25-OH Vitamin D (25OHVD) ELISA Kit

ELISA Microplate (Dismountable)

8×12 strips

-20°C

Lyophilized Standard

2

-20°C

Sample Diluent

20mL

-20°C

Assay Diluent A

10mL

-20°C

Assay Diluent B

10mL

-20°C

Detection Reagent A

60µL

-20°C

Detection Reagent B

120µL

-20°C

Wash Buffer

30mL

4°C

Substrate

10mL

4°C

Stop Solution

10mL

4°C

Plate Sealer

5

-

1.

Add 50µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible.

2.

Immediately add 50µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.

3.

Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette, manifold dispenser or automated washer are needed) and let it sit in the well for 1-2 minutes. Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.

4.

Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 45 minutes at 37°C.

5.

Repeat the wash process for five times as conducted in step 3.

6.

Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminate the reaction.

7.

Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.

8.

Determine the optical density (OD value) of each well at once, using amicro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.

9.

After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

Serum

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid