Theoretical limitations of quantification for noncompetitive sandwich immunoassays.
Immunoassays exploit the highly selective interaction between antibodies and antigens to provide a vital method for biomolecule detection at low concentrations. Developers and practitioners of immunoassays have long known that non-specific binding often restricts immunoassay limits of quantification (LOQs). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improving the signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single-fluorescence molecules, this approach is unlikely to lead to dramatic improvements in the future. Here, fundamental interactions based on the law of mass action are analytically connected to signal generation, replacing the four- and five-parameter fittings commercially used to approximate sigmoidal immunoassay curves and allowing quantitative consideration of non-specific binding and statistical limitations in order to understand the ultimate detection capabilities of immunoassays. The restrictions imposed on limits of quantification by instrumental noise, non-specific binding, and counting statistics are discussed based on equilibrium relations for a sandwich immunoassay. Understanding the maximal capabilities of immunoassays for each of these regimes can greatly assist in the development and evaluation of immunoassay platforms. While many studies suggest that single molecule detection is possible through immunoassay techniques, here, it is demonstrated that the fundamental limit of quantification (precision of 10 % or better) for an immunoassay is approximately 131 molecules and this limit is based on fundamental and unavoidable statistical limitations.
Department of Chemistry and Biochemistry, Arizona State University, Main Campus, P. O. Box 871604, Tempe, AZ, 85287-1604, USA.
Christine F
CF
Woolley
Department of Chemistry and Biochemistry, Arizona State University, Main Campus, P. O. Box 871604, Tempe, AZ, 85287-1604, USA. MHayes@asu.edu.
Mark A
MA
Hayes
School of Earth and Space Exploration, Arizona State University, 781 S Terrace Rd, Tempe, AZ, 85281, USA.
Prasun
P
Mahanti
Department of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA, 70803, USA.
S
S
Douglass Gilman
Department of Mathematics and Statistical Sciences, Arizona State University, Tempe, AZ, 85287, USA.
Tom
T
Taylor
D000906:Antibodies / Q000737:chemistry*; D000941:Antigens / Q000737:chemistry*; D006801:Humans; D007118:Immunoassay / Q000706:statistics & numerical data*; D057230:Limit of Detection; D015999:Multivariate Analysis; D011485:Protein Binding; D059629:Signal-To-Noise Ratio
D000906:Antibodies; D000941:Antigens
Detection limit; Immunoassay; Molecular shot noise; Single molecule