Analytical Sciences

Validation of a Method for Predicting the Precision, Limit of Detection and Range of Quantitation in Competitive ELISA

Dong Hwan CHOI,* Yoshio KATAKURA,* Rieko MATSUDA,** Yuzuru HAYASHI,** Masato HIROBE,*** Yasuhiro GODA,*** Kazuaki NINOMIYA,* and Suteaki SHIOYA*

*Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan
**National Institute of Health Sciences, 1-18-1 Kami-Yoga, Setagaya, Tokyo 158-8501, Japan
***Japan EnviroChemicals, Ltd., 2-17-85 Juso-Honmachi, Yodogawa, Osaka 532-0024, Japan

The mathematical model for predicting the precision, limit of detection (LOD) and range of quantitation (ROQ) in a competitive enzyme-linked immunosorbent assay (ELISA) proposed by Hayashi et al. (Anal. Chem., 2004, 76, 1295) was validated. The model describes the relative standard deviation (RSD) of concentration estimates by the RSDs of pipetting volumes of analyte, enzyme-conjugated antigen, antibody and substrate solutions, and the standard deviation (SD) of inherent absorbances between the wells in an ELISA plate. For 6 kinds of direct competitive ELISA kits, the LOD and ROQ predicted by the model agreed well with those obtained by experiments with real samples. It was also confirmed that the model is applicable to the prediction of uncertainty that depends on the pipetting error of the viscous antiserum solution. The model was demonstrated to be useful for estimating the LOD and ROQ of competitive ELISA.

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