A single domain antibody-based Luminex assay for the detection of SARS-CoV-2 in clinical samples.

Within the past decade, single domain antibodies (sdAbs) have been recognized as unique affinity binding reagents that can be tailored for performance in a variety of immunoassay formats. Luminex MagPlex color-coded magnetic microspheres provide a high-throughput platform that enables multiplexed immunoassays. We developed a MagPlex bead-based assay for the detection of SARS-CoV-2, using sdAbs against SARS-CoV-2 nucleocapsid (N) protein in which we engineered the sdAb capture reagents to orient them on the beads. The oriented sdAbs provided an increase in sensitivity over randomly oriented sdAbs for samples of N diluted in buffer, which also translated into better detection of SARS-CoV-2 in clinical samples. We assessed the specificity of the assay by examining seasonal coronavirus clinical samples. In summary, we provide a proof-of-concept that a bead-based assay using sdAbs to detect SARS-CoV-2 is feasible and future research combining it with other sdAb-coated beads that can detect other viruses may provide a useful diagnostic tool.

Accessing Antibody Reactivities in Serum or Plasma to (Auto-)antigens Using Multiplexed Bead-Based Protein Immunoassays.

Antibody (AB) testing or serotesting for reactive ABs against antigenic proteins is broadly used. Parallel examination of many antigens is of high interest to identify autoantibodies (AAB) or differential antigenic reactivities in many biological settings like allergy and infectious autoimmune, cancerous, or systemic disease. The resulting AAB profiles can be used for diagnosis, prognosis, and monitoring of such conditions. Protein microarrays have been used for AB profiling over the past decade but show some significant limitations which make them unsuitable for clinical applications. Alternative multiplexing platforms such as bead arrays were shown to provide a versatile tool for the confirmation and efficient analysis of high numbers of biological samples. Luminex' bead-based xMAP technology combines advantages such as multiplexing and lower demand for sample volume and at the same time overcomes the challenges of microarrays. It works faster, shows better antigen stability, is more reproducible, and allows the analysis of up to 500 analytes in one sample well. In this chapter we introduce our established workflow for the use of the xMAP technology for AB profiling including an overview of the method principle and protocols for the covalent immobilization of proteins to the MagPlex beads, confirmation of protein coupling, the execution of a multiplexed bead-based protein immunoassay, and subsequent data handling.

Multiplexed Bead-Based Peptide Immunoassays for the Detection of Antibody Reactivities.

Antigenic peptides are commonly used in serological test settings such as enzyme-linked immunosorbent assays (ELISA) to determine reactive antibodies (ABs) from serum or plasma samples. The use of synthetic peptides provides advantages like lower production effort and easier incorporation of specific chemical modifications compared to full-length antigenic proteins. Multiplexed antibody (AB) profiling methods such as microarray technologies enable the simultaneous identification of multiple novel biomarkers for the use in early disease diagnostics, vaccine development, or monitoring of immune responses. Despite various benefits they still show major limitations which can be overcome with bead-based assay technologies like the multi-analyte profiling (xMAP) technology developed by Luminex. In this chapter we introduce our established workflow for AB profiling with a multiplexed bead-based peptide immunoassay. The workflow is based on copper-catalyzed click chemistry to immobilize designed synthetic peptides onto uniquely color-coded paramagnetic beads in an orientation-specific manner. The individual peptide-coupled beads can be distinguished by their unique emission spectra during readout in the xMAP instrument and therefore allow testing of up to 500 different antigenic peptides in one multiplexed reaction. The multistep process described in this chapter is divided into separate sections for peptide design, coupling of functionalized peptides to MagPlex beads via click chemistry, confirmation of successful peptide immobilization, processing of serum or plasma samples, or preferably purified IgG thereof, with the multiplexed bead-based peptide immunoassay and subsequent data export and analysis.

Multiplex Microsphere PCR (mmPCR) Allows Simultaneous Gram Typing, Detection of Fungal DNA, and Antibiotic Resistance Genes.

OBJECTIVE: To show the high analytical specificity of our multiplex microsphere polymerase chain reaction (mmPCR) method, which offers the simultaneous detection of both general (eg, Gram type) and specific (eg, Pseudomonas species) clinically relevant genetic targets in a single modular multiplex reaction. MATERIALS AND METHODS: Isolated gDNA of 16S/rRNA Sanger-sequenced and Basic Local Alignment Tool-identified bacterial and fungal isolates were selectively amplified in a custom 10-plex Luminex MagPlex-TAG microsphere-based mmPCR assay. The signal/noise ratio for each reaction was calculated from flow cytometry standard data collected on a BD LSR Fortessa II flow cytometer. Data were normalized to the no-template negative control and the signal maximum. The analytical specificity of the assay was compared to single-plex SYBR chemistry quantitative PCR. RESULTS: Both general and specific primer sets were functional in the 10-plex mmPCR. The general Gram typing and pan-fungal primers correctly identified all bacterial and fungal isolates, respectively. The species-specific and antibiotic resistance-specific primers correctly identified the species- and resistance-carrying isolates, respectively. Low-level cross-reactive signals were present in some reactions with high signal/noise primer ratios. CONCLUSION: We found that mmPCR can simultaneously detect specific and general clinically relevant genetic targets in multiplex. These results serve as a proof-of-concept advance that highlights the potential of high multiplex mmPCR diagnostics in clinical practice. Further development of specimen-specific DNA extraction techniques is required for sensitivity testing.

Selection and Characterization of Single-Domain Antibodies for Detection of Lassa Nucleoprotein.

Lassa virus is the etiologic agent of Lassa fever, an acute and often fatal illness endemic to West Africa. It is important to develop new reagents applicable either for the specific diagnosis or as improved therapeutics for the treatment of Lassa fever. Here, we describe the development and initial testing of llama-derived single-domain antibodies that are specific for the Lassa virus nucleoprotein. Four sequence families based on complementarity-determining region (CDR) homology were identified by phage-based enzyme-linked immunosorbent assays, however, the highest affinity clones all belonged to the same sequence family which possess a second disulfide bond between Framework 2 and CDR3. The affinity and thermal stability were evaluated for each clone. A MagPlex-based homogeneous sandwich immunoassay for Lassa virus-like particles was also demonstrated to show their potential for further development as diagnostic reagents.

Multiplexed Microsphere Suspension-Array Assay for Urine Mitochondrial DNA Typing by C-Stretch Length in Hypervariable Regions.

BACKGROUND: The standard method for personal identification and verification of urine samples in doping control is short tandem repeat (STR) analysis using nuclear DNA (nDNA). The DNA concentration of urine is very low and decreases under most conditions used for sample storage; therefore, the amount of DNA from cryopreserved urine samples may be insufficient for STR analysis. We aimed to establish a multiplexed assay for urine mitochondrial DNA typing containing only trace amounts of DNA, particularly for Japanese populations. METHODS: A multiplexed suspension-array assay using oligo-tagged microspheres (Luminex MagPlex-TAG) was developed to measure C-stretch length in hypervariable region 1 (HV1) and 2 (HV2), five single nucleotide polymorphisms (SNPs), and one polymorphic indel. Based on these SNPs and the indel, the Japanese population can be classified into five major haplogroups (D4, B, M7a, A, D5). The assay was applied to DNA samples from urine cryopreserved for 1 - 1.5 years (n = 63) and fresh blood (n = 150). RESULTS: The assay with blood DNA enabled Japanese subjects to be categorized into 62 types, exhibiting a discriminatory power of 0.960. The detection limit for cryopreserved urine was 0.005 ng of nDNA. Profiling of blood and urine pairs revealed that 5 of 63 pairs showed different C-stretch patterns in HV1 or HV2. CONCLUSIONS: The assay described here yields valuable information in terms of the verification of urine sample sources employing only trace amounts of recovered DNA. However, blood cannot be used as a reference sample.

Selection of Single-Domain Antibodies towards Western Equine Encephalitis Virus.

In this work, we describe the selection and characterization of single-domain antibodies (sdAb) towards the E2/E3E2 envelope protein of the Western equine encephalitis virus (WEEV). Our purpose was to identify novel recognition elements which could be used for the detection, diagnosis, and perhaps treatment of western equine encephalitis (WEE). To achieve this goal, we prepared an immune phage display library derived from the peripheral blood lymphocytes of a llama that had been immunized with an equine vaccine that includes killed WEEV (West Nile Innovator + VEWT). This library was panned against recombinant envelope (E2/E3E2) protein from WEEV, and seven representative sdAb from the five identified sequence families were characterized. The specificity, affinity, and melting point of each sdAb was determined, and their ability to detect the recombinant protein in a MagPlex sandwich immunoassay was confirmed. Thus, these new binders represent novel recognition elements for the E2/E3E2 proteins of WEEV that are available to the research community for further investigation into their applicability for use in the diagnosis or treatment of WEE.

Development of multiplexed bead arrays for the simultaneous detection of nucleic acid from multiple viruses in bat samples.

Virus surveillance of wildlife populations is important for identifying, monitoring, and predicting the emergence of pathogens that pose a potential threat to animal and human health. Bats are identified as important wildlife hosts of many viruses capable of causing fatal human disease, including members of the henipaviruses, coronaviruses, rhabdoviruses and filoviruses. As global warming and habitat change are thought to impact upon pathogen transmission dynamics and increase the risk of spillover, virus surveillance in bat populations remains a significant component of efforts to improve the prediction and control of potential future disease outbreaks caused by bat-borne viruses. In this study we have developed two fluid bead array assays containing customized panels that target multiple bat-borne viruses. These assays detect up to 11 viral RNA's simultaneously in urine samples collected from wild bat populations in Australia and Bangladesh. The assays developed show high specificity for the target viruses and the analytical sensitivity compares favorably to qRT-PCR. These assays enhance the ability to monitor multi-pathogen dynamics and identify patterns of virus shedding from bat populations, thus informing key approaches to outbreak response and control.