Duck DVh, duck viral hepatitis (DVh) ELISA kit instruction manual - Database & Sql Blog Articles

Duck Viral Hepatitis

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DVh

ELISA Kit Instruction Manual

This kit is for research use only.

Experimental Principle

Duck Viral Hepatitis (DVh) ELISA Kit

The presence of Duck Viral Hepatitis (DVh) in the samples is detected using a double-antibody sandwich method. A solid-phase antibody, specific to DVh, is coated onto a microplate. The antigen from the sample binds to this antibody, and after washing, an HRP-labeled antibody is added. This forms a complex of antibody-antigen-HRP-labeled antibody. After washing, TMB substrate is added, which turns blue under HRP catalysis and then yellow when acid is introduced. The optical density (OD) at 450 nm is measured using a microplate reader. The result is compared with the CUTOFF value to determine if DVh is present in the sample.

Duck Viral Hepatitis (DVh) ELISA Kit – Composition

1. 30× Washing Solution: 20 ml × 1 bottle

7. Stop Solution: 6 ml × 1 bottle

2. Enzyme Standard Reagent: 6 ml × 1 bottle

8. Positive Control: 0.5 ml × 1 bottle

3. Enzyme-Labeled Coated Plate: 12 wells × 8 strips

9. Negative Control: 0.5 ml × 1 bottle

4. Sample Diluent: 6 ml × 1 bottle

10. Instructions: 1 copy

5. Reagent A: 6 ml × 1 bottle

11. Sealing Film: 2 sheets

6. Developer B: 6 ml × 1 bottle

12. Sealed Bag: 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. If not tested immediately, store at -20°C, avoiding repeated freezing and thawing.

2. Samples containing NaN3 cannot be tested, as it inhibits HRP activity.

Duck Viral Hepatitis (DVh) ELISA Kit – Procedure

1. Label the microwells according to the sample number. Each plate should include 2 negative control wells, 2 positive control wells, and 1 blank well.

2. Add 50 µL of negative and positive controls to their respective wells. Add 40 µL of sample diluent to the sample wells, followed by 10 µL of the sample. Avoid touching the walls of the wells.

3. Seal the plate and incubate at 37°C for 30 minutes.

4. Prepare the 30× washing solution by diluting it 30 times with distilled water.

5. Wash the plate 5 times with the diluted washing solution, ensuring each well is filled and drained thoroughly.

6. Add 50 µL of enzyme-labeled reagent to all wells except the blank ones.

7. Incubate again at 37°C for 30 minutes.

8. Wash the plate again following step 5.

9. Add 50 µL of developer A and 50 µL of developer B to each well. Mix gently and incubate at 37°C for 15 minutes.

10. Add 50 µL of stop solution to each well to halt the reaction. The color will change from blue to yellow.

11. Measure OD values at 450 nm using a microplate reader within 15 minutes of adding the stop solution.

Calculation and Result Interpretation

Test Validity: Positive control OD ≥ 1.00; Negative control OD ≤ 0.10

CUTOFF Value = Average of Negative Control + 0.15

Negative Result: Sample OD < CUTOFF → Negative for DVh

Positive Result: Sample OD ≥ CUTOFF → Positive for DVh

Precautions

1. Follow instructions carefully. Do not mix reagents from different batches.

2. Allow the kit to reach room temperature (15–30 minutes) before use. Store unsealed enzyme-labeled plates in a sealed bag.

3. The concentrated washing solution may crystallize. Heat it in a water bath if needed.

4. Use a new sealing film for each experiment to prevent contamination.

5. Keep the substrate away from light.

6. Use a microplate reader for results. For dual-wavelength detection, use 630 nm as the reference wavelength.

7. Treat all samples, washes, and waste as biohazardous materials. Handle the stop solution (2M sulfuric acid) with care.

Storage Conditions and Expiration

1. Store the kit at 2–8°C.

2. Shelf life: 6 months.