PRINCIPLE OF TEST
The kit uses a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To
the coated microwells pre-coated with the Inco virus antibody, the specimen, the HRP-labeled
detection antibody were sequentially added, and the wells were thoroughly washed. Using the
substrate TMB to develop color, TMB is converted to blue under the catalysis of peroxidase and
converted to the final yellow color by the action of an acid. The absorbance (OD value) was
measured at 450 nm using a microplate reader and compared with the CUT OFF value to determine
the presence or absence of human Inco virus antigen in the specimen.
SAMPLE COLLECTION AND STORAGES
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
Note: The samples shoule be centrifugated dequately and no hemolysis or granule was allowed.
MATERIALS REQUIRED BUT NOT SUPPLIED
- Standard microplate reader(450nm)
- Precision pipettes and Disposable pipette tips.
- 37 ℃ incubator
- 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.
- 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.
- Mix all reagents before using.Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C)
20X Wash solution
Chromogen Solution A
Chromogen Solution B
Closure plate membrane
20×wash solution:Dilute with Distilled or deionized water 1:20.
- Prepare all reagents before starting assay procedure. It is recommended that all Standards and
Samples be added in duplicate to the Microelisa Stripplate.
- 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.
- Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for
60 minutes at 37°C.
- Aspirate each well and wash, repeating the process four times for a total of five washes. Wash
by filling each well with Wash Solution (400 μ l) 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.
- 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.
- 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.
- Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
CALCULATION OF RESULTS
- Test validity: the average of Positive control well≥1.00; the average of Negative control well ≤
- Calculate Critical (CUT OFF): Critical= the average of Negative control well + 0.15.
Negative Result: sample OD< Calculate Critical (CUT OFF) is Negative.
Positive Result: sample OD≥ Calculate Critical (CUT OFF) is Positive.
Twelve months [see label on the outer box for the specific date].
- The kit takes out from the refrigeration should be balanced 15-30 minutes in the room
temperature, if the coated ELISA plates have not been used up after opening, the plate should
be stored in sealed bag.
- Washing buffer will Crystallization separation, it can be heated the water helps dissolve when
dilution. Washing does not affect the result.
- Pipette sample with pipettors each step, and proofread its accuracy frequently, avoids the
experimental error. Pipette sample within 5 min, if the number of sample is much, recommend
using multichannel pipettor.
- If the testing material content is excessively high (The sample OD is higher than the first
standard well)，please dilute sample (n-fold).
- Adhesive Strip only limits the disposable use to avoid cross-contamination.
- The substrate should be preserved evade the light.
- Please refer to use instruction strictly. The test result determination must take the microtiter
plate reader as a standard.
- All samples, washing buffer and each kind of reject should refer to infective material process.
- Do not mix reagents with those from other lots.