4.32: Spectrophotometry- ELISA Reader

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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 (cuvette) by recording the light passing through the solution from the top to the bottom of each well. The assay’s color, when tested in a standard spectrophotometer, is linear to an absorbance of 2.3. The ELISA reader has a range of linearity which extends to an absorbance of 1.2. When a microtiter plate run 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.

QUESTIONS

Why didn’t the absorbance value change?
The technologist consults with the clinical chemist who suggests that she remove half of the solution from each well and recalculate. Why should this step now bring the absorbances within the linear range of the ELISA reader?

Questions to Consider

What is the equation, which relates absorbance to concentration?
What happens to each of the parameters in the equation in question 1 when buffer is added to the microtiter well to double the sample volume?

Answer

In this case, diluting the solution increased the path length through which the ELISA reader measures by a factor of two. This exactly counters the halving of the concentration by doubling the volume in the well, so in the above equation as c decreased by two-fold, the path length increased by a factor of 2, and the net absorbancy remained the same. This is in contrast to the usual spectrophotometry reading which is made through a cell of fixed path length and is independent of the volume in the cuvette.
Removing half the volume of the diluted reaction mixture from the well reduces the path length by one half. Since the solution has been diluted, the measured absorbance will be reduced by a factor of 2, the pathlength is restored to the initial value, and the absorbance of the solution will now be in the instrument’s range. The technologist must simple multiply the calculated concentration by a the dilution factor of 2.

Answers to Questions to Consider

The equation which relates concentration to absorbance is Beer’s law: $$A = abc$$where A is absorbance; a, absorptivity; b, light path of the solution in centimeters; and c, concentration of the substance of interest (p. 88).
When buffer is added to a microtiter plate well, both b and c in the Beer’s law equation change in inverse proportion to each other. The concentration decreases to one-half its initial value because the solution is being diluted. However, the path length (b) is increased because the volume in the well has doubled and the height of the solution in the well is approximately doubled. The configuration of the reader is such that the path length is proportional to the amount of solution in each well.