagent with a sensitivity of 0.06EU / ml was tested, and the corresponding standard curve was obtained as (5)y =-0.377x+3.48;

[0020] 2) Sensitivity determination of Limu...

Embodiment 2

[0023] 1) Standard curves of different limulus reagents used for endotoxin determination: use a standard endotoxin concentration of 0.22-22EU / ml to test the standard limulus reagent with a sensitivity of 0.125EU / ml, based on the standard curve LogT(OD0.02)= a+b LogC, the corresponding standard curve obtained is (6) y=-0.4204x+3.1932;

[0024]2) Sensitivity determination of Limulus reagent 3: The standard endotoxin of 4.4EU / ml was used to test Limulus reagent 3, and the T (OD0.02) value was 820 seconds, respectively using standard curves (1) and (2) , (4) Calculate the recovery rate of endotoxin concentration and endotoxin, the recovery rate is 58%, 303%, 111% successively, and the recovery rate is the result calculated according to the standard curve (6) between 75-120%, so The sensitivity of Limulus Reagent 3 is 0.125EU / ml;

[0025] The sensitivity determination of Limulus reagent 3: adopt the standard endotoxin of 22EU / ml, test Limulus reagent 3 to be tested, obtain T (OD0....

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Abstract

The invention relates to the detection for bacterial endotoxin, concretely a limuloid reagent sensitivity determining method, its character: 1) adopt standard endotoxins at 3-6 concentrations to test standard limuloid reagents at different sensitivities on the endotoxin determiner, make linear regression on the logarithms of reacting time and endotoxin concentration, to obtain the corresponding standard curves at different sensitivities as the justification basis; 2) make the limuloid reagent to be measured react with standard endotoxins at 1-6 concentrations, on the corresponding condition of reacting time or solution absorbency, use the standard curves to calculate endotoxin concentration and recovery, according to the sensitivity corresponding to the standard curve at the recovery 75-120%, determine it as the sensitivity of the limuloid reagent to be measured.

Description

technical field [0001] The invention relates to the detection of bacterial endotoxin, in particular to a method for measuring the sensitivity of Limulus reagent. Background technique [0002] Bacterial endotoxin (Bacterial Endotoxin) is a macromolecular substance with various biological activities produced by Gram-negative bacteria, and its main chemical component is lipopolysaccharide (LPS); endotoxin is the main pollution in injection medicine (raw materials, etc.) substance. [0003] The detection methods of bacterial endotoxin include rabbit test method and limulus test method. Limulus Amebocyte Lysate (LAL, or Tachypleus Amebocyte Lysate, TAL) is an in vitro detection method that uses the mechanism of agglutination reaction between Limulus Amebocyte Lysate and endotoxin to qualitatively or quantitatively detect drug or bacterial endotoxin in the body's blood. . Use chromogenic or fluorescent peptide compounds for the analysis of endotoxins, compound structure R1-A1-A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/579G01N33/52G01N33/50
Inventor 李京华邵英光王俊德
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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