Rotavirus Antigen (RV Ag) BioAssay™ Qualitative ELISA Kit (Bovine)

Intended Use:
This Rotavirus Antigen (RV Ag) ELISA Kit is for the qualitative determination of RV Ag in bovine feces. This kit is intended for research use only and may not be used for therapeutic or diagnostic applications

Principle of the Test:
This assay employs the qualitative enzyme immunoassay technique.
The microtiter plate provided in this kit has been pre-coated with an antibody specific to RV Ag. Samples are pipetted into the wells with Horseradish Peroxidase (HRP)-conjugated antibody. Any virus antigen present in the samples will bind to the pre-coated specific antibody. Following a wash to remove any unbound reagent, a substrate solution is added to the wells and color develops in proportion to the amount of RV Ag bound in the initial step. The color development is stopped and the intensity of the color is measured.

Kit Components:
351689A: Microtiter Strips, 1x96 wells. Pre-coated.
351689B: Negative Control, 1x800ul
351689C: Positive Control, 1x00ul
351689D: HRP Conjugate, 1x126ml
351689E: Wash Buffer, 30X, 1x6mll
351689F: Substrate A, 1x5ml
351689G: Substrate B, 1x5ml
351689H: Stop Solution, 1x5ml

Storage and Stability:
Store kit at 4°C. The unopened kit is stable for 6 months after receipt. Once opened, the kit is stable for up to 2 weeks at 4°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

Assay Procedure:
Bring all reagents and samples to room temperature before use. Centrifuge the samples again after thawing before the assay. It is recommended that all samples be assayed in duplicate.

1. Prepare all reagents, and *samples as directed in the previous sections.

2. Determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc; store unused wells at 4°C.

3. Set a Blank well (without any solution), three Negative Control wells and two Positive Control wells.

4. Add 50ul of Negative Control, Positive Control or Sample per well, then add 50ul of HRP Conjugate to each well except the Blank well. Cover with the provide adhesive strip. Incubate for 15 minutes at room temperature.

5. Aspirate each well. Add 50ul Wash Buffer to each well.

6. Wash by filling each well with 250ul ddH2O using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it stand for 30 seconds; complete removal of liquid at each step is essential to good performance. Repeat the process ten times for a toal of ten washes. After the last wash, remove any remaining liquid by aspirating or decanting. Invert the plate and blot it against clean paper towels.

7. Add 50ul of Substrate A and 50ul Substrate B to each well. Incubate for 10 minutes at room temperature. Protect from light.

8. Add 50ul of Stop Solution to each well; gently tap the plate to ensure thorough mixing.

9. Determine the optical density of each well within 10 minutes using a microplate reader set to 450nm. Take blank well as zero.

Interpretation of Results:
The presence or absence of Rotavirus Antigen is determined by comparing the absorbance of the samples with those of the controls.

A Cut-off value is defined as the average Negative Control OD value plus 0.1. If the average Negative Control OD valuel is <0.05, calculate it as 0.05.

1. The Negative Control OD value should be less than 0.1 and the Positive Control OD value should be greater than 0.8. Otherwise, the test must be repeated.

2. Determination of results:

If ODsample is ≥ Cut-off Value: Test is Positive
If ODsample is < Cut-off Value: Test is Negative
If OD sample is close to the Cut-off Value, we recommend repeating the experiment.