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.
