AffiVET® ASFV Indirect Antibody ELISA Kit

AffiVET® ASFV Indirect Antibody ELISA Kit

Overview: 

African Swine Fever (ASF) is a highly contagious and lethal viral illness that impacts both domestic and wild pigs across all age groups. Crucially, ASF poses no risk to human health and cannot be transmitted from pigs to humans, making it a non-food safety concern.

In the effort to eradicate ASF, the early and ongoing use of antibody or antigen testing is paramount. These tests must possess exceptional sensitivity, reproducibility, and stability to accurately detect antibodies or antigens in swine serum and plasma samples throughout the eradication process.

Test Principal:

The AffiVET® ASFV Indirect Antibody ELISA Kit is used to detect ASFV specific antibody level in serum.

The 96-well microtiter plate is pre-coated with ASFV antigen. When ASFV antibodies are present in the serum sample, they bind to the ASFV antigen in the wells, forming an antigen-antibody complex. After washing away any unbound components, a specific enzyme-labeled antibody is added, which attaches to the antigen-antibody complex. Following another wash to remove any excess enzyme-labeled antibody, a substrate is added to induce color development. The intensity of the color produced is directly proportional to the amount of antibody present in the sample.

Material Required:

The AffiVET® ASFV Indirect Antibody ELISA Kit contains the following items:

Materials Required But Not Provided : 

1. ELISA reader

2. Micropipette 20-200ul

3. Micropipette Multi-Channel 50-300ul

Storage and Shelf Life : 

• Storage: Storing at 2-8℃, in the dark.

• Expiry date: 12 months.

Reagent and Sample Preparation: 

Before proceeding with the test, ensure that both the kit and all test samples have reached room temperature (18-26°C). Prior to adding them to the well, gently shake each reagent.

1.Preparation of Wash Buffer: Dilute the 25X wash buffer with either sterile or ionized water at a ratio of 1:24. For example, mix 10ml of the 25X wash buffer with 240ml of water.

2. Sample Treatment: Start with whole blood and centrifuge it for 10 minutes at 4000 revolutions per minute (r/min). Then, collect the supernatant and dilute it with sample buffer in a ratio of 1:49 (e.g., 5 μl of serum + 245 μl of sample buffer). Ensure thorough mixing before use.

Test procedure : 

1. Prepare Microplate:
- Add 100 μl of positive control to two wells (100 μl/well).
- Add 100 μl of negative control to another two wells (100 μl/well).
2. Add Serum Samples:
- Add 100 μl of prepared serum samples to the remaining wells (100 μl/well).
- Incubate the plate at room temperature (20-25°C) for 30 minutes.
3. Wash the Plate:
- Discard the liquid from the wells.
- Wash each well with wash buffer (300 μl per well). Ensure thorough washing by repeating this step five times.
- Tap the plate against absorbent paper to remove any excess liquid and ensure the plate is dry.
4. Add Enzyme Conjugate:
- Add 100 μl of HRP enzyme conjugate to each well.
- Cover the plate and incubate at room temperature (20-25°C) for 30 minutes.
5. Repeat Washing:
- Repeat the washing step as described in step 3.
6. Add Substrate Solution:
- Add 100 μl of substrate solution to each well.
7. Incubate:
- Cover the plate and incubate at room temperature for 15 minutes.
8. Stop the Reaction:
- Add 50 μl of stop solution to each well to halt the reaction.
9. Read the Plate:
- Use an ELISA reader to measure the absorbance of the plate at 450 nm.

Results : 

Results Evaluation:

The test results are valid only if the Average OD value of negative control is below 0.30, and mean OD of positive control must be no less than 0.80. 

Calculation Method:

Compute the S/P value using the formula :

S/P = (OD of sample – average OD of negative control) / (average OD of positive control –average OD of negative control)

Interpretation of Results :

S/P ≥ 0.4 indicates a Positive result.

0.3 ≤ S/P < 0.4 indicates a Suspected result.

S/P < 0.3 indicates a Negative result.

NOTE : Based on field tests involving over 500 samples, the kit demonstrates a sensitivity of 96.8% and a specificity of 99.7%.

Precautions : 

1. Store the kit at 2-8°C and ensure to check the lot number and expiration date before use. Before utilizing the test kit, acclimate it to room temperature by leaving it at room temperature for a minimum of 30 minutes after removing it from cold storage.
2. Exercise caution to avoid contact with the acidic stop solution in the kit. Do not touch it with your hands or skin.
   
3. While the components of the kit are noninfectious, treat the field sample as potentially infectious. Adhere to your lab regulations to handle all materials properly.
   
4. Following the experiment, dispose of all lab materials in accordance with local regulations.

Limitations : 

This ELISA kit is primarily intended for veterinary diagnostic purposes. We advise validating its performance in your own laboratory using various methodologies to ensure accuracy. If you intend to use it for a different purpose, please reach out to us for assistance.