Investigation of the “Antigen Hook Effect” in Lateral Flow Sandwich Immunoassay: The Case of Lumpy Skin Disease Virus Detection

Abstract

Lumpy skin disease (LSD) is an infectious disease affecting bovine with severe symptomatology. The implementation of effective control strategies to prevent infection outbreak requires rapid diagnostic tools. Two monoclonal antibodies (mAbs), targeting different epitopes of the LSDV structural protein p32, and gold nanoparticles (AuNPs) were used to set up a colorimetric sandwich-type lateral flow immunoassay (LFIA). Combinations including one or two mAbs, used either as the capture or detection reagent, were explored to investigate the hook effect due to antigen saturation by the detector antibody. The mAb-AuNP preparations were optimized by a full-factorial design of experiment to achieve maximum sensitivity. Opposite optimal conditions were selected when one Mab was used for capture and detection instead of two mAbs; thus, two rational routes for developing a highly sensitive LFIA according to Mab availability were outlined. The optimal LFIA for LSDV showed a low limit of detection (103.4 TCID50/mL), high inter- and intra-assay repeatability (CV% < 5.3%), and specificity (no cross-reaction towards 12 other viruses was observed), thus proving to be a good candidate as a useful tool for the point-of-need diagnosis of LSD.

Lumpy Skin Disease (LSD): Etiology, Pathogenesis, Prevention and Control

“…The gold standard for diagnosing LSD virus is virus isolation, however using this method requires time to isolate and culture the virus from tissue or chorioallantoic membranes from embryonated chicken eggs (Amin et al, 2021). This is in accordance with the statement of Cavalera et al (2022) which stated that virus isolation is the gold standard for diagnosing LSD, but it takes several weeks to isolate the virus. However, according to the World Organization for Animal Health (WOAH) recommends using the VNT method as the gold standard in detecting antibodies to the LSD virus.…”

Development of Gold-Nanoparticle-Based Lateral Flow Immunoassays for Rapid Detection of TB ESAT-6 and CFP-10

“…In cases where the test line was visualized but with the faint control line, the antigen could be causing steric hindrance, blocking the binding site for the immobilized secondary antibody of the control line. The kinetics of solid-phase immobilized antibodies have demonstrated that equilibrium between antigen, capture and detection antibodies do not occur simultaneously, suggesting that steric hindrance plays a role in the detection-antigen-capture sandwich formation [ 22 ]. However in some instances, antibodies are able to re-organize to allow for further binding, and a high binding affinity may prevent this re-organization, causing saturation of binding sites at a faster rate [ 22 , 23 , 24 ].…”

“…The kinetics of solid-phase immobilized antibodies have demonstrated that equilibrium between antigen, capture and detection antibodies do not occur simultaneously, suggesting that steric hindrance plays a role in the detection-antigen-capture sandwich formation [ 22 ]. However in some instances, antibodies are able to re-organize to allow for further binding, and a high binding affinity may prevent this re-organization, causing saturation of binding sites at a faster rate [ 22 , 23 , 24 ]. Thus, in addition to target epitope compatibility, the association (k ass ) and dissociation (k off ) rate constants of the detection and capture antibodies may have a crucial role in defining suitable binding pairs.…”

Understanding the research advances on lumpy skin disease: A comprehensive literature review of experimental evidence

“…There is no cross-reactivity with other common bovine viruses ( Jiang et al, 2022 ). A rapid diagnostic tool of colorimetric sandwich-type lateral flow immunoassay (LFIA) was established using two monoclonal antibodies against different epitopes of P32 structural protein of LSDV and gold nanoparticles ( Cavalera et al, 2022 ). The sensitivity of this new method is similar to that of ELISA, but it has not been widely used in clinical diagnosis, and its specificity needs to be determined after clinical trials.…”