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Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA) remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as "disease-free". However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR) disease. The avidin-nucleic-acid-nanoassembly (ANANAS) is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA sensitivity limits without the need for new hardware investments.

The Avidin-Nucleic-Acids-Nano-Assembly (ANANAS) is a kind of soft poly avidin nanoparticle originating from the high affinity interaction between avidin and the nucleic acids. In this work we investigated the possibility of transforming ANANAS cores into stoichiometrically controlled multifunctional nanoparticles through a "one-pot" procedure, and we measured in a quantitative way their ability to work as reagents for enhanced immunodiagnostic detection. Initially, we measured the ANANAS loading capability for biotinylated proteins of different nature. About 200 molecules of biotin-horseradish-peroxidase (40KDa b-HRP) and 60 molecules of biotin-immunoglobulin-G (150KDa b-IgG) could be accommodated onto each nanoparticle, showing that steric limitations dictate the number of loadable entities. Stoichiometrically controlled functional assemblies were generated by mixing core particles with subsaturating amounts of b-HRP and b-IgG. When applied as detection reagents in an Enzyme-Linked-ImmunoSorbed-Assay (ELISA), these assemblies were up to two-orders of magnitude more sensitive than commercial HRP-based reagents. Assemblies of different composition displayed different efficacy, indicating that the system functionality can be fine-tuned. Within-assay variability (CV%), measured to assess if the assembly procedure is reproducible, was within 10%. Stability experiments demonstrated that the functionalyzed assemblies are stable in solution for more than one week. In principle, any biotinylated function can be loaded onto the core particle, whose high loading capacity and tunability may open the way toward further application in biomedicine.