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

EL-C1600N100013-B

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Duck Viral Hepatitis (DVH) ELISA Kit Instruction Manual

This kit is for research use only. It should not be used for diagnostic purposes or in clinical settings.

Experimental Principle

The Duck Viral Hepatitis (DVH) ELISA Kit uses a double-antibody sandwich assay to detect the presence of DVH antigens in biological samples. A solid-phase antibody specific to DVH is coated on a microplate, and when the sample is added, any DVH present will bind to this antibody. After washing away unbound substances, an HRP-labeled anti-DVH antibody is added, forming a complex of antibody-antigen-enzyme-labeled antibody. TMB substrate is then added, and under the catalysis of HRP, it turns blue and eventually yellow upon acid addition. The absorbance at 450 nm is measured using a microplate reader, and compared with a cutoff value to determine whether DVH is present in the sample.

Kit Composition

1. 30× Washing Solution – 20ml × 1 bottle
2. Stop Solution – 6ml × 1 bottle
3. Enzyme Standard Reagent – 6ml × 1 bottle
4. Positive Control – 0.5ml × 1 bottle
5. Enzyme-Labeled Coated Plate – 12 wells × 8 strips
6. Negative Control – 0.5ml × 1 bottle
7. Sample Diluent – 6ml × 1 bottle
8. Instructions – 1 copy
9. Reagent A – 6ml × 1 bottle
10. Color Developer B – 6ml × 1 bottle
11. Sealing Film – 2 sheets
12. Sealed Bag – 1

Sample Requirements

1. Samples should be processed as soon as possible after collection, following relevant protocols. If testing cannot be performed immediately, store at -20°C and avoid repeated freeze-thaw cycles.

2. Samples containing NaN3 should not be used, as they may inhibit horseradish peroxidase (HRP) activity.

Testing Procedure

1. Plate Setup: Label each well accordingly. Include 2 negative control wells, 2 positive control wells, and 1 blank control well.
2. Loading: Add 50 µl of negative and positive controls to their respective wells. Add 40 µl of sample diluent to the test wells, followed by 10 µl of the sample. Avoid touching the walls of the wells.
3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.
4. Washing: Discard liquid, add washing solution, let stand for 30 seconds, discard, repeat 5 times, and pat dry.
5. Add Enzyme: Add 50 µl of enzyme-labeled reagent to all wells except the blank.
6. Incubation: Repeat the incubation step as above.
7. Color Development: Add 50 µl of developer to each well and incubate at 37°C for 15 minutes.
8. Stop Reaction: Add 50 µl of stop solution to each well to terminate the reaction.
9. Measurement: Read the absorbance at 450 nm 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: Cutoff = Average of negative control + 0.15

Interpretation: OD < Cutoff = Negative for DVH; OD ≥ Cutoff = Positive for DVH

Precautions

1. Follow instructions strictly. Do not mix components from different batches.
2. Allow the kit to reach room temperature before use. Store unsealed enzyme plates in a sealed bag.
3. Concentrated washing solution may crystallize. Heat 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. Results must be read using a microplate reader. Use 630nm as reference wavelength for dual-wavelength detection.
7. Treat all samples, washes, and waste as infectious materials. Handle stop solution (2M sulfuric acid) with care.

Storage and Shelf Life

1. Store the kit at 2–8°C.

2. Shelf life: 6 months from the date of manufacture.