Abstract:

Objective To establish sensitive method for early diagnosis of Trichinella spiralis infection, polymeric gold nanorods were used to label the excretion and secretion antigens of T. spiralis. Methods Crude and purified antigens were extracted from adult T. spiralis, newborn larvae, and encapsulated larvae, and excretory and secreted antigens were prepared from larvae in muscle tissue of infected mice. We optimized the amount of cetyltrimethylammonium bromide (CTAB), ascorbic acid (AA), and silver nitrate (AgNO₃) in the procedure of gold crystal seed growth to yield of gold nanorods with stable length-diameter ratio. The gold nanorods were functionalized by coupling with different concentrations (10, 20, 30, 40, and 50 μg/ml) of mercaptosylated excreted/secreted antigens, worm crude antigen and worm purified antigen. Screening for optimum labeling concentrations and diagnostic target antigens was based on observation of the changes of surface plasma resonance absorption peak of labeled antigens by ultraviolet spectroscopic scanning. The serum antibody of mice with different infection intensity of encysted larvae (light: 50/mouse, medium: 100/mouse, severe: 300/mouse) was examined using optimal nanoro-labeled diagnostic antigen on 5, 8, 11, 17, and 23 d post-infection. Meantime, the serum antibody of severely infected mice were detected at different serum dilutions (1 ∶ 300, 1 ∶ 400, 1 ∶ 500, 1 ∶ 600, 1 ∶ 700, 1 ∶ 800); the results were compared with those assayed by ELISA with optimum coating antigen to evaluate the sensitivity in detecting early infection with low worm burden. Results Using 11.875 ml 0.2 mol/L CTAB, 160 μl 100 mmol/L AA, and 150 μl 10 mmol/L AgNO₃ in the growth media produced high length-diameter ratio and most stable solution of nanorods. The spectrophotometric spectrum of different concentrations of gold nanorod-labeled worm crude, purified, and excreted/secreted antigens showed a highest plasma resonance absorption peak of 87 nm, indicating the optimum antigen for coating gold-nanorod surface being excreted/secreted antigens. Using gold nanorod-labeled excreted/secreted antigens, serum antibody in mice with light, medium, and severe infection of encysted larvae could be detected on 5 d post-infection at 1 ∶ 800 dilution. By contrast, ELISA could detecte serum antibodies of mice with light and medium infection rate of encysted larvae on 11 d post-infection, while the serum antibody of mice with severe infection could be detected on 8 d post-infection, at 1 ∶ 600 dilution. Conclusion Gold nanorods can effectively label T. antigens, and the excreted/secreted antigen-functionalized gold nanorods show significant superiority in diagnosis of early infection and low worm burden of T. spiralis.

Key words: Trichinella spiralis, Trichinella spiralis worm antigens, Gold nanorod, Labeling technology, Diagnosis