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The Brucellosis-Ag ELISA Kit operates based on a double-antibody one-step sandwich ELISA principle. The microplates are pre-coated with specific antibodies against Brucellosis-Antigen (Brucellosis-Ag). After adding the sample, standard, and HRP-labeled detection antibody, the plate is incubated, washed thoroughly, and then developed using TMB substrate. Under the action of peroxidase, TMB turns blue and then yellow when an acid is added. The intensity of the color is directly proportional to the amount of Brucellosis-Ag in the sample. The absorbance at 450 nm is measured using a microplate reader, allowing for the quantification of the antigen concentration.
Sample Collection and Preparation:
1. **Serum**: Use endotoxin-free tubes. After blood collection, centrifuge at 3000 rpm for 10 minutes to separate serum from red blood cells.
2. **Plasma**: Use anticoagulants like EDTA, citrate, or heparin. Centrifuge at 3000 rpm for 30 minutes and collect the supernatant.
3. **Cell Supernatant**: Centrifuge at 3000 rpm for 10 minutes to remove debris.
4. **Tissue Homogenate**: Mix tissue with physiological saline, homogenize, and centrifuge at 3000 rpm for 10 minutes to obtain the supernatant.
5. **Storage**: If not tested immediately, aliquot the samples and store at -20°C. Avoid repeated freeze-thaw cycles. Thaw at room temperature before use.
Required Equipment:
- Microplate reader (450 nm)
- Precision pipettes (0.5–10 μL, 2–20 μL, 20–200 μL, 200–1000 μL)
- 37°C incubator
Precautions:
- Store the kit at 2–8°C. Allow it to equilibrate at room temperature for 20 minutes before use.
- The concentrated washing buffer may crystallize after refrigeration; dissolve by water bath before use.
- Unused wells should be returned to the ziplock bag and stored at low temperature.
- No dilution is needed for pre-treated samples; add 10 μL directly.
- Follow the instructions strictly regarding incubation time, volume, and order.
- Shake all reagents well before use.
Kit Components:
| Component | 96-well | 48-well | Notes |
|---------------------------|---------|---------|-------|
| Microporous plates | 12×8 | 12×4 | |
| Standards (500 ng/ml) | 0.5 mL | 0.5 mL | |
| Diluted standards | 6 mL | 3 mL | As instructed |
| Sample diluent | 6 mL | 3 mL | As instructed |
| Detection antibody-HRP | 6 mL | 3 mL | |
| 20× Wash buffer | 20 mL | 20 mL | Dilute as instructed |
| Substrate A | 6 mL | 3 mL | |
| Substrate B | 6 mL | 3 mL | |
| Stop solution | 6 mL | 3 mL | |
| Seal film | 2 sheets| 2 sheets| |
| Instructions | 1 part | 1 part | |
| Zipper bag | 1 piece | - | |
Reagent Preparation:
- **Wash Buffer (20×)**: Dilute 1 part of 20× wash buffer with 19 parts of distilled water.
Washing Procedure:
- Manual: Fill each well with wash buffer, let stand for 1 minute, discard, and repeat 5 times.
- Automatic: Add 350 μL wash buffer, soak for 1 minute, and repeat 5 times.
Procedure:
1. Remove the required number of wells from the foil pouch after 20-minute equilibration at room temperature.
2. Set up standard, sample, and blank wells. Add 50 μL of standard solutions at different concentrations.
3. Add 10 μL of sample and 40 μL of diluent to each sample well.
4. Add 50 μL of HRP-labeled detection antibody to each well. Seal and incubate at 37°C for 60 minutes.
5. Wash 5 times with wash buffer.
6. Add 50 μL of TMB A and B, incubate in the dark for 15 minutes.
7. Add 50 μL stop solution and measure OD at 450 nm within 15 minutes.
Result Interpretation:
Plot standard curve using Excel: X-axis = standard concentration, Y-axis = OD values. Calculate sample concentrations using the regression equation.
Kit Performance:
- Accuracy: R ≥ 0.9900
- Sensitivity: <1.0 ng/mL
- Specificity: No cross-reactivity with other antigens
- Repeatability: CV <15% between plates
- Storage: 2–8°C, away from light and moisture
- Shelf Life: 6 months
- Detection Range: 15.6–500 ng/mL
Disclaimer:
- This kit is for research purposes only. Not suitable for clinical trials or animal testing. The company assumes no responsibility for misuse.
- Follow instructions carefully. Do not mix reagents from different batches. Any deviation is the user’s responsibility.
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