Clinical Biochemicals> HCY
Total Homocysteine Enzymatic Microtiter-Plate Assay

For 100 tests:

Regents (refrigerate all regents in 40 C)
Reaction buffer 100 ml
Reagent I (reducing agent) 800 ul
Reagent II (oxidant agent) 8 ml
Reagent III (chromophore agent) 450 ul
Enzyme (freeze dry) 1 bottle
Calibrator 1,2,3,4,5,6 (0, 6.1, 10.5, 13.5, 22.1, 39.4μM) 250 ul each

Before assay, adding 7 ml Reaction buffer to freeze dry enzyme to become the enzyme solution. (for 100 tests).

Before assay, adding 360μl reagent I to 50 ml buffer to become the assay buffer. (for 100 tests).

Before assay, adding 200μl reagent III to 5 ml reagent II and mixing well to become the fluorophore agent (for 100 tests).


Assay Procedure: (in a 96-well plate)

1. For each sample, add 20μl sample and 170μl of assay buffer to 1 well as the sample well, and add the same solution to the next well for the blank well.

2. Incubate at 370 C for 20 minutes.

3. Add 30μl enzyme solution to the sample well and 30μl of assay buffer to the
blank well.

4.Incubate at room temperature for 5 minutes.

5.Add 30μl fluorophore agent to each well, and incubate at room temperature for 10 minutes.

6. Read fluorescence at EX 660/EM 710.

Subtract the reading of blank well from reading of sample well for all samples and calibrators.

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