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A method for detecting pyrogens by cell line method and its kit

A cell line and cell technology, applied in the field of medicine, can solve the problems of difficult to obtain cells, poor dose dependence, low sensitivity, etc., and achieve the effects of good dose dependence, easy operation and high sensitivity

Active Publication Date: 2018-04-27
SHANGHAI INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims at the defects that the method for detecting pyrogens in the prior art can only detect bacterial endotoxin derived from Gram-negative bacteria, cells are difficult to obtain, low sensitivity, poor dose dependence, etc., and provides a method for detecting pyrogens by cell line method and its kit, the method can qualitatively and quantitatively analyze pyrogens derived from Gram-negative bacteria, Gram-positive bacteria and yeast, with high sensitivity and good dose dependence, and HL-60 cells Commercially available and easy to obtain, suitable for application promotion

Method used

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  • A method for detecting pyrogens by cell line method and its kit
  • A method for detecting pyrogens by cell line method and its kit
  • A method for detecting pyrogens by cell line method and its kit

Examples

Experimental program
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Embodiment 1

[0039] Example 1 The method for detecting pyrogens based on the cell line method of HL-60 cells

[0040] 1.1 Materials and reagents

[0041] Cell culture medium: 400 mL of IMDM culture medium (purchased from Gibco), add 100 mL of fetal bovine serum (purchased from Gibco) and mix well.

[0042] Cells: HL-60 were purchased from the Institute of Biochemistry and Cells, Academy of Life Sciences, Chinese Academy of Sciences; passage: F10.

[0043] ELISA kit: human interleukin-6 (IL-6) enzyme-linked immunosorbent (ELISA) kit (purchased from BD OptEIA TM )

[0044] Consumables: cell counting plate, sterile plastic centrifuge tube (1.5mL, 50mL), disposable plastic pipette (sterile, single package, 10mL), 25cm 2 Culture flasks, 24-well or 96-well cell culture plates.

[0045] Diluent: IMDM culture medium (purchased from Gibco) 490mL, add fetal bovine serum (purchased from Gibco) 10mL and mix well.

[0046] 1.2 Instruments

[0047] Enzyme label detector (model: Molecular Devices M...

Embodiment 2

[0071] The method for the detection pyrogen of embodiment 2 different cell densities

[0072] The cell suspension prepared in Example 1 was diluted to the following concentrations respectively (i.e. the cell density was ): 1×10 5 pcs / mL, 5×10 5 pcs / mL, 1×10 6 pcs / mL, 5×10 6 pcs / mL, 1×10 7 / mL and 5×10 7 individual / mL. Add 400 μl / well to 24-well culture plate respectively.

[0073] Add the 0.5Eu / mL LPS standard solution described in Example 1 into a 24-well cell culture plate at 100 μL / well for pyrogen stimulation. 2 , 37 ° C incubator to continue culturing for 2 days, the percentage is the volume percentage of fetal bovine serum in the IMDM culture medium. The cell supernatant was taken, and the amount of IL-6 was determined according to the instructions of the IL-6 ELISA kit. The results are shown in Table 1.

[0074] Table 1 Detection results of different cell densities

[0075] Cell density (unit / mL)

[0076] The results in Table 1 show that after the pyr...

Embodiment 3

[0077] The method for the detection pyrogen of embodiment 3 different cultivation time

[0078] The cell suspension prepared in Example 1 was adjusted to a cell concentration of 5 × 10 6 cells / mL, added to 24-well culture plate at 400 μl / well.

[0079] Add the 0.5Eu / mL LTA standard solution described in Example 1 into a 24-well cell culture plate at 100 μL / well for pyrogen stimulation. 2 , 37 ° C incubator to continue culturing for 2 days, the percentage is the volume percentage of fetal bovine serum in the IMDM culture medium. The cell supernatant was taken, and the amount of IL-6 was determined according to the instructions of the IL-6 ELISA kit, and the results are shown in Table 2.

[0080] Table 2 Test results at different times

[0081]

[0082] The results in Table 2 show that the secretion of IL-6 is the highest after 2 days of pyrogen-stimulated cells, so it is determined that 2 days of pyrogen-treated cells is the best action time.

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Abstract

The invention discloses a cell line method pyrogen detection method and a kit. The method includes following steps: (1) a pyrogen material standard solution and a to-be-detected sample solution is prepared; (2) the pyrogen material standard solution or the to-be-detected sample solution are respectively added into a HL-60 cell-containing cell culture solution for culture, and cultured cell supernatant is taken; and (3) the content of cytokine IL-6 in the cultured cell supernatant of the step (2). According to the method, human body pyrogen reaction can be simulated, Gram-negative bacterium sourced, Gram-positive bacterium sourced and yeast sourced pyrogen materials can be qualitatively and quantitatively analyzed, sensitivity is high, and the method is simple and easy. The kit includes the pyrogen material standard solution, a cytokine IL-6 detection agent and HL-60 cells, the kit has the advantages of simple operation, capability of pyrogen material qualitative and quantitative analysis, and high sensitivity.

Description

technical field [0001] The invention belongs to the field of medicine, and in particular relates to a method for detecting pyrogens by a cell line method and a kit thereof. Background technique [0002] Pyrogen refers to a pyrogenic substance produced by microorganisms that can cause an abnormal increase in the body temperature of warm-blooded animals. The pyrogen detection methods recorded in the pharmacopoeias of various countries include the rabbit pyrogen test method and the bacterial endotoxin test method (bacterial endotoxins test, BET). The rabbit method was first included in the United States Pharmacopoeia in 1942, and has been considered as the "gold standard" for detecting pyrogens, but it also has some shortcomings: such as only negative or positive qualitative results, low sensitivity, and poor repeatability of results , The accuracy of the results depends on the selection of rabbit strains and the control of experimental conditions; the cost is high; live anima...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/02C12N5/09
Inventor 陈钢王灿邵泓吴利红董闪闪
Owner SHANGHAI INST FOR FOOD & DRUG CONTROL
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