2019-04-17
In the work, the author sometimes receives a consultation call - why is the positive control not condensable in the bacterial endotoxin test? After a detailed understanding of the specific situation, we found that similar problems have become an experimental phenomenon that often troubles us. The author analyzed the causes of this problem, summarized and provided relevant solutions for your reference.
The author believes that there are three main reasons for the non-coagulation of positive control in bacterial endotoxin test.
Reagent problems (including endotoxin and tachypleus amebocyte lysate)
When the labeling values of limulus lysate and endotoxin standard products exceed the deviation range, or the endotoxin dilution is improperly used, or the bacterial endotoxin and test water are used interchangeably for products from different manufacturers, the positive control may not be established. Some users use the standard of bacterial endotoxin provided by the manufacturers of limulus reagent as positive control, but the limulus reagent and water used in the test are not the same manufacturer, which is one of the main reasons why the positive control is not established at present. In our work, we found that the bacterial endotoxin standard, inspection water and test consumables provided by Zhanjiang Bokang Marine Biology Co., Ltd. are suitable for tachypleus amebocyte lysate reagents produced by all domestic and foreign suppliers. Many customers neglected this problem in the experiment, which led to the failure of positive control or negative sensitivity test. The reason was found that the bacterial endotoxin standard, inspection water and test consumables provided by some limulus reagent suppliers were not suitable for the Limulus reagents of other manufacturers, especially for the inspection water of joint ventures of Limulus reagents, which was the most prominent phenomenon.
2. Problem of experimental conditions
2.1 The "experimental preparation" of bacterial endotoxin test for utensils in China Standard Operational Code for Drug Inspection, 2010 edition, page 311 stipulates that "the utensils shall be fully immersed in chromic acid lotion or other pyrogen inactivator or cleaning liquid, then the washing liquid shall be extracted and dried, the residual washing liquid shall be thoroughly washed with tap water, and then rinsed repeatedly with distilled water for more than three times, and then dried by air. Then put it in a suitable closed metal container or wrapped in tin foil paper, then put it in a metal container and put it in an oven to remove the exogenous endotoxins that may exist on the surface of glassware.
According to Article 22 of the 2010 edition of the Chinese Pharmacopoeia, "Test water, unless otherwise specified, means purified water." Special attention should be paid to the fact that the final rinse of glassware must be "rinsed with distilled water". Some customers used glass containers for rinsing test of purified water prepared by reverse osmosis. The results showed that all positive controls in the test were negative. There was a big error in the test results when the sensitivity of tachypleus amebocyte lysate reagent was checked. This was mainly due to the different preparation methods of purified water and distilled water, and their water quality was different from each other. Because of the different preparation methods, the anions and cations in the purified water can not be destroyed by drying at 250 C. These ions have strong inhibitory effect on the enzyme reaction of Limulus reagent, leading to the failure of positive control and the validity of sensitivity review results. This is the main reason why the known positive control is not established and the results of sensitivity review are not established.
2.2 Washing solution problem 5% potassium dichromate sulfuric acid lotion configuration should be sealed and preserved. Direct exposure to air is prohibited. Once the lotion turns green, it indicates that chromic acid has been reduced, lost oxidation capacity, and is not suitable for reuse.
2.3 After some units found that the positive control tube was not condensable, the tachypleus amebocyte lysate and endotoxin were taken to the laboratory of our company for further test, and the results showed that the positive control tube was well coagulated. However, when we went outside to carry out the bacterial endotoxin test in situ, we found that the positive control tube did not coagulate in two laboratories. The reason is that the most common problems occur in the insulation links. One is that the power socket of the water bath is not well contacted during the heat preservation process, and the constant temperature can not be guaranteed; the other is that a unit uses a capless constant temperature water bath pot instead of a capped electrothermal constant temperature water bath tank, and the temperature of the water surface may not reach 37 C. The gelling time of tachypleus amebocyte lysate is negatively correlated with temperature in a certain range. Low temperature will prolong the gelling time, so it is not surprising that the positive control tube will not coagulate in the prescribed time. Another reason for the actual low temperature of the water bath may be that the experimenter placed the thermometer near the heating pipe to monitor the reaction temperature. We advocate the use of mercury thermometers on an electrothermal thermostat to monitor the reaction temperature. Accurate temperature control is also an important guarantee for accurate results.
2.4 The 2010 edition of the Standard Operational Code for Pharmaceutical Inspection in China requires that the thermostat for bacterial endotoxin test can be used as a "thermostat or a suitable thermostat, and the temperature of the thermostat can be set at 37 +1". In some laboratories, tachypleus amebocyte lysate was placed in microbial incubator or blast thermostat at 37 for 60 minutes. The positive control was not established and the results of sensitivity review were not valid. The 2010 edition of the China pharmaceutical inspection standard operation standard prompts that "since the agglutination reaction is irreversible, it should be noted that during the reaction and observation results, the test tube should not be vibrated, so that the gel will break and produce false negative results." Some customers insist that they did not make the test tube vibrate during the reaction and during the observation, but neglected that the hot air in the microbial incubator or the blast thermostat was always in the circulating state, and that the ampoule was very light, and that the ampoule was always in the vibrating state in the hot air circulating state, which was a typical false negative result. We recommend an instrument, ET-96 endotoxin gel analyzer, which is specially used for the gel test of bacterial endotoxin test. It is fully in line with the requirements of the Chinese Pharmacopoeia. The instrument provides 96 detection holes, which can be directly inserted into the ampoule of Tal, and the 96 detection holes are divided into 12 groups. Each group is individually timed, the sample is placed directly into the instrument after sample addition, and the time is 60 minutes to keep warm. It avoids the inaccuracy caused by the time difference when different samples are added to the insulation device.
2.5 In order to ensure the accuracy of the experimental results and prevent pollution, the endotoxin test should be carried out in a sealed clean room or under a super-clean workbench in a sealed laboratory. The bacterial endotoxin test should set up independent laboratories as far as possible, and should not share the same laboratory with other tests.
3. Operational problems.
3.1 Endotoxin can only open the endotoxin bottle at the top of the breaking point of the ampoule, but not along the breaking point, so as to prevent endotoxin from sticking to the sealing film during the oscillation process. After contact with endotoxin solution, some of the inner solutes of sealing film or medical adhesive tape can dissolve in the solution, which can enhance or inhibit the growth of endotoxin.
3.2 The problem of endotoxin oscillation. In addition, we should pay attention to the process of dissolution and dilution of endotoxin according to pharmacopoeia. The time and intensity of oscillation should be guaranteed to make the endotoxin solution uniform and stable and the endotoxin molecules fully dispersed.
3.3 The opening time of tachypleus amebocyte lysate should be operated strictly according to the operating rules. Before opening the ampoule, the neck should be wiped with alcohol cotton ball to break the neck and prevent debris from falling into the ampoule. Sampling should be accurate in the dissolution of tachypleus amebocyte lysate, dilution of sample and addition of sample. If one person completes the test independently, the standard solution and sample solution of endotoxin can be diluted and prepared first, and then the lysate reagent can be opened to shorten the exposure time of the lysate solution to air as much as possible (preferably within 15 minutes). The alcohol cotton ball wipes the neck and kneads the alcohol cotton ball. The neck of the ampoule is not left with obvious alcohol. The alcohol has strong inhibition in the ampoule of the limulus reagent and the alcohol can dissolve the gel. This is also a factor leading to the failure of positive.
To sum up, the author makes an analysis of the three reasons for the non-condensation of positive control in bacterial endotoxin test. As the author's level is limited, the conclusion may not be comprehensive. Readers are invited to understand and put forward valuable suggestions.