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Rat NADPH oxidase (NADPH-OX) enzyme-linked immunosorbent assay kit instruction manual
**Rat NADPH Oxidase (NADPH-OX) ELISA Kit Instructions for Use**
Before using this enzyme-linked immunosorbent assay (ELISA) kit, please read the instructions carefully. This kit is designed to detect rat NADPH oxidase (NADPH-OX) using a biotin double-antibody sandwich ELISA method. It is strictly for research purposes and not intended for medical diagnosis or clinical use.
**Application:**
This kit is suitable for the quantitative determination of NADPH-OX in rat serum, plasma, and other liquid samples.
**Working Principle:**
The kit employs a biotin-labeled double-antibody sandwich ELISA technique. The microplate is pre-coated with anti-NADPH-OX antibodies. After adding the sample, NADPH-OX binds to the immobilized antibodies. A biotinylated anti-NADPH-OX antibody is then added, followed by streptavidin-HRP. After incubation and washing, the substrate solution (A and B) is added, resulting in a color change from blue to yellow. The intensity of the color is directly proportional to the concentration of NADPH-OX in the sample.
**Kit Components:**
1. Standard (3200 pg/ml) – 0.5 ml
2. Standard Diluent – 3 ml
3. Enzyme-labeled Coating Plate – 12 wells × 4 strips
4. Streptavidin-HRP – 3 ml
5. Biotinylated Anti-NADPH-OX Antibody – 1 ml
6. 20× Washing Solution – 20 ml
7. Developer A – 3 ml
8. Developer B – 3 ml
9. Stop Solution – 3 ml
10. Sealing Film – 2 sheets
11. Sealed Bag – 1
12. Manual – 1 part
**Required Reagents and Equipment Not Included:**
- Incubator at 37°C
- Microplate reader
- Precision pipettes and disposable tips
- Distilled water
- Disposable test tubes
- Absorbent paper
**Notes:**
- Allow the kit to equilibrate at room temperature for at least 30 minutes after removing from 2–8°C storage.
- Ensure accuracy by calibrating pipettes regularly.
- Never reuse tips or caps to avoid cross-contamination.
- Store unused plate wells in a sealed bag.
- Avoid mixing reagents from different batches.
- Protect Developer B from light exposure.
**Washing Procedure:**
Manual: Remove liquid from the plate, blot with absorbent paper, and add 0.35 ml of diluted washing solution per well. Soak for 1–2 minutes, repeat as needed.
Automatic: Use an automatic washer after proper training.
**Sample Requirements:**
- Avoid using samples containing NaN3, as it may inhibit HRP activity.
- Process samples immediately after collection. If not tested right away, store at -20°C and avoid repeated freeze-thaw cycles.
**Standard Dilution:**
Follow the provided instructions to prepare standards from the original stock.
**Procedure Summary:**
Prepare reagents, samples, and standards. Add samples and standards to the plate, along with biotinylated antibody and enzyme reagent. Incubate at 37°C for 60 minutes. Wash the plate five times. Add developer A and B, incubate in the dark for 10 minutes. Stop the reaction with stop solution and measure OD at 450 nm within 10 minutes.
**Detection Range:** 10 pg/ml – 300 pg/ml
**Specifications:** 48 tests per box
**Storage:** 2–8°C
**Shelf Life:** 6 months (2–8°C)
This kit provides a reliable and sensitive method for measuring NADPH-OX levels in rat biological samples. Always follow the protocol closely to ensure accurate results.