Basic knowledge of reagents used in bacterial endotoxin testing
Bacterial endotoxin
In the clinical use of drug injections in hospitals, there are often cold sensations, chills, fever, headache, nausea, vomiting, gray complexion, shock, and serious death. This symptom is called pyrogen reaction.
What is a pyrogen? At present, there is still no unified understanding at home and abroad, but from the domestic and foreign literature reports, a common opinion is generally believed: it refers to bacterial endotoxin. J. Van Noordwijk, Vice-Chairman of the European Pharmacopoeia Commission, said: "Strictly speaking, not every pyrogen has a lipopolysaccharide structure, but all known bacterial endotoxin lipopolysaccharides have pyrogen activity." Under the conditions of Good Manufacturing Practice (GMP), the quality control of pharmaceutical production is generally acceptable: the absence of bacterial endotoxin means the absence of pyrogens.
Bacterial endotoxin is a complex of a lipopolysaccharide (Lipoply Saccharide) and a microprotein (Protein) on the cell wall of Gram-negative bacteria. Its specificity is not a bacterial or bacterial metabolite, but a bacterial death or disintegration. A substance with endotoxin biological activity released. Its chemical composition is widely distributed in the cell wall layer of Gram-negative bacteria (such as Escherichia coli, Brucella, Salmonella typhi, Proteus, Salmonella, etc.) and other microorganisms (such as Chlamydia, Rickettsia, spirochete, etc.) The chemical composition of polysaccharides is mainly composed of O-specific chain, core polysaccharide and lipid A.
Endotoxin chemical structure (LPS)
Chemical structure of bacterial endotoxin
Bacterial endotoxin standard
Bacterial Endotoxin National Standard (RSE): Lot Number: 981 Potency: 9000EU
Bacterial Endotoxin Working Standard (CSE): calibrating its potency based on bacterial endotoxin national standards
Bacterial endotoxin status in aqueous solution
Bacterial endotoxin is easily adsorbed on the container wall or gathered together. The 2005 Chinese Pharmacopoeia clearly states that the bacterial endotoxin standard solution should be mixed for 15 minutes and mixed for 30 seconds per dilution step to fully mix.
Heat resistance of bacterial endotoxin
Since the bacterial endotoxin can be completely inactivated at a high temperature of 250 ° C for 30 minutes, it is a headache in the pharmaceutical industry and disposable medical equipment and blood bags. The various glass equipment used for testing should be strict. After the endotoxin is removed, it can be used. The finished test equipment we provide must pass 7 strict procedures to meet the requirements.
鲎 reagent
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鲎Reagents - biological reagents extracted from the blood of marine organisms, only the specific gel reaction between sputum and endotoxin. The reagent has reached international standardization, and the reagents are used worldwide to detect bacterial endotoxin. Manufacture of biosynthetic methods. Other detection methods are limited and the operation is too complicated or the cost is too high to be popular.
Principle of biochemical reaction of bacterial endotoxin and sputum reagent
The guanidine reagent has two reaction pathways for detecting bacterial endotoxin produced by Gram-negative bacteria and 1,3-(β, D)-glucan produced by fungi, which can indirectly detect Gram-negative bacteria and fungi. Currently, reagent manufacturers can provide specific guanidine reagents for each of these two substances.
Observation of gel reaction process of hydrazine reagent
Using the principle of photodetection to detect the reaction curve, quantitative detection can be performed. At present, a complete methodology and stable detection instrument have been established.
Detection principle
Optical density reaction time (t 0.02 )
The BET series instrument detects the optical density OD value of the reactant in the test tube, and takes the time when the OD rises to 0.02 as the reaction time. The position on the reaction curve is a period in which the turbidity starts to change rapidly, and the value can be quickly Stable time to obtain reaction.
Absorbance limit method reaction time
Theoretical analysis and a large number of experiments have shown that no matter which method determines the reaction time, it is a marker of endotoxin content, and the endotoxin concentration and the gelation time are negatively correlated, that is, the higher the endotoxin concentration, the shorter the gelation time. Experiments have confirmed that there is also a negative correlation between the critical time t 0.02 and the endotoxin concentration Ct 0.02 . By experimenting with the standard, a C-relationship curve can be obtained, and the curve is fitted by least squares method, and the values of a 0 and b 0 can be solved . When testing the test article , the endotoxin concentration value [1] can be obtained based on the critical reaction time t 0.02 and the formula (2.3-2) of the test article .
Reaction time method
Reaction rate determination and concentration calculation method
A large number of experiments have shown that the higher the endotoxin concentration, the faster the gelation time, that is, the faster the reaction rate, and the kinetic reaction curve is converted into a differential curve, that is, a reaction rate curve.
                                                   Reaction rate curve
The point at which the reaction rate is the highest is the inflection point of the reaction curve. The time at this point as the reaction time is t50 in the turbidity reaction time method, and the maximum reaction rate is also related to the endotoxin content, which can be obtained by experimenting with the standard product. The C-Vmax relationship curve is fitted to the curve by least squares method, and the values ​​of a0 and b0 can be solved. When the test article is tested, the endotoxin concentration value can be obtained by substituting the maximum reaction rate Vmax of the test article into the standard curve equation.
Reaction rate method
The instrument directly identifies the principle of the presence of glucan
According to the biochemical reaction principle of sputum reagent, dextran reacts directly with G factor in sputum reagent, and then gel is produced, while bacterial endotoxin reacts with C factor of sputum reagent, and activated C factor reacts with factor B and then produces condensate. Glue, due to the different progress of the reaction, a clear difference can be found from the reaction curve. The endotoxin curve is a distinct S-shaped curve, while the dextran curve is a straight line close to a straight line, which is analyzed by the reaction time method and the reaction rate method. It was found that the results of the samples containing dextran were more than 10 times different between the two methods, and the results of endotoxin detection were not significantly different between the two methods.
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