Multiplexed rapid antigen tests developed using multicolored nanoparticles and cross-reactive antibody pairs: Implications for pandemic preparedness

Description

We explore use of cross-reactive monoclonal antibody pair generated from a single immunization regimen, along with multicolored nanoparticles, to create rapid antigen diagnostics that specifically detect and distinguish related viruses.

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In Step 1, blue and gold nanoparticles were generated from gold salts and characterized by electron microscopy, zeta potential, and hydrodynamic diameter analyses. In step 2, mice were immunized with the Dengue virus serotype 3 NS1 protein, followed by creating hybridomas from spleen or lymph node cells, and testing hybridoma supernatants containing monoclonal antibodies in an ELISA format. Step 3 of the workflow confirms ELISA antibody-antigen binding data using the lateral flow chromatography format. In the example of the Dengue 3 NS1 immunization, three monoclonal antibodies (323, 411, 55) bound differentially to the viral NS1 proteins, as indicated by the serotype number (1-4) bound to the immobilized antibody. In Step 4, optimal monoclonal antibody pairs are used to create multiplexed tests where flow phase antibodies are conjugated to red nanospheres or blue nanostars, and membrane-adsorbed antibodies are applied at the test areas of the strip membrane. Chromatographing the test strip with ligand generates signal patterns corresponding to the red and blue nanoparticle-antibody conjugates captured at the two test areas. By using two test areas and two colored nanoparticles, the theoretical total number of antigens that could be distinguiushed in the assays is 16 (red, blue, red/blue or no color) for each of the two test areas. In Step 5, the combinations of test area red and blue nanoparticle colors, representing antibody binding, are deconvoluted into their red/green/blue (RGB) color components using the open-source imaging software ImageJ, followed by principal component analysis and data clustering in Step 6 to distinguish the ligands. Step 7 is data analysis using a confusion matrix to assess the efficacy of the approach in detecting and distinguishing the NS1 proteins of Dengue virus serotypes 1-4, using antibodies raised to a single antigen, i.e. the NS1 proteins of Dengue virus serotype 3. Methods used were lateral flow chromatography and principal component analysis to disambiguate color signals in each positive test.

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