Development of a Gold Nanoparticle-labeled Sandwich Format Lateral Flow Immunoassay Kit for the Detection of Tropical House Dust Mite Suidasia pontifica

Abstract

Background: The house dust mite Suidasia pontifica (Sp) is an important source of allergens in tropical regions that trigger IgE-mediated allergic reactions such as allergic asthma, atopic dermatitis and allergic rhinitis. Detection of Sp-specific proteins are important in the management and prevention of allergic diseases. Objective: The study aimed to provide a proof of concept for a gold nanoparticle-labeled sandwich format Lateral Flow Immunoassay (LFIA) kit for the detection of Sp-specific proteins. Methods: Protein A chromatography-purified rabbit anti-Sp polyclonal antibodies were labeled with gold nanoparticles (AuNP) synthesized from chloroauric acid using the citrate reduction method, then dispensed on a glass fiber pad. Unlabeled antibodies and anti-rabbit IgG were immobilized onto nitrocellulose membrane as test line and control line respectively. Cellulose fiber pad, glass fiber, and the nitrocellulose membrane pad were then assembled as LFIA kit. Results: Protein-A affinity chromatography purification with pre-concentration yielded 1.45 mg/mL of anti-Sp polyclonal antibodies. Synthesized AuNPs with ~20 nm sizes observed under transmission electron microscope were used for antibody conjugation at an optimal pH of 8.5 (borate buffer) and an optimal ratio of 10 µ L 50µg/mL antibody:100 µ L AuNP. Optimal color intensity and fastest migration time were observed with the treatment of 0.05% Tween20 and 10% sucrose in the conjugate pads; 5% BSA and 0.05% Tween20 in the sample pads, and 1% BSA in the test pads. The limit of detection of the LFIA Sp-specific proteins is 0.076 µg/mL. The sensitivity of the Sp LFIA kit is 83% while the specificity is 100%. Conclusion: This is the first report of a prototype for a cost-effective, rapid, and equipment-free detection of the house dust mite Suidasia pontifica.