ELISA rescue protocol: Recovery of sample concentrations from an assay with an unsuccessful standard curve

The Enzyme-linked Immunosorbant Assay (ELISA) is a method commonly used to measure proteins in various biological matrices, due to its ease of performance and relatively low cost. In order for quantitative data to be generated, a reference standard curve must be prepared for each assay; however, due to investigator error or standard protein degradation, otherwise representative experimental sample data are rendered useless. Herein, we describe a protocol by which sample concentrations can be recovered from assays in which the standard curve fails. The ΔOD values of the experimental samples are used to generate a new standard curve, which is applied back to the original plate. For validation of this method, experimental sample concentrations obtained using acceptable standard curves were potted against those calculated using this new method. Using linear regression analysis, we show a near 1:1 correlation between sample concentrations, with r2 values between 0.98 and 0.99 and slopes between 0.97 and 1.10. This method demonstrates that assays resulting in unusable standard curves do not require re-assay of all samples. Instead, the experimental sample concentrations can be retrieved saving the investigator the time and resources required to rerun samples or repeat entire experiments.