Verify all reagents and samples are at room temperature (15-25°C) before commencing the assay. It is recommended that all assays be performed in duplicate.

1. According to the [Reagent Preparations], prepare every required reagents, STD solutions and samples^{*1, *2}
2. Set Antibody-coated Microplate (A) as required in the Frame (I).
3. Add 100μL of Reaction Buffer to each well.
4. And then add 20μL of KS Standard Solutions (4, 2, 1, 0.5, 0.25ng/mL), blanks (0ng/mL), or samples to each well. Then Gently shake the microplate on a plate shaker or equivalent to assure proper mixing for approximately 30sec (DO NOT splash). Incubate them for 60min at room temperature. (Primary Reaction)^*3
5. Discard the well contents and wash the wells of microplate 5 times with 300μL of the Washing Solution^*4
6. Add 100μL of HRP-Conjugated Antibody Solution to each well. Incubate them for 60 min at room temperature. (Secondary Reaction)^*3
7. Discard the well contents and wash the wells of microplate 5 times with 300μL of the Washing Solution^*4
8. Add 100μL of Substrate Solution (D) to each well. Incubate them for 30min at room temperature (protect from light). (Color Development)^*3
9. Add 100μL of Stop Solution (E) to each well and then mix gently for a several seconds.
10. Gently shake the microplate on a plate shaker or equivalent to assure proper mixing for approximately 30sec (DO NOT splash). Then measure the absorbance at 450nm in microplate reader (reference wavelength, 630nm) within 30 min of adding stopping solution.
    
    *1 Remove particulates by centrifugation, and dilute the obtained supernatant with Reaction Buffer as needed.
    *2 Examples of sample dilution
    

    Sample	Serum
    	Mouse	Rat	Guinea pig	Rabbit	Dog	Human
    Dilution	x5	x50	x200	x50	x500	x1000

    *3 Seal the microplate with Plastic Film (J) during the incubation to avoid drying out.
    *4 For wash step, discard the well contents to an appropriate receptacle and rap the inverted microplate on absorbent paper towels to remove the remainder. Multi-channel manifold dispenser or equivalent is recommended to add Washing Solution into wells.

[ Calculation of Keratan Sulfate concentrations ]

1. Average duplicate values for each KS standard and sample.
2. To make the KS standard curve (quadratic curve), plot the KS concentration on the x-axis (log) and absorbance (blank subtracted) on the y-axis (log) using graph paper or appropriate software.
3. Determine the KS concentration for each sample using the generated KS standard curve.^*
4. To obtain the KS concentration in each sample, multiply the above value by the appropriate dilution factor.

* As to (diluted) samples that exceeded 4ng/mL, repeat the assay with proper dilution in Reaction Buffer.

[ Typical Standard Curve ]