Dissertation

Scalable Affinity-Proteomics on Microparticles

Dagher, Milad

2018

Overview

The immunoassay is a core method for basic and clinical research. Owing to the requirements of dual-antibody binding, the sandwich immunoassay provides exquisite specificity, sensitivity, and cost-efficiency in the measurement of proteins within plasma. Fluorescently-barcoded microparticles are a popular format for immunoassays as they allow multiplexing with high sample throughput and rapid read-out by cytometry. However, this widely used format suffers from two shortcomings that limit its scalability and applicability. First and foremost, the specificity and sensitivity of multiplexed sandwich assays (MSAs) is severely hindered with multiplexing due to cross-reactivity between antibodies applied as a mixture. This reagent-driven cross-reactivity (rCR) continues to be the main obstacle towards increased multiplexing in microparticle-based MSAs. The second challenge is the generation of large numbers of spectrally-distinct microparticles for use in barcoding. Spectral barcoding calls for microparticle functionalization with distinct proportions of multicolour dyes to create a unique barcode; however, multicolour Förster resonance energy transfer (mFRET) leads to hitherto unpredictable shifts in emission intensities thus confounding the barcodes. Thus, current methods for spectral barcoding do not tolerate mFRET, are constrained to using specialized dyes and cytometers that minimize the spectral overlap, and employ lengthy trial-and-error optimization protocols to experimentally determine the barcode-specific dye ratios.Here, we introduce a platform for (i) rCR-free multiplexing of sandwich assays on microparticles with (ii) mFRET-tolerating high-capacity barcoding. Building on the work of Förster, we introduce an ensemble multicolour FRET (emFRET) model that can accurately predict the ensemble spectral profiles of any combination of dyes. We also developed a facile, proportional microparticle labelling method to achieve proportional labeling, conjugating the dyes to DNA oligonucleotides, which served as chemically-homogeneous cross-linkers. The emFRET model enabled in silico design of 580 distinguishable barcodes using dyes with strong spectral overlap (FAM, Cy3, Cy5, Cy5.5), and with inter-dye FRET efficiency reaching up to 0.76. The emFRET also allowed robust and fully-automated decoding without need for manual calibration. Taken together, the results established a platform for rapid, high-capacity microparticle barcoding using common dyes, and fully automated decoding using commonly used cytometers, and which can be used for immunoassays.linkage assay on microparticles\" (CLAMP). In our approach both members of an antibody pair are pre-immobilized to the surface of micron-sized microparticles, with one of the members tethered via a cleavable DNA linker. Different microparticle sets can be prepared, each containing both affinity reagents for specific detection of their target analyte. Importantly, only complete protein-antibody sandwich complexes on each bead are labeled using a DNA-displacement strategy, prior to rapid read-out of CLAMPs by flow cytometry. The CLAMP assay was optimized by tuning the valency and surface density of antibody conjugates, and was shown to eliminate rCR from microparticlebased MSAs. The CLAMP assay holds potential as the first scalable MSA on microparticles, and paves the way for simple, rapid, and high-throughput MSAs. By pre-assembling antibody pairs, this immunoassay concept represents a departure from typical immunoassays, and offers the possibility to design the multivalent sensors a priori, which could enable higher sensitivity assays.In this dissertation, we have presented an integrated platform that addresses two of the most important and highly sought-for scaling challenges for MSAs. These results set the foundation for the next-generation of protein assays that can be multiplexed while maintaining the excellent sensitivity and specificity of the single-plex ELISAs. Importantly, the approach taken in this work is cost-efficient and can be immediately deployed, enabling application in large population-wide studies and promising to meet the increasing demands in precision medicine.

• Publisher: ProQuest Dissertations & Theses
• Subject: Analytical chemistry / Antibodies / Bioinformatics / Biomarkers / Chemistry / Drug dosages / Flow cytometry / Immunoassay / Mass spectrometry / Medical research / Medicine / Precision medicine / Proteomics / Reagents / Scientific imaging
• ISBN: 9798426880863