scholarly journals Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

Sensors ◽  
2021 ◽  
Vol 21 (14) ◽  
pp. 4814
Author(s):  
Shira Roth ◽  
Amos Danielli
Keyword(s):  
Neutralizing Antibodies ◽  
Screening Tool ◽  
Dynamic Range ◽  
Specific Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Angiotensin Converting Enzyme 2 ◽  
Inhibitor Screening ◽  
Drug Candidates ◽  
Potential Inhibitors

Inhibitor screening is an important tool for drug development, especially during the COVID-19 pandemic. The most used in vitro inhibitor screening tool is an enzyme-linked immunosorbent assay (ELISA). However, ELISA-based inhibitor screening is time consuming and has a limited dynamic range. Using fluorescently and magnetically modulated biosensors (MMB), we developed a rapid and sensitive inhibitor screening tool. This study demonstrates its performance by screening small molecules and neutralizing antibodies as potential inhibitors of the interaction between the spike protein 1 (S1) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the angiotensin-converting enzyme 2 (ACE2) receptor. The MMB-based assay is highly sensitive, has minimal non-specific binding, and is much faster than the commonly used ELISA (2 h vs. 7–24 h). We anticipate that our method will lead to a remarkable advance in screening for new drug candidates.

scholarly journals Viral Protein VP4 Is a Target of Human Antibodies Enhancing Coxsackievirus B4- and B3-Induced Synthesis of Alpha Interferon

2005 ◽  
Vol 79 (22) ◽  
pp. 13882-13891 ◽  
Author(s):  
Wassim Chehadeh ◽  
Pierre-Emmanuel Lobert ◽  
Pierre Sauter ◽  
Anne Goffard ◽  
Bernadette Lucas ◽  
...  
Keyword(s):  
Healthy Subjects ◽  
Neutralizing Antibodies ◽  
Mononuclear Cells ◽  
Insulin Dependent Diabetes Mellitus ◽  
Alpha Interferon ◽  
Dependent Diabetes Mellitus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peripheral Blood Mononuclear ◽  
Coxsackievirus B4

ABSTRACT Coxsackievirus B4 (CVB4)-induced production of alpha interferon (IFN-α) by peripheral blood mononuclear cells (PBMC) is enhanced in vitro by nonneutralizing anti-CVB4 antibodies from healthy subjects and, to a higher extent, from patients with insulin-dependent diabetes mellitus. In this study, we focused on identification of the viral target of these antibodies in CVB systems. High levels of IFN-α were obtained in supernatants of PBMC incubated with CVB4E2 or CVB3 and plasma from healthy subjects and, to a higher extent, from patients. The VP4 capsid proteins dissociated by heating at 56°C from CVB4E2 (VP4CVB4) and CVB3 (VP4CVB3) but not H antigen preincubated with plasma from healthy subjects or patients inhibited the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis. There was no cross-reaction between VP4CVB4 and VP4CVB3 in the inhibiting effect. IFN-α levels in culture supernatants showed dose-dependent correlation with anti-VP4 antibodies eluted from plasma specimens using VP4-coated plates. There were higher index values for anti-VP4 antibodies detected by enzyme-linked immunosorbent assay (ELISA) and higher proportions of positive detection in 40 patients than in 40 healthy subjects (80% versus 15% for anti-VP4CVB4). There was no relationship between the levels of anti-CVB neutralizing antibodies and the detection of anti-VP4 antibodies by ELISA. The CVB plasma-induced IFN-α levels obtained in PBMC cultures in the anti-VP4 antibody-positive groups were significantly higher than those obtained in the anti-VP4 antibody-negative groups regardless of the titers of anti-CVB neutralizing antibodies. These results show that VP4 is the target of antibodies involved in the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis by PBMC.

scholarly journals Distinct Contributions of Vaccine-Induced Immunoglobulin G1 (IgG1) and IgG2a Antibodies to Protective Immunity against Influenza

2006 ◽  
Vol 13 (9) ◽  
pp. 981-990 ◽  
Author(s):  
Victor C. Huber ◽  
Raelene M. McKeon ◽  
Martha N. Brackin ◽  
Laura A. Miller ◽  
Rachael Keating ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Neutralizing Antibodies ◽  
Protective Immunity ◽  
Influenza Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Replicon Particle ◽  
Antibody Isotypes ◽  
The Individual

ABSTRACT Vaccination represents the most effective form of protection against influenza infection. While neutralizing antibodies are typically measured as a correlate of vaccine-induced protective immunity against influenza, nonneutralizing antibodies may contribute to protection or amelioration of disease. The goal of this study was to dissect the individual contributions of the immunoglobulin G1 (IgG1) and IgG2a antibody isotypes to vaccine-induced immunity against influenza virus. To accomplish this, we utilized an influenza vaccine regimen that selectively enhanced IgG1 or IgG2a antibodies by using either DNA or viral replicon particle (VRP) vectors expressing influenza virus hemagglutinin (HA) (HA-DNA or HA-VRP, respectively). After HA-DNA vaccination, neutralizing antibodies were detected by both in vitro (microneutralization) and in vivo (lung viral titer) methods and were associated with increased IgG1 expression by enzyme-linked immunosorbent assay (ELISA). Vaccination with HA-VRP did not strongly stimulate either neutralizing or IgG1 antibodies but did induce IgG2a antibodies. Expression of IgG2a antibodies in this context correlated with clearance of virus and increased protection against lethal influenza challenge. Increased induction of both antibody isotypes as measured by ELISA was a better correlate for vaccine efficacy than neutralization alone. This study details separate but important roles for both IgG1 and IgG2a expression in vaccination against influenza and argues for the development of vaccine regimens that stimulate and measure expression of both antibody isotypes.

scholarly journals Generation of a Candidate Live Marker Vaccine for Equine Arteritis Virus by Deletion of the Major Virus Neutralization Domain

2003 ◽  
Vol 77 (15) ◽  
pp. 8470-8480 ◽  
Author(s):  
Javier Castillo-Olivares ◽  
Roeland Wieringa ◽  
Tamás Bakonyi ◽  
Antoine A. F. de Vries ◽  
Nick J. Davis-Poynter ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Rna Virus ◽  
Viral Envelope ◽  
Equine Arteritis Virus ◽  
Wild Type Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Challenge Virus ◽  
Marker Vaccine

ABSTRACT Equine arteritis virus (EAV) is an enveloped plus-strand RNA virus of the family Arteriviridae (order Nidovirales) that causes respiratory and reproductive disease in equids. Protective, virus-neutralizing antibodies (VNAb) elicited by infection are directed predominantly against an immunodominant region in the membrane-proximal domain of the viral envelope glycoprotein GL, allowing recently the establishment of a sensitive peptide enzyme-linked immunosorbent assay (ELISA) based on this particular domain (J. Nugent et al., J. Virol. Methods 90:167-183, 2000). By using an infectious cDNA we have now generated, in the controlled background of a nonvirulent virus, a mutant EAV from which this immunodominant domain was deleted. This virus, EAV-GLΔ, replicated to normal titers in culture cells, although at a slower rate than wild-type EAV, and caused an asymptomatic infection in ponies. The antibodies induced neutralized the mutant virus efficiently in vitro but reacted poorly to wild-type EAV strains. Nevertheless, when inoculated subsequently with virulent EAV, the immunized animals, in contrast to nonvaccinated controls, were fully protected against disease; replication of the challenge virus occurred briefly at low though detectable levels. The levels of protection achieved suggest that an immune effector mechanism other than VNAb plays an important role in protection against infection. As expected, infection with EAV-GLΔ did not induce a measurable response in our GL-peptide ELISA while the challenge infection of the animals clearly did. EAV-GLΔ or similar mutants are therefore attractive marker vaccine candidates, enabling serological discrimination between vaccinated and wild-type virus-infected animals.

scholarly journals A New Strategy for Rapidly Screening Natural Inhibitors Targeting the PCSK9/LDLR Interaction In Vitro

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2397 ◽  
Author(s):  
Li Li ◽  
Chen Shen ◽  
Ya-Xuan Huang ◽  
Ya-Nan Li ◽  
Xiu-Feng Liu ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Lipoprotein Receptor ◽  
Drug Candidates ◽  
Promising Target ◽  
New Strategy ◽  
Density Lipoprotein Receptor ◽  
Potential Inhibitors ◽  
Natural Inhibitors

The interaction between proprotein convertase subtilisin/kexin type 9 (PCSK9) and the low-density lipoprotein receptor (LDLR) is a promising target for the treatment of hyperc-holesterolemia. In this study, a new method based on competitive affinity and tag detection was developed, which aimed to evaluate potent natural inhibitors preventing the interaction of PCSK9/LDLR directly. Herein, natural compounds with efficacy in the treatment of hypercholesterolemia were chosen to investigate their inhibitory activities on the PCSK9/LDLR interaction. Two of them, polydatin (1) and tetrahydroxydiphenylethylene-2-O-glucoside (2), were identified as potential inhibitors for the PCSK9/LDLR interaction and were proven to prevent PCSK9-mediated LDLR degradation in HepG2 cells. The results suggested that this strategy could be applied for evaluating potential bioactive compounds inhibiting the interaction of PCSK9/LDLR and this strategy could accelerate the discovery of new drug candidates for the treatment of PCSK9-mediated hypercholesterolemia.

scholarly journals Mucosal but Not Parenteral Immunization with Purified Human Papillomavirus Type 16 Virus-Like Particles Induces Neutralizing Titers of Antibodies throughout the Estrous Cycle of Mice

1999 ◽  
Vol 73 (11) ◽  
pp. 9609-9613 ◽  
Author(s):  
Denise Nardelli-Haefliger ◽  
Richard Roden ◽  
Carole Balmelli ◽  
Alexandra Potts ◽  
John Schiller ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Estrous Cycle ◽  
Neutralizing Antibodies ◽  
Female Genital Tract ◽  
Neutralization Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Virus Like Particles ◽  
Human Papillomavirus Type 16 ◽  
Parenteral Immunization

ABSTRACT We have recently shown that nasal immunization of anesthetized mice with human papillomavirus type 16 (HPV16) virus-like particles (VLPs) is highly effective at inducing both neutralizing immunoglobulin A (IgA) and IgG in genital secretions, while parenteral immunization induced only neutralizing IgG. Our data also demonstrated that both isotypes are similarly neutralizing according to an in vitro pseudotyped neutralization assay. However, it is known that various amounts of IgA and IgG are produced in genital secretions along the estrous cycle. Therefore, we have investigated how this variation influences the amount of HPV16 neutralizing antibodies induced after immunization with VLPs. We have compared parenteral and nasal protocols of vaccination with daily samplings of genital secretions of mice. Enzyme-linked immunosorbent assay analysis showed that total IgA and IgG inversely varied along the estrous cycle, with the largest amounts of IgA in proestrus-estrus and the largest amount of IgG in diestrus. This resulted in HPV16 neutralizing titers of IgG only being achieved during diestrus upon parenteral immunization. In contrast, nasal vaccination induced neutralizing titers of IgA plus IgG throughout the estrous cycle, as confirmed by in vitro pseudotyped neutralization assays. Our data suggest that mucosal immunization might be more efficient than parenteral immunization at inducing continuous protection of the female genital tract.

scholarly journals Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

2020 ◽  
Author(s):  
Hua-Long Xiong ◽  
Yang-Tao Wu ◽  
Jia-Li Cao ◽  
Ren Yang ◽  
Jian Ma ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Neutralizing Antibodies ◽  
Neutralization Assay ◽  
Cell Number ◽  
New Drugs ◽  
Human Society ◽  
Angiotensin Converting Enzyme 2 ◽  
Global Pandemic ◽  
Vesicular Stomatitis

AbstractThe global pandemic of Coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable in vitro neutralization assay is very important for the development of neutralizing antibodies, vaccines and other inhibitors. In this study, G protein-deficient vesicular stomatitis virus (VSVdG) bearing full-length and truncated spike (S) protein of SARS-CoV-2 were evaluated. The virus packaging efficiency of VSV-SARS-CoV-2-Sdel18 (S with C-terminal 18 amino acid truncation) is much higher than VSV-SARS-CoV-2-S. A neutralization assay for antibody screening and serum neutralizing titer quantification was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and human angiotensin-converting enzyme 2 (ACE2) overexpressed BHK21 cell (BHK21-hACE2). The experimental results can be obtained by automatically counting EGFP positive cell number at 12 hours after infection, making the assay convenient and high-throughput. The serum neutralizing titer of COVID-19 convalescent patients measured by VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with live SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting receptor binding domain (RBD) of SARS-CoV-2-S were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

Immunosurveillance of Alglucerase Enzyme Therapy for Gaucher Patients: Induction of Humoral Tolerance in Seroconverted Patients After Repeat Administration

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 2081-2088 ◽  
Author(s):  
Mireille Rosenberg ◽  
Wytske Kingma ◽  
Mary Anne Fitzpatrick ◽  
Susan M. Richards
Keyword(s):  
Neutralizing Antibodies ◽  
Clinical Symptoms ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inhibitory Antibody ◽  
Enzyme Replacement ◽  
Repeat Administration ◽  
Number Of Patients ◽  
Initiation Of Therapy ◽  
Humoral Tolerance

Alglucerase, a macrophage-targeted enzyme replacement therapy for Gaucher disease, has been successfully used for several years to improve clinical symptoms and reverse disease progression. As part of an immunosurveillance program, 1,122 Gaucher patients were monitored for antibody response to glucocerebrosidase, the active component of alglucerase. Seroconversion was detected in 142 patients (12.8%) by enzyme-linked immunosorbent assay (ELISA) and confirmed by radioimmunoprecipitation. The majority (75%) of the seroconverted population had no detectable levels of circulating inhibitory antibody as assessed by in vitro inhibition of enzymatic activity of the therapeutic molecule. Of the remaining patients with putative inhibitory antibodies, the majority had only low levels of serum inhibitory activity, which was transient. A very small number of patients were identified as developing true neutralizing antibodies, as defined by the development of antibodies that impacted clinical efficacy. Many of the patient antibody responses were also diminished with time. Eighty-two of the 142 seroconverted patients have stopped producing antibody to the molecule and appear tolerized. The mean time for humoral tolerization was 28 months from initiation of therapy. Of 64 seroconverted patients followed for at least 30 months of therapy, the tolerization rate was 93%. These results show that although 12.8% of the patients on therapy developed antibodies to the molecule, 90% of these patients became tolerized over time.

The human anti-CD30 antibody 5F11 shows in vitro and in vivo activity against malignant lymphoma

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3737-3742 ◽  
Author(s):  
Peter Borchmann ◽  
John F. Treml ◽  
Hinrich Hansen ◽  
Claudia Gottstein ◽  
Roland Schnell ◽  
...  
Keyword(s):  
Cell Lines ◽  
Specific Binding ◽  
Severe Combined Immunodeficiency ◽  
Scid Mice ◽  
Growth Delay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Complete Regression ◽  
Term Survival

AbstractCD30 is a promising target for antibody-based immunotherapy of Hodgkin lymphoma (HL) and anaplastic large cell lymphoma. To overcome the limitations from currently available murine anti-CD30 monoclonal antibodies (mAbs), a new fully human anti-CD30 antibody was generated. Binding properties were evaluated by recombinant CD30 capture enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell-sorter (FACS) flow cytometry. Activity of this new mAb was assessed in vitro using growth inhibition and antibody-dependent cellular cytotoxicity (ADCC) assays on several cell lines. In vivo activity was determined in a solid as well as in a disseminated xenografted model of HL in severe combined immunodeficiency (SCID) mice. The mAb 5F11 showed specific binding to CD30 (cluster A). The ADCC assays indicated dose-dependent lysis of L540 cells when 5F11 was combined with human effector cells. Upon cross-linking in vitro, 5F11 inhibited the growth of CD30-expressing cell lines. In vivo, treatment with 5F11 induced a marked growth delay or even a complete regression of established xenografted HL in SCID mice. In the disseminated HL model, a high proportion of 5F11-treated mice experienced long-term survival. The new human anti-CD30 monoclonal antibody 5F11 shows promise as a means of CD30-targeted immunotherapy of malignant lymphomas. Based on these results, a clinical phase 1 study in patients with refractory CD30+ lymphoma has been initiated. (Blood. 2003;102:3737-3742)

In-Vitro Diagnosis of Colon Cancer Using Bio-Functionalized Magnetic Nanoparticles

2011 ◽  
Author(s):  
Chin-Yih Hong ◽  
Shieh-Yueh Yang ◽  
K. W. Huang ◽  
Herng-Er Horng ◽  
Hong-Chang Yang
Keyword(s):  
Colon Cancer ◽  
Magnetic Nanoparticles ◽  
Detection Limit ◽  
Dynamic Range ◽  
Risk Level ◽  
Enzyme Linked Immunosorbent Assay ◽  
Detection Range ◽  
Low Detection Limit ◽  
In Vitro Diagnosis

The popular bio-marker for colon cancer is carcinoembryonic antigen (CEA). By conjugating anti-CEA onto magnetic nanoparticles, CEA can be specially labeled and detected by measuring magnetic signals via immunomagnetic reduction (IMR). The low detection limit and detection range of IMR on CEA are investigated. The results are compared with those by using the existing assay, such as enzyme-linked immunosorbent assay (ELISA). It is evidenced that IMR has sensitivity much higher than that of ELISA. The low detection limit is below the normal level of CEA concentration of clinic practice and is suitable for early-stage in-vitro diagnosis for colon cancer. Furthermore, the dynamic range of detection for the CEA concentration using IMR extends well above the threshold of high risk level of colorectal carcinoma in current diagnosing practice. Therefore, IMR is also suitable in other stages diagnosis for colon cancer development.

scholarly journals Computational drug repurposing strategy predicted peptide-based drugs that can potentially inhibit the interaction of SARS-CoV-2 spike protein with its target (humanACE2)

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245258
Author(s):  
Samuel Egieyeh ◽  
Elizabeth Egieyeh ◽  
Sarel Malan ◽  
Alan Christofells ◽  
Burtram Fielding
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Protein Interaction ◽  
Drug Repurposing ◽  
Spike Protein ◽  
Converting Enzyme ◽  
Peptide Drugs ◽  
Angiotensin Converting Enzyme 2 ◽  
Protein Protein Interaction ◽  
Potential Inhibitors

Drug repurposing for COVID-19 has several potential benefits including shorter development time, reduced costs and regulatory support for faster time to market for treatment that can alleviate the current pandemic. The current study used molecular docking, molecular dynamics and protein-protein interaction simulations to predict drugs from the Drug Bank that can bind to the SARS-CoV-2 spike protein interacting surface on the human angiotensin-converting enzyme 2 (hACE2) receptor. The study predicted a number of peptide-based drugs, including Sar9 Met (O2)11-Substance P and BV2, that might bind sufficiently to the hACE2 receptor to modulate the protein-protein interaction required for infection by the SARS-CoV-2 virus. Such drugs could be validated in vitro or in vivo as potential inhibitors of the interaction of SARS-CoV-2 spike protein with the human angiotensin-converting enzyme 2 (hACE2) in the airway. Exploration of the proposed and current pharmacological indications of the peptide drugs predicted as potential inhibitors of the interaction between the spike protein and hACE2 receptor revealed that some of the predicted peptide drugs have been investigated for the treatment of acute respiratory distress syndrome (ARDS), viral infection, inflammation and angioedema, and to stimulate the immune system, and potentiate antiviral agents against influenza virus. Furthermore, these predicted drug hits may be used as a basis to design new peptide or peptidomimetic drugs with better affinity and specificity for the hACE2 receptor that may prevent interaction between SARS-CoV-2 spike protein and hACE2 that is prerequisite to the infection by the SARS-CoV-2 virus.

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