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Medicilon Bacterial Endotoxin Testing Solutions

2022-01-04
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What are Endotoxins?

Endotoxin is a complex of lipopolysaccharide (LPS) and protein on the cell wall of Gram-negative bacteria, which is released when the bacteria die or autolyse. A large amount of endotoxin into the blood will cause a fever reaction - "pyrogenic reaction". Endotoxin is closely related to a variety of infectious diseases. The deterioration of the disease is often accompanied by an increase in the content of endotoxin, and the remission of the disease is often accompanied by a decrease in the content of endotoxin. Therefore, rapid detection (1 hour) of endotoxin content in blood and organs can provide a reference for the diagnosis and prognosis of clinically relevant diseases.

The Effect of Endotoxin

The active center of endotoxin is lipid A, one of the strongest known immune stimulators. Can stimulate monocytes and macrophages to produce interleukin-1 (IL-1), tumor necrosis factor (TNF), interferon (IFN), macrophage inflammatory protein-1 (MIP-1) , leukocyte pyrogen (LP) and other inflammatory mediators and cytokines. Endotoxin has a wide range of biological activities, mainly by interacting with target cells to induce these cells to produce a series of inflammatory mediators and cytokines, which can cause fever response, promote prostaglandin synthesis, promote the release of vasoactive substances, and reduce blood pressure. Activation of the coagulation system and complement, causing local allergies, affecting or participating in immune responses, affecting blood sugar and tumor necrosis, etc., and causing death in severe cases.

The Endotoxin Receptor

Currently known endotoxin receptors are mainly divided into the following categories [1]: scavenger receptor (SR), lipopolysaccharide binding protein (LBP), cluster of differentiation antigen 14 , CD14), integrin family (CD11/CD18), Toll-like receptor family (TLR), lectin S domain receptor kinase (LORE) proteins. Endotoxin test method is a method that uses Limulus reagent to detect or quantify bacterial endotoxin produced by Gram-negative bacteria, so as to judge whether the limit of bacterial endotoxin in the test product meets the regulations.

Bacterial Endotoxin Test Procedure

  •      •Bacterial endotoxin method development (sample volume 3 bottles/batch, one batch)

  •      •Bacterial endotoxin method verification (sample volume 3 bottles/batch, three batches)

  •      •Bacterial endotoxin test (sample volume 3 bottles/batch, three batches)

  •      •Bacterial endotoxin is one of the components of the cell wall of Gram-negative bacteria: LPS

  •      •Released after the bacteria die

Bacterial endotoxin characteristics: heat resistance, heat resistance, high pyrogenicity

Medicilon provides endotoxin testing for the pharmaceutical, biopharmaceutical and medical device industries.

Endotoxin Testing Solutions.jpg

Bacterial endotoxin experiment involves content and related requirements

• Personnel:
• Reagents, consumables
• Instruments
• Test environment

Bacterial endotoxin inspection methods include: gel method (limit experiment, semi-quantitative experiment), photometric method (turbidimetric method, chromogenic matrix method). You can use any of these methods to test. When the measurement result is in dispute, unless otherwise specified, the result of the gel method shall prevail.

Bacterial Endotoxin Test Method

Bacterial endotoxin examination includes two methods, namely gel method and photometric method, the latter includes turbidimetric method and chromogenic matrix method. When testing the test article, any one of these methods can be used for the test. When the determination results are in dispute, unless otherwise specified, the results of the gel method shall prevail.
The experimental operation process should prevent the contamination of microorganisms and endotoxins.
Limulus Reagent Principle of Gel Method Limulus Reagent is the freeze-dried product of the lysate of Limulus orientalis amebocyte lysate, which contains progelenzyme and coagulation protein which can be activated by trace bacterial endotoxin. Under suitable conditions (temperature, pH value and no interfering substances), bacterial endotoxin can activate the coagulase in the Limulus reagent, so that the Limulus reagent can agglutinate and form a gel. The gel method Limulus reagent is based on the firmness of the gel formed by the agglutination reaction to limit the detection of bacterial endotoxin.

①Bacterial Endotoxin Test-Gel Method

The gel method uses the principle that the bacterial endotoxin reacts with the limulus reagent to form a gel to determine whether the endotoxin content in the test product meets the requirements.
Interference effect:
• Most of the interference effects can be eliminated by diluting the test product with bacterial endotoxin test water.
• When some interference effects cannot be eliminated by using only the dilution method, other methods can be used to eliminate the interference factors, and then the test can be carried out.

Inspection method-gel limit test


• Determination of limit value and calculation of maximum effective dilution factor (MVD)
• Preparation of test solution
• Inspection method-limit experiment

Suspicious result processing procedure

Processing program

• The experimenter first reviews the experimental project:

  1.      1.Standards for inspection

  2.      2.Inspection operation

  3.      3.Recording and calculation

  4.      4.Instrument status

  5.      5.Standard material

  6.      6.Reagent quality

  7.      7.Other abnormal phenomena (whether there may be pollution, environment, equipment…)

• If an abnormality is found in the inspection process of the inspector, the reviewer repeats the experiment once with the correct inspection process to replace the original experimental result.

• If no abnormality is found in the inspection process of the inspector, in order to determine whether the unqualified result is caused by the test or analysis error, the reviewer can repeat the experiment.

• Eliminate false positive interference (dextran substances)! Anti-enhancement solution, specific limulus reagent

• Quantitative test provides data reference

• Use of different limulus reagents

②Bacterial Endotoxin Inspection-Photometric Method

The photometric method is a method for determining the content of endotoxin by detecting the change in turbidity or color during the reaction between the limulus reagent and endotoxin.
Four detection methods: end point turbidity method, dynamic turbidity method, end point color method, dynamic color method
The essence of the photometric method: Use endotoxin standard materials to prepare a standard curve, and reversely calculate the endotoxin content in the sample.
Photometric method experiment part: reliability experiment, interference experiment, inspection method of standard curve

Other Bacterial Endotoxin Test Metohd

Dynamic turbidity method; 

the turbidity method is a method for measuring the content of endotoxin by detecting the turbidity change during the reaction between the Limulus reagent and the endotoxin. The coagulase in the endotoxin and the Limulus reagent is activated to form a coagulase, and the coagulase can turn the coagulation protein into a coagulation protein (ie, a gel), which changes the turbidity of the liquid, which can be detected by dynamically observing the turbidity change rate.

End-point chromogenic method:

 Through the activation of Limulus reagent (the Limulus reagent contains C factor, B factor, coagulase, coagulation protein, etc.) factor C due to bacterial endotoxin, a series of enzymatic reactions are caused to activate the formation of coagulase. Coagulase, coagulase decomposes artificially synthesized chromogenic substrates (containing N-α-benzoyl-DL-arginyl-4-nitroaniline hydrochloride) into polypeptides and yellow p-nitroaniline (pNA, λmax = 405 nm). At the same time, p-nitroaniline (pNA) can also be dyed rose red (λmax = 545nm) with an azo reagent, which avoids the interference of the color of the test substance itself on the absorption peak at 405nm. Judging the endotoxin concentration according to the color of the product, also known as colorimetry.

Dynamic chromogenic method: 

Endotoxin can activate the Limulus reagent to cause a series of enzymatic reactions to produce yellow p-nitroaniline (pNA, λmax = 405nm). The amount produced is positively correlated with the bacterial endotoxin concentration. However, this method requires a dynamic photometric instrument with an incubation system and supporting software.

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