Highly sensitive ligand-binding assays in pre-clinical and clinical applications: immuno-PCR and other emerging techniques

Abstract

Recombinant DNA technology and corresponding innovations in molecular biology, chemistry and medicine have led to novel therapeutic biomacromolecules as lead candidates in the pharmaceutical drug development pipelines. While monoclonal antibodies and other proteins provide therapeutic potential beyond the possibilities of small molecule drugs, the concomitant demand for supportive bioanalytical sample testing creates multiple novel challenges. For example, intact macromolecules can usually not be quantified by mass-spectrometry without enzymatic digestion and isotopically labeled internal standards are costly and/or difficult to prepare. Classical ELISA-type immunoassays, on the other hand, often lack the sensitivity required to obtain pharmacokinetics of low dosed drugs or pharmacodynamics of suitable biomarkers. Here we summarize emerging state-of-the-art ligand-binding assay technologies for pharmaceutical sample testing, which reveal enhanced analytical sensitivity over classical ELISA formats. We focus on immuno-PCR, which combines antibody specificity with the extremely sensitive detection of a tethered DNA marker by quantitative PCR, and alternative nucleic acid-based technologies as well as methods based on electrochemiluminescence or single-molecule counting. Using case studies, we discuss advantages and drawbacks of these methods for preclinical and clinical sample testing.

From DNA Nanotechnology to Material Systems Engineering

“…The merging of the subareas is already evident from many examples in this article, such as the use of tile‐based assembly methods for the production of origami superstructures (Section ), the exploitation of recombinant protein technology to enable functional DNA nanostructures for live science applications (Section ), the use of DNA nanostructures for creating colloidal superstructures that in turn can be fine tuned and functionalized with proteins (Section ), the implementation of functional nucleic acid motifs into polymeric materials (Section ), or the integration of DNA‐surfaces to enable efficient manufacture of heterogeneous superstructures applicable in biology and material sciences (Section ). Based on these examples, we have illustrated that research on DNA‐based materials has already produced a first generation of applications in sensing and medicine[12a,67,185] and the second generation, is already emerging in the form of, e.g., advanced material surfaces, smart carriers, and robots for biomedicine or devices for plasmonics. [50a,96] In order to open up these and other fields of application, the increasing implementation of engineering sciences is of great importance.…”

Diagnosing COVID-19: The Disease and Tools for Detection

“…To improve the assay readout signal, researchers have developed a variety of signal amplifying techniques (e.g., thermal imaging and assembly of multiple gold nanoparticles). 79 Another approach for use at the point-of-care is microfluidic devices. These devices consist of a palm-sized chip etched with micrometer-sized channels and reaction chambers.…”

A simple and rapid LC-MS/MS method for therapeutic drug monitoring of cetuximab: a GPCO-UNICANCER proof of concept study in head-and-neck cancer patients

“…To date, little is known about the pharmacokinetics of monoclonal antibodies 20 and developing specific bioanalytical methods to measure plasma concentrations in routine patient has remained a challenging task for years 21 . Most of the methods currently used, either as part of registration clinical trials or academic studies are based upon immuno-assay such as canonical ELISA methods 22 , although other emerging immune-based techniques have been recently proposed 23 . Regarding cetuximab, all clinical reports published thus far were based upon ELISA measurement of drug levels in plasma using frequently in-house methods 3–5 .…”