A direct, competitive enzyme-linked immunosorbent assay (ELISA) as a quantitative technique for small molecules

Jennifer L. Powers, Karen Duda Rippe, Kelly Imarhia, Aileen Swift, Melanie Scholten, Naina Islam

Research output: Contribution to journalArticlepeer-review

14 Scopus citations


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.

Original languageEnglish
Pages (from-to)1587-1590
Number of pages4
JournalJournal of Chemical Education
Issue number12
StatePublished - Nov 13 2012


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