Bovine Brucellosis Antibody Enzyme-Linked Immunoassay Kit Instruction Manual

**Brucellosis Ab Enzyme-Linked Immunosorbent Assay (ELISA) Kit Instruction Manual** This reagent is for research use only. It is designed to detect the presence of Brucellosis Antibodies (Brucellosis Ab) in bovine serum, plasma, and related fluid samples, as well as to aid in the diagnosis of brucellosis in humans. **Experimental Principle:** The Brucellosis Ab ELISA Kit utilizes a double antigen sandwich ELISA method to detect the presence of Brucellosis antibodies in the sample. The microplate is pre-coated with purified bovine Brucellosis antigens. These antigens bind to specific antibodies present in the sample. After washing to remove unbound components, an HRP-labeled secondary antibody is added, forming an antigen-antibody-enzyme complex. Following another wash, the substrate TMB is added, which turns blue under the action of HRP and then changes to yellow when an acid stop solution is applied. The absorbance at 450 nm is measured using a microplate reader. A cutoff value is calculated based on the negative control, and the sample is considered positive if its OD value exceeds this threshold. **Kit Composition:** - **Instruction Manual:** 1 copy - **Sealing Film:** 2 pieces (for 48-well configuration), 2 pieces (for 96-well configuration) - **Sealed Bag:** 1 - **Enzyme-Labeled Plate:** 1×48 (for 48-well), 1×96 (for 96-well) - **Negative Control:** 0.5 ml × 1 bottle - **Positive Control:** 0.5 ml × 1 bottle - **Enzyme Standard Reagent:** 3 ml × 1 bottle (for 48-well), 6 ml × 1 bottle (for 96-well) - **Sample Diluent:** 3 ml × 1 bottle (for 48-well), 6 ml × 1 bottle (for 96-well) - **Developer A Solution:** 3 ml × 1 bottle (for 48-well), 6 ml × 1 bottle (for 96-well) - **Developer B Solution:** 3 ml × 1 bottle (for 48-well), 6 ml × 1 bottle (for 96-well) - **Stop Solution:** 3 ml × 1 bottle (for 48-well), 6 ml × 1 bottle (for 96-well) - **Concentrated Washing Solution:** (20 ml × 20 times) × 1 bottle (for 48-well), (20 ml × 30 times) × 1 bottle (for 96-well) All reagents should be stored at 2–8°C. **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. If precipitation occurs during storage, re-centrifuge. 2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant. 3. **Urine:** Collect in a sterile tube 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 repeated freeze-thaw cycles and centrifuge again. 5. **Tissue Sample:** Homogenize in PBS (pH 7.4), centrifuge at 2000–3000 rpm for 20 minutes. Store the supernatant at 2–8°C or freeze for later use. 6. **Storage:** Process samples immediately after collection. If not tested right away, store at -20°C. Avoid repeated freezing and thawing. 7. **Avoid NaN3:** Samples containing sodium azide may interfere with HRP activity and should not be used. **Procedure:** 1. **Labeling:** Number the wells accordingly. Include 2 negative control, 2 positive control, and 1 blank control per plate. 2. **Loading:** Add 50 µl of negative and positive controls. Add 40 µl of sample diluent, followed by 10 µl of the sample into each test well. 3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes. 4. **Washing:** Prepare the diluted washing solution (30 ml concentrated washing solution + 570 ml distilled water). Wash 5 times. 5. **Add Enzyme:** Add 50 µl of enzyme-labeled reagent to each well except the blank. 6. **Incubation:** Incubate at 37°C for 30 minutes. 7. **Washing:** Repeat the washing step. 8. **Color Development:** Add 50 µl of Developer A, then 50 µl of Developer B. Incubate at 37°C for 15 minutes. 9. **Stop Reaction:** Add 50 µl of stop solution to each well. 10. **Measurement:** Measure OD at 450 nm within 15 minutes of adding the stop solution. **Result Interpretation:** - **Validity Check:** Positive control OD ≥ 1.00; Negative control OD ≤ 0.10. - **Cutoff Value:** Cutoff = Negative control average + 0.15. - **Negative Result:** Sample OD < Cutoff → Negative for Brucellosis Ab. - **Positive Result:** Sample OD ≥ Cutoff → 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 before use. Store unused strips in sealed bags. 3. The concentrated washing solution may crystallize. Heat it gently in a water bath 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. Dual-wavelength detection can use 630 nm as reference. 7. All waste materials should be treated as biohazardous. Handle the stop solution (2M sulfuric acid) with care. **Storage Conditions and Expiration Date:** - **Storage:** 2–8°C - **Expiration:** 6 months from the date of manufacture Always refer to the latest version of the instruction manual for any updates or additional information.

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