Theoretical limitations of quantification for noncompetitive sandwich immunoassays

Abstract

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 nonspecific binding often restricts immunoassay limits of quantification (LOQ). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improved signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single fluorescent 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.

Selectivity enhancements in gel‐based DNA‐nanoparticle assays by membrane‐induced isotachophoresis: thermodynamics versus kinetics

“…Therefore, given similar initial concentrations, the selectivity will be C tar C mis = e −(k d,tar −k d,mis )t (5) If we substitute in the definitions of the rate constants, Eq. (6) reduces to…”

“…technique, reverse transcriptase polymerase chain reaction (RT-PCR), possesses the desirable characteristics of large dynamic range, sequence-specific amplification, and low limits of detection, it does not actually quantify the concentration of RNA but rather determines the number of cycles required for specific amplification [5,6]. Thus, relative measures such as cycle of quantitation are reported instead of concentrations.…”

Fortschritte in der optischen Einzelmoleküldetektion: Auf dem Weg zu höchstempfindlichen Bioaffinitätsassays

“…Beispielsweise wurde geschätzt, dass ein Molekül in einer Konzentration von 1f m durchschnittlich mehr als zehn Minuten bençtigt, um durch Diffusion ein Detektionsvolumen von 10 fL zu erreichen. [26] Stochastische Fluktuationen sind das zweite Problem, [27] denn bei niedrigen Analytkonzentrationen ist ein kleines Beobachtungsvolumen zufällig zu einem Zeitpunkt von einem einzelnen Analytmolekülb esetzt und zu einem anderen Zeitpunkt leer. Das so genannte Poisson-Rauschen (( p n)/n)h ängt von der Anzahl der gezählten Ereignisse (n)abund kann bei herkçmmlichen analogen Assays,b ei denen n sehr groß ist, vernachlässigt werden.…”

Advances in Optical Single‐Molecule Detection: En Route to Supersensitive Bioaffinity Assays

“…[26] Stochastic fluctuations are the second problem. [27] At low analyte concentrations,asmall observation volume is randomly at one time occupied by as ingle analyte molecule and, at another time,e mpty.T he so-called Poisson noise (( p n)/n) depends on the number of counted events (n)and is negligible in conventional analog assays where n is very large.For digital assays,h owever, it presents ap roblem because as ingle detection event of an analyte molecule does not contain enough analytical information. Therefore,i ti sn ecessary to make either many parallel measurements on alarger area or many sequential measurements in the same detection volume.…”