ABSTRACT
The 12 distinct subtypes that comprise the interferon alpha (IFNα) family of cytokines possess anti-viral, anti-proliferative and immunomodulatory activities. They are implicated in the etiology and progression of many diseases, and also used as therapeutic agents for viral and oncologic disorders. However, a deeper understanding of their role in disease is limited by a lack of tools to evaluate single subtypes at the protein level. Antibodies that selectively inhibit single IFNα subtypes could enable interrogation of each protein in biological samples and could be used for characterization and treatment of disease. Using phage-displayed synthetic antibody libraries, we have conducted selections against 12 human IFNα subtypes to explore our ability to obtain fine-specificity antibodies that recognize and antagonize the biological signals induced by a single IFNα subtype. For the first time, we have isolated antibodies that specifically recognize individual IFNα subtypes (IFNα2a/b, IFNα6, IFNα8b and IFNα16) with high affinity that antagonize signaling. Our results show that highly specific antibodies capable of distinguishing between closely related cytokines can be isolated from synthetic libraries and can be used to characterize cytokine abundance and function.
Subject(s)
Antibodies, Immobilized/chemistry , Antibodies/chemistry , Immunoglobulin Fab Fragments/chemistry , Interferon-alpha/chemistry , Peptide Library , Amino Acid Sequence , Antibodies/genetics , Antibodies, Immobilized/biosynthesis , Antibodies, Immobilized/genetics , Antibody Specificity , Bacteriophages/genetics , Bacteriophages/metabolism , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genes, Reporter , Humans , Immunoglobulin Fab Fragments/biosynthesis , Immunoglobulin Fab Fragments/genetics , Interferon-alpha/antagonists & inhibitors , Interferon-alpha/metabolism , Kinetics , Luciferases/genetics , Luciferases/metabolism , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
A combined capture and detection method comprising of nano-immunomagnetic separation (NIMS) and surface enhanced Raman spectroscopy (SERS) was developed to detect Escherichia coli O157 from liquid media including apple juice. The capture antibodies (cAbs) were immobilized on magnetite-gold (Fe3O4/Au) magnetic nanoparticles (MNPs) which were used for separation and concentration of the E. coli O157 cells from model liquid food matrix. The capture efficiency (CE) for E. coli O157 using MNP was found to be approximately 84-94%. No cross reactivity was observed with background non-target organisms. There was a significant difference in the mean CE of bacteria captured by MNP and commercially sourced immunomagnetic microbeads (p<0.05). For the detection of target pathogen, SERS labels were prepared by conjugating gold nanoparticles with Raman reporter molecules and the detector antibody (dAb). Au-Raman label-dAb was interacted with gold coated MNP-cAb-E. coli O157 complex. The ability of this immunoassay to detect E. coli O157 in apple juice was investigated. We have successfully applied the synthesized Fe3O4/Au nanoclusters to E. coli O157 detection in apple juice using the SERS method. The lowest detectable bacterial cell concentration in apple juice was 10(2)CFU/mL with a total analysis time of less than an hour. This method presents a convenient way of preconcentration, separation, and detection of low levels of target pathogen from liquid food matrix.
Subject(s)
Beverages/microbiology , Escherichia coli O157/isolation & purification , Immunoassay/methods , Immunomagnetic Separation/methods , Malus/microbiology , Antibodies, Immobilized/biosynthesis , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/isolation & purification , Ferrosoferric Oxide/chemistry , Gold/chemistry , Limit of Detection , Magnetite Nanoparticles/chemistry , Spectrum Analysis, RamanABSTRACT
A highly robust immunoassay applicable for the detection of aflatoxin B1 (AFB1) using a Fab antibody fragment was developed. A key factor was the use of covalently immobilized AFB1 which allowed an almost three fold increase in sensitivity, reduced assay time and regeneration with retention of binding capacity. Various factors that might affect the sensitivity of the assay such as pH, organic solvents, storage stability and wash stringency were critically evaluated. It was also demonstrated that the assay was applicable for determination of AFB1 in corn samples at concentration within the European union regulatory limits.
Subject(s)
Aflatoxin B1/analysis , Antibodies, Immobilized/chemistry , Antibodies/chemistry , Immunoassay , Immunoglobulin Fab Fragments/chemistry , Zea mays/chemistry , Antibodies, Immobilized/biosynthesis , Antibody Affinity , Antibody Specificity , European Union , Hydrogen-Ion Concentration , Immunoglobulin Fab Fragments/biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Reproducibility of Results , Sensitivity and Specificity , SolventsABSTRACT
Botulinum neurotoxins (BoNTs) are classified as category A biological threat agents by the Centers for Disease Control and Prevention (CDC) in the United States for its hazardous and potential bioterrorist threat to the public. About 1% naturally occurring botulisms are caused by Botulinum neurotoxin serotype F (BoNT/F). Most of the immunoassays for detecting BoNTs focus on the serotypes A and B, but few methods have been established for the detection of BoNT/F. Recently, the recombinant Hc subunit of botulinum neurotoxin type F (rFHc) was expressed as an effective vaccine against BoNT/F, indicating that this rFHc could be an effective immunogen to raise monoclonal antibodies (MAbs) for the detection and neutralization of BoNT/F. Here we present a novel sandwich enzyme-linked immunosorbent assay (ELISA) based on two MAbs against rFHc, which were FMMU-BTF-8 and FMMU-BTF-29 as capture antibody and detection antibody, respectively. The limit of detection (LOD) of this ELISA reached 12.09 pg/mL, much less than that of the other reported immunoassays. A simple, sensitive ELISA for detecting and quantifying BoNT/F was established, which can be used as a valuable method to detect and quantify BoNT/F.
