Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
2.
Sci Transl Med ; 13(588)2021 04 07.
Article in English | MEDLINE | ID: mdl-33827978

ABSTRACT

Ebola virus (EBOV) hemorrhagic fever outbreaks have been challenging to deter due to the lack of health care infrastructure in disease-endemic countries and a corresponding inability to diagnose and contain the disease at an early stage. EBOV vaccines and therapies have improved disease outcomes, but the advent of an affordable, easily accessed, mass-produced rapid diagnostic test (RDT) that matches the performance of more resource-intensive polymerase chain reaction (PCR) assays would be invaluable in containing future outbreaks. Here, we developed and demonstrated the performance of a new ultrasensitive point-of-care immunoassay, the EBOV D4 assay, which targets the secreted glycoprotein of EBOV. The EBOV D4 assay is 1000-fold more sensitive than the U.S. Food and Drug Administration-approved RDTs and detected EBOV infection earlier than PCR in a standard nonhuman primate model. The EBOV D4 assay is suitable for low-resource settings and may facilitate earlier detection, containment, and treatment during outbreaks of the disease.


Subject(s)
Hemorrhagic Fever, Ebola , Point-of-Care Systems , Animals , Ebolavirus , Glycoproteins , Hemorrhagic Fever, Ebola/diagnosis , Immunoassay , Polymerase Chain Reaction
3.
J Immunol Methods ; 340(2): 144-53, 2009 Jan 30.
Article in English | MEDLINE | ID: mdl-19017532

ABSTRACT

The generation of recombinant single-chain antibodies from either non-immune or immune phage display antibody libraries is an effective means to obtain high affinity antibodies against a specific target. Non-immune libraries contain a wide variety of antibodies but these are often low affinity. Immune libraries contain a high frequency of high affinity antibodies, but are typically limited to a single antigen. Due to the V(H) and V(L) recombination that occurs during antibody library construction, we hypothesized that an immune antibody library produced against one member of a protein family would contain antibodies specific for other members of the same protein family. Here, we tested this hypothesis by mining an existing anti-human Toll-like receptor-2 (hTLR2) antibody library for antibodies specific for other members of the TLR family. This procedure, referred to as homolog mining, proved to be effective. Using a cell-based system to pan and screen the anti-TLR2 library, we identified single chain antibodies specific for three of the four hTLR2 homologs we targeted. The antibodies identified, anti-murine TLR2, anti-hTLR5, and anti-hTLR6, bind specifically to their target, with no cross-reactivity to hTLR2 or other TLRs tested. These results demonstrate that combinatorial re-assortment of V(H) and V(L) fragments from multiple sources during Ab library construction increases Ab repertoire complexity, allowing antibody libraries produced by immunization with one antigen to be used to obtain antibodies specific to related antigens. The principle of homolog mining may be extended to other protein families and will facilitate and accelerate antibody production processes.


Subject(s)
Antibodies/analysis , Antibodies/immunology , Immunoglobulin Variable Region/analysis , Immunoglobulin Variable Region/immunology , Peptide Library , Toll-Like Receptors/immunology , Animals , Antibodies/genetics , Antibodies/metabolism , Antibody Specificity/immunology , Cells, Cultured , Humans , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/metabolism , Mice , Mice, Inbred C57BL , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism
4.
J Mol Biol ; 379(2): 261-72, 2008 May 30.
Article in English | MEDLINE | ID: mdl-18455737

ABSTRACT

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.


Subject(s)
Biological Assay/methods , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/metabolism , Receptors, Cell Surface/metabolism , Toll-Like Receptor 2/metabolism , Animals , Antibody Affinity , Antibody Specificity , Cell Line , Drosophila melanogaster/physiology , Humans , Immunoglobulin Fragments/genetics , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Peptide Library , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Toll-Like Receptor 2/chemistry , Toll-Like Receptor 2/genetics
5.
Cancer Res ; 67(10): 4716-24, 2007 May 15.
Article in English | MEDLINE | ID: mdl-17510399

ABSTRACT

The antiangiogenic protein angiostatin inhibits ATP synthase on the endothelial cell surface, blocking cellular proliferation. To examine the specificity of this interaction, we generated monoclonal antibodies (mAb) directed against ATP synthase. mAb directed against the beta-catalytic subunit of ATP synthase (MAb3D5AB1) inhibits the activity of the F(1) domain of ATP synthase and recognizes the catalytic beta-subunit of ATP synthase. We located the antibody recognition site of MAb3D5AB1 in domains containing the active site of the beta-subunit. MAb3D5AB1 also binds to purified Escherichia coli F(1) with an affinity 25-fold higher than the affinity of angiostatin for this protein. MAb3D5AB1 inhibits the hydrolytic activity of F(1) ATP synthase at lower concentrations than angiostatin. Like angiostatin, MAb3D5AB1 inhibits ATP generation by ATP synthase on the endothelial cell surface in acidic conditions, the typical tumor microenvironment where cell surface ATP synthase exhibits greater activity. MAb3D5AB1 disrupts tube formation and decreases intracellular pH in endothelial cells exposed to low extracellular pH. Neither angiostatin nor MAb3D5AB1 showed an antiangiogenic effect in the corneal neovascularization assay; however, both were effective in the low-pH environment of the chicken chorioallantoic membrane assay. Thus, MAb3D5AB1 shows angiostatin-like properties superior to angiostatin and may be exploited in cancer chemotherapy.


Subject(s)
Angiostatins/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Mitochondrial Proton-Translocating ATPases/immunology , Adenosine Triphosphate/biosynthesis , Animals , Antibodies, Monoclonal/metabolism , Binding Sites, Antibody , Biomimetic Materials , Catalytic Domain/immunology , Cattle , Chorioallantoic Membrane/blood supply , Corneal Neovascularization/drug therapy , Endothelial Cells/cytology , Endothelial Cells/drug effects , Epitope Mapping , Female , Humans , Hydrogen-Ion Concentration , Mice , Mice, Inbred BALB C , Mitochondrial Proton-Translocating ATPases/antagonists & inhibitors , Mitochondrial Proton-Translocating ATPases/metabolism , Models, Molecular , Neovascularization, Physiologic/drug effects , Rats , Rats, Inbred F344
6.
RNA ; 8(6): 762-71, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12088149

ABSTRACT

Patients with systemic autoimmune disorders produce autoantibodies against sequence-specific conformational RNA epitopes on U1 snRNA, 28S rRNA, and transfer RNAs. The molecular basis for immunological reactivity with these highly abundant and stable RNAs is not understood. Here, we report the existence of discrete RNA epitopes in messenger RNAs that are generally less abundant and less stable than snRNAs and tRNAs. An iterative selection and amplification procedure using pooled autoimmune patient sera identified immunoreactive mRNA species. Following deconvolution of the pools to identify the reactive sera, several mRNAs recognized by these autoantibodies were cloned and sequenced. Detailed analysis using one particular serum indicated reactivity against the messages encoding alternative splicing factor (ASF/SF2) and calmodulin. Deletion and site-directed mutagenesis determined that an epitope recognized by this serum is located in a 17-base stem-loop structure common to both messages. This serum was then used to immunoprecipitate native mRNAs encoding ASF/SF2 and calmodulin from total HeLa cell RNA. Our results demonstrate that despite its low abundance and instability, messenger RNA is capable of reacting with autoantibodies generated during an autoimmune response. These data are consistent with direct presentation as a model to explain the generation of RNA conformation-specific autoantibodies.


Subject(s)
Autoantibodies/chemistry , Epitopes/analysis , RNA, Messenger/chemistry , Autoantibodies/immunology , Base Sequence , Calmodulin/genetics , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Precipitin Tests , Protein Conformation
SELECTION OF CITATIONS
SEARCH DETAIL
...