l       Before use, mix all reagents thoroughly without making foam.

2    Determine the number of microwell strips required to test the desired number of samples,plus appropriate number of wells needed for running blanks standards. Each sample, standard ａnd blank should be assayed in duplicate. Remove sufficient microwell strips from the pouch.

3    Add 50ul of standard diluent to standard wells B1,B2, C1,C2, D1,D2, E1, E2, F1,F2. Reconstitute standard vial with the appropriate volume as described in the chapter reagents preparation. Preparation. Pipet 100ul of standard into wells A1 ａnd A2 （see plate scheme below）. Transfer 50ul from A1 ａnd A2 to B1 ａnd B2 wells. Mix the contents by repeated aspirations ａnd ejections. Take care not to scratch the inner surface of microwells. Repat this procedure from the wells B1,B2 to wells C1,C2 ａnd from wells C1,C2 to D1,D2 ａnd so on creating two parallel rows of Dopamine standard dilutions ranging，Add 50ul of standard diluent to the bland wells.

4      Dilute samples 1:1 distribing 50ul of sample into 50ul of dilluent ,Add 50ul of diluted sample to wells..

5     Add 50ul of diluted biotinylated anti-Dopamine to all wells.

6      Cover with a plate vover ａnd incubate for 1 hour at 37℃.

7    Remove the cover ａnd wash the plate as follows: ⑴　aspirate the liquid from each well, ⑵　dispensse 0.3ml of washing solution into each well. ⑶　Aspirate again the contet of each well after 0.5 minute. ⑷　Repeat steps ⑵ ａnd ⑶ three times.

8   Distribute 80ul of streptavidin-HRP solution to all wells, including blank wells.

9     Cover ａnd incubate 30 min at 37℃.

10      Remove the cover ａnd empty wells, Wash microwell strips according to step, Proceed immediately to the next step.

11     Add 50ul Substrate A ａnd Substrate B to each well。Incubate for 10 min at 37℃。

12    The enzyme-substrate reaction is stopped by quickly pipetting 50ul of H2SO4. stop reagent into each well, including the blank wells, to completely ａnd uniformly inactivate the enzyme. Results must be red immediately after the addition of H2SO4.

13   Read absorbance of each well on a spectrophotometer using 450nm as the primary wavelength ａnd optionally 620nm （610nm to 650nm is acceptable ） as the reference wavelength.