Research test kit CFTK-HMM-0076

for medical researchcoagulationTMB

Product Name
Human Activated Coagulation Factor VIIa (FVIIa) ELISA Kit (Catalog No.: CFTK-HMM-0076)

Application
In vitro quantitative determination of human activated coagulation factor VIIa (FVIIa) in serum, plasma, tissue homogenates, cell culture supernatants and related biological fluids for research and diagnostic support.

Detection Principle
The kit is based on a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). Samples, standards and HRP-labeled detection antibody are added to microtiter wells pre-coated with capture antibody against human FVIIa, incubated and washed. Color development uses TMB substrate catalyzed by HRP (blue) and stopped to yellow by acid. The color intensity is proportional to FVIIa concentration and is measured at 450 nm to calculate sample concentration.

Sample Processing

• Serum: Collect blood in serum separator tubes; clot at room temperature for 2 hours or at 4°C overnight, centrifuge at 1000×g for 20 min, collect supernatant; store at -20°C or -80°C if required. Avoid repeated freeze–thaw cycles.
• Plasma: Collect with EDTA or heparin, centrifuge at 1000×g for 15 min at 2–8°C within 30 min of collection, collect supernatant; store at -20°C or -80°C. Avoid repeated freeze–thaw cycles.
• Tissue homogenate: Rinse tissue with cold PBS (0.01 M, pH 7.4), weigh and mince, add PBS (typ. 1:9 w/v), optionally add protease inhibitors, homogenize on ice, sonicate or perform freeze–thaw if needed, centrifuge at 5000×g for 5–10 min and collect supernatant.
• Cell culture supernatant/other specimens: Centrifuge at 1000×g for 20 min and use supernatant for assay or store at -20°C/-80°C. Avoid hemolyzed samples.

Self-contained Reagents / Instruments / Consumables

• Microplate reader (450 nm)
• High-precision pipettes and tips: 0.5–10 µL, 2–20 µL, 20–200 µL, 200–1000 µL
• 37°C incubator or thermostat
• Distilled or deionized water

Standard Concentrations

• 16 ng/mL
• 8 ng/mL
• 4 ng/mL
• 2 ng/mL
• 1 ng/mL
• 0.5 ng/mL

Reagent Preparation
Allow kit reagents to equilibrate to room temperature before use. Prepare 20× Wash Buffer by diluting 1:20 with distilled water (1 part 20× Wash Buffer to 19 parts water).

Procedures (summary)

• 1. Equilibrate plate at room temperature for 20 min; use required wells and reseal remaining plates for 4°C storage.
• 2. Add 50 µL standards to standard wells.
• 3. Add 50 µL sample to sample wells (do not add to blank wells).
• 4. Add 100 µL HRP-labeled detection antibody to each standard and sample well (except blank), seal and incubate 60 min at 37°C.
• 5. Discard liquid and wash each well with 350 µL washing solution, let stand 1 min; repeat wash 5 times (or use plate washer).
• 6. Add 50 µL substrate A and 50 µL substrate B to each well, incubate 15 min at 37°C.
• 7. Add 50 µL stop solution to each well and measure OD at 450 nm within 15 min.

Calculation of Results
Plot OD (x-axis) of standards versus concentration (y-axis) to construct a standard curve, obtain the regression equation and calculate sample concentrations by substituting sample OD into the equation.

Performance

• Detection Range: 0.5–16 ng/mL
• Sensitivity: < 0.1 ng/mL
• Specificity: No cross-reactivity with related soluble analogs
• Repeatability: Intra-assay CV < 10%; inter-assay CV < 15%

Notes / Important Considerations

• 1. Dilute samples exceeding the highest standard with sample diluent before assay.
• 2. Adhere strictly to specified incubation times and temperatures; equilibrate reagents to 20–25°C before use and refrigerate after use.
• 3. Inadequate washing or drying of wells may cause inaccurate results; ensure proper washing and avoid drying during incubations.
• 4. Remove liquid residues and fingerprints from the plate bottom to prevent OD interference.
• 5. Use colorless or very pale substrate solution only; discard if it has turned blue.
• 6. Avoid cross-contamination between reagents and samples.
• 7. Protect reagents and reactions from strong light during storage and incubation.
• 8. Equilibrate sealed reagent bags to room temperature before opening to prevent condensation.
• 9. Keep reaction reagents away from bleaching agents and strong solvent fumes.
• 10. Do not use expired products.
• 11. Handle potentially infectious samples in accordance with biosafety procedures.

Technical Specifications

• Catalog No.: CFTK-HMM-0076
• Test Species: Human
• Shelf Life: 6 months
• Storage: 2–8°C
• Detection Principle: Double-antibody one-step sandwich ELISA; HRP/TMB detection; OD measured at 450 nm
• Standard Curve Points: 16, 8, 4, 2, 1, 0.5 ng/mL
• Detection Range: 0.5–16 ng/mL
• Sensitivity: < 0.1 ng/mL
• Intra-assay CV: < 10%; Inter-assay CV: < 15%