Product Overview: Why Measure Fish IL‑4?
Interleukin 4 (IL‑4) is a key Th2‑type cytokine that regulates immune responses in teleost fish, including B‑cell activation, antibody class switching (IgT/IgM), and differentiation of T helper cells. It plays a critical role in defence against extracellular parasites, bacterial infections, and in the resolution of inflammation. Accurate quantification of Fish IL‑4 is essential for:
- Aquaculture immunology and disease resistance studies
- Vaccine efficacy evaluation in fish
- Understanding host‑pathogen interactions in farmed species
- Environmental immunotoxicology
This Inflammatory factor ELISA kit is specifically validated for a wide range of fish species (e.g., zebrafish, salmon, seabass, carp) and uses a high‑binding microplate to ensure maximum sensitivity and reproducibility.
Why Choose Yanda Bio for Your Fish Cytokine Assays?
| EEAT Factor | Our Commitment |
|---|---|
| Experience | 6,000+ factors developed; 60+ university collaborations; years of expertise in aquatic species immunoassays. |
| Expertise | Proprietary antibody pairs validated for fish IL‑4; sandwich ELISA format eliminates matrix interference. |
| Authoritativeness | Kits used in aquaculture research; ISO‑aligned manufacturing; batch‑to‑batch consistency. |
| Trustworthiness | Rapid custom development (3–7 days); unconditional quality guarantee; free shipping on 3+ kits. |
Superior Quality Features
- High‑binding microplate – specially treated bottom for enhanced adsorption of capture antibodies, resulting in stronger signals and lower backgrounds.
- High specificity – no cross‑reactivity with fish IL‑1β, TNF‑α, IL‑10, or other common cytokines.
- High stability – validated for long‑term storage at 2–8 °C.
Kit Components (96‑Well Configuration)
| Component | Quantity |
|---|---|
| High‑Binding Microplate (Pre‑coated) | 8 wells × 12 strips |
| Standards | 0.3 mL × 6 tubes |
| Sample Diluent | 6 mL |
| HRP‑Conjugated Detection Antibody | 6 mL |
| 20× Wash Buffer | 25 mL |
| Substrate A | 6 mL |
| Substrate B | 6 mL |
| Stop Solution | 6 mL |
| Plate Sealer | 2 sheets |
| Manual | 1 copy |
48‑well configuration available on request (8 wells × 6 strips).
Assay Principle: Sandwich ELISA
This kit employs a solid‑phase sandwich enzyme‑linked immunosorbent assay. The high‑binding microplate is pre‑coated with a monoclonal antibody specific for Fish IL‑4. Standards and samples are added, allowing IL‑4 to bind to the immobilised antibody. After washing, a Horseradish Peroxidase (HRP)‑conjugated detection antibody is added, forming an antibody‑antigen‑antibody “sandwich”. Following a second wash, TMB substrate is added. The HRP enzyme catalyses a blue colour that turns yellow upon acidification. The optical density (OD) at 450 nm is directly proportional to the IL‑4 concentration in the sample.
Sample Dilution Note: For serum/plasma/culture supernatants, samples are pre‑diluted 5‑fold during the procedure (10 μL sample + 40 μL diluent). Multiply final results by 5. For tissue homogenates, a separate validated dilution may be required – refer to the product datasheet.
Sample Collection & Handling
| Sample Type | Procedure |
|---|---|
| Serum | Allow blood to clot at room temperature for 10–20 minutes. Centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant. If precipitate forms, re‑centrifuge. |
| Plasma | Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant. |
| Cell Culture Supernatant | For secreted IL‑4: collect supernatant and centrifuge at 2000–3000 rpm for 20 minutes. For intracellular: adjust cell concentration to ~1×10⁶/mL in PBS (pH 7.2–7.4), then lyse by repeated freeze‑thaw cycles. Centrifuge and collect supernatant. |
| Tissue Homogenate | Weigh tissue, add PBS (pH 7.4), homogenise on ice, then centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant. |
| Storage | Extract as soon as possible. Store at –20 °C (≤ 1 month) or –80 °C (≤ 3 months). Avoid repeated freeze‑thaw cycles. |
Important: Do not use samples containing sodium azide (NaN₃), as it inhibits HRP activity.
Materials Required But Not Provided
- Microplate reader (450 nm)
- Precision pipettes (0.5–10 μL, 2–20 μL, 20–200 μL, 200–1000 μL)
- 37 °C incubator
- Distilled or deionised water
Assay Procedure (Step‑by‑Step)
- Equilibration: Bring all reagents to room temperature (15–30 minutes). Unused strips must be resealed and stored at 2–8 °C.
- Standard dilution: Prepare serial dilutions of the stock standard in clean tubes according to the lot‑specific datasheet.
- Add standards: Add 50 μL of each standard concentration to standard wells.
- Add samples: Add 40 μL sample diluent to sample wells, then 10 μL test sample. Blank wells: leave empty.
- Add detection antibody: Add 100 μL HRP‑conjugated detection antibody to all wells except blanks. Seal and incubate at 37 °C for 30 minutes.
- Prepare wash buffer: Dilute the concentrated wash buffer 30‑fold (or 20‑fold for 48T) with distilled water.
- Wash (first): Remove seal, discard liquid, tap dry. Fill each well with wash buffer, stand for 30 seconds, then discard. Repeat 5 times.
- Add enzyme: Add 50 μL enzyme reagent to all wells except blanks.
- Incubate again: Seal and incubate at 37 °C for 30 minutes.
- Wash (second): Repeat the same washing step (5 times).
- Colour development: Add 50 μL Substrate A then 50 μL Substrate B to each well. Gently tap to mix. Incubate at 37 °C in the dark for 10 minutes.
- Stop reaction: Add 50 μL stop solution (blue turns yellow).
- Read OD: Measure absorbance at 450 nm within 15 minutes using a blank well as zero.
Data Analysis
Plot standard curve: OD values (y‑axis) vs. standard concentrations (x‑axis) on linear graph paper or using curve‑fitting software (linear regression recommended, R ≥ 0.990). Calculate sample concentrations from the curve, then multiply by the dilution factor (5× for serum/plasma; for diluted samples multiply by the total dilution factor, e.g., n × 5).
Note: If the sample OD value exceeds that of the highest standard, dilute the sample further and repeat the assay, multiplying the final result by the additional dilution factor.
Performance Specifications
| Parameter | Performance |
|---|---|
| Accuracy | Standard curve linear regression coefficient (R) ≥ 0.990 |
| Detection Range | 12 ng/L – 400 ng/L |
| Sensitivity | Minimum detectable concentration < 5 ng/L |
| Specificity | No cross‑reactivity with fish IL‑1β, TNF‑α, IFN‑γ, or IL‑10 |
| Intra‑assay CV | < 9 % |
| Inter‑assay CV | < 11 % |
| Storage | 2–8 °C |
| Shelf Life | 6 months |
Important Notes
- Allow the kit to equilibrate to room temperature for 15–30 minutes before opening.
- Concentrated wash buffer may crystallise – warm in a water bath to dissolve; this does not affect performance.
- For large numbers of samples, keep the addition time within 5 minutes to avoid drift. Use a multi‑channel pipette if possible.
- Run standards and samples in duplicate (recommended).
- If the sample OD value is higher than that of the highest standard, dilute the sample with sample diluent (n‑fold) and re‑test. Multiply the final result by the total dilution factor (n × 5).
- The plate sealer is single‑use only – do not reuse to avoid cross‑contamination.
- Substrate solution is light‑sensitive – store in the dark.
- All samples, wash solutions, and waste should be treated as potentially infectious.
- Do not mix components from different lot numbers.
Storage & Stability
- Store the complete kit at 2–8 °C.
- Do not freeze.
- Under proper storage, the kit is stable for 6 months from the date of manufacture.
Related Products (Recommended for Cross‑Species Inflammatory Profiling)
Expand your inflammation research with these complementary Inflammatory factor ELISA kits (available from Yanda Bio – internal links to be added by user):
- Mouse Interleukin 6 (IL-6) ELISA Kit – for murine models of acute phase inflammation
- Human Interleukin 1α (IL-1α) ELISA Kit – for human pro‑inflammatory cytokine studies
Our extensive portfolio includes over 6,000 factors covering more than 15 species, including fish, shrimp, chicken, pig, horse, rabbit, rat, mouse, and human. All kits are manufactured on high‑binding plates for superior performance.
Pricing & Special Offers
| Quantity | Price per Kit |
|---|---|
| 1–2 kits | Standard retail – contact us for current pricing |
| 3 or more kits | Wholesale price – significant discounts available |
Free shipping on all orders of three or more ELISA kits.
Free technical support provided from sample collection to final data analysis.
Why Choose Yanda Bio?
- Manufacturer direct – over 6,000 factors, 60+ university partners
- High‑binding microplates – specially treated for maximum protein adsorption → stronger signals, lower background
- Rapid custom development – 3–7 days for new targets or species
- Reliable quality – strict QC at each batch, high specificity, excellent reproducibility
- Cost‑effective – premium performance without premium pricing
- Free shipping on 3+ kits
Disclaimer
This product is for research use only. Not for diagnostic or therapeutic use. Strict adherence to the protocol is required; Yanda Bio is not responsible for results obtained from deviations. The user assumes all responsibility for any misuse.
Ordering & Contact Information
To order this Fish Interleukin 4 (IL-4) ELISA Kit – a dedicated Inflammatory factor ELISA kit for aquaculture immunology – or to inquire about volume discounts, custom species validation, and bulk orders, please contact our sales team.
