elisa For Nitric Oxide Synthase 3, Endothelial （NOS3）-shuo ming shu

 [ MATERIALS REQUIRED BUT NOT SUPPLIED ]

1. Microplate reader with 450 ± 10nm filter.

2. Precision single or multi-channel pipettes and disposable tips.

3. Eppendorf Tubes for diluting samples.

4. Deionized or distilled water.

5. Absorbent paper for blotting the microtiter plate.

6. Container for Wash Solution

[ STORAGE OF THE KITS ]

1. For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection

Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20oC upon receipt while the

others should be at 4 oC.

2. For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above

storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and

reseal along entire edge of zip-seal.

Note:

It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date

of the kit.

[ SAMPLE COLLECTION AND STORAGE ]

Serum - Allow samples to clot for two hours at room temperature or overnight at 4oC before centrifugation for 20

minutes at approximay 1000×g. Assay immediay or store samples in aliquot at -20oC or -80oC. Avoid

repeated freeze/thaw cycles.

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at

1000×g within 30 minutes of collection. Remove plasma and assay immediay or store samples in aliquot at

-20oC or -80oC. Avoid repeated freeze/thaw cycles.

Other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay

immediay or store samples in aliquot at -20oC or -80oC. Avoid repeated freeze/thaw cycles.

Note:

1. Samples to be used within 5 days may be stored at 4oC, otherwise samples must be stored at -20oC （􀀀1

month） or -80oC （􀀀2 months） to avoid loss of bioactivity and contamination.

2. Sample hemolysis will influence the result, so hemolytic specimen can not be detected.

3. When performing the assay, bring samples to room temperature.

[ REAGENT PREPARATION ]

1. Bring all kit components and samples to room temperature （18-25oC） before use.

2. Standard - Reconstitute the Standard with 1.0mL of Standard Diluent, kept for 10 minutes at room

temperature, shake gently（not to foam）. The concentration of the standard in the stock solution is

1,000pg/mL. Please prepare 7 tubes containing 0.5mL Standard Diluent and produce a double dilution series

according to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 7 points of

diluted standard such as 1,000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.2pg/mL, 15.6pg/mL,

and the last EP tubes with Standard Diluent is the blank as 0pg/mL.