Description

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 Sindbis 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 the human Sindbis virus antigen(SINV-Ag) 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

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.

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

MATERIALS SUPPLIED

Name

96T

Microelisa stripplate

12*8strips

Negative control

1x0.5ml

Positive control

1x0.5ml

HRP-Conjugate reagent

1x10.0ml

20X Wash solution

1x25ml

Sample Diluent

1x6.0ml

Chromogen Solution A

1x6.0ml

Chromogen Solution B

1x6.0ml

Stop Solution

1x6.0ml

Closure plate membrane

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User manual

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Sealed bags

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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 Standards and

Samples 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.

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 (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.

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 OF RESULTS

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

0.15.

2. 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.

EXPIRATION

Twelve months [see label on the outer box for the specific date].

ATTENTION

1. 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.

2. Washing buffer will Crystallization separation, it can be heated the water helps dissolve when

dilution. Washing does not affect the result.

3. 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.

4. If the testing material content is excessively high (The sample OD is higher than the first

standard well)，please dilute sample (n-fold).

5. Adhesive Strip only limits the disposable use to avoid cross-contamination.
6. The substrate should be preserved evade the light.
7. Please refer to use instruction strictly. The test result determination must take the microtiter

plate reader as a standard.

8. All samples, washing buffer and each kind of reject should refer to infective material process.
9. Do not mix reagents with those from other lots.

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