Sheep Epidermal Growth Factor (EGF) Enzyme-Linked Immunosorbent Assay (ELISA) technology, provided by Guangrui Bio — a trusted supplier of high-quality ELISA kits in China. Proper sample handling and preparation are essential for accurate results. Here’s a detailed guide on sample processing and requirements:
1. **Serum**: Allow blood to clot at room temperature for 10–20 minutes. Centrifuge at 2000–3000 rpm for approximately 20 minutes. Carefully collect the supernatant. If precipitation occurs during storage, re-centrifuge before use.
2. **Plasma**: Use EDTA or sodium citrate as an anticoagulant, depending on your needs. Mix gently for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant carefully. If precipitates form during storage, re-centrifuge.
3. **Urine**: Collect using a sterile tube. Centrifuge at 2000–3000 rpm for 20 minutes. Carefully remove the supernatant. If any precipitate forms during storage, re-centrifuge before testing. Similar procedures apply to pleural fluid, ascites, and cerebrospinal fluid.
4. **Cell Culture Supernatant**: For secreted components, collect the supernatant in a sterile tube and centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, dilute the cell suspension with PBS (pH 7.2–7.4) to about 1 million cells/mL. Freeze-thaw the cells several times to lyse them, then centrifuge again at 2000–3000 rpm for 20 minutes. Collect the supernatant carefully and re-centrifuge if needed.
5. **Tissue Specimens**: Weigh the tissue after cutting, then add PBS (pH 7.4). Rapidly freeze in liquid nitrogen. After thawing, keep the sample at 2–8°C. Add more PBS and homogenize manually or with a homogenizer. Centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant and test immediately, storing the rest at -20°C for future use.
6. **Sample Handling**: Process samples as soon as possible after collection. Follow standard protocols from relevant literature. Perform the assay promptly after extraction. If not tested immediately, store at -20°C but avoid repeated freeze-thaw cycles.
7. **Avoid NaN3**: Samples containing sodium azide (NaN3) should not be used, as it can inhibit horseradish peroxidase (HRP) activity, affecting the ELISA results.
**Procedure Steps**:
1. **Labeling**: Assign numbers to each well on the microplate. Include 2 negative control wells, 2 positive control wells, and 1 blank control well. The blank control contains no sample or enzyme reagent, but follows all other steps.
2. **Loading**: Add 50 μL of negative and positive controls to their respective wells. In each sample well, add 40 μL of sample diluent, followed by 10 μL of the sample. Gently mix without touching the walls of the wells.
3. **Incubation**: Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing Solution Preparation**: Dilute the concentrated washing solution 48 times with distilled water (total volume: 600 mL).
5. **Washing**: Remove the sealing film, discard the liquid, and dry the plate. Fill each well with washing solution, let stand for 30 seconds, and repeat this process 5 times. Pat dry thoroughly.
6. **Enzyme Addition**: Add 50 μL of enzyme-labeled reagent to each well except the blank ones.
7. **Second Incubation**: Repeat the same incubation step as in point 3 (37°C for 30 minutes).
8. **Second Washing**: Follow the same washing procedure as in step 5.
9. **Color Development**: Add 50 μL of developer A, then 50 μL of developer B to each well. Mix gently and incubate at 37°C for 15 minutes.
10. **Stop Reaction**: Add 50 μL of stop solution to each well to terminate the reaction. The color will change from blue to yellow.
11. **Measurement**: Measure the absorbance (OD value) at 450 nm using a microplate reader. Ensure the measurement is completed within 15 minutes after adding the stop solution. Always zero the instrument with the blank well first.
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