**Human MTB ESAT6 ELISA Kit – For the Quantitative In Vitro Determination of Human M. tuberculosis ESAT6 in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
*For Laboratory Research Use Only – Not for Diagnostic Procedures*
This ELISA kit is designed for the quantitative detection of Mycobacterium tuberculosis ESAT6 in biological samples such as serum, plasma, cerebrospinal fluid, tissue homogenates, and other body fluids. The assay is based on a sandwich ELISA principle, where specific antibodies bind to ESAT6, and the signal is detected using a horseradish peroxidase (HRP)-conjugated secondary antibody. A chromogenic substrate is then added, producing a color change that is measured at 450 nm. The intensity of the color correlates with the concentration of ESAT6 in the sample.
The kit includes a set of calibration standards to generate a standard curve, allowing for accurate quantification of ESAT6 levels in unknown samples. This procedure ensures reliable and reproducible results when performed according to the instructions provided.
**Sample Collection and Storage:**
- **Serum:** Use a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation at 2000×g for 20 minutes. Remove serum and analyze immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma:** Collect using heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g, 2–8°C. Store at -20°C. Avoid repeated freezing.
- **Tissue Homogenate & Other Biological Fluids:** Centrifuge to remove particulates and analyze immediately or store at -20°C. Thaw only once.
**Materials Required (Not Supplied):**
1. Incubator at 37°C
2. Microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes, disposable tips, and absorbent paper
4. Distilled or deionized water
**Reagents Provided (Stored at 2–8°C):**
- 96-well microplate (12 × 8 strips)
- Standards (6 vials, 0.5 ml/vial)
- Sample Diluent (6.0 ml / 3.0 ml)
- HRP-Conjugate Reagent (10.0 ml / 5.0 ml)
- 20X Wash Solution (25 ml / 15 ml)
- Chromogen A (6.0 ml / 3.0 ml)
- Chromogen B (6.0 ml / 3.0 ml)
- Stop Solution (6.0 ml / 3.0 ml)
- Closure Plate Membrane (2 units)
- User Manual (1 copy)
- Sealed Bags (1 unit)
**Precautions:**
- Do not mix reagents from different kits.
- Allow all reagents to reach room temperature (20–25°C) before use.
- Do not use reagents beyond their expiration date.
- Store unused strips in the sealed bag with desiccant.
- Handle all samples as potentially infectious. Wear gloves and follow biosafety protocols.
- Dispose of waste properly, including inactivating viruses by adding 1% sodium hypochlorite.
- Keep chromogen solutions away from heat and flame.
**Assay Procedure:**
1. Prepare all reagents and add 50 µL of standard or sample in duplicate to the wells.
2. Add 100 µL of HRP-conjugate reagent to each well (except blank), cover, and incubate for 60 minutes at 37°C.
3. Wash the plate 4 times with 1X wash buffer.
4. Add 50 µL of Chromogen A and 50 µL of Chromogen B, incubate for 15 minutes at 37°C, protected from light.
5. Add 50 µL of Stop Solution and measure OD at 450 nm.
6. Plot the average OD values against the standard concentrations to create a standard curve.
7. Calculate sample concentrations based on the standard curve.
**Performance Characteristics:**
- Sensitivity: <10 pg/mL
- Dynamic Range: 62.5 pg/mL – 2000 pg/mL
- Specificity: No significant cross-reactivity observed
- Storage: 2–8°C (for frequent use); -20°C for long-term storage (up to 6 months)
**Important Notes:**
- Always read the entire protocol before starting.
- Ensure proper calibration and quality control.
- Maintain consistent timing and technique for accurate results.
- Follow all safety guidelines when handling biological materials.
This kit is intended solely for research purposes and should not be used for diagnostic or clinical applications.
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