Description
GeniePlex MagPro Human GvHD Monitoring 8-Plex is a magnetic bead-based multiplex immunoassay kit for the simultaneous quantitative detection of 8 analytes from a single sample using flow cytometry. Antibody-conjugated beads with distinct fluorescence intensities capture target antigens, which are then detected via biotinylated antibodies and streptavidin-PE to produce a fluorescent signal proportional to each analyte concentration.
| Product Name: | GeniePlex MagPro Human GvHD Monitoring 8-Plex (96 Tests) |
| Product Code: | MPES0022 |
| Reactivity: | Human |
| Analytes (8): | IL-6, TNF-Alpha, IFN-Gamma, IL-2, ST2, IP-10, IL-10, and sCD25 |
| Range of Linearity: | IL-6: 5–5,000 pg/mL, TNF-Alpha: 5–5,000 pg/mL, IFN-Gamma: 5–5,000 pg/mL, IL-2: 5–5,000 pg/mL, ST2: 20–10,000 pg/mL, IP-10: 10–5,000 pg/mL, IL-10: 5–5,000 pg/mL, sCD25: 10–5,000 pg/mL |
| Sensitivity (LODs): | IL-6: < 3 pg/mL, TNF-Alpha: < 3 pg/mL, IFN-Gamma: < 3 pg/mL, IL-2: < 3 pg/mL, ST2: < 10 pg/mL, IP-10: < 5 pg/mL, IL-10: < 3 pg/mL, sCD25: < 5 pg/mL |
| Sample Types: | Serum, EDTA plasma, cell culture supernatants, and other biological fluids |
| Assay Type: | Magnetic bead-based multiplex immunoassay |
| Detection Method: | Flow cytometry (PE, APC, and APC/Cy7 channels) |
| Sample Volume: | 50 µL/well |
| Assay Time: | ~2.5 hours |
| Kit Size: | 96 tests |
| Component | Quantity (96T) | Storage |
| Premixed Antibody-Conjugated Beads | 2.4 mL × 2 | 2–8 °C, protected from light |
| Biotinylated Detection Antibodies | 4.8 mL × 2 | 2–8 °C |
| SA-PE (ready to use) | 4.8 mL × 2 | 2–8 °C, protected from light |
| Lyophilized Standard | 2 vials | 2–8 °C |
| Assay Buffer | 5 mL × 1 | 2–8 °C |
| Wash Buffer | 30 mL × 2 | 2–8 °C |
| Plate Sealing Film | 5 pieces | — |
| Manual | 1 copy | — |
Storage & Stability
| Condition | Details |
| Unopened kit | 2–8 °C, protected from light — stable for 12 months |
| Opened kit | 2–8 °C, protected from light — use within 30 days |
| Reconstituted standard | 2–8 °C — use within 24 hours |
Materials Required but Not Supplied
- U-bottom 96-well transparent plates
- Vortex mixer
- Incubator / microplate shaker
- Magnetic separator for 96-well plates
- Flow cytometer with PE, APC, and APC/Cy7 detection channels
| Range of Linearity: | IL-6: 5–5,000 pg/mL, TNF-Alpha: 5–5,000 pg/mL, IFN-Gamma: 5–5,000 pg/mL, IL-2: 5–5,000 pg/mL, ST2: 20–10,000 pg/mL, IP-10: 10–5,000 pg/mL, IL-10: 5–5,000 pg/mL, sCD25: 10–5,000 pg/mL |
| Limit of Blank (LoB): | ≤ 8 pg/mL for all analytes |
| Recovery: | 70–120% |
| Intra-assay CV: | < 15% |
| Inter-assay CV: | < 15% |
| Cross-reactivity: | No significant cross-reactivity among panel analytes |
*Note: The below is a summary protocol. Protocols may vary by lot. Always follow the instructions included with your kit.
Standard Preparation
- Label eight 0.6 mL microcentrifuge tubes 0–7. Add 150 µL Assay Buffer to tubes 0–6. Leave tube 7 empty.
- Reconstitute the lyophilized standard: centrifuge briefly (500 × g, 10 s), add 500 µL Assay Buffer, stand 5 min, mix gently until dissolved. Transfer to tube 7 (highest standard).
- Serial dilution: transfer 50 µL from tube 7 → tube 6 (1:3.2), then 100 µL from tube 6 → 5 → 4 → 3 → 2 → 1, mixing at each step. Tube 0 = zero standard (buffer only).
Assay Procedure
| Step | Protocol |
| 1. Bead & Sample Incubation | Add 50 µL Premixed Antibody-Conjugated Beads (vortex ≥ 15 s) and 50 µL sample or standard to each well. Seal plate. Incubate on shaker at RT, 600 rpm, protected from light, 1 hour. |
| 2. Detection Antibody | Place plate on magnetic separator for 1 min, remove supernatant. Add 100 µL Biotinylated Detection Antibodies. Seal and incubate at RT, 600 rpm, protected from light, 1 hour. |
| 3. SA-PE & Wash | Magnetic separate 1 min, remove supernatant. Wash once with 200 µL Wash Buffer. Add 100 µL SA-PE. Seal and incubate at RT, 600 rpm, protected from light, 30 min. Wash twice with 200 µL Wash Buffer. |
| 4. Acquisition | Resuspend beads in 200 µL Wash Buffer. Acquire on flow cytometer using PE, APC, and APC/Cy7 channels. |
Data Analysis
- Collect at least 200 beads per analyte gate (minimum 1,600 total beads for this 8-plex panel).
- Calculate median fluorescence intensity (MFI) for standards and samples. Subtract blank MFI.
- Plot standard curve (concentration vs. MFI, log-log) and fit with a 4-parameter logistic (4-PL) model.
- Determine sample concentrations from the standard curve. If MFI exceeds the upper limit, dilute and re-measure.
