GAG Assay Kits
Heparan Sulfate ELISA Kit
- Assay Procedure
Verify all reagents are at room temperature before commencing assay. It is recommended that all standards and samples be assayed in duplicate.
  1. According to the [Reagent Preparations], prepare diluted Reaction Buffer, Washing Solution, samples *1 and Biotinylated-Antibody (reconstituted).
  2. Set Antibody-coated Microstrips (A) as required in the Frame (J).
  3. Wash wells of microstrips 5 times with 300µL of the Washing Solution.*2
  4. Add 100µL of Reaction Buffer in each well. And then 20µL of HS Standard Solutions (8, 4, 2, 1, 0.5, 0.25µg/mL), blanks (0µg/mL), or samples and then mix gently.*3 Incubate them for 18-24hrs. at 2-8 °C .*4 (Primary Reaction)
  5. Discard the well contents*2 and wash the wells in the same manner as in the step 3 above.
  6. Add 100µL of HRP-Conjugated Streptavidin (C) and 100µL of the Biotinylated-Antibody (reconstituted) into each well and then mix gently.*3 Incubate them for 60 min. at RT*4. (Secondary Reaction)
  7. Repeat the step 5.
  8. Add 100µL of Substrate (F) into each well and then mix gently.*3 Incubate them for 30min. at RT in dark.*4 (Color Development)
  9. Add 100µL of Stopping Solution (G) into each well and then mix gently.*3
  10. Measure the absorbance at 450nm in microplate reader (reference wavelength, 630nm) within 30 minutes of adding stopping solution.
*1 Samples with suspensions should be centrifuged to remove particles and then use the supernatant. Use the Reaction Buffer for sample dilution.
< Examples of sample dilution >
Sample Urine Culture
medium
Rat Human
Dilution x1 / 2 x 1 x 1
*2 For wash step, discard the well contents to an appropriate receptacle and rap the inverted microstrip in the Frame (J) on absorbent paper towels to remove the remainder. Multi-channel manifold dispenser or equivalent is recommended to add Washing Solution into wells.
*3 Gently shake the microstrips in the Frame (J) on a plate shaker or equivalent for a several seconds to assure proper mixing (DO NOT splash).
*4 Seal the microstrips with Plastic Film (K) during incubation.

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