Pork Swine Fever Virus Antibody Detection Kit (ELISA) Instruction Manual

Product Code:
E-CSFV-05

Product Name:
Pork Swine Fever Virus Antibody Detection Kit (ELISA)

Packaging Specification:
96T/box, 480T/box

Intended Use:
This kit is designed for the detection of antibodies to swine fever virus (CSFV) in pig serum.

Experimental Principle:
The kit employs a double-antigen sandwich method to determine the presence of CSFV antibodies in samples. Recombinant CSFV antigen is used to coat the microplate wells, forming a solid phase. Samples are added to the antigen-coated wells, where they bind with HRP (Horseradish Peroxidase)-labeled CSFV antigen, forming an antigen-antibody-enzyme-labeled antigen complex. After washing, the substrate TMB (3,3',5,5'-Tetramethylbenzidine) is added and converted into blue under the catalysis of HRP, which then turns yellow under the action of acid. The intensity of the color is positively correlated with the amount of CSFV antibodies in the sample. The absorbance (OD value) at 450nm wavelength is measured using an ELISA reader, and the presence of CSFV antibodies in the sample is determined based on the OD value.

Kit Components:

Reagent Preparation:

• 1x Wash Solution: Dilute the 20x concentrated wash solution 20-fold with deionized water.
• Sample Preparation: Dilute the samples 10-fold with 1x sample diluent before use (e.g., add 20μL of serum to 180μL of sample diluent).
• Substrate Solution (Prepare Freshly): Mix equal volumes of Substrate A and Substrate B, protect from light.
• Experimental Procedure:

• Before use, equilibrate all components of the kit and the samples to be tested at room temperature for 30 minutes.

  1. 1.Prepare all reagents, negative and positive controls, and samples to be tested according to the previous reagent preparation instructions.

  2. 2.Calculate the number of enzyme-labeled strips required for the test samples. Remove the required number of strips from the aluminum foil bag, and return the remaining strips to the bag, seal it properly, and store at 2-8°C.

  3. 3.Sample Incubation: Add negative control, positive control, and samples to be tested separately, making sure to record the additions. Use 100 μL per well. Mix well. Then add enzyme-labeled antigen to the microplate wells, 50 μL per well. Mix well, cover the plate with a film, and incubate at 37°C for 30 minutes.

  4. 4.Plate Washing: Discard the liquid from the wells and wash the plate five times with 1x wash buffer (300 μL per well). Pat dry.

  5. 5.Color Development: Add the pre-prepared substrate solution to the microplate wells, 100 μL per well. Mix well and incubate at 37°C in the dark for 10 minutes.

  6. 6.Stopping the Reaction: Add 50 μL of stop solution to each well of the microplate. Gently tap the plate to ensure even color development.

  7. 7.Reading: Read the optical density (OD) value at 450 nm within 20 minutes.

  8. Result Interpretation:

     1. The OD value of the CSFV negative control is recorded as ODN; the OD value of the CSFV positive control is recorded as ODP; and the OD value of the sample to be tested is recorded as ODS.

     2. Calculation of Cut Off Value: Cut Off Value = ODN + 0.15.

     3. Validity Conditions of the Test: The test results are valid if the ODN value is ≤ 0.15 and the ODP value is ≥ 0.5. If these conditions are not met, the test should be repeated.

     4. Result Determination:

        • If the OD value of the sample to be tested (ODS) is ≤ the Cut Off Value, the result is considered negative.
        • If the OD value of the sample to be tested (ODS) is > the Cut Off Value, the result is considered positive.

        • Precautions:

          1. Please follow the instructions strictly.

          2. Do not mix reagent kits from different batches.

          3. The entire experimental process should be conducted in a clean and dust-free environment as much as possible.

          4. The kit and samples to be tested must be equilibrated to room temperature for 30 minutes before use.

          5. The concentrated wash buffer should be diluted 20 times before use. If crystals appear in the wash buffer, place it at 37°C to dissolve them.

          6. Calibrate the dispensing volume and residual volume of the plate washer daily, and ensure that the pipes are unobstructed. During washing, confirm that each well is filled with wash buffer. After each wash, pat dry the droplets in the wells on a dust-free absorbent paper. Proceed to the next step immediately after washing to prevent the microplate from drying. Avoid long interruptions in the experimental steps to ensure uniform experimental conditions in each well.

          7. If the measured values of the quality control samples are not within the expected range, the experiment is invalid, and it should be repeated.

          8. All reagents in this kit should be stored at 2-8°C and have a shelf life of 12 months. If the strips cannot be used up at once, seal the remaining strips in plastic bags and store them at 2-8°C.

          9. To avoid any potential biological hazards in the samples, test samples should be treated as infectious materials and avoided contact with skin and mucous membranes.