Highly sensitive detection of species-specific malaria antigens using a cellulose nanobead-based lateral flow immunoassay.

Accurate and species-specific diagnosis of Plasmodium vivax malaria remains a significant challenge in malaria management due to the insufficient sensitivity and selectivity of existing rapid antigen tests. In this study, a highly sensitive and selective lateral flow immunoassay (LFIA) platform was developed utilizing blue-colored cellulose nanobeads (CNBs) as high-contrast colorimetric reporters for the detection of P. vivax lactate dehydrogenase (PvLDH). Six novel monoclonal antibodies with sub-nanomolar dissociation constants and exceptional specificity toward PvLDH were generated and characterized. Systematic screening identified an optimal antibody pair-clone #6 for detection (probe) and clone #4 for capture-which was subsequently incorporated into the CNB-based LFIA. The developed assay achieved limit of detection values of 5 ng/mL in buffer and 10 ng/mL in blood sample, representing a 50- to 100-fold improvement in sensitivity compared with conventional gold nanoparticle-based commercial RDTs. Notably, the distinct blue signal produced by the CNBs provided superior visual contrast against the red background of the blood sample, enabling clear, instrument-free interpretation. The assay exhibited no cross-reactivity with P. falciparum LDH or human LDH, thereby ensuring robust species-specific discrimination. Given its high sensitivity, portability, and strong performance in complex biological matrices, this CNB-based LFIA represents a promising next-generation point-of-care diagnostic tool for effective P. vivax malaria detection in endemic settings.