CASE#1 A technologist is performing a colorimetric assay using 96-well ELISA microtiter plates The ELISA plate reader measures the absorbance of the solution in each well ( recording the light passing through the solution from the top to the b well. The assay\'s color, when tested in a standard spectrophotometer, is linear to an absorbance of 4.0 however the ELISA reader has a range of linearity which only extends to an absorbance of 1.2. When a microtiter plate is placed in the ELISA reader, the technologist notes that the solution in several wells has an approximate absorbance of 1.5. The assay material is very expensive and each assay takes one day to perform. Considerable effort is made to get as much data as possible from each run. Therefore, in order to measure the absorbance of the solution in these wells, the technologist dilutes the samples by adding a volume of buffer equal to the volume in the well. To her surprise, the absorbance values remain the same. cuvet) by ottom of each 1. Considering Beer\'s law, why didn\'t the absorbance value change? (2 pts) 2. Are there any steps that can be taken to bring the absorbance within the linear range? (1 pt) Solution 1. According to Beer\'s law if there is dilution i.e) concentration decreases then the absorbance value also tends to decrease. However to keep the absorbance value constant the optical density of the spectrophotometer should be kept smaller if the dilution factor is great and greater if the dilution factor is small. 2. To bring the absorbance within the linear range linear regression methods like least squares fitting can be adopted. .

1. CASE#1 A technologist is performing a colorimetric assay using 96-well ELISA microtiter
plates The ELISA plate reader measures the absorbance of the solution in each well ( recording
the light passing through the solution from the top to the b well. The assay's color, when tested
in a standard spectrophotometer, is linear to an absorbance of 4.0 however the ELISA reader has
a range of linearity which only extends to an absorbance of 1.2. When a microtiter plate is placed
in the ELISA reader, the technologist notes that the solution in several wells has an approximate
absorbance of 1.5. The assay material is very expensive and each assay takes one day to perform.
Considerable effort is made to get as much data as possible from each run. Therefore, in order to
measure the absorbance of the solution in these wells, the technologist dilutes the samples by
adding a volume of buffer equal to the volume in the well. To her surprise, the absorbance values
remain the same. cuvet) by ottom of each 1. Considering Beer's law, why didn't the absorbance
value change? (2 pts) 2. Are there any steps that can be taken to bring the absorbance within the
linear range? (1 pt)
Solution
1. According to Beer's law if there is dilution i.e) concentration decreases then the absorbance
value also
tends to decrease. However to keep the absorbance value constant the optical density of the
spectrophotometer should be kept smaller if the dilution factor is great and greater if the dilution
factor is small.
2. To bring the absorbance within the linear range linear regression methods like least squares
fitting can be adopted.