Design of multiplexing lateral flow immunoassay for detection and typing of foot-and-mouth disease virus using pan-reactive and serotype-specific monoclonal antibodies: Evidence of a new hook effect

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

“…This was made by mapping the intensity of the test line as a function of the mAb-to-AuNP ratio and optical density. As previously observed for a similar LFIA for FMDV detection, the SE showed a saturation behavior, where the intensity of the test line decreased by increasing the 2F10* optical density ( Figure S3 and Table S2 ) [ 23 ]. The decrease in the signal, while increasing antigen concentration, occurs for sandwich-type immunoassays when the antigen amount exceeds the capture and/or detection antibodies.…”

“…We had previously observed a peculiar hook effect in the LFIA [ 23 ] and made the hypothesis that it was due to the antigen saturation caused by the excess of the labeled antibody. Therefore, we compared the cases of one antibody used as a capture and detector ligand (i.e., one epitope of the antigen is involved in the binding with both ligands, so that the saturation of the antigen by the detector impedes its subsequent capture by the reagent deposed on the test line) and two antibodies targeting different epitopes (where we expected that the two would not interfere reciprocally and the capture would not be hampered by the excess of the detector).…”

“…Though apparently antibodies can be employed for capture or detection of target antigens, optimizing the role of each bioreagent modulated the performance of the LFIA device [ 22 ]. In addition, other parameters, such as the antibody-to-label ratio, the amount of the labeled antibody, the concentration of the capture antibody, and the position of the test line play a crucial role in developing effective LFIA devices [ 22 , 23 ]. Typical sandwich-type immunoassays rely on the use of two antibodies, which specifically target different epitopes of the antigen.…”

“…In the eventuality that the pathogen shows repeated copies of the same antigen or that the antigen is composed of repeated molecular domains, a single antibody targeting the repeated epitope can be employed successfully to develop a sandwich-type immunoassay. In a previous work, we evidenced a different behavior between resorting to a “single epitope” (SE) targeting sandwich assay, i.e., one antibody for capture and detection, and using a “double epitope” (DE) targeting sandwich assay, where the capture and detection antibodies differed [ 23 ]. Indeed, we observed that for DE assays, the increase in the antibodies led to an increase in the analytical signal.…”

Identification of faecal extracellular vesicles as novel biomarkers for the non‐invasive diagnosis and prognosis of colorectal cancer

“…Nevertheless, with the increase in EV amount, the detection signal of A33 rose at first and then dropped when the concentration of fEVs exceeded 40 μg/100 μl (Supplementary Figure S2C). This phenomenon probably results from the “high‐dose hook effect” when the antigen concentration exceeds the antibody concentration (Cavalera et al., 2022; Erickson & Grenache, 2016). Accordingly, the protein dosage for unified EV detection was set to be 40 μg/100 μl in the subsequent clinical sample detection and analysis to avoid this hook effect.…”