Bovine reovirus type 3 (RV-3) ELISA kit operating instructions - Database & Sql Blog Articles

Bovine reovirus type 3 (RV-3)

ELISA

Kit

Instructions

This kit is intended for research use only and not for diagnostic or therapeutic purposes.

Experimental Principle

The Bovine Reovirus Type 3 (RV-3) ELISA Kit utilizes a double-antibody sandwich immunoassay to detect the presence of RV-3 in biological samples. The microtiter plate is pre-coated with specific antibodies against RV-3, forming a solid-phase antibody. When the sample is added, any RV-3 present will bind to these immobilized antibodies. After washing away unbound substances, HRP-conjugated secondary antibodies are added, forming a complex of antibody-antigen-enzyme-labeled antibody. Following another wash, TMB substrate is introduced, which changes color under the action of HRP. The reaction is stopped by adding an acidic solution, causing the color to shift from blue to yellow. The absorbance at 450 nm is measured using a microplate reader, and results are interpreted based on a calculated CUTOFF value to determine whether the sample is positive or negative for RV-3.

Bovine reovirus type 3 (RV-3)

ELISA

Kit

Composition

1. 130× 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 Coating Plate – 12 wells × 8 strips

6. Negative Control – 0.5ml × 1 bottle

7. Sample Diluent – 6ml × 1 bottle

8. TMB Substrate A – 6ml × 1 bottle

9. TMB Substrate B – 6ml × 1 bottle

10. Instructions – 1 copy

11. Sealing Film – 2 sheets

12. Sealed Bag – 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. Follow standard protocols for extraction and testing. If not tested immediately, store at -20°C. Avoid repeated freeze-thaw cycles.

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

Kit Steps

1. Label: Assign unique 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 to their respective wells. Add 40 µL of sample diluent to the test wells, followed by 10 µL of the sample. Carefully pipette the sample to the bottom of the well without touching the sides.

3. Incubation: Seal the plate with the provided film and incubate at 37°C for 30 minutes.

4. Washing: Remove the seal, discard the liquid, and add washing solution. Let stand for 30 seconds, then discard. Repeat this process 5 times and gently pat dry.

5. Enzyme Addition: Add 50 µL of enzyme-labeled reagent to each well except the blank one.

6. Incubation: Repeat the 37°C incubation for 30 minutes.

7. Washing: Repeat the washing steps as above.

8. Color Development: Add 50 µL of TMB Substrate A and B to each well. Mix gently and incubate at 37°C for 15 minutes.

9. Stop: Add 50 µL of stop solution to each well to terminate the reaction. The color should turn yellow.

10. Measurement: Measure the OD450 values using a microplate reader within 15 minutes of adding the stop solution.

Calculation and Result Interpretation

Test Validity: The average of the positive control must be ≥ 1.00, and the average of the negative control must be ≤ 0.10.

CUTOFF Value: CUTOFF = Average of Negative Control + 0.15

Interpretation:

- Negative: Sample OD < CUTOFF

- Positive: Sample OD ≥ CUTOFF

Precautions

1. Always follow the 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 reagents in a sealed bag.

3. The concentrated washing solution may crystallize; if so, warm it in a water bath before dilution. This will not affect the results.

4. Use the sealing film only once to prevent cross-contamination.

5. Keep the substrate away from light to maintain stability.

6. Results must be read using a microplate reader. For dual-wavelength detection, use 630 nm as the reference wavelength.

7. All samples, waste, and wash solutions should be treated as biohazardous materials. The stop solution contains 2M sulfuric acid, which requires careful handling.

Storage Conditions and Expiration

1. Store the kit at 2–8°C.

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