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Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

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Development of a Sensitive Lateral Flow Immunoassay Using Recombinant Rabbit Single-Chain Variable Fragments Fused to

Ngoc Minh Nguyen¹, Ryosuke Mukai¹, Makoto Nita¹

¹Faculty of Molecular Chemistry and Engineering, Kyoto Institute of Technology, Kyoto 606-8585, Japan.

Analytical Chemistry

|December 15, 2025

Summary

We developed a novel lateral-flow immunoassay (LFIA) platform using recombinant antibody fragments fused with binding proteins for enhanced diagnostic performance. This innovation improves antigen detection sensitivity and reproducibility in point-of-care testing.

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Cell-Free Dot Blot as a Practical and Adaptable Immunoassay Platform for the Detection of Antibody Response in Human and Animal Sera

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Published on: May 23, 2025

Area of Science:

Biotechnology
Immunotechnology
Materials Science

Background:

Lateral-flow immunoassays (LFIAs) are crucial for rapid point-of-care diagnostics.
Traditional antibody immobilization on nitrocellulose (NC) membranes has limitations.
Enhanced immobilization strategies are needed for improved LFIA performance.

Purpose of the Study:

To develop an innovative LFIA platform using antibody fragments genetically fused with NC-binding proteins (NBPs).
To enhance antibody immobilization stability and orientation on NC membranes.
To improve the sensitivity and reproducibility of LFIAs.

Main Methods:

Screening of 21 candidate NBPs, identifying lactoferrin (LF) as superior.
Construction and expression of single-chain variable fragment-LF (scFv-LF) fusion proteins.
Utilizing complementarity-determining region (CDR)-grafting for scFv-LF variants.
Evaluating performance in dot blot, ELISA, and lateral flow assays.

Main Results:

Lactoferrin demonstrated robust and stable adsorption onto NC membranes.
scFv-LF fusion proteins showed high antigen-binding activity across a wide pH range.
CDR-grafted scFv-LF variants (e.g., B1R/C2R) significantly enhanced antigen activity and signal intensity.
The B1R/C2R scFv-LF fusion achieved a 100-fold lower detection limit for influenza B nucleocapsid protein.

Conclusions:

The scFv-LF fusion platform enables stable and oriented antibody immobilization on NC membranes.
This approach substantially enhances LFIA sensitivity and reproducibility.
The modular CDR-grafting strategy facilitates rapid customization for next-generation diagnostics.