Cross-Platform Comparison of Highly Sensitive Immunoassays for Inflammatory Markers in a COVID-19 Cohort.

A variety of commercial platforms are available for the simultaneous detection of multiple cytokines and associated proteins, often employing Ab pairs to capture and detect target proteins. In this study, we comprehensively evaluated the performance of three distinct platforms: the fluorescent bead-based Luminex assay, the proximity extension-based Olink assay, and a novel proximity ligation assay platform known as Alamar NULISAseq. These assessments were conducted on human serum samples from the National Institutes of Health IMPACC study, with a focus on three essential performance metrics: detectability, correlation, and differential expression. Our results reveal several key findings. First, the Alamar platform demonstrated the highest overall detectability, followed by Olink and then Luminex. Second, the correlation of protein measurements between the Alamar and Olink platforms tended to be stronger than the correlation of either of these platforms with Luminex. Third, we observed that detectability differences across the platforms often translated to differences in differential expression findings, although high detectability did not guarantee the ability to identify meaningful biological differences. Our study provides valuable insights into the comparative performance of these assays, enhancing our understanding of their strengths and limitations when assessing complex biological samples, as exemplified by the sera from this COVID-19 cohort.

Cross-platform comparison of highly-sensitive immunoassays for inflammatory markers in a COVID-19 cohort 1.

A variety of commercial platforms are available for the simultaneous detection of multiple cytokines and associated proteins, often employing antibody pairs to capture and detect target proteins. In this study, we comprehensively evaluated the performance of three distinct platforms: the fluorescent bead-based Luminex assay, the proximity extension-based Olink assay, and a novel proximity ligation assay platform known as Alamar NULISAseq. These assessments were conducted on serum samples from the NIH IMPACC study, with a focus on three essential performance metrics: detectability, correlation, and differential expression. Our results reveal several key findings. Firstly, the Alamar platform demonstrated the highest overall detectability, followed by Olink and then Luminex. Secondly, the correlation of protein measurements between the Alamar and Olink platforms tended to be stronger than the correlation of either of these platforms with Luminex. Thirdly, we observed that detectability differences across the platforms often translated to differences in differential expression findings, although high detectability did not guarantee the ability to identify meaningful biological differences. Our study provides valuable insights into the comparative performance of these assays, enhancing our understanding of their strengths and limitations when assessing complex biological samples, as exemplified by the sera from this COVID-19 cohort.

NULISA: a proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing.

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range of the plasma proteome. Here we address these challenges with NUcleic acid Linked Immuno-Sandwich Assay (NULISA™), which improves the sensitivity of traditional proximity ligation assays by ~10,000-fold to attomolar level, by suppressing assay background via a dual capture and release mechanism built into oligonucleotide-conjugated antibodies. Highly multiplexed quantification of both low- and high-abundance proteins spanning a wide dynamic range is achieved by attenuating signals from abundant targets with unconjugated antibodies and next-generation sequencing of barcoded reporter DNA. A 200-plex NULISA containing 124 cytokines and chemokines and other proteins demonstrates superior sensitivity to a proximity extension assay in detecting biologically important low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA makes broad and in-depth proteomic analysis easily accessible for research and diagnostic applications.

NULISA: a novel proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing.

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range across the proteome. We report a novel proteomic technology - NUcleic acid Linked Immuno-Sandwich Assay (NULISA™) - that incorporates a dual capture and release mechanism to suppress the assay background and improves the sensitivity of the proximity ligation assay by over 10,000-fold to the attomolar level. It utilizes pairs of antibodies conjugated to DNA oligonucleotides that enable immunocomplex purification and generate reporter DNA containing target- and sample-specific barcodes for a next-generation sequencing-based, highly multiplexed readout. A 200-plex NULISA targeting 124 cytokines and chemokines and 80 other immune response-related proteins demonstrated superior sensitivity for detecting low-abundance proteins and high concordance with other immunoassays. The ultrahigh sensitivity allowed the detection of previously difficult-to-detect, but biologically important, low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA addresses longstanding challenges in proteomic analysis of liquid biopsies and makes broad and in-depth proteomic analysis accessible to the general research community and future diagnostic applications.

The RNA-binding protein Swm is critical for Drosophila melanogaster intestinal progenitor cell maintenance.

The regulation of stem cell survival, self-renewal, and differentiation is critical for the maintenance of tissue homeostasis. Although the involvement of signaling pathways and transcriptional control mechanisms in stem cell regulation have been extensively investigated, the role of post-transcriptional control is still poorly understood. Here, we show that the nuclear activity of the RNA-binding protein Second Mitotic Wave Missing is critical for Drosophila melanogaster intestinal stem cells and their daughter cells, enteroblasts, to maintain their progenitor cell properties and functions. Loss of swm causes intestinal stem cells and enteroblasts to stop dividing and instead detach from the basement membrane, resulting in severe progenitor cell loss. swm loss is further characterized by nuclear accumulation of poly(A)+ RNA in progenitor cells. Second Mitotic Wave Missing associates with transcripts involved in epithelial cell maintenance and adhesion, and the loss of swm, while not generally affecting the levels of these Second Mitotic Wave Missing-bound mRNAs, leads to elevated expression of proteins encoded by some of them, including the fly ortholog of Filamin. Taken together, this study indicates a nuclear role for Second Mitotic Wave Missing in adult stem cell maintenance, raising the possibility that nuclear post-transcriptional regulation of mRNAs encoding cell adhesion proteins ensures proper attachment of progenitor cells.

Coordinated repression of pro-differentiation genes via P-bodies and transcription maintains Drosophila intestinal stem cell identity.

The role of processing bodies (P-bodies), key sites of post-transcriptional control, in adult stem cells remains poorly understood. Here, we report that adult Drosophila intestinal stem cells, but not surrounding differentiated cells such as absorptive enterocytes (ECs), harbor P-bodies that contain Drosophila orthologs of mammalian P-body components DDX6, EDC3, EDC4, and LSM14A/B. A targeted RNAi screen in intestinal progenitor cells identified 39 previously known and 64 novel P-body regulators, including Patr-1, a gene necessary for P-body assembly. Loss of Patr-1-dependent P-bodies leads to a loss of stem cells that is associated with inappropriate expression of EC-fate gene nubbin. Transcriptomic analysis of progenitor cells identifies a cadre of such weakly transcribed pro-differentiation transcripts that are elevated after P-body loss. Altogether, this study identifies a P-body-dependent repression activity that coordinates with known transcriptional repression programs to maintain a population of in vivo stem cells in a state primed for differentiation.

I-KCKT allows dissection-free RNA profiling of adult Drosophila intestinal progenitor cells.

The adult Drosophila intestinal epithelium is a model system for stem cell biology, but its utility is limited by current biochemical methods that lack cell type resolution. Here, we describe a new proximity-based profiling method that relies upon a GAL4 driver, termed intestinal-kickout-GAL4 (I-KCKT-GAL4), that is exclusively expressed in intestinal progenitor cells. This method uses UV crosslinked whole animal frozen powder as its starting material to immunoprecipitate the RNA cargoes of transgenic epitope-tagged RNA binding proteins driven by I-KCKT-GAL4 When applied to the general mRNA-binder, poly(A)-binding protein, the RNA profile obtained by this method identifies 98.8% of transcripts found after progenitor cell sorting, and has low background noise despite being derived from whole animal lysate. We also mapped the targets of the more selective RNA binder, Fragile X mental retardation protein (FMRP), using enhanced crosslinking and immunoprecipitation (eCLIP), and report for the first time its binding motif in Drosophila cells. This method will therefore enable the RNA profiling of wild-type and mutant intestinal progenitor cells from intact flies exposed to normal and altered environments, as well as the identification of RNA-protein interactions crucial for stem cell function.

Identification of Split-GAL4 Drivers and Enhancers That Allow Regional Cell Type Manipulations of the Drosophila melanogaster Intestine.

The Drosophila adult midgut is a model epithelial tissue composed of a few major cell types with distinct regional identities. One of the limitations to its analysis is the lack of tools to manipulate gene expression based on these regional identities. To overcome this obstacle, we applied the intersectional split-GAL4 system to the adult midgut and report 653 driver combinations that label cells by region and cell type. We first identified 424 split-GAL4 drivers with midgut expression from ∼7300 drivers screened, and then evaluated the expression patterns of each of these 424 when paired with three reference drivers that report activity specifically in progenitor cells, enteroendocrine cells, or enterocytes. We also evaluated a subset of the drivers expressed in progenitor cells for expression in enteroblasts using another reference driver. We show that driver combinations can define novel cell populations by identifying a driver that marks a distinct subset of enteroendocrine cells expressing genes usually associated with progenitor cells. The regional cell type patterns associated with the entire set of driver combinations are documented in a freely available website, providing information for the design of thousands of additional driver combinations to experimentally manipulate small subsets of intestinal cells. In addition, we show that intestinal enhancers identified with the split-GAL4 system can confer equivalent expression patterns on other transgenic reporters. Altogether, the resource reported here will enable more precisely targeted gene expression for studying intestinal processes, epithelial cell functions, and diseases affecting self-renewing tissues.

Canonical nucleators are dispensable for stress granule assembly in Drosophila intestinal progenitors.

Stressed cells downregulate translation initiation and assemble membrane-less foci termed stress granules (SGs). Although SGs have been extensively characterized in cultured cells, the existence of such structures in stressed adult stem cell pools remains poorly characterized. Here, we report that the Drosophila orthologs of the mammalian SG components AGO1, ATX2, CAPRIN, eIF4E, FMRP, G3BP, LIN-28, PABP and TIAR are enriched in adult fly intestinal progenitor cells, where they accumulate in small cytoplasmic messenger ribonucleoprotein complexes (mRNPs). Treatment with sodium arsenite or rapamycin reorganized these mRNPs into large cytoplasmic granules. Formation of these intestinal progenitor stress granules (IPSGs) depended on polysome disassembly, led to translational downregulation and was reversible. Although the canonical SG nucleators ATX2 and G3BP were sufficient for IPSG formation in the absence of stress, neither of them, nor TIAR, either individually or collectively, were required for stress-induced IPSG formation. This work therefore finds that IPSGs do not assemble via a canonical mechanism, raising the possibility that other stem cell populations employ a similar stress-response mechanism.