Cat#:	KITG-123
Description/Features:	The Recombinant Factor C Endotoxin Detection Kit is a non-shorecrab-derived pre-assembled kit for in vitro quality control during the manufacturing process, as well as final product endotoxin quantitative determination of parenteral drugs, biological products, infusion cells, medical devices and media for tissue culture. The kit is for research use only and is NOT for use in diagnostic or therapeutic applications. Pack Size: 48 Tests / 96 Tests. Endotoxins, also called lipopolysaccharides (LPS), are the component of the outer membrane of gram-negative bacteria. They are released into the surrounding environment when the intact bacteria are disrupted (either by death or cell lysis). It is known that endotoxin can trigger reactions in animals, with symptoms including high fever, vasodilation, and diarrhea. In extreme cases, it may lead to fatal shock. Endotoxin content is a critical quality control parameter for raw materials, biological products, and medical devices. It directly impacts product release and compliance. The Recombinant Factor C Endotoxin Detection Kit represents a novel approach for endotoxin detection, leveraging recombinant technology. Recombinant Factor C, which is the key component in the horseshoe crab coagulation cascade reaction, gets activated upon exposure to endotoxin. Once activated, Factor C cleaves the fluorogenic substrate, thereby generating a fluorescent signal. There exists a positive correlation between the increment in the fluorescence signal and the amount of endotoxin. The experiment is conducted on a nontransparent 96-well plate, with measurements taken at the start (time zero) and after a one-hour incubation at 37°C. A fluorescence microplate reader is employed to measure at the excitation/emission wavelength of 380/440 nm to assess whether the sample is contaminated with endotoxin. The Limulus Amoebocyte Lysate test (LAL) has been widely used for decades for the quality assurance of injectable drugs and medical devices for endotoxin detection. However, variations in the LAL's sensitivity and specificity towards endotoxin, along with the decreasing supply of horseshoe crabs, are presenting the biotechnology industry with growing challenges. The recombinant factor C (rFC) assay was developed as a reliable alternative to the LAL test.
Formulation:	Bacterial Endotoxin Standard: Powder Recombinant Factor C Protein: Powder Fluorogenic Substrate: Powder Water for Bacterial Endotoxins Test: Liquid 96 well plate: Plastic plate
Detection Range:	0.005-5 EU/mL
Product Sensitivity:	0.005 EU/mL (Limit of quantification, LoQ)
Kit Composition:	Bacterial Endotoxin Standard: 1 vial, Powder, Storage: 2-8°C Recombinant Factor C Protein: 48 tests / 96 tests, Powder, Storage: 2-8°C Fluorogenic Substrate: 48 tests / 96 tests, Powder, Storage: 2-8°C Water for Bacterial Endotoxins Test: 50 mL, Liquid, Storage: 2-8°C 96 well plate: 1 set, Plastic plate, Storage: ambient
Package:	48 Tests / 96 Tests
Usage:	This kit is for research use only and is NOT for use in diagnostic or therapeutic applications. This kit must be utilized strictly in accordance with the provided instructions to ensure accurate and reliable results. Do NOT mix reagents from different lots. Bring all reagents and samples to room temperature (20-25°C) before use. Store the kit at 2-8°C to maintain the performance and stability. Each reconstituted component must be pipetted with a pyrogen-free pipette tip, and the rest should be dispensed in a pyrogen-free glass bottle or not dispensed at all, sealed and stored at 2-8°C to avoid introducing exogenous contamination, please use it up within 4 weeks. Please prepare the working solutions of each component in accordance with the protocol and experimental requirements. All prepared working solutions are intended for single use only and cannot be stored for future use. All reagents and consumables used in the endotoxin testing process must be explicitly defined sterile and pyrogen free to prevent the contamination of testing reagents. Bring all reagents and samples to room temperature (20-25°C) before use. Each reconstituted component must be pipetted with a pyrogen-free pipette tip, and the rest should be dispensed in a pyrogen-free glass bottle or not dispensed at all, sealed and stored at 2-8°C. Please use it up within 4 weeks. All prepared working solutions are intended for single use only and cannot be stored for future use. In order to ensure the accuracy of the detection of endotoxin, the operation process must be conducted in a manner to prevent the introduction of additional endotoxin. If the open lab environment cannot be confirmed to meet the requirements, the operation should be carried out in a biological safety cabinet. Depyrogenate all glassware and other heat-stable materials in a hot air oven following a validated process. A commonly employed minimum time and temperature is 30 minutes at 250°C. If using plastic labware such as microplates and pipette tips, select labware that is proven to be free of detectable endotoxin and does not interfere with the test.
Application:	In vitro quality control during the manufacturing process, as well as final product endotoxin quantitative determination of parenteral drugs, biological products, infusion cells, medical devices and media for tissue culture. Applicable in endotoxin detection of injectable drugs such as Recombinant Human Interferon α-1b and Human insulin injection. Compatible sample types include parenteral drugs, biological products, infusion cells, medical devices, media for tissue culture, injectable drugs such as Recombinant Human Interferon α-1b and Human insulin injection. Various buffer systems including: 1M NaCl, 20 mM CaCl₂, 20 mM MgCl₂, 1M Sodium acetate pH 5.0, 50 mM Sodium acetate pH 3.5, 100 mM Tris pH 10.9, 100 mM Glycine pH 3.5, 1× PBS pH 7.5, 1× PBS pH 6.0, RPMI 1640 Medium, DMEM Medium, Essential 8 Flex Basal Medium, mTeSR Plus, CelThera GMP T Cell Expansion Medium, CTS OpTmizer T-Cell Expansion SFM, 12.5 mM Histidine Buffer pH 6.5, Keytruda Formulation, Hemlibra Formulation, 30% DMSO, HSA (25 mg/mL), Toripalimab (40 mg/mL), Multiple Electrolytes Injection, 100% FBS.
Storage:	Store the kit at 2-8°C to maintain the performance and stability. The kit is shipped with blue ice. Store reconstituted components at 2-8°C, use within 4 weeks. 96 well plate: ambient storage. The kit is shipped with blue ice and must be stored at 2-8°C upon receiving. The expiration date is attached on the exterior packaging. Do not use expired reagents. The components can be stable at 2-8°C for 4 weeks after reconstitution. All prepared working solutions are intended for single use only and cannot be stored for future use.
Notes:	1. When establishing the endotoxin standard curve, prior to each experiment, the endotoxin stock solution should be serially diluted to obtain each concentration point required for the standard curve. Diluting the concentration points of the standard curve in advance and storing them for later use is not recommended. 2. In order to counteract any standard sticking, we recommend changing tips between each dilution. 3. To prevent adsorption, it is recommended to prepare endotoxin standards in endotoxin-free glass tubes. Plastic tubes are not recommended to prepare endotoxin standards. 4. The sample to be tested cannot be adjusted by pH electrode directly, to avoid the samples are contaminated by pH electrode resulting in false positive endotoxin. It is recommended to separate a part of the sample for pre-experiment to select an appropriate pH adjustment method, and the actual test sample should be adjusted by using endotoxin-free buffer as per the pre-experiment results. 5. Do not use vortex to mix the Mixture of Substrate working solution. Just gently shake and mix. It is required to prepare and use the mixture of Substrate working solution freshly. 6. The standards and all test samples should be measured at the same gain setting for the same experiment. 7. Every time samples test requires simultaneous testing of the endotoxin working standard solution and the creation of a new standard curve. Fluorescence microplate reader parameter settings: Mode - Fluorescence. Excitation/emission: 380/440 nm. Gain: Fluorescence signals are usually recorded as Relative Fluorescence Units (RFU). As the actual fluorescence signal is converted into an electronic signal that can be adjusted via the gain or sensitivity setting, RFU is an arbitrary unit. Different microplate readers and gain values can yield different fluorescence signals. Adjust the parameters according to the equipment manual. Increase the gain if the signal of the lowest concentration point can't be measured. Reduce it when the background signal is too high. In the fixed Gain value mode, the fluorescence range is 0-260,000. It's advisable that the RFU value of the highest concentration point not exceed 80% of the maximum 260,000 (i.e., 208,000) to prevent signal overflow. If there are no special requirements for the signal value and your instrument has an automatic calibration mode, you can also choose to automatically calibrate the Gain value based on your experiment to ensure no signal overflow. The setting of instruments is not the same across different brands, please consult with the instrument vendor for details. Appropriate parameters setting is essential before conducting the experiment. Sample storage: If the samples are not tested promptly, they must be stored under conditions where all bacteriological activity is halted or endotoxin levels will not escalate over time. To inhibit bacteriological activity, it is advisable to store the samples at a temperature range of 2-8°C for a duration of less than 24 hours. End-user is responsible for ensuring that the containers and storage conditions are suitable for their specific samples. Sample interference evaluation: To determine whether there is interference or not, the recovery rate of endotoxin detection in each test sample after adding the standard substance should be examined. When the detected value of the spiked sample minus the detected value of the sample without added endotoxin is divided by the known content of the added endotoxin, if the recovery rate of the added endotoxin standard substance falls within the range of 50%-200%, it can be considered that there is no interference in the sample solution under these experimental conditions. This experiment can be performed by adding 1 part of the 0.5 EU/mL standard to 1 part of the test sample. This yields an added spike of 0.25 EU/mL. Any endogenous endotoxins from the sample itself determined prior to spiking and corrected for by the 50% dilution of that sample should be subtracted from the value determined for the spiked sample, then calculate the concentration of endotoxins to give the recovery rate. Maximum Valid Dilution (MVD): MVD is the formula for calculating the maximum allowable dilution of the test sample for the endotoxin limit. MVD = (endotoxin limit × concentration of Sample Solution)/λ, where λ represents the lowest concentration adopted in the standard curve of the kit, that is, 0.005 EU/mL. pH adjustment: Given that the rFC endotoxin assay reagent employed for endotoxin determination involves an enzymatic reaction, it might be requisite to adjust the pH of the reaction system to fall within the range of 6.0-8.0. Should the pH of the reaction system deviate from this range, it can be adjusted using endotoxin-free hydrochloric acid, sodium hydroxide, or other buffer solutions. The sample to be tested cannot be adjusted by pH electrode directly, to avoid the samples are contaminated by pH electrode resulting in false positive endotoxin. It is recommended to separate a part of the sample for pre-experiment to select an appropriate pH adjustment method.
Highlights:	Comparable to Limulus Amoebocyte Lysate (LAL) based method - Endpoint fluorescent assay, and other chromogenic quantitative LAL methods. High specificity - Unlike LAL Assay, as Factor G is absent from the rFC test kit, false-positive results due to β-glucan activation are not expected to occur. Accuracy - Traceability of endotoxin standards against USP Standard. Short time to obtain results - 1 hour. High sensitivity - 0.005-5 EU/mL. Extensive validation - Verify in accordance with the EUROPEAN PHARMACOPOEIA 11.0 and USP chapter <1085.1> pharmacopoeia, including specificity, sensitivity, precision, accuracy, applicability, and other aspects such as biological products, microplate readers and various buffer systems. Sustainable resource - This approach reduces dependence on horseshoe crab populations, alleviates fishing pressure, and ensures a consistent and long-term supply of materials, supporting both sustainability and ecological balance. Good interbatch consistency - Batch consistency of products is guaranteed due to the use of genetic recombination technology for kit production.