- Prepare all reagents, Keratan Sulfate Standard Solutions and samples. * **
- Set the necessary number of strips of Antibody-coated
Microplate (A) in the Frame (I).
- Wash each well 4 times with 200 µL of Wash
Solution.
- Add 50 µL of Keratan Sulfate Standard Solutions (40, 20, 10, 5, 2.5ng/mL),
blanks and/or samples. Incubate for 60 min. at 37°C. (Primary
Reaction) ***
- After removing the reaction solution, wash each well 4 times with
200 µL of Wash Solution.
- Add 25 µL of HRP-Conjugated Streptavidin
Solution (C) and 25 µL of Biotinylated
Antibody Solution (D) into each well and then mix gently. Incubate for 60 min. at 37°C. (Secondary Reaction) ***
- After removing the reaction solution, wash each well 4 times with 200 µL of Wash
Solution.
- Add 50 µL of Substrate Solution
(E) into each well. Incubate for 10 min. at room temperature
in dark (15 - 25°C). (Development Reaction)***
- Add 50 µL of Stop Solution (F) into each well and then mix gently.
- Measure absorbance at 450 nm using a microplate reader (reference wavelength,
630-650 nm).
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