Pair Of Antibodies Research Articles

The Effects of Different Antigen-Antibody Pairs on the Results of 20 Min ELISA and 8 Min Chromatographic Paper Test for Quantitative Detection of Acetamiprid in Vegetables.

To establish rapid, high-sensitive, quantitative detection of ACP residues in vegetables. A 1G2 cell clone was selected as the most sensitive for anti-ACP antibody production following secondary immunization, cell fusion, and screening. The affinity of the 1G2 antibody to each of the four coating agents (imidacloprid–bovine serum albumin [BSA], thiacloprid–BSA, imidaclothiz–BSA, and ACP-BSA) was determined using a 20 min enzyme-linked immunosorbent assay (ELISA). The half maximal inhibitory concentration (IC50) was 0.51–0.62 ng/mL, showing no significant difference in affinity to different antigens. However, we obtained IC50 values of 0.58 and 1.40 ng/mL on the linear regression lines for 1G2 anti-ACP antibody/imidacloprid–BSA and 1G2 anti-ACP antibody/thiacloprid–BSA, respectively, via quantum dot (QD)-based immunochromatography. That is, the 1G2 antibody/imidacloprid–BSA pair (the best combination) was about three times more sensitive than the 1G2 antibody/thiacloprid–BSA pair in immunochromatographic detection. The best combination was used for the development of an 8 min chromatographic paper test. With simple and convenient sample pretreatment, we achieved an average recovery of 75–117%. The coefficient of variation (CoV) was <25% for all concentrations tested, the false–positive rate was <5%, the false–negative rate was 0%, and the linear range of the method was 50–1800 μg/kg. These performance metrics met the ACP detection standards in China, the European Union (EU), and the United States (US). In summary, in this study, we established an 8 min QD-based immunochromatographic stripe for the rapid and accurate quantitative determination of ACP residues in vegetables.

Quantitation of Pax-6 protein in ocular impression cytology samples using an electrochemiluminescence immunoassay

Paired box protein Pax-6 (oculothrombin) is a transcription factor that plays an important regulatory role in ocular, brain, and pancreatic development. Mutations of the PAX6 gene cause aniridia and Peters anomaly. Reduction in Pax-6 protein is also associated with ocular diseases such as dry eye. An electrochemiluminescence immunoassay method using the Meso Scale Discovery platform was developed to measure Pax-6 protein levels in corneal epithelial cells obtained by impression cytology. Impression cytology involves harvesting ocular epithelial cells by applying a polyethersulfone membrane patch briefly to the ocular surface using a commercially available EYEPRIM™ device. The epithelial cells that adhere to the membrane patch of the EYEPRIM™ device provide a biological sample which can be assayed for Pax-6 protein levels. Assay development identified an antibody pair capable of detecting purified recombinant Pax-6 protein produced in mammalian cells. The optimized assay has a dynamic range of 24 pg mL−1 to 100,000 pg mL−1 and a lower limit of quantification of 24 pg mL−1. Assay selectivity was demonstrated using either HeLa or HEK293 cells transfected with inhibitory RNA. Finally, the method was validated by measuring Pax-6 protein levels in impression cytology acquired samples obtained using the EYEPRIM™ device from rabbit cornea.

Dual detection of COVID-19 antigens and antibodies using nanoscale fluorescent plasmonic substrates.

There is a continuing need for biosensors that can be used in the diagnosis of COVID-19 infection and to measure a subject's immune response to the virus itself (SARS-CoV-2). In this study, grating-coupled fluorescent plasmonic (GC-FP)-based detection of SARS-CoV-2 antigens was coupled with antibody detection to yield a dual-mode detection assay. Pairs of capture and detection antibodies were screened for direct detection of the SARS-CoV-2 nucleocapsid (Nuc) antigen, which were then combined with an existing GC-FP antibody detection assay. Nuc could be detected as low as 1 µg/mL concentrations, while antibodies were detectable to 50 ng/mL. The dual detection assay was tested by adding purified Nuc antigen to serum from a polymerase chain reaction (PCR)-positive COVID-19-infected individual. Using this sample, co-detection of Nuc antigen and anti-spike protein antibodies was successfully performed on a single GC-FP chip. Total assay time was 1 h, making this the first known example of rapid dual antibody and antigen detection on the same biosensor chip.

Potential of antibody pair targeting conserved antigenic sites in diagnosis of SARS-CoV-2 variants infection

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has become disaster for human society. As the pandemic becomes more regular, we should develop more rapid and accurate detection methods to achieve early diagnosis and treatment. Antigen detection methods based on spike protein has great potential, however, it has not been effectively developed, probably due to the torturing conformational complexity. By utilizing cross-blocking data, we clustered SARS-CoV-2 receptor binding domain (RBD)-specific monoclonal antibodies (mAbs) into 6 clusters. Subsequently, the antigenic sites for representative mAbs were identified by RBDs with designed residue substitutions. The sensitivity and specificity of selected antibody pairs was demonstrated using serial diluted samples of SARS-CoV-2 S protein and SARS-CoV S protein. Furthermore, pseudovirus system was constructed to determine the detection capability against SARS-CoV-2 and SARS-CoV. 6 RBD-specific mAbs, recognizing different antigenic sites, were identified as potential candidates for optimal antibody pairs for detection of SARS-CoV-2 S protein. By considering relative spatial position, accessibility and conservation of corresponding antigenic sites, affinity and the presence of competitive antibodies in clinical samples, 6H7–6G3 was rationally identified as optimal antibody pair for detection of both SARS-CoV-2 and SARS-CoV. Furthermore, our results showed that 6H7 and 6G3 effectively bind to SARS-CoV-2 variants of concern (VOCs). Taken together, we identified 6H7–6G3 antibody pair as a promising rapid antigen diagnostic tool in containing COVID-19 pandemic caused by multiple VOCs. Moreover, our results also provide an important reference in screening of antibody pairs detecting antigens with complex conformation.

Precision cancer targeting with antibody pairs.

Precision cancer targeting with antibody pairs.

Logic-gated antibody pairs that selectively act on cells co-expressing two antigens

The use of therapeutic monoclonal antibodies is constrained because single antigen targets often do not provide sufficient selectivity to distinguish diseased from healthy tissues. We present HexElect®, an approach to enhance the functional selectivity of therapeutic antibodies by making their activity dependent on clustering after binding to two different antigens expressed on the same target cell. lmmunoglobulin G (lgG)-mediated clustering of membrane receptors naturally occurs on cell surfaces to trigger complement- or cell-mediated effector functions or to initiate intracellular signaling. We engineer the Fc domains of two different lgG antibodies to suppress their individual homo-oligomerization while promoting their pairwise hetero-oligomerization after binding co-expressed antigens. We show that recruitment of complement component C1q to these hetero-oligomers leads to clustering-dependent activation of effector functions such as complement mediated killing of target cells or activation of cell surface receptors. HexElect allows selective antibody activity on target cells expressing unique, potentially unexplored combinations of surface antigens.

Simple, Rapid Chemical Labeling and Screening of Antibodies with Luminescent Peptides.

Sensitive and selective detection assays are essential for the accurate measurement of analytes in both clinical and research laboratories. Immunoassays that rely on nonoverlapping antibodies directed against the same target analyte (e.g., sandwich enzyme-linked immunosorbent assays (ELISAs)) are commonly used molecular detection technologies. Use of split enzyme reporters has simplified the workflow for these traditionally complex assays. However, identifying functional antibody pairs for a given target analyte can be cumbersome, as it generally involves generating and screening panels of antibodies conjugated to reporters. Accordingly, we sought a faster and easier reporter conjugation strategy to streamline antibody screening. We describe here the development of such a method that is based on an optimized ternary NanoLuc luciferase. This bioluminescence complementation system is comprised of a reagent-based thermally stable polypeptide (LgTrip) and two small peptide tags (β9 and β10) with lysine-reactive handles for direct conjugation onto antibodies. These reagents enable fast, single-step, wash-free antibody labeling and sensitive functional screening. Simplicity, speed, and utility of the one-pot labeling technology are demonstrated in screening antibody pairs for the analyte interleukin-4. The screen resulted in the rapid development of a sensitive homogeneous immunoassay for this clinically relevant cytokine.

Development of a sensitive sandwich ELISA with broad species specificity for improved fish allergen detection

A sensitive Enzyme-linked Immunosorbent Assay (ELISA) with improved broad species specificity was developed for the detection of southern hemisphere fish residues in processed foods. The polyclonal antibodies were raised against parvalbumins from 13 fish species representing 7 fish orders selected for their molecular diversity and immunoreactivity profile. The optimized ELISA-2 (based on the rabbit capture antibody (RB#4) - sheep detection antibody (S2#4) pair) displayed an improved detection limit of 0.6 μg/L (3.7 μg of /kg). Our immunoreactivity-directed species selection approach in the strategized antibody production significantly improved the detection of no or weakly immunoreactive fish species previously not detected immunochemically. Of 37 commercially important fish species tested, the ELISA-2 could detect 28 fish species (76%). The optimized sample extraction with a buffer additive achieved good protein recoveries of 87.2 – 117.3% (within the AOAC recommended range). The ELISA-2 was able to detect fish residues in five highly processed food products.

Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development.

Nanobodies (VHH) from camelid antibody libraries hold great promise as therapeutic agents and components of immunoassay systems. Synthetic antibody libraries that could be designed and generated once and for various applications could yield binders to virtually any targets, even for non-immunogenic or toxic ones, in a short term. One of the most difficult tasks is to obtain antibodies with a high affinity and specificity to polyglycosylated proteins. It requires antibody libraries with extremely high functional diversity and the use of sophisticated selection techniques. Here we report a development of a novel sandwich immunoassay involving a combination of the synthetic library-derived VHH-Fc fusion protein as a capture antibody and the immune single-chain fragment variable (scFv) as a tracer for the detection of pregnancy-associated glycoprotein (PAG) of cattle (Bos taurus). We succeeded in the generation of a number of specific scFv antibodies against PAG from the mouse immune library. Subsequent selection using the immobilized scFv-Fc capture antibody allowed to isolate 1.9 nM VHH binder from the diverse synthetic library without any overlapping with the capture antibody binding site. The prototype sandwich ELISA based on the synthetic VHH and the immune scFv was established. This is the first successful example of the combination of synthetic and immune antibody libraries in a single sandwich immunoassay. Thus, our approach could be used for the express isolation of antibody pairs and the development of sandwich immunoassays for challenging antigens. KEY POINTS: • Heavily glycosylated PAG Bos Taurus were used for immune library construction and specific scFv isolation by phage display. • Nanomolar affinity VHH for PAG was selected from the original synthetic nanobodies library. • A novel VHH/scFv-based immunoassay for Bos Taurus pregnancy determination was developed.

Abstract 1874: Use of high throughput SPR to characterize antibodies to common cancer biomarkers

Abstract As a disease of mis-regulation, cancer is characterized both genetically and proteomically, requiring high quality tools to dissect the change difference between cell states. Among the reagents used to characterize biological systems at the protein level, antibodies are routinely applied, and engineered versions have proven useful for treating multiple therapeutic indications. We have generated a human proteome wide toolbox of polyclonal antibodies, identifying three antigenic 14mer sequences within each of the 20,000+ open reading frames at N-terminal, middle and C-terminal regions. Using a high throughput SPR system, Carterra LSA, we are able to define average on-rates, off-rates, and KD’s for pAb’s generated to important cancer regulators and biomarkers, including KRAS, STAT3, and Gli1. Due to the small size of the peptide targets, the kinetics of the observed interactions with polyclonal antibodies exhibit remarkable 1:1 fit typically observed when measuring monoclonal antibody affinities. The platform is a sensitive system capable of both identifying multiple antibody-antigen interactions with KD in the nanomolar to picomolar range, but also as a mass screening tool for developing de novo antibody pairs. We define the kinetic characteristics of these antibody reagents relative to performance in western blotting specifically. Improving the characterization of such widely used tools is critical for advancing translational opportunities into use in the clinic. Citation Format: Sergei Bibikov, John Rosenfeld, Kevin J. Harvey. Use of high throughput SPR to characterize antibodies to common cancer biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1874.

Abstract 3919: MosaicNeedles: A tool for large-scale proteomics combining antibodies and aptamers

Abstract Proteomics have the potential to transform biomarker discovery, diagnostics, and personalized medicine as it provides functional and actionable information linked to disease phenotypes. However, mass spectrometry (MS) – based proteomics has not been possible at large scale due to limited dynamic range, complex workflow and high experimental cost per sample. Antibody-based methods, although widely used in targeted proteomics for their better sensitivity, have been limited to the profiling of a few hundred selected proteins, partly due to the limited availability of antibody pairs. NanoMosaic has demonstrated antibody-aptamer sandwich assays on the MosaicNeedle࣪ platform. This expands the content size from a few hundred to a few thousand, achievable in one reaction with high sensitivity and specificity and low cross-reactivity. MosaicNeedles࣪ are densely integrated nanoneedle sensors fabricated on a planar substrate. 94,000 sensors with more than 2 billion total nanoneedles can be integrated on to a standard SBS plate, which can also be grouped into 96, 384 or 1536 formats. Each sensor contains an array of nanoneedles, dedicated to detecting one analyte of interest. All nanoneedles comprising the same sensor are functionalized with the same capture antibodies or aptamers. At low analyte concentration, the binding of proteins to the nanoneedles follows a Poisson distribution. Therefore, statistically, no more than one molecule is bound per nanoneedle. A further addition of aptamers or antibodies will form a sandwich complex with the target analyte. Since each of the nanoneedles has an intrinsic optical resonance spectrum and will red-shift as the sandwich complex forms on its surface, the number of analytes can be quantitated by simply counting the number of nanoneedles that display a color change. At high analyte concentration, each nanoneedle has more than one analyte, so the number of analytes can be calculated by averaging the spectrum shifts of all nanoneedles. This combined single molecule counting (digital) and spectrum shift (analog) analysis allows the platform to detect both high abundance and low abundance protein analytes in one reaction. We have demonstrated that the aptamer-antibody assay on the MosaicNeedle࣪ platform achieves better sensitivity than the well-developed antibody-pair assay. It also achieves larger dynamic range and alleviates the antibody cross-reactivity by refining aptamers during the SELEX process. The scalability in manufacturing nanoneedles combined with the low sample requirement and no fluorescent labeling all help to drive down the cost-per-protein to enable large-scale, unbiased proteomics. We envision researchers will use MosaicNeedle࣪ platform for both the upstream target discovery phase and then transfer seamlessly to the downstream validation phase by building customized and targeted panels for proteins of high interest that emerge from the discovery phase. Citation Format: Qimin Quan, Alaina Kaiser, Joshua Ritchey, Joe Wilkinson, John Geanacopoulos, John Boyce. MosaicNeedles: A tool for large-scale proteomics combining antibodies and aptamers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3919.

Abstract 2902: Development of PSB202, a bifunctional antibody pair that target CD20 and CD37, for the treatment of B-cell malignancies

Abstract CD20 and CD37 are co-expressed on most B-NHL and CLL tumors. Systemic depletion of normal and malignant B-cells by anti-CD20 antibodies (e.g. rituximab and obinutuzumab) and anti-CD37 antibodies (e.g. otlertuzumab and BI 836826) has been well tolerated in clinical studies. Depletion of B-cells is reversible, with the ability to repopulate B-cells from hematopoietic stem cells. Dual targeting CD20 and CD37 may improve the clinical efficacy and decrease drug resistance. PSB202 is a novel biological entity composed of two engineered monoclonal antibodies which are produced from a single stable CHO cell line - a humanized anti-CD20 antibody (PSB102) and a humanized anti-CD37 antibody (PSB107) at a 1:1 molecular ratio. In vitro binding studies demonstrated that PSB202 specifically binds to human CD20 and CD37. The antibodies’ binding to FcγRIIIa was enhanced by reducing the fucose level on each antibody component. PSB202 directly induced potent autonomous killing via target engagement. PSB202 mediated depletion of human and monkey blood B-cells and malignant human B-cell lines through ADCC activity at EC50 of double digit pM and CDC activity at EC50 of low single digit nM. Importantly, PSB202 effectively depleted B-cells in ex vivo cultures of blood cells from CLL and NHL patients who were refractive to rituximab treatment. The antitumor activity of PSB202 was demonstrated in human xenograft models in mice. PSB202 was able to reduce different human lymphoma tumors by up to ~97.9% in a dose-dependent manner, suggesting that PSB202 might be potentially efficacious against multiple types of B-cell malignancies. Synergy was demonstrated for PSB202 in combination with lenalidomide. Toxicokinetic assessment in cynomolgus monkeys showed a dose proportional exposure for both antibody components, with mean terminal elimination half-life (T1/2) of ~60 and ~68 hours for the anti-CD20 and anti-CD37 component, respectively. PBS202 was well tolerated, with observed toxicities mostly related to its activity as a B-cell depletion agent. As expected, PSB202 when administered at ≥ 10 mg/kg resulted in nearly complete depletion of B lymphocytes 24 hours following the 1st dose. B-cells returned to near baseline within 2 months after the last dose at 10 mg/kg. PSB202 can deplete normal and malignant B-cells by targeting two clinically validated targets through multiple distinct mechanisms including direct B-cell killing, enhanced ADCC activity, and CDC activity. PSB202 offers the potential to improve clinical efficacy, decrease drug resistance, provide administration convenience especially when combining with additional therapeutics, lower the cost for treatment, and reduce the unwanted side effects from chemotherapy. PSB202 is currently being evaluated in patients with previously treated, relapsed, indolent B-cell malignancies (NCT05003141). Citation Format: Paul Algate, Zhi Liu, Saying Yuan, Hua Liu, Cristina Domeier, Haiming Jiang, Yuanzhi Lao, Yanling Xu, William C. Fanslow, Ron Schoner, Wei Yan. Development of PSB202, a bifunctional antibody pair that target CD20 and CD37, for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2902.

A fluorescent immunosensor on optical fibre for the multiplex detection of proinflammatory cytokines

Cytokines are typical mediators of the immune response. Single cytokine measurement is unable to reflect the true complexity of these physiological processes and multiplex detection is required. In this work, we developed an optical fibre based biosensor for multiplex detection of cytokines. This multiplex biosensor has been successfully applied for the measurement of proinflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the linear range of 12.5–200 pg mL−1 with the limit of detection (LOD) of 12.5 pg mL−1. Additionally, it has compatible performance with ELISA kit for the monitoring of cytokines from rat PBMC cell culture mediums stimulated by LPS or UV radiation. Moreover, this universal platform could be applied for multiplex detection of other proteins by simply changing the capture/detection antibody pairs. This fibre based multiplex biosensor is designed for in-situ measurement of target cytokines, which is significant for the investigation of inflammatory processes or disease prognosis and treatment monitoring.

Highly sensitive pregnancy test kit via oriented antibody conjugation on brush-type ligand-coated quantum beads

Lateral flow assays (LFA) enable development of portable and rapid diagnostic kits; however, their capacity to detect low levels of disease markers remains poor. Here, we report a highly sensitive pregnancy test kit as a proof of concept, by combining brush-type ligand-coated quantum beads (B-type QBs) and nanobody, which can control the antibody orientation and enhance sensitivity. The brush-type ligand provided excellent dispersion stability and high-binding capacity toward antibody. Fc-binding nanobody increased the antigen-binding capacity of conjugated antibodies on the B-type QBs. To facilitate convenient acquisition of the LFA results, we developed a smartphone-based reader with a 3D-printed optical imaging module, and validated the diagnostic performance of the sensing platform. The pregnancy test kit achieved a 5.1 pg mL−1 limit of detection, corresponding to the levels for early-stage detection of heart disease and malaria. Our LFA application can potentially be expanded to diagnosis other diseases by simply changing the antibody pair in the kit.

Identification and application of a pair of noncompeting monoclonal antibodies broadly binding to the nucleocapsid proteins of SARS-CoV-2 variants including Omicron

The SARS-CoV-2 nucleocapsid protein (NP) is an important indicator for the virus infection, highlighting the crucial role of NP-specific monoclonal antibodies (mAbs) used in multiple biochemical assays and clinical diagnosis for detecting the NP antigen. Here, we reported a pair of noncompeting human NP-specific mAbs, named P301-F7 and P301-H5, targeting two distinct linear epitopes on SARS-CoV-2 or SARS-CoV. We evaluated the application of P301-F7 in the analysis of enzyme linked immunosorbent assay, western blot, flow cytometry, immunofluorescence, and focus reduction neutralization test. We for the first time report a broad mAb effectively recognizing various live viruses of SARS-CoV-2 variants including Alpha, Beta, Delta, and Omicron, indicating a wide range of application prospects.

Simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodies

Amplification of immunofluorescence (IF) signals is becoming increasingly critical in cancer research and neuroscience. Recently, we put forward a new signal amplification technique, which we termed fluorescent signal amplification via cyclic staining of target molecules (FRACTAL). FRACTAL amplifies IF signals by repeatedly labeling target proteins with a pair of secondary antibodies that bind to each other. However, simultaneous amplification of multiple IF signals via FRACTAL has not yet been demonstrated because of cross-reactivity between the secondary antibodies. In this study, we show that mutual cross-adsorption between antibodies can eliminate all forms of cross-reactions between them, enabling simultaneous amplification of multiple IF signals. First, we show that a typical cross-adsorption process—in which an antibody binds to proteins with potential cross-reactivity with the antibody—cannot eliminate cross-reactions between antibodies in FRACTAL. Next, we show that all secondary antibodies used in FRACTAL need to be mutually cross-adsorbed to eliminate all forms of cross-reactivity, and then we demonstrate simultaneous amplification of multiple IF signals using these antibodies. Finally, we show that multiplexed FRACTAL can be applied to expansion microscopy to achieve higher fluorescence intensities after expansion. Multiplexed FRACTAL is a highly versatile tool for standard laboratories, as it amplifies multiple IF signals without the need for custom antibodies.

Rapid Optical Biosensing of SARS-CoV-2 Spike Proteins in Artificial Samples.

Tests for SARS-CoV-2 are crucial for the mass surveillance of the incidence of infection. The long waiting time for classic nucleic acid test results highlights the importance of developing alternative rapid biosensing methods. Herein, we propose a fiber-optic biolayer interferometry-based biosensor (FO-BLI) to detect SARS-CoV-2 spike proteins, extracellular domain (ECD), and receptor-binding domain (RBD) in artificial samples in 13 min. The FO-BLI biosensor utilized an antibody pair to capture and detect the spike proteins. The secondary antibody conjugated with horseradish peroxidase (HRP) reacted with the enzyme substrate for signal amplification. Two types of substrates, 3,3′-diaminobenzidine (DAB) and an advanced 3-Amino-9-ethylcarbazole (i.e., AMEC), were applied to evaluate their capabilities in enhancing signals and reaching high sensitivity. After careful comparison, the AMEC-based FO-BLI biosensor showed better assay performance, which detected ECD at a concentration of 32–720 pM and RBD of 12.5–400 pM in artificial saliva and serum, respectively. The limit of detection (LoD) for SARS-CoV-2 ECD and RBD was defined to be 36 pM and 12.5 pM, respectively. Morphology of the metal precipitates generated by the AMEC-HRP reaction in the fiber tips was observed using field emission scanning electron microscopy (SEM). Collectively, the developed FO-BLI biosensor has the potential to rapidly detect SARS-CoV-2 antigens and provide guidance for “sample-collect and result-out on-site” mode.

Development and performance of a point-of-care rapid antigen test for detection of SARS-COV-2 variants

BackgroundSARS-CoV-2 antigen-based tests are well-calibrated to infectiousness and have a critical role to play in the COVID-19 public health response. We report the development and performance of a unique lateral flow immunoassay (LFA). MethodsCombinations of several monoclonal antibodies targeting multiple antigenic sites on the SARS-CoV-2 nucleocapsid protein (NP) were isolated, evaluated, and chosen for the development of a LFA termed CoV-SCAN (BioMedomics, Inc.). Clinical point-of-care studies in symptomatic and asymptomatic individuals were conducted to evaluate positive predictive agreement (PPA) and negative predictive agreement (NPA) with RT-PCR as comparator. ResultsIn laboratory testing, CoV-SCAN detected 14 recombinant N-proteins of SARS-CoV-2 variants with sensitivity in the range of 0.2–3.2 ng/mL, and 10 authentic SARS-CoV-2 variants with sensitivity in the range of 1.6–12.5 TCID50/swab. No cross reactivity was observed with other human coronaviruses or other respiratory pathogens. In clinical point-of-care testing on 148 individuals over age 2 with symptoms of ≤5 days, PPA was 87.2% (CI 95: 78.3–94.8%) and NPA was 100% (CI 95: 94.2–100%). In another 884 asymptomatic individuals, PPA was 85.7% (CI 95: 42.1–99.6%) and 99.7% (99.0–99.9%). Overall, CoV-SCAN detected over 97.2% of specimens with CT values <30 and 93.8% of nasal swab specimens with the Omicron variant, even within the first 2 days after symptom onset. ConclusionsThe unique construction of CoV-SCAN using two pairs of monoclonal antibodies has resulted in a test with high performance that remains durable across multiple variants in both laboratory and clinical evaluations. CoV-SCAN should identify almost all individuals harboring infectious SARS-CoV-2. SummaryUnique construction of a point-of-care rapid antigen test using two pairs of monoclonal antibodies has led to good performance that remained durable across multiple variants in laboratory and clinical evaluations. Test should identify almost all individuals harboring infectious SARS-CoV-2.

Healthy humans can be a source of antibodies countering COVID-19

ABSTRACT Here, we describe the isolation of 18 unique anti SARS-CoV-2 human single-chain antibodies from an antibody library derived from healthy donors. The selection used a combination of phage and yeast display technologies and included counter-selection strategies meant to direct the selection of the receptor-binding motif (RBM) of SARS-CoV-2 spike protein’s receptor binding domain (RBD2). Selected antibodies were characterized in various formats including IgG, using flow cytometry, ELISA, high throughput SPR, and fluorescence microscopy. We report antibodies’ RBD2 recognition specificity, binding affinity, and epitope diversity, as well as ability to block RBD2 binding to the human receptor angiotensin-converting enzyme 2 (ACE2) and to neutralize authentic SARS-CoV-2 virus infection in vitro. We present evidence supporting that: 1) most of our antibodies (16 out of 18) selectively recognize RBD2; 2) the best performing 8 antibodies target eight different epitopes of RBD2; 3) one of the pairs tested in sandwich assays detects RBD2 with sub-picomolar sensitivity; and 4) two antibody pairs inhibit SARS-CoV-2 infection at low nanomolar half neutralization titers. Based on these results, we conclude that our antibodies have high potential for therapeutic and diagnostic applications. Importantly, our results indicate that readily available non immune (naïve) antibody libraries obtained from healthy donors can be used to select high-quality monoclonal antibodies, bypassing the need for blood of infected patients, and offering a widely accessible and low-cost alternative to more sophisticated and expensive antibody selection approaches (e.g. single B cell analysis and natural evolution in humanized mice).

Arterial antibodies promote atherosclerotic plaque development in mice

Abstract Atherosclerosis is characterized by the progressive buildup of cholesterol-rich plaques within the arterial wall. Plaques activate the immune system, of which B cells play a major role. Antibodies bind autoantigens released from necrotic cells and form immune complexes to increase inflammation. We isolated aorta-associated B cells from atherosclerotic ApoE-deficient mice on a high-fat diet and performed single cell sequencing of the B cell receptors to obtain antibody pairs. 32 over-represented antibodies were identified and then expressed in Expi293F cells. Epitope determination of these antibodies was performed against a human proteome array, and 15 potential autoantigens bound the monoclonal antibodies with significant affinity and specificity. The 12 most-promising antigens were scrutinized for their location in plaque tissue by immunohistochemistry using the cognate antibodies. Nine of these relevant antigens were then successfully synthesized using the pET bacterial expression system, and purified proteins were used to interrogate antibodies in human and mouse sera by ELISA. Mice were then immunized with the 5 best antigens to confirm their immunogenicity. The relevant antigens and antibodies are now being tested to see if they will protect or promote atherosclerosis development, and if they can be used as biomarkers in human disease. This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.