**Human Arginase (Arg) ELISA Kit Procedure:**
Before starting the experiment, make sure all reagents are properly prepared and stored as per the kit instructions. When diluting samples or reagents, mix thoroughly but avoid excessive shaking to prevent air bubbles. Always include a standard curve in each run to ensure accurate quantification. If the sample concentration is above the detection range of the kit, dilute it with the provided sample diluent before proceeding.
1. **Sample Addition:**
Add 100 µL of sample diluent to the blank well, and 100 µL of either standard or test sample into the corresponding wells. Ensure no air bubbles are introduced during this step. Carefully pipette the sample to the bottom of the well without touching the sides. Gently mix and cover the plate with a lid. Incubate at 37°C for 120 minutes. It’s important to prepare a fresh standard solution for each experiment to maintain accuracy.
2. **Add Biotin-Labeled Antibody:**
After incubation, discard the liquid from the wells and dry them. Then add 100 µL of biotin-labeled antibody working solution to each well. Prepare the working solution by mixing 1 µL of biotin-labeled antibody with 99 µL of diluent, and use it within one hour. Incubate at 37°C for 60 minutes.
3. **Washing Step:**
After the incubation, remove the liquid and dry the plate. Wash the microplate three times with wash buffer, soaking each well for 1–2 minutes. After washing, dry the plate completely.
4. **Add HRP-Conjugated Avidin:**
Add 100 µL of horseradish peroxidase (HRP)-labeled avidin working solution to each well. Incubate at 37°C for 60 minutes.
5. **Second Washing:**
Discard the liquid, dry the plate, and wash it five times with wash buffer. Soak each well for 1–2 minutes and dry thoroughly after each wash.
6. **Substrate Addition:**
Add 90 µL of substrate solution to each well. Incubate at 37°C for up to 30 minutes. Observe the color change; the first few standard wells should show a clear blue gradient, while the blank or negative wells remain uncolored. Stop the reaction once the desired color development is achieved.
7. **Stop Solution Addition:**
Add 50 µL of stop solution to each well to terminate the reaction. The color will change from blue to yellow. Ensure that the stop solution is added in the same order as the substrate solution to maintain consistency and accuracy.
8. **OD Measurement:**
Measure the optical density (OD) at 450 nm using an ELISA reader. Perform the measurement within 15 minutes after adding the stop solution to ensure accurate results.
**Notes for Using the Human Arginase (Arg) ELISA Kit:**
1. Before first use, centrifuge all reagent tubes briefly to collect any precipitated material at the bottom of the tube.
2. Set aside one well as a blank zero hole. This well should not contain any reagents except the final substrate and stop solution. Use this to set the baseline OD value during measurement.
3. To prevent evaporation, cover the plate with a lid or film and place it in a sealed container with a damp cloth.
4. Store unused plates and reagents at 2–8°C. Do not reuse diluted standards, biotin-labeled antibodies, or HRP-avidin solutions.
5. For better accuracy, it is recommended to run samples in duplicate.
This procedure ensures reliable and reproducible results when measuring human arginase levels. Always follow the manufacturer's instructions carefully and maintain proper lab practices throughout the process.
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