A new generation of bacterial endotoxin detection technology----Application of quantitative method test

Bacterial endotoxin (both pyrogen) is the main component of the cell wall of Gram-negative bacteria. A small amount of endotoxin can cause fever or "pyrogenic reaction" by intravenous injection, and high-dose injection can cause blood circulation disorder and endotoxin shock. In severe cases, it even leads to death. Therefore, preparations such as biological products, injectable pharmaceuticals, chemicals, radiopharmaceuticals, antibiotics, vaccines, dialysate, and implantable medical devices must be tested after passing the bacterial endotoxin test.

The sputum test method is the best method for detecting endotoxin in the world. It is simple, rapid, sensitive and accurate. Therefore, it has been designated as the statutory endotoxin test by the European and American Pharmacopoeia and the Chinese Pharmacopoeia. The method has the characteristics of high sensitivity, strong specificity and simple and convenient operation, and gradually replaces the rabbit pyrogen test, and becomes the best method for detecting endotoxin in injection, intravenous medicine, medical equipment, food and water source.

The reagent used in the oxime test method is a guanidine reagent extracted from a deformed cell lysate of marine animal mites. At present, most of the bismuth reagents on the domestic market are gel method hydrazine reagents. The gel method is based on the reaction of blood stasis cell lysate and bacterial endotoxin to form a gel to visually determine whether there is bacterial endotoxin. This method cannot be accurately quantified. Only qualitative results greater than or equal to the endotoxin limit or less than the endotoxin limit can be obtained.

In recent years, the application range of the sputum test method has been continuously developed, and various reasons have led to the failure of the gel method 鲎 reagent to meet the needs of the development of the 鲎 test method:

First, the dynamic monitoring of bacterial endotoxin levels in the pharmaceutical industry production process must use quantitative sputum reagents. At present, most of the gel method ruthenium reagents are used in the endotoxin limit test before the factory outlet of the pharmaceutical industry. If the endotoxin content is found to be equal to or greater than the endotoxin limit, the batch is unqualified and must be destroyed. The emergence of non-conforming products will cause significant economic losses to pharmaceutical companies. The use of quantitative method for the dynamic monitoring of endotoxin levels in the production process, the endotoxin level is elevated, but the problem has been found in time when the endotoxin limit has not been reached or exceeded, and the economic loss of the enterprise can be avoided.

The pharmaceutical industry implements the requirements of the new GMP:
(1) Quality control of the pharmaceutical production process is ex ante control in quality control.
(2) Dynamic control of the production process.
(3) It is required to be convenient, small, easy to operate, fast, accurate and reliable, and can automatically process data.

The quantitative method has high sensitivity, automatic operation, accurate quantification, and is most suitable for monitoring the change trend of endotoxin content. It can effectively control the operation of each link, and timely feedback information to relevant operation departments, which can control endotoxin more intuitively and better. Can greatly reduce unnecessary losses.

Second, in clinical medicine, quantitative detection of bacterial endotoxin levels in patients' body fluids is of great significance. The bacterial endotoxin content of the patient's body fluid directly reflects the extent of Gram-negative infection. Body fluid endotoxin levels can be used to rapidly diagnose sepsis and endotoxemia caused by Gram-negative infections. Endotoxemia and sepsis caused by Gram-negative bacteria are still one of the leading causes of death in the clinic. Bacteriological culture as a clinically early diagnosis of Gram-negative bacterial infection takes several days, not only takes a long time, but also has a low culture positive rate. The endotoxin test 鲎 kit (Dynamic Turbidimetry) of Xiamen City 鲎 reagent Experimental Factory Co., Ltd. uses a unique formula sample treatment solution, which effectively eliminates the interference of various interference factors in plasma on the sputum test, and accurately detects plasma within 2 hours. Bacterial endotoxin levels are one of the best methods for early diagnosis of Gram-negative infections.

3. The fungal (1,3)-β-D-glucan quantitative test for sputum test based on the G-factor bypass reaction in sputum reagent is a good method for early diagnosis of deep fungal infection. In recent years, with the widespread use of broad-spectrum antibiotics, hormones and immunosuppressive agents, and various catheter intubations, the incidence of deep fungal infections has increased. Patients with low immune function, such as HIV-infected patients, diabetic patients, and patients undergoing major surgery, are also prone to deep fungal infections. At present, deep fungal infections have become the most common cause of morbidity and mortality in immunocompromised patients. The diagnosis of mycosis requires fungal culture, which takes a long time and the culture positive rate is very low. Xiamen Fungal Reagent Experimental Co., Ltd. produces a fungal glucan detection kit (Dynamic Turbidimetry) that can quantitatively detect (1,3)-β-D-glucan in human plasma within 2 hours. Provide accurate basis for early diagnosis of deep fungal infections.

The quantitative method is called photometry in the pharmacopoeia. Divided into turbidity method and chromogenic substrate method. The turbidity method hydrazine reagent is an optical detection system that reads the relationship between the speed of the turbidity increase of the reaction liquid and the inverse ratio of the endotoxin concentration in the process of forming the gel, and is converted into endotoxin by software. The chromogenic substrate method is to add a synthetic chromogenic substrate to the reaction solution, and quantitatively detect the inside of the sample according to the ratio between the endotoxin concentration in the reaction solution and the amount of chromogenic groups released at the end of the incubation. Toxin concentration.

Both the dynamic turbidity method and the chromogenic assay require a dynamic photometric instrument with a good incubation system and a professional endotoxin and fungal glucan detection software. The lonza brand endotoxin is a microbial detection system designed for the bacterial endotoxin and fungal glucan quantitative test. Supporting lonza brand WinKQCL endotoxin detection and analysis software, LONZA endotoxin detector and other auxiliary instruments and pyrogen consumables, suitable for all quantitative methods, is the preferred recommended model for endotoxin testing.

For more information on 鲎 test and related products, please contact Beijing Bitbetter Biotechnology Co., Ltd.

Betbo Bio (Beilong Transgenic http:// ) , which started in 1994, is the first agent in mainland China to introduce LONZA (formerly Cambrex) into the Chinese mainland market. We have been working hard to introduce Lonza products to Chinese research institutions.