ELISA in the multiplex era: Potentials and pitfalls

Abstract

Multiplex immunoassays confer several advantages over widely adopted singleplex immunoassays including increased efficiency at a reduced expense, greater output per sample volume ratios and higher throughput predicating more resolute, detailed diagnostics and facilitating personalised medicine. Nonetheless, to date, relatively few protein multiplex immunoassays have been validated for in vitro diagnostics in clinical/point-of-care settings. This review article will outline the challenges, which must be ameliorated prior to the widespread integration of multiplex immunoassays in clinical settings: (i) biomarker validation; (ii) standardisation of immunoassay design and quality control (calibration and quantification); (iii) availability, stability, specificity and cross-reactivity of reagents; (iv) assay automation and the use of validated algorithms for transformation of raw data into diagnostic results. A compendium of multiplex immunoassays applicable to in vitro diagnostics and a summary of the diagnostic products currently available commercially are included, along with an analysis of the relative states of development for each format (namely planar slide based, suspension and planar/microtitre plate based) with respect to the aforementioned issues.

Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays

“…Selected reaction monitoring (SRM) and parallel reaction monitoring (PRM) assays have high specificity, dynamic range of six orders of magnitude, and limits of detection (LOD) and quantification (LOQ) in the low ng/ml level. Unlike multiplex ELISA, which allows for the detection of up to 25 analytes in a single assay (20), multiplex SRM assays facilitate measurements of tens to hundreds of proteins in a single run without compromising assay sensitivity and accuracy (21)(22)(23)(24).…”

Simultaneous capture and sequential detection of two malarial biomarkers on magnetic microparticles

“…A multiplexed immunoassay utilizes the same “sandwich” format (capture antibody, sample, detection antibody) as a conventional singleplex ELISA, except the former usually adopts fluorescent or chemiluminescent reporter systems rather than amplification of a colorimetric substrate by enzymes [1]. Two common formats for multiplexed immunoassays include planar arrays and bead-based suspension assays [2].…”

“…The functionalized supports are then treated with sample, followed by reporter-labeled antibody. Signal is detected using a high-resolution scanner or fluorescence microscope [1]. In addition to multiplexing capabilities, planar micro-array immunoassays benefit from ambient analyte theory.…”

Reduction of dilution error in ELISAs using an internal standard

“…ELISAs and related assay formats provide a degree of specificity and sensitivity that is difficult to achieve with other analytical techniques [1][2][3][4]. However, immunoassays often suffer from relatively poor precision.…”