Bovine Plg ELISA Kit
Bovine Plg ELISA Kit
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Product Specification
| Usage | Required experimental equipment: 1. Microplate reader (450nm) 2. High-precision pipettes and pipette tips: 0.5-10uL, 5-50uL, 20-100uL, 20-1000uL 3. 37°C incubator 4. Distilled or deionized water Sample handling and requirements: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge at 1000×g for 15 at 2-8°C within 30 minutes of collection. Remove the supernatant for testing or store at -20°C or -80°C, but avoid repeated freezing and thawing. Pre-test: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare the standard working solution: Add 1 mL of universal diluent to the lyophilized standard. Stand for 15 minutes to equilibrate, then gently mix (concentration 400 ng/mL). Then prepare the following concentrations: 400 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 0 ng/mL. Standard dilution method: Label 7 EP tubes and add 500 μL of universal diluent to each. Pipette 500 μL of the 400 ng/mL standard working solution to the first EP tube and mix thoroughly to make 200 ng/mL. Repeat the serial dilution to prepare the remaining standard working solutions. The 0 ng/mL is the blank well; no need to add any liquid. Refer to the figure below for details. 3. Prepare biotinylated detection antibody working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 15 seconds before use. Dilute the 100× concentrated biotinylated antibody to 1× working concentration with universal diluent (10 μL concentrate + 990 μL universal diluent). Mix and use immediately. 4. Prepare the enzyme conjugate working solution: Centrifuge the 100× concentrated enzyme conjugate at 1000×g for 15 seconds before use. Dilute the 100× HRP enzyme conjugate to 1× working concentration with universal diluent (10 μL concentrate + 990 μL universal diluent). Mix and use immediately. 5. Prepare the wash solution: Dispense 10 mL of 20× wash solution with 190 mL of distilled water. (Note: The concentrated wash solution removed from the refrigerator may crystallize; this is normal). Procedure: 1. Remove the strips from the aluminum bag after equilibration at room temperature for 10 minutes. Reseal the remaining strips in a ziplock bag and store at 4°C. 2. Sample addition: Add 100 μL of sample or standard at different concentrations to the wells. Add 100 μL of universal diluent to the blank wells. Cover with a film and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample 1:1 with universal diluent before adding to the ELISA plate. Multiply the dilution factor when calculating the final concentration. Run replicates for all samples and standards.) 3. Add Biotinylated Antibody: Remove the ELISA plate and discard the liquid without washing. Add 100 μL of Biotinylated Antibody Working Solution directly to each well. Cover with a film and incubate at 37°C for 60 minutes. 4. Wash: Discard the liquid and add 300 μL of Wash Solution to each well. Let stand for 1 minute, shake off, and pat dry on absorbent paper. Repeat three times (a plate washer can be used). 5. Add Enzyme Conjugate Working Solution: Add 100 μL of Enzyme Conjugate Working Solution to each well. Cover with a film and incubate at 37°C for 30 minutes. 6. Wash: Discard the liquid and repeat the wash step five times. 7. Add substrate: Add 90 μL of substrate (TMB) to each well. Cover with a film and incubate at 37°C in the dark for 15 minutes. 8. Stop the reaction: Remove the ELISA plate and add 50 μL of stop solution to each well. Immediately measure the OD value at 450 nm. Calculating experimental results: 1. Calculate the average OD values of the standards and samples. Subtract the OD value of the blank well for correction. Plot the standard curve using the four-parameter logistic function on double-logarithmic graph paper. Concentration is the horizontal axis; OD is the vertical axis. 2. If the sample OD exceeds the upper limit of the standard curve, dilute the sample and retest. Multiply the concentration by the dilution factor. |
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| Theory | This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a plasminogen (PLg) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of plasminogen (PLg) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Bovine | |||||||||||||||||||||||||||||||||
| Synonym | Bovine Plasminogen ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Plasminogen (Plg) is an important enzyme present in the blood (EC 3.4.21.7) that dissolves fibrin clots. In addition to fibrinolysis, plasminogen activators also proteolyze proteins in various other systems. They activate collagenases and several mediators of the complement system, weakening the walls of the follicles of the graffiti, leading to ovulation. They can cleave fibrin, fibronectin, thrombochondrin, laminin, and von Willebrand factor. Like trypsin, they belong to the serine protease family. | |||||||||||||||||||||||||||||||||
| General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use. 2. Improper plate washing may result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly before adding substrate. Do not allow the wells to dry out during incubation. 3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value. 4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue. 5. Avoid cross-contamination of reagents and specimens to prevent erroneous results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching agent will destroy the biological activity of the reagents in the kit. 8. Do not use expired products, and do not mix components with different product numbers and batches. 9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly and samples and testing devices should be handled according to prescribed procedures. |
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| Storage Temp. | If the unopened kit is stored at 4°C, the shelf life is 6 months. | |||||||||||||||||||||||||||||||||
| Test Range | 6.25-400 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | plasma |
