Fig. 1

Serological immunoassays with conventional detection techniques. (A) Colorimetric indirect ELISA: antigens of interest are immobilized on the microplate surface and incubated with diluted blood serum samples. Specific human antibodies (blue) are captured, and non-specific antibodies and proteins are removed with microplate washing. Enzyme-conjugated secondary anti-human antibodies (green) oxidize the substrate (yellow), and the absorbance of the product is measured by a spectrophotometer. Relative antibody titers are determined by the highest dilution of a positive blood serum sample that provided a positive result. (B) Lateral flow immunoassay: Specific antibodies in patient samples bind to an antigen immobilized on colloidal gold nanoparticles on a sample pad (S). Capillary flow transfers complexes to the conjugation pad, where the complexes interact with the immobilized anti-human secondary antibodies, aggregate, and precipitate at the test line (T). Precipitation of gold nanoparticles results in color change (red stripe). As a control for test completion, rabbit antibodies conjugated to gold nanoparticles travel along through the T region, interact with the goat-anti-rabbit antibodies at the control line C, precipitate, and result in color change. (C) Multiplex particle-based flow cytometry: Each antigen is conjugated to a bead of a unique “color” which is predetermined by a unique combination of ten infrared dyes at different concentrations. Beads are mixed and incubated with serum, and the antigens capture corresponding human antibodies. Secondary anti-human IgG antibodies are conjugated to a fluorescent dye (green) used for quantification. Particle-based flow cytometry utilizes two different lasers to detect the bead identity (red laser) and signal intensity (green) of a single bead passing through the detection region. To map antibody isotypes and subclasses across multiple antigens, the analysis is repeated with the secondary antibodies specific for human IgM, IgA, or IgG1-4