CAT. NO: EKHU-0574

Please read this insert completely prior to using the product. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Intended Use

This assay employs the qualitative enzyme immunoassay technique (double-antibody sandwich) to analyze the existence or not of Rotavirus Antigen (RV-Ag) in human serum, blood plasma, stool, urine, and other biological fluids.

Test principle

The microtiter plate provided in this kit has been pre-coated with antibody. Add samples, positive control, negative control and HRP conjugated antibody to wells. After incubation and washing to remove the uncombined enzyme, add Chromogen Solution A and B. The color of the liquid will change into blue. At the effect of acid, the color finally becomes yellow. The color change is measured spectrophotometrically at a wavelength of 450 nm. The existence or not of Rotavirus Antigen (RV-Ag) in the samples is then determined by comparing the O.D. of the samples to the CUT OFF.

Sample collection and storage

Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 10 minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Cell culture supernates and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Note: The samples shoule be centrifugated dequately and no hemolysis or granule was allowed.

Materials required but not supplied

1.  Standard microplate reader(450nm)

2.  Precision pipettes and Disposable pipette tips.

3.  37 ℃ incubator

Precautions

1.  Do not substitute reagents from one kit to another. Standard, conjugate and microplates are matched for optimal performance. Use only the reagents supplied by manufacturer.

2.  Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.

3.  Mix all reagents before using.

Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C)

Materials supplied

Reagent preparation

20×wash solution:Dilute with Distilled or deionized water 1:20.

Assay procedure

1.  Prepare all reagents before starting assay procedure. It is recommended that all reagent should be added in duplicate to the Microelisa Stripplate.

2.  Separately add Positive control and Negative control 50μl to the Positive and Negative well. Add testing sample 10μl. Then add sample diluent 40μl to testing sample well; Blank well doesn’t add anything.

3.  Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C.

4.  Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.

5.  Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.

6.  Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not

appear uniform, gently tap the plate to ensure thorough mixing.

7.  Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.

CALCULATION RESULTS

Test validity: the average of Positive control well≥1.00;the average of Negative control well≤0.15

Calculation of the Cut-off value (C.O.)= the absorbance value for of Negative control wells + 0.15.

Negative control: sample (Rotavirus Antigen (RV-Ag) )OD< Calculate Critical(CUT OFF), the result is Negative.

Positive control: sample (Rotavirus Antigen (RV-Ag) )OD≥ Calculate Critical(CUT OFF), the result is Positive.

Expiration Date

6 months from the date of manufacture

Storage

2-8℃