Bovine Ferritin FE ELISA Kit
Introduction
Bovine ferritin is a heavy and light chain protein complex involved in intracellular iron storage. It is primarily localized in the cytosol and stores iron in a bioavailable and non-toxic form. Ferritin levels in serum can reflect iron metabolism status and are altered in infection, inflammation, and iron deficiency states. The Bovine Ferritin FE ELISA Kit is designed for quantitative detection of ferritin in bovine biological samples using a sandwich enzyme-linked immunosorbent assay (ELISA) method.
Assay Principle
This ELISA uses a double-antibody sandwich format. The capture antibody is pre-coated on a 96-well microplate and binds bovine ferritin in the sample. A biotin-conjugated detection antibody specific for a different epitope of bovine ferritin is then added. Streptavidin-HRP binds to biotin, and a TMB substrate is added to develop a color signal. The intensity of the signal at 450 nm is proportional to the concentration of ferritin in the sample.
Key Features
- Target Protein: Ferritin (iron-storage protein)
- Species Reactivity: Bos taurus (cattle)
- Assay Type: Quantitative sandwich ELISA
- Detection Range: 7.8–500 ng/mL (typical)
- Sensitivity: <3.2 ng/mL
- Assay Duration: ~3 hours
- Detection Method: TMB chromogenic, read at 450 nm
- Calibration: Standard curve based on recombinant bovine ferritin
Sample Types and Volume
- Recommended Samples: Serum, plasma (EDTA, citrate, heparin), tissue lysates, and cell culture supernatants
- Minimum Volume Required: 50–100 µL per well
- Sample Dilution: Typically 1:5 to 1:20, depending on ferritin levels
Kit Components
| Component | Description |
|---|---|
| 96-well ELISA plate | Pre-coated with anti-bovine ferritin antibody |
| Ferritin standards | 7 concentrations, lyophilized |
| Detection antibody | Biotin-conjugated anti-bovine ferritin |
| HRP-Streptavidin | For signal amplification |
| Substrate solution (TMB) | Chromogen for color development |
| Stop solution | Usually 1N H₂SO₄ or equivalent |
| Sample diluent | Protein-stabilized PBS buffer |
| Wash buffer (20×) | Concentrated phosphate buffer with Tween-20 |
| Instruction manual | Protocol and troubleshooting guide |
| Adhesive plate sealers | For minimizing evaporation |
Assay Procedure Summary
- Add Standards/Samples: 100 µL per well
- Incubate: 1 hour at 37°C
- Add Detection Antibody: 100 µL, incubate 1 hour at 37°C
- Wash: 3–5× with diluted wash buffer
- Add HRP-Streptavidin: 100 µL, incubate 30 minutes at 37°C
- Wash Again: 5×
- Add TMB Substrate: 90 µL, incubate 10–15 minutes in dark
- Add Stop Solution: 50 µL
- Read Absorbance: Immediately at 450 nm
Performance Characteristics
- Precision (Intra-assay): CV ≤ 10%
- Precision (Inter-assay): CV ≤ 12%
- Linearity: Recovery 88–101% across bovine sample types
- Cross-reactivity: Negligible with non-bovine ferritins
- Interference: Hemolysis and lipemia should be avoided; chelators may reduce signal
Storage Conditions
- Unopened Kit: Store at 2–8°C
- Opened/Reconstituted Components: Use within 1–2 weeks if refrigerated
- Avoid Freeze-Thaw: Critical for antibodies and enzyme reagents
- Shelf Life: 12 months from manufacture under proper conditions
Applications
- Veterinary Diagnostics: Ferritin quantification in cattle serum
- Nutritional Studies: Monitor iron metabolism and deficiency in bovine
- Pathophysiological Research: Inflammatory and hepatic markers
- Iron Supplementation Monitoring: Ferritin as surrogate iron marker
