ABSTRACT
Enzyme immunoassays (EIAs) are widely used in the clinical laboratory and research institutes for the detection of biologically relevant analytes. Almost all EIAs are heterogeneous in nature and require multiple steps of process. In contrast, homogeneous immunoassays (HA) offer a simplified one-step approach with a number of potential advantages over contemporary heterogeneous EIAs such as higher throughput and greater clinical utility. Utilizing TEM-1 beta-lactamase as a reporter enzyme, we have developed HAs based on in vitro protein fragment complementation (PCA) for the detection of antibodies and potentially be used for antigens or other biomarkers. In this proof-of-principle study we demonstrate the successful in vitro differentiation of anti-herpes simplex virus (HSV) type-1 and type-2 Immunoglobulin G (IgG) in human serum with high sensitivity and specificity.
Subject(s)
Antibodies, Viral/blood , Herpesvirus 1, Human/isolation & purification , Herpesvirus 2, Human/isolation & purification , Immunoenzyme Techniques , Immunoglobulin G/blood , Amino Acid Sequence , Antigens, Viral/chemistry , Antigens, Viral/immunology , Herpesvirus 1, Human/immunology , Herpesvirus 2, Human/immunology , Humans , Molecular Sequence Data , Peptides/chemistry , Peptides/immunology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Sensitivity and Specificity , Zinc/chemistry , beta-Lactamases/chemistry , beta-Lactamases/geneticsABSTRACT
We demonstrate a functional in vitro proof-of-principle homogeneous assay capable of detecting small (<1kDa) to large (150kDa) analytes using TEM-1 beta-lactamase protein fragment complementation. In the assays reported here, complementary components are added together in the presence of analyte and substrate resulting in colorimetric detection within 10-min. We demonstrate the use of functional mutations leading to either increased enzymatic activity, reduced fragment self-association or increased inhibitor resistance upon analyte driven fragment complementation. Kinetic characterization of the resulting reconstituted enzyme illustrates the importance of balancing increased enzyme activity with fragment self-association, producing diagnostically relevant signal-to-noise ratios. Complementation can be utilized as a homogeneous immunoassay platform for the potential detection of a range of analytes including, antibodies, antigens and biomarkers.
Subject(s)
Immunoenzyme Techniques , Peptide Fragments/chemistry , beta-Lactamases/chemistry , Calorimetry , Enzyme Inhibitors/pharmacology , Humans , Kinetics , Mutation , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/genetics , Protein Engineering , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , beta-Lactam Resistance/genetics , beta-Lactamase Inhibitors , beta-Lactamases/geneticsABSTRACT
The expression of human neuronal protein 22 (hNP22) is up-regulated in the superior frontal cortex of chronic alcoholics. hNP22 shares significant homology with a number of proteins implicated in bundling of actin filaments. In addition, it contains domains similar to those found in microtubule-associated proteins. We investigated the ability of hNP22 to induce cytoskeletal changes by overexpression in Chinese hamster ovary cells. Overexpression of hNP22 resulted in process formation in these cells that increased upon treatment with cytochalasin D, an actin depolymerising agent. Transfection of mutant hNP22 containing either a deletion of the putative actin-binding domain or deletion of a consensus protein kinase C (PKC) phosphorylation site (Ser-180) failed to induce process formation. In contrast, a mutation to mimic persistent PKC phosphorylation resulted in a cellular morphology similar to that seen in wild-type hNP22 transfections. This observation suggests that hNP22 requires phosphorylation at Ser-180 by PKC to induce cytoskeletal rearrangements. hNP22 was also observed to colocalise with actin and tubulin in processes of transfected cells. An hNP22-specific antibody specifically immunoprecipitated a complex including tubulin from human brain indicating that hNP22 binds directly to microtubules. Taken together, this data suggests that NP22 is part of a signaling complex that associates with cytoskeletal elements to regulate neuronal morphology.
Subject(s)
Actin Cytoskeleton/metabolism , Microfilament Proteins/metabolism , Microtubules/metabolism , Nerve Tissue Proteins/metabolism , Neurites/metabolism , Actin Cytoskeleton/drug effects , Animals , Blotting, Western/methods , CHO Cells/cytology , Cricetinae , Cricetulus , Cytochalasin D/pharmacology , Immunohistochemistry/methods , Immunoprecipitation/methods , Mutagenesis/physiology , Nerve Tissue Proteins/pharmacology , Neurites/drug effects , Nucleic Acid Synthesis Inhibitors/pharmacology , Protein Kinase C/chemistry , Protein Kinase C/metabolism , Serine/metabolism , Transfection/methodsABSTRACT
The action of alcohol on neuronal pathways has been an issue of increasing research focus, with numerous findings contradicting the previously accepted idea that its effect is nonspecific. The human NP22 (hNP22) gene was revealed by its elevated expression in the frontal cortex of the human alcoholic. The sequences of hNP22 and the rat orthologue rNP22 contain a number of domains consistent with those of cytoskeletal-interacting proteins. Localization of rNP22 is restricted to the cytoplasm and processes of neurons and it colocalizes with elements of the microfilament and microtubule matrices including filamentous actin (F-actin), alpha-tubulin, tau, and microtubule-associated protein 2 (MAP2). Withdrawal of Wistar rats after alcohol dependence induced by alcohol vapor produced elevated levels of rNP22 mRNA and protein in the cortex, CA2, and dentate gyrus regions of the hippocampus. In contrast, there was decreased rNP22 expression in the striatum after chronic ethanol exposure. Chronic ethanol exposure did not markedly alter rNP22 colocalization with F-actin, alpha-tubulin, or MAP2, although colocalization at the periphery of the neuronal soma with F-actin was observed only after chronic ethanol exposure and withdrawal. Rat NP22 colocalization with MAP2 was reduced during withdrawal, whereas association with alpha-tubulin and actin was maintained. These findings suggest that the effect of chronic ethanol exposure and withdrawal on rNP22 expression is region selective. Rat NP22 may affect microtubule or microfilament function, thereby regulating the neuroplastic changes associated with the development of alcohol dependence and physical withdrawal.
