This kit is intended for in vitro research only and is not intended for clinical diagnosis.
This kit is used for the quantitative detection of human gastric lipase (LIPF) content in serum, plasma, tissue homogenates and related liquid samples in vitro.
Expiration date: 6 months.
Storage conditions: 2-8
Kit composition.
Remarks:1(96t 48t) After opening the package, please check whether all items are complete in time.
2.The standards are at pg ml concentrations
3.After a large number of normal specimens were tested, the normal concentration values of the specimens were within the detection range provided by the kit, and 50 L samples could be directly taken and loaded during the experiment. When some sample values exceed the maximum standard concentration, the sample can be appropriately diluted with sample diluent before experimentation.
Pre-test preparation:
1.Please remove the kit from the refrigerator 20 minutes in advance and equilibrate to room temperature.
2.The concentrated washing solution taken out of the refrigerator may be crystallized, which is normal; Place at room temperature, shake gently, and wait until the crystals are completely dissolved before preparing the washing solution. 20ml of concentrated washing solution can be diluted with distilled water or deionized water to prepare 400ml of working concentration washing solution, and the unused one can be put back to 4
3.20 Dilution of wash buffer: Distilled water is diluted 1:20, i.e., 1 part of 20 wash buffer plus 19 parts of distilled water.
Experimental principle: The kit uses a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). Specimens, standards, and HRP-labeled detection antibodies were added to the coated microwells pre-coated with human gastric lipase (LIPF) capture antibodies, incubated, and thoroughly washed. Color is developed with the substrate TMB, which is transformed into blue by peroxidase, and to its final yellow color by acid. The shade of color was positively correlated with the human gastric lipase (LIPF) in the sample. Measure the absorbance (OD value) with a microplate reader at a wavelength of 450 nm and calculate the sample concentration.
Prepare your own test equipment for the experiment:
1.Microplate reader (450 nm).
2.High-precision dispenser and tip: 05-10ul、2-20ul、20-200ul、200-1000ul
3.37 incubator.
4.Distilled or deionized water.
Sample Handling & Requirements:
1.Serum: Whole blood specimens collected in a serum separator were left at room temperature for 2 hours or 4 overnight, then centrifuged at 1000 g for 20 minutes, and the supernatant could be taken or stored at -20 or -80, but repeated freeze-thaw cycles should be avoided.
2.Plasma: Collect specimens with EDTA or heparin as anticoagulants, centrifuge the specimens at 2-8 1000 g for 15 minutes within 30 minutes of collection, take the supernatant for detection, or store the supernatant at -20 or -80, but avoid repeated freeze-thaw cycles.
3.Tissue homogenate: Homogenize with pre-chilled PBS (001m,ph=7.4) Rinse the tissue, remove residual blood (lysed red blood cells in the homogenate will affect the measurement results), and shred the tissue after weighing. The minced tissue is compared to the corresponding volume of PBS (generally according to the weight to volume ratio of 1:9, for example, 1g of tissue sample corresponds to 9ml of PBS, and the specific volume can be appropriately adjusted according to the needs of the experiment, and a record should be made. It is recommended to add protease inhibitors to PBS) to a glass homogenizer and grind well on ice. To further lyse the histiocytes, the homogenate can be sonicated, or repeatedly freeze-thaw. Finally, the homogenate was centrifuged at 5000 g for 5 for 10 minutes, and the supernatant was taken for detection.
4.Cell culture supernatant: Centrifuge at 1000 g for 20 minutes and remove the supernatant for detection, or store the supernatant at -20 or -80, but avoid repeated freeze-thaw cycles.
5.Other biological specimens: 1000 g centrifugation for 20 minutes, take the supernatant for detection.
6.Sample appearance: The sample should be clear and transparent, and the suspended solids should be centrifuged and removed.
7.Sample preservation: If the sample is collected and tested within 1 week, it can be stored in 4, if it cannot be tested in time, please divide it according to the amount of one use, and freeze it in -20 (tested within 1 month), or -80 (tested within 6 months), to avoid repeated freezing and thawing, and the hemolysis of the specimen will affect the final test result, so the hemolytic specimen should not be tested for this test.
Notes:1Incubation is carried out in strict accordance with the specified time and temperature to guarantee accurate results. All reagents must be brought to room temperature 20-25 before use. Refrigerate reagents immediately after use.
2.Incorrect plate washing can lead to inaccurate results. Make sure to blot up as much liquid as possible from the wells before adding the substrate. Do not allow the micropores to dry out during the incubation process.
3.Eliminate residual liquid and finger marks from the bottom of the plate, otherwise the OD value will be affected.
4.The substrate chromogenic solution should be colorless or very light in color, and the substrate solution that has turned blue should not be used.
5.Avoid cross-contamination of reagents and specimens that can lead to false results.
6.Avoid direct exposure to strong light during storage and incubation.
7.Equilibrate to room temperature before opening the sealed bag to prevent water droplets from condensing on the cold slats.
8.None of the reaction reagents should come into contact with the bleaching solvent or the strong gases emitted by the bleaching solvent. Any bleaching component will destroy the biological activity of the reaction reagent in the kit.
9.Expired products cannot be used.
10.If there is a risk of disease transmission, all samples should be managed and samples and detection devices should be handled in accordance with the prescribed procedures.
Operation steps: 1 Remove the required slats from the aluminum foil bag after 60min of room temperature equilibration, and seal the remaining slats with a ziplock bag and put them back 4.
2. Set up standard wells and sample wells, and add 50 l of standard with different concentrations to each standard well.
3. Add 50 L of sample to be tested in sample well a; Blank holes are not added.
4 Except for the blank wells, 100 L of horseradish peroxidase (HRP)-labeled detection antibody was added to each well of the standard well and sample well, the reaction well was sealed with a sealing film, and 37 years were incubated in a water bath or incubator for 60 min.
5 Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 l), let stand for 1min, shake off the washing liquid, pat dry on absorbent paper, and repeat the washing plate 5 times (you can also use a microplate washer to wash the plate).
6 Add 50 L of substrate A and 50 L of B to each well, and incubate for 15min in 37 dark.
7 Add 50 L of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min.
Calculation of experimental results:
Draw the standard curve: In the excel worksheet, the concentration of the standard is used as the abscissa and the corresponding OD value is used as the ordinate, the linear regression curve of the standard is drawn, and the concentration value of each sample is calculated according to the curve equation.
Performance: 1Detection range: 25 pg ml 800 pg ml.
2.Sensitivity: The lowest detection concentration is less than 10 pg/ml。
3.Specificity: No cross-reactivity with other soluble structural analogues.
4.Repeatability: The coefficient of variation within the plate is less than 10%, and the coefficient of variation between plates is less than 15%.
Tech Tips:
1.When mixing or resolubilizing protein solutions, try to avoid foaming.
2.In order to avoid cross-infection, the pipette tips need to be changed when configuring standards of different concentrations, loading samples, and adding different reagents. In addition, please use different pipettes for different reagents.
3.Correct use of sealing gel during each incubation will ensure the accuracy of the results.
4.The mixed chromogenic substrate should be colorless before being plated, and should be stored in the dark; If the microplate is posted, it will change from the color to a different depth of blue.
5.The order of destining the liquid should be the same as that of the chromogenic substrate; After adding the stop solution, the color in the well changed from blue to yellow; If there is green in the well, it means that the liquid in the well is not mixed; Mix thoroughly.
Instructions:1Due to the existing conditions and the level of science and technology, all raw materials provided by all suppliers cannot be comprehensively identified and analyzed, and this product may have certain quality and technical risks.
2.The final experimental results are closely related to the validity of the reagents, the relevant operations of the experimenter, and the experimental environment at that time, so please be sure to prepare sufficient specimen backup.
3.There may be slight differences between different batches of the same product, such as: detection limit, sensitivity and color development time, etc., please follow the instructions in the kit for experimental operation, **The electronic version of the manual is for reference only.
4.Only the use of all the reagents associated with this kit can ensure the detection effect, and products from other manufacturers cannot be mixed. The best results will be obtained only if the experimental instructions of this kit are strictly followed.