Last data update: May 06, 2024. (Total: 46732 publications since 2009)
Records 1-10 (of 10 Records) |
Query Trace: Goldstein JM[original query] |
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Rapid Development of Neutralizing and Diagnostic SARS-COV-2 Mouse Monoclonal Antibodies (preprint)
Chapman AP , Tang X , Lee JR , Chida A , Mercer K , Wharton RE , Kainulainen M , Harcourt JL , Martines RB , Schroeder M , Zhao L , Bryksin A , Zhou B , Bergeron E , Bollweg BC , Tamin A , Thornburg N , Wentworth DE , Petway D , Bagarozzi DA Jr , Finn MG , Goldstein JM . bioRxiv 2020 2020.10.13.338095 The need for high-affinity, SARS-CoV-2-specific monoclonal antibodies (mAbs) is critical in the face of the global COVID-19 pandemic, as such reagents can have important diagnostic, research, and therapeutic applications. Of greatest interest is the ~300 amino acid receptor binding domain (RBD) within the S1 subunit of the spike protein because of its key interaction with the human angiotensin converting enzyme 2 (hACE2) receptor present on many cell types, especially lung epithelial cells. We report here the development and functional characterization of 29 nanomolar-affinity mouse SARS-CoV-2 mAbs created by an accelerated immunization and hybridoma screening process. Differing functions, including binding of diverse protein epitopes, viral neutralization, impact on RBD-hACE2 binding, and immunohistochemical staining of infected lung tissue, were correlated with variable gene usage and sequence.Competing Interest StatementThe authors have declared no competing interest. |
High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay (preprint)
Kainulainen MH , Bergeron E , Chatterjee P , Chapman AP , Lee J , Chida A , Tang X , Wharton RE , Mercer KB , Petway M , Jenks HM , Flietstra TD , Schuh AJ , Satheshkumar PS , Chaitram JM , Owen SM , Finn MG , Goldstein JM , Montgomery JM , Spiropoulou CF . medRxiv 2020 2020.09.16.20195446 SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.One sentence summary Protein complementation enables mix-and-read SARS-CoV-2 serology that rivals sensitivity and specificity of ELISA but excels in throughput and quantitation.Competing Interest StatementThe authors have declared no competing interest.Funding StatementThis research was funded by the Centers for Disease Control and Prevention.Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Residual specimen materials were used for diagnostics development under a protocol that was reviewed and approved by the CDC Institutional Review Board (See 45 C.F.R. part 46; 21 C.F.R. part 56)All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesNo external data links |
Analysis of the initial lot of the CDC 2019-Novel Coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel.
Lee JS , Goldstein JM , Moon JL , Herzegh O , Bagarozzi DAJr , Oberste MS , Hughes H , Bedi K , Gerard D , Cameron B , Benton C , Chida A , Ahmad A , Petway DJJr , Tang X , Sulaiman N , Teklu D , Batra D , Howard D , Sheth M , Kuhnert W , Bialek SR , Hutson CL , Pohl J , Carroll DS . PLoS One 2021 16 (12) e0260487 At the start of the COVID-19 pandemic, the Centers for Disease Control and Prevention (CDC) designed, manufactured, and distributed the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel for SARS-CoV-2 detection. The diagnostic panel targeted three viral nucleocapsid gene loci (N1, N2, and N3 primers and probes) to maximize sensitivity and to provide redundancy for virus detection if mutations occurred. After the first distribution of the diagnostic panel, state public health laboratories reported fluorescent signal in the absence of viral template (false-positive reactivity) for the N3 component and to a lesser extent for N1. This report describes the findings of an internal investigation conducted by the CDC to identify the cause(s) of the N1 and N3 false-positive reactivity. For N1, results demonstrate that contamination with a synthetic template, that occurred while the "bulk" manufactured materials were located in a research lab for quality assessment, was the cause of false reactivity in the first lot. Base pairing between the 3' end of the N3 probe and the 3' end of the N3 reverse primer led to amplification of duplex and larger molecules resulting in false reactivity in the N3 assay component. We conclude that flaws in both assay design and handling of the "bulk" material, caused the problems with the first lot of the 2019-nCoV Real-Time RT-PCR Diagnostic Panel. In addition, within this study, we found that the age of the examined diagnostic panel reagents increases the frequency of false positive results for N3. We discuss these findings in the context of improvements to quality control, quality assurance, and assay validation practices that have since been improved at the CDC. |
High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay.
Kainulainen MH , Bergeron E , Chatterjee P , Chapman AP , Lee J , Chida A , Tang X , Wharton RE , Mercer KB , Petway M , Jenks HM , Flietstra TD , Schuh AJ , Satheshkumar PS , Chaitram JM , Owen SM , McMullan LK , Flint M , Finn MG , Goldstein JM , Montgomery JM , Spiropoulou CF . Sci Rep 2021 11 (1) 12330 SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA. |
Rapid development of neutralizing and diagnostic SARS-COV-2 mouse monoclonal antibodies.
Chapman AP , Tang X , Lee JR , Chida A , Mercer K , Wharton RE , Kainulainen M , Harcourt JL , Martines RB , Schroeder M , Zhao L , Bryksin A , Zhou B , Bergeron E , Bollweg BC , Tamin A , Thornburg N , Wentworth DE , Petway D , Bagarozzi DA Jr , Finn MG , Goldstein JM . Sci Rep 2021 11 (1) 9682 The need for high-affinity, SARS-CoV-2-specific monoclonal antibodies (mAbs) is critical in the face of the global COVID-19 pandemic, as such reagents can have important diagnostic, research, and therapeutic applications. Of greatest interest is the ~ 300 amino acid receptor binding domain (RBD) within the S1 subunit of the spike protein because of its key interaction with the human angiotensin converting enzyme 2 (hACE2) receptor present on many cell types, especially lung epithelial cells. We report here the development and functional characterization of 29 nM-affinity mouse SARS-CoV-2 mAbs created by an accelerated immunization and hybridoma screening process. Differing functions, including binding of diverse protein epitopes, viral neutralization, impact on RBD-hACE2 binding, and immunohistochemical staining of infected lung tissue, were correlated with variable gene usage and sequence. |
Comparison of Zika virus inactivation methods for reagent production and disinfection methods
Chida AS , Goldstein JM , Lee J , Tang X , Bedi K , Herzegh O , Moon JL , Petway D , Bagarozzi DAJr , Hughes LJ . J Virol Methods 2020 287 114004 Zika virus (ZIKV) infection remains a public health concern necessitating demand for long-term virus production for diagnostic assays and R&D activities. Inactivated virus constitutes an important component of the Trioplex rRT-PCR assay and serological IgM assay (MAC-ELISA). The aim of our study is to establish standard methods of ZIKV inactivation while maintaining antigenicity and RNA integrity. We tested viral supernatants by four different inactivation methods: 1. Heat inactivation at 56 °C and 60 °C; 2. Gamma-Irradiation; 3. Chemical inactivation by Beta-propiolactone (BPL) and 4. Fast-acting commercial disinfecting agents. Effectivity was measured by cytopathic effect (CPE) and plaque assay. RNA stability and antigenicity were measured by RT-PCR and MAC-ELISA, respectively. Results: Heat inactivation: Low titer samples, incubated at 56 °C for 2 hrs, showed neither CPE or plaques compared to high titer supernatants that required 2.5 hrs. Inactivation occurred at 60 °C for 60 min with all virus titers. Gamma irradiation: Samples irradiated at ≥3 Mrad for low virus concentrations and ≥5Mrad for high virus titer completely inactivated virus. Chemical Inactivation: Neither CPE nor plaques were observed with ≥0.045% BPL inactivation of ZIKV. Disinfectant: Treatment of viral supernatants with Micro-Chem Plus(TM), inactivated virus in 2 min, whereas, Ethanol (70%) and STERIS Coverage® Spray TB inactivated the virus in 5 min. |
Novel graphene-based biosensor for early detection of Zika virus infection
Afsahi S , Lerner MB , Goldstein JM , Lee J , Tang X , Bagarozzi DA Jr , Pan D , Locascio L , Walker A , Barron F , Goldsmith BR . Biosens Bioelectron 2017 100 85-88 We have developed a cost-effective and portable graphene-enabled biosensor to detect Zika virus with a highly specific immobilized monoclonal antibody. Field Effect Biosensing (FEB) with monoclonal antibodies covalently linked to graphene enables real-time, quantitative detection of native Zika viral (ZIKV) antigens. The percent change in capacitance in response to doses of antigen (ZIKV NS1) coincides with levels of clinical significance with detection of antigen in buffer at concentrations as low as 450pM. Potential diagnostic applications were demonstrated by measuring Zika antigen in a simulated human serum. Selectivity was validated using Japanese Encephalitis NS1, a homologous and potentially cross-reactive viral antigen. Further, the graphene platform can simultaneously provide the advanced quantitative data of nonclinical biophysical kinetics tools, making it adaptable to both clinical research and possible diagnostic applications. The speed, sensitivity, and selectivity of this first-of-its-kind graphene-enabled Zika biosensor make it an ideal candidate for development as a medical diagnostic test. |
Phage display analysis of monoclonal antibody binding to anthrax toxin lethal factor
Goldstein JM , Lee J , Tang X , Boyer AE , Barr JR , Bagarozzi DA Jr , Quinn CP . Toxins (Basel) 2017 9 (7) AVR1674 and AVR1675 are monoclonal antibodies (mAbs) that bind with high specificity to anthrax toxin lethal factor (LF) and lethal toxin (LTx). These mAbs have been used as pivotal reagents to develop anthrax toxin detection tests using mass spectrometry. The mAbs were demonstrated to bind LF with good affinity (KD 10−7–10−9 M) and to enhance LF-mediated cleavage of synthetic peptide substrates in vitro. Sequence analysis indicated that the mAbs shared 100% amino acid identity in their complementarity determining regions (CDR). A phage display library based on a combinatorial library of random heptapeptides fused to the pIII coat protein of M13 phage was enriched and screened to identify peptide sequences with mAb binding properties. Selection and sequence analysis of 18 anti-LF-reactive phage clones identified a 7-residue (P1–P7) AVR1674/1675 consensus target binding sequence of TP1-XP2-K/RP3-DP4-D/EP5-ZP6-X/ZP7 (X = aromatic, Z = non-polar). The phage peptide sequence with highest affinity binding to AVR1674/1675 was identified as T-F-K-D-E-I-V. Synthetic oligopeptides were designed based on the phage sequences and interacted with mAbs with high affinity (KD~ 10−9 M). Single amino acid substitutions of A, H, or Q in the peptides identified positions P1–P5 as critical residues for mAb-peptide interactions. CLUSTALW alignment of phage sequences with native LF implicated residues 644–650 (sequence T-H-Q-D-E-I-Y) as a putative linear epitope component located within a structural loop (L2) of LF Domain IV. The activation effects of these mAbs contribute to the analytic sensitivity of function-based LF detection assays. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. |
The salt-sensitive structure and zinc inhibition of Borrelia burgdorferi protease BbHtrA
Russell TM , Tang X , Goldstein JM , Bagarozzi D , Johnson BJ . Mol Microbiol 2015 99 (3) 586-96 HtrA serine proteases are highly conserved and essential ATP-independent proteases with chaperone activity. Bacteria express a variable number of HtrA homologs which contribute to the virulence and pathogenicity of bacterial pathogens. Lyme disease spirochetes possess a single HtrA protease homolog, Borrelia burgdorferi HtrA (BbHtrA). Previous studies established that, like the human orthologue HtrA1, BbHtrA is proteolytically active against numerous extracellular proteins in vitro. In this study, we utilized size exclusion chromatography and blue native polyacrylamide gel electrophoresis (BN-PAGE) to demonstrate BbHtrA oligomeric structures which were substrate-independent and salt sensitive. Examination of the influence of transition metals on the activity of BbHtrA revealed that this protease is inhibited by Zn2+ >Cu2+ >Mn2+ . Extending this analysis to two other HtrA proteases, E. coli DegP and HtrA1, revealed that all three HtrA proteases were reversibly inhibited by ZnCl2 at all micro molar concentrations examined. Commercial inhibitors for HtrA proteases are not available and physiologic HtrA inhibitors are unknown. Our observation of conserved zinc inhibition of HtrA proteases will facilitate structural and functional studies of additional members of this important class of proteases. |
In vitro growth, cytopathic effects and clearance of monolayers by clinical isolates of Balamuthia mandrillaris in human skin cell cultures
Yera H , Dupouy-Camet J , Jackson JW , Sriram R , Sweat S , Goldstein JM , Visvesvara GS . Exp Parasitol 2015 156 61-7 Balamuthia mandrillaris is a free-living ameba (FLA) that has been isolated or its DNA identified in soil, dust and water. It causes a fatal central nervous system infection in humans and animals. Although it is environmental as Acanthamoeba and Naegleria fowleri, the two other free-living amebae that also cause CNS infections in humans and other animals, Balamuthia does not feed on bacteria as the other FLA. In the laboratory, it can be grown on a variety of mammalian cell cultures. In this study we examined the ability of three different Balamuthia isolates to grow on several different human skin cell cultures including the WT/A keratinocyte cell cultures. A corneal isolate of Acanthamoeba castellanii was used for comparison. |
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