Silent Antibodies Start Talking: Enhanced Lateral Flow Serodiagnosis with Two-Stage Incorporation of Labels into Immune Complexes

Abstract

The presence of pathogen-specific antibodies in the blood is widely controlled by a serodiagnostic technique based on the lateral flow immunoassay (LFIA). However, its common one-stage format with an antigen immobilized in the binding zone of a test strip and a nanodispersed label conjugated with immunoglobulin-binding proteins is associated with risks of very low analytical signals. In this study, the first stage of the immunochromatographic serodiagnosis was carried out in its traditional format using a conjugate of gold nanoparticles with staphylococcal immunoglobulin-binding protein A and an antigen immobilized on a working membrane. At the second stage, a labeled immunoglobulin-binding protein was added, which enhanced the coloration of the bound immune complexes. The use of two separated steps, binding of specific antibodies, and further coloration of the formed complexes, allowed for a significant reduction of the influence of non-specific immunoglobulins on the assay results. The proposed approach was applied for the serodiagnosis using a recombinant RBD protein of SARS-CoV-2. As a result, an increase in the intensity of test zone coloration by more than two orders of magnitude was demonstrated, which enabled the significant reduction of false-negative results. The diagnostic sensitivity of the LFIA was 62.5% for the common format and 100% for the enhanced format. Moreover, the diagnostic specificity of both variants was 100%.

Immunochromatographic enhancement strategy for SARS-CoV-2 detection based on nanotechnology

“…Sotnikov et al enhanced the coloration of immune complexes by using two separation steps based on conventional AuNP–LFIA. 75 The proposed method had a LOD of 10 ng mL −1 for IgG and the results demonstrated that the diagnostic sensitivity was 100% in the enhanced format. Thompson et al proposed a colorimetric LFIA for detecting SARS-CoV-2 IgG antibodies by using saliva samples with a LOD of 4 ng mL −1 , thereby providing a new approach for the non-invasive detection of SARS-CoV-2 antibodies.…”

Antibodies, repertoires and microdevices in antibody discovery and characterization

“…This allows readout by eye without the need for further equipment but does not enable quantification of antibody concentration in the sample and can yield ambiguous results at low antibody concentrations. 69,70 One recent approach to better quantify antibody concentrations was developed by preloading and immobilizing the antibodies in the testing chamber of a microfluidic chip and subsequently adding fluorescent polystyrene particles. 71 Using a smartphone-based optical readout, antibody concentration in saliva was quantified by either counting the number of unbound free-floating particles or measuring the capillary flow velocity affected by the number of unbound particles.…”

Electrophoretic Assembly of Antibody–Antigen Complexes Facilitates 1000 Times Improvement in the Limit of Detection of Serological Paper-Based Assay

“…As a result of these confines, most of IgG sp in conventional serological LFIA remains unbound to P GNP . A few enhancement and optimization strategies for serological LFIA have been proposed, but they greatly complicate the procedure and/or do not achieve the required LOD and, thus, the needed diagnostic sensitivity. − …”