Table 1.

Comparison of the two different assay procedures.

	R5 Method	G12 Method
Addition of standard/samples to wells	100 µL of each standard solution or sample	Add 100 µL of each standard solution or sample
1st incubation	30 min at room temperature (RT)	20 min at room temperature (RT)
Washing	Empty the contents of the microwell strips	Empty the contents of the microwell strips
	Wash by filling each microwell with 250 μL diluted wash buffer, and then emptying the buffer from the microwell strips.	Wash by filling each microwell with 300 µL diluted wash buffer, and then emptying the buffer from the microwell strips.
	Repeat this step a total of 3 times.	Repeat this step a total of 5 times.
Addition of conjugate and 2nd incubation	Add 100 μL of the diluted enzyme conjugate to each well and incubate for 30 min at room temperature (20–25 °C/68–77 °F).	Add 100 μL of the diluted enzyme conjugate to each well and incubate for 20 min at room temperature.
Washing	Repeat washing step as described above	Repeat washing step as described above
Addition of substrate and 3rd incubation	Add 50 μL of substrate and 50 μL of chromogen to each well. Mix gently by shaking the plate manually and incubate for 30 min at room temperature (20–25 °C/68–77 °F) in the dark.	Pipette 100 µL of the Substrate into each microwell and incubate at room temperature for 20 min in the dark.
Additions of stop solution and measurement	Add 100 μL of the stop reagent to each well. Mix gently by shaking the plate manually and measure the absorbance at 450 nm. Read within 30 min after addition of stop solution.	Pipette 100 µL of Stop Solution into each microwell. The color should change from blue to yellow. Read the strips with a microwell reader using a 450 nm filter. Record OD readings for each microwell.