Bovine Brucellosis Antibody Enzyme-Linked Immunoassay Kit Instruction Manual

**Brucellosis Ab Enzyme-Linked Immunosorbent Assay (ELISA) Kit Instruction Manual** This reagent is intended for research use only. The kit is designed to detect the presence of Brucellosis Antibodies (Brucellosis Ab) in bovine serum, plasma, and other related fluid samples. It can also be used for diagnosing brucellosis in human patients. **Experimental Principle:** The Brucellosis Ab ELISA kit utilizes a double-antigen sandwich ELISA method to detect the antibody levels in the sample. The microtiter plate is pre-coated with purified Brucellosis antigens. These antigens bind to specific antibodies present in the sample. After washing to remove unbound components, an enzyme-labeled secondary antibody is added. This forms a complex of antigen-antibody-enzyme conjugate. Following another wash, the substrate TMB is added, which changes color under the action of HRP. The reaction is stopped with a stop solution, and the absorbance at 450 nm is measured using a microplate reader. The results are compared against a cut-off value to determine whether the sample is positive or negative for Brucellosis Ab. **Kit Composition:** | Component | 48-well Configuration | 96-well Configuration | |----------|------------------------|------------------------| | Instruction Manual | 1 piece | 1 piece | | Sealing Film | 2 pieces | 2 pieces | | Sealed Bag | 1 | 1 | | Enzyme-Labeled Plate | 1×48 | 1×96 | | Negative Control | 0.5 mL × 1 bottle | 0.5 mL × 1 bottle | | Positive Control | 0.5 mL × 1 bottle | 0.5 mL × 1 bottle | | Enzyme Standard Reagent | 3 mL × 1 bottle | 6 mL × 1 bottle | | Sample Diluent | 3 mL × 1 bottle | 6 mL × 1 bottle | | Developer A Solution | 3 mL × 1 bottle | 6 mL × 1 bottle | | Developer B Solution | 3 mL × 1 bottle | 6 mL × 1 bottle | | Stop Solution | 3 mL × 1 bottle | 6 mL × 1 bottle | | Concentrated Washing Solution | 20 mL × 20 times × 1 bottle | 20 mL × 30 times × 1 bottle | **Sample Preparation and Requirements:** 1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant carefully. 2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge for 20 minutes at 2000–3000 rpm. Collect the supernatant. 3. **Urine:** Collect using a sterile container and centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant. 4. **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes after collection. For intracellular components, lyse cells by freezing and thawing, then centrifuge again. 5. **Tissue Specimen:** Homogenize in PBS (pH 7.4), centrifuge, and collect the supernatant. Store at 2–8°C if not used immediately. 6. **Storage:** Samples should be processed as soon as possible. If not tested immediately, store at -20°C and avoid repeated freeze-thaw cycles. 7. **Avoid NaN3:** Sodium azide inhibits horseradish peroxidase activity and should not be present in samples. **Procedure:** 1. **Labeling:** Assign numbers to each well. Include 2 negative control wells, 2 positive control wells, and 1 blank control well. 2. **Loading:** Add 50 µL of negative and positive controls. Add 40 µL of diluent and 10 µL of sample to each test well. 3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes. 4. **Washing:** Prepare diluted washing solution (30 mL concentrated solution + 570 mL distilled water). Wash 5 times. 5. **Enzyme Addition:** Add 50 µL of enzyme-labeled reagent to each well except the blank. 6. **Second Incubation:** Repeat the incubation step at 37°C for 30 minutes. 7. **Second Washing:** Repeat the washing procedure. 8. **Color Development:** Add 50 µL of developer A and 50 µL of developer B. Incubate at 37°C for 15 minutes. 9. **Stop Reaction:** Add 50 µL of stop solution to each well to terminate the reaction. 10. **Measurement:** Read the OD values at 450 nm within 15 minutes of adding the stop solution. **Result Interpretation:** - **Validity Check:** Positive control mean ≥ 1.00; Negative control mean ≤ 0.10 - **Cut-off Value:** Cut-off = Mean of Negative Control + 0.15 - **Negative Result:** Sample OD < Cut-off → Negative for Brucellosis Ab - **Positive Result:** Sample OD ≥ Cut-off → Positive for Brucellosis Ab **Precautions:** 1. Follow instructions strictly. Do not mix reagents from different batches. 2. Allow the kit to equilibrate at room temperature for 15–30 minutes before use. Store unused strips in a sealed bag. 3. The concentrated washing solution may crystallize; warm it gently if needed. 4. Use a new sealing film for each experiment to prevent contamination. 5. Protect the substrate from light. 6. Use a microplate reader for accurate results. If using dual-wavelength detection, set the reference wavelength to 630 nm. 7. Treat all waste materials as biohazardous. Handle the stop solution (2M sulfuric acid) with care. **Storage Conditions and Expiration:** - **Storage:** Keep the kit at 2–8°C. - **Expiration:** 6 months from the date of manufacture. This manual provides detailed guidance for the proper use of the Brucellosis Ab ELISA kit. Always refer to the latest version of the instruction manual and follow standard laboratory safety protocols.

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