Product Name: Porcine Reproductive and Respiratory Syndrome Virus Antibody Detection Kit (ELISA Method)

Product Number: E-PRRSV-01

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

Intended Use:
This kit is designed for the detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) antibodies in pig serum.

Experimental Principle:
The kit utilizes a sandwich assay to determine the presence of PRRSV antibodies in samples. Recombinant PRRSV antigens are used to coat the microplate wells, forming a solid phase. Samples are added to the antigen-coated wells and then bind to HRP-labeled PRRSV antigens, forming an antigen-antibody-enzyme-labeled antigen complex. After washing, a substrate TMB is added, which turns blue under the catalysis of HRP enzyme and eventually yellow under acidic conditions. The intensity of the color is positively correlated with the PRRSV antibody content in the sample. The absorbance (OD value) is measured at 450 nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader, and the OD value is used to determine the presence of PRRSV antibodies in the sample.

Major Components of the Kit:

Reagent Preparation:

1. 1x Wash Solution Preparation: Dilute the 20x concentrated wash solution 20-fold with deionized water.
2. Sample Preparation: Dilute the sample 10-fold with 1x sample diluent before use (e.g., add 20 μL serum to 180 μL sample diluent).
3. Substrate Solution Preparation (prepare freshly): Mix Substrate A and Substrate B in a 1:1 ratio immediately before use, and keep it in the dark.

4. Experimental Procedure:

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

   1. 1.Prepare various reagents, negative and positive quality control materials, and samples to be tested according to the previous reagent preparation instructions.

   2. 2.Calculate the number of enzyme-labeled strips required for testing the samples. Remove the required strips from the aluminum foil pouch, and place the remaining strips back into the pouch, seal it well, and store at 2-8°C.

   3. 3..Sample Incubation: Add negative quality control material, positive quality control material (duplicate wells for quality controls), and samples to be tested into the designated wells. Record the additions. Mix thoroughly and cover the wells with a film. Incubate at 37°C for 30 minutes. Note: The negative and positive quality control materials do not require dilution.

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

   5. 5.Enzyme-Labeled Antigen Incubation: Add the enzyme-labeled antigen to the wells of the microplate, ensuring 100μL per well. Mix thoroughly, cover the wells with a film, and incubate at 37°C for 30 minutes.

   6. 6.(Repeated "0." is assumed to be a typo; assuming it should be step 6 or a continuation of the previous step.) Plate Washing (Repeated Step): Discard the liquid from the wells, and wash the plate five times with 1x wash buffer (300μL per well), patting dry after each wash.

   7. 7.Color Development: Add the pre-prepared substrate solution to the wells of the microplate, ensuring 100μL per well. Mix thoroughly and incubate at 37°C in the dark for 15 minutes.

   8. 8.Stopping Reaction: Add 50μL of stop solution to the wells of the microplate, gently shaking the plate to ensure even color development.

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

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

5. Result Interpretation:
   

6. 1.The OD value of PRRSV negative control is denoted as ODN; the OD value of PRRSV positive control is denoted as ODP; the OD value of the sample to be tested is denoted as ODS.

7. 2.Establishment Criteria of the Test: The ODN value must be ≤0.3 and the ODP value must be ≥0.5 for the test results to be considered valid; otherwise, the test must be repeated

8. 3.Calculation Method:

9.    Calculation Method: NC1 + NC2, where NC1 and NC2 represent the OD values of PRRSV negative and positive controls, respectively, and NCOD450m mean represents the average OD value of all samples.

10.    Sample Cutoff Value Calculation Formula: Cutoff Value = Average of NCOD450m + 0.3.

11. 4.Judgment Criteria: If the OD450 of the tested sample is ≥ the Cutoff Value, the result is judged as positive. If the OD450 of the tested sample is < the Cutoff Value, the result is judged as negative.

12. [Precautions]

    1. 1.Please strictly follow the instructions for operation.
    2. 2.Do not mix reagent kits of different batch numbers.
    3. 3.The entire experimental process should be conducted in a clean and dust-free environment as much as possible.
    4. 4.The reagent kit and the samples to be tested must be equilibrated at room temperature for 30 minutes before use.
    5. 5.The concentrated wash solution needs to be diluted 20 times before use. If crystals appear in the wash solution, it can be placed at 37°C to dissolve them.
    6. 6.The plate washer should be calibrated for injection volume and residual volume daily, paying attention to whether the pipes are unobstructed. During washing, confirm that the wash solution is filled in each well. After each wash, the droplets in the wells should be patted dry on a dust-free absorbent paper. Proceed to the next step immediately after washing, without allowing the enzyme-labeled plate to dry. Avoid prolonged interruptions in experimental steps to ensure uniform experimental conditions in each well.
    7. 7.If the measured value of the quality control sample is not within the expected range, the experiment is invalid, and should be repeated.
    8. 8.All reagents in this kit should be stored at 2-8°C with a validity period of 12 months. If the strips cannot be used up at once, the remaining strips should be sealed in plastic bags and stored at 2-8°C.
    9. 9.To avoid any potential biological hazards in the samples, the test samples should be treated as infectious materials and avoided from contacting skin and mucous membranes.