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

Experimental Principle

The Bovine Reovirus Type 3 (RV-3) ELISA Kit employs 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 capture reagent. When the sample is added, any RV-3 present binds to these immobilized antibodies. After washing away unbound components, a horseradish peroxidase (HRP)-labeled secondary antibody is introduced, forming a complex of antibody-antigen-enzyme-labeled antibody. Following another wash step, the substrate TMB is added, which turns blue under HRP catalysis and then changes to yellow when an acidic stop solution is applied. The absorbance at 450 nm is measured using a microplate reader, and results are interpreted by comparing the OD values against a calculated cutoff value to determine the presence or absence of RV-3 in the sample.

Bovine Reovirus Type 3 (RV-3)

ELISA

Kit

Composition

1. 130x Wash 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 6. Negative Control – 0.5ml × 1 bottle 7. Sample Diluent – 6ml × 1 bottle 8. Instructions – 1 copy 9. TMB A Solution – 6ml × 1 bottle 10. TMB B Solution – 6ml × 1 bottle 11. Sealing Film – 2 sheets 12. Aluminum Seal Bag – 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. If testing cannot be performed immediately, store at -20°C, avoiding repeated freeze-thaw cycles. 2. Samples containing sodium azide (NaN₃) should not be used, as it may inhibit the activity of horseradish peroxidase (HRP).

Bovine Reovirus Type 3 (RV-3)

ELISA

Kit

Procedure

1. Labeling: Assign unique numbers to each well for the test samples. Each plate should include 2 negative control wells, 2 positive control wells, and 1 blank control well (no sample or enzyme standard). 2. Loading: Add 50 µL of negative and positive controls to their respective wells. Then add 40 µL of sample diluent to the test wells, followed by 10 µL of the actual sample. Carefully pipette the sample into the bottom of each well without touching the walls. 3. Incubation: Cover the plate with the sealing film and incubate at 37°C for 30 minutes. 4. Washing: Dilute the 30x wash solution with distilled water (1:30), and use it for washing. 5. Washing Steps: Remove the sealing film, discard the liquid, and rinse each well with the diluted wash solution. Let sit for 30 seconds, then discard. Repeat this process 5 times, and gently pat dry. 6. Enzyme Addition: Add 50 µL of enzyme-labeled reagent to all wells except the blank control. 7. Incubation: Repeat the incubation step at 37°C for 30 minutes. 8. Washing: Follow the same washing procedure as above. 9. Color Development: Add 50 µL of TMB A and 50 µL of TMB B to each well. Mix gently and incubate at 37°C for 15 minutes. 10. Stop Reaction: Add 50 µL of stop solution to each well to terminate the reaction. The color should turn from blue to yellow. 11. Measurement: Measure the OD450 values using a microplate reader. Ensure measurements are taken within 15 minutes after adding the stop solution.

Calculation and Interpretation

Test Validity: - Positive Control Mean ≥ 1.00 - Negative Control Mean ≤ 0.10

Cutoff Value: Cutoff = Average of Negative Control + 0.15

Interpretation: - If Sample OD < Cutoff → Negative for RV-3 - If Sample OD ≥ Cutoff → Positive for RV-3

Bovine Reovirus Type 3 (RV-3)

ELISA

Kit

Precautions

1. Always follow the instructions provided. Do not mix reagents from different batches. 2. Allow the kit to reach room temperature (15–30 minutes) before use. If the enzyme-labeled plate is opened, store it in a sealed bag. 3. The concentrated wash solution may crystallize; if so, warm it in a water bath before use. This will not affect the test results. 4. Use a new sealing film for each experiment to prevent cross-contamination. 5. Keep the TMB substrate away from light. 6. Results must be read on a microplate reader. For dual-wavelength detection, use 630 nm as the reference wavelength. 7. Treat all samples, washes, and waste materials as biohazardous. The stop solution contains 2M sulfuric acid and should be handled with care.

Storage Conditions and Expiry

1. Store the kit at 2–8°C. 2. Shelf life: 6 months from the date of manufacture.