Dynamic chromogenic endotoxin detection recombinant tachypleus amebocyte lysate Kit,specification,price,image-Bio-Equip in China

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Usage	materials required: endotoxin working standard, water for bacterial endotoxin test, pyrogen removal test tube, pyrogen Removal Tip, pyrogen removal 96 well microplate (or detachable strip), vortex mixer, pipette, test tube rack, dynamic photometric instrument with incubation system and supporting software 1The setting of the dynamic photometric instrument preheat the instrument and set the incubation temperature to 37º C. Set up templates and procedures. Set the plate reading wavelength to 405 nm, and set the kinetic parameters: read the plate for 60 minutes, once every 30 seconds 2Preparation of endotoxin standard solution a Concentration of dynamic color development method: 10, 1, 0.1, 0.01 eu/ml or 5, 0.5, 0.05, 0.005 eu/ml b Concentration by rate method: 0.1, 0.05, 0.025, 0.0125, 0.00625 eu/ml 2.1Take one endotoxin working standard, scratch it along the neck of the ampoule with a grinding wheel, open it, add an appropriate amount of water for bacterial endotoxin test, and place it on the vortex mixer to shake vigorously for 2 minutes 2.2Further dilute the above endotoxin solution with water for bacterial endotoxin test to the required concentration, and shake vigorously on the vortex mixer for 1 minute at each dilution step. The diluted endotoxin solution shall be left standing for more than 10 minutes. Before use, it shall be vigorously shaken on the vortex mixer for 1 minute. The endotoxin solution placed for more than 4 hours shall be discarded 3The negative control was water for bacterial endotoxin test 4Configuration of recombinant tachypleus amebocyte lysate: pipette 3 ml of the complex solution into the lyophilized recombinant tachypleus amebocyte lysate, gently shake or blow with a pyrogen Removal Tip to completely mix the recombinant tachypleus amebocyte lysate. The dissolved recombinant tachypleus amebocyte lysate should be used within 20 minutes 5In the experimental operation, remove the pyrogen microplate, and add 100 UL of bacterial endotoxin test water, endotoxin standard solution, or test article into each well. Add 100 UL of recombinant tachypleus amebocyte lysate per well with a pipette or multi-channel pipette (to avoid bubbles ), mix with medium speed vortex for 15 seconds, and put it into the microplate reader to read according to the set parameters 6Data processing 6.1Threshold method: set the threshold to 0.1 (which can be adjusted according to the result), paste the data into the calculation table according to the specification, and the calculation table will automatically give its start time (CT). According to the requirements of the calculation table, fill in the concentration value of the working calibrator, and automatically establish the standard curve: lgct = B LGC + A, where CT is the start-up time, C is the concentration of endotoxin, B is the linear slope, and a is the y-axis intercept. The experiment is valid only when the experimental data meet the following three conditions at the same time: ① the concentration point of the standard curve ≥ 4, correlation coefficient of standard curve \| R \| ≥ 0.980, ② the T value of the lowest point of the standard curve is less than that of the negative control, ③ the average value of the parallel tubes of the test article is within the range of the standard curve 6.2Rate method: for samples with concentration below 0.1 eu/ml, the rate method can also be used, and the reading time is 30 minutes. The rate method calculation table is used for data processing, and the reaction time parameters can be adjusted within 900-1800 seconds interference test of test article ① when endotoxin test method is established for new drugs or varieties without endotoxin test items, interference test must be carried out ② when the source, formula and production process of tachypleus amebocyte lysate and test article are changed, or any change that may affect the test results occurs in the test environment, the interference test must be repeated ③ when there may be interfering substances of Limulus amebocyte lysate test in the test article, the interference test must be carried out the steps are as follows: 1Select the midpoint of the standard curve or an endotoxin concentration close to the midpoint (set as λ m) as the endotoxin concentration added in the test article interference test. If the standard curve is 10, 1, 0.1, 0.01eu/ml series, 0.1eu/ml endotoxin concentration can be prepared with the test article as the positive control of the test article 2The concentration of the test solution is λ M endotoxin solution (i.e., the positive control of the test sample containing λ m endotoxin), and the endotoxin concentration of the solution is measured, which is called CS 3Measure the endotoxin concentration of the test solution without exogenous endotoxin, which is called C0 4Calculate the recovery rate r = (cs– C0) /λ M × 100% 5When R is between 50% and 200%, it is considered that the test solution has no obvious interference effect under this test condition 6When R is beyond 50% - 200%, serial dilution or other treatment shall be carried out on the test article to eliminate interference. Repeat steps 2-4 for each diluted solution until the recovery rate of endotoxin R is between 50% - 200%. The dilution multiple with the recovery r closest to 100% was selected for endotoxin detection. (refer to bacterial endotoxin test method in Pharmacopoeia of the People'sRepublic ofChina, 2020 Edition)
Theory	The recombinant horseshoe crab reagent contains three recombinant proteins: factor C, factor B, and prothrombin. The reaction principle is shown in Figure 1. Bacterial endotoxins activate factor C, causing a series of enzymatic reactions, activating coagulase to form coagulase, which decomposes the artificially synthesized chromogenic matrix into peptides and yellow p-nitroaniline (pNA), λ Max=405). The higher the concentration of endotoxin, the faster the release of pNA and the faster the absorbance change.
Detection Type	Endotoxin testing
Description	Recombinant tachypleus amebocyte lysate is an alternative to pharmacopoeia testing, which can be used for the final product testing of human injected drugs (including biological products), animal injected drugs and medical equipment products. Guidance on the validation of alternative test methods can be found at USP <1223>And <1225>Found in. Such methods should prove to be equivalent or better than pharmacopoeial methods the test method of this kit can also be used for quantitative detection of endotoxin in non pharmacopoeial substances (for example, raw materials including water and in-process monitoring), without method validation recombinant tachypleus amebocyte lysate is not suitable for detecting endotoxin in clinical samples, such as those in clinical samples for the diagnosis of human diseases (such as endotoxemia) product features: 1The traditional Limulus amebocyte lysate has no sensory substitution: the operation process is unchanged, the detection equipment is the same, and the analysis method is the same 2No animal ingredients - no tachypleus amebocyte lysate blood required 3The same cascade reaction process 4With 1, 3-&beta-D glucan did not react.
Composition	Two bottles of freeze-dried recombinant limulus lysate, two bottles of compound solution, and two bottles of endotoxin working standard
General Notes	1. this kit is used for quantitative detection of bacterial endotoxin in vitro. It is strictly forbidden for reagents to enter the body in any way 2All vessels in contact with reagents and test articles must be pyrogen free. Microbial contamination should be prevented during the test. The pyrogen in this manual refers to endotoxin concentration less than 0.005 eu/ml 3The pH value of the test article should be 6.0 – If it is beyond this range, it needs to be adjusted with pyrogen removal buffer, 0.1M sodium hydroxide or 0.1M hydrochloric acid 4In the interference test of the test article, if there may be interference substances of the recombinant tachypleus amebocyte lysate test in the test article, the interference test must be carried out.
Application	Quantitative testing of endotoxin quantification for non pharmacopoeial items (such as raw materials including water and monitoring during the process)
Storage Temp.	2-8° Cprotected from light, 2 years