Title
A Direct, Competitive Enzyme-Linked Immunosorbent Assay (ELISA) as a Quantitative Technique for Small Molecules
Document Type
Article
Publication Date
12-2012
Abstract
ELISA (enzyme-linked immunosorbent assay) is a widely used technique with applications in disease diagnosis, detection of contaminated foods, and screening for drugs of abuse or environmental contaminants. However, published protocols with a focus on quantitative detection of small molecules designed for teaching laboratories are limited. A competitive, direct ELISA used to detect and quantify levels of digoxin, a cardiac glycoside, is described. Unique features of this lab include collecting data in quadruplicate followed by statistical analysis of replicates using a Q-test. Use of a microplate reader for measuring absorbances makes data collection extremely quick. Students plot their average absorbance versus log concentration digoxin and fit data to a third- or fourth-order polynomial. They also examine the maximum and minimum absorbance for the assay, determine the region of linearity, and then fit the linear region to a straight-line equation that can be used to determine the concentration of an unknown. The experiment can be completed in a 3-h period and is suitable for upper-level biochemistry, chemistry, and biology students. Although students find understanding a competitive ELISA more challenging than some other experiments, they enjoy learning about this commonly used laboratory technique.
Recommended Citation
Powers JL, Rippe KD, Imarhia K, Swift A, Scholten M, Islam N. 2012. A direct, competitive enzyme-linked immunosorbent assay (ELISA) as a quantitative technique for small molecules. J Chem Educ 89(12):1587-90.