The Effect of Nitrocellulose Membrane Pore Size of Lateral Flow Immunoassay on Sensitivity for Detection of Shigella sp. in Milk Sample

Abstract

Shigella flexneri is commonly detected in contaminated food and drinking water. Currently, a rapid and simple detection method to detect Sh. flexneri is lateral flow immunoassays (LFIAs). Nitrocellulose (NC) membrane is commonly used as a platform for reaction to occur in LFIAs and is classified according to their pore size. Thus, suitable pore size must be determined to produce the highest sensitivity of the test. In this study, three types of NC membrane were optimized for detection of Sh. flexneri. The citrate reduction method was used to synthesize 15 nm AuNPs seed followed by growth to 40 nm AuNPs. Transmission electron microscope (TEM) was used to confirm this monodisperse AuNPs and then conjugated with anti-gram-negative endotoxin monoclonal antibody. Three sets of NC membrane, HF90, HF120 and HF135 were used. These membranes were then immobilized with anti-Shigella sp. polyclonal antibody at 3.0 mg/mL as the test line and compared to0.5 mg/mL anti-mouse IgG monoclonal antibody. The spiked sample was prepared by adding 10-fold dilution of Sh. flexneri ATCC strain to milk samples. These LFIA was able to detect Sh. flexneri in milk sample as low as 3 x 106 CFU/mL whereby NC HF135 gave better line intensity and shape followed by HF120 and HF90, respectively. In HF90, the lowest detectable test line was hardly observed by naked eyes. The HF135 was the best choice as it gave better colour intensity and lines shape for detection of Sh. flexneri to monitor contamination in food.