A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk

Abstract

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.

Improvement and extension of anti-EGFR targeting in breast cancer therapy by integration with the Avidin-Nucleic-Acid-Nano-Assemblies

“…A single NP contains ~ 50 avidins and 1200 biotin-binding sites (BBS) and has ~6000 nm 2 available for surface functionalization. 11 – 13 They can be functionalized with stoichiometric control by simply mixing with biotinylated functional elements at desired biotin:BBS molar ratios and no purification is necessary if neither the BBS become saturated nor the surface area available is exceeded 11 – 13 . Provided these two requirements are fulfilled, an infinite number of surface composition combinations can be conveniently generated and screened so that, in principle, selection of the best performing composition is relatively easy to perform.…”

“…In the two biotin-doxorubicin conjugates the drug was linked through an acid-reversible hydrazone bond and the derivatives differ for the length of the spacer between biotin and the drug (no spacer— biotin-Hz-doxo —or a 5 KDa PEG— biotin-PEG-Hz-doxo ). The 5 KDa PEG spacer in both the Atto488 and doxorubicin reagents was selected to maximize the NP PEG surface protection to guarantee long-term colloidal stability 10 , whereas the use of biotin-Hz-doxo permits to maximize drug loading by exploiting all of the available BBS, as steric impediments do not permit to saturate all of the available BBS with the bulky PEG reagent 11 – 13 . Finally, the 5 KDa spacer in biotin-PEG-cetux was selected to guarantee exposure of the targeting moiety at the NP outer surface.…”

“…ANANAS are nanosized ( Ø = 120 nm) poly-avidin toroids generated from the condensation of a nucleic acid (NA) filament by the high-affinity interaction with egg-white avidin (1 avidin each 14 ± 4 NA base pairs) 9 , colloidally stabilized by the presence at their surface of a controlled amount of biotinylated poly(ethylene glycol) (PEG) 10 . Thanks to their intact biotin-binding capability, ANANAS have found application as in vitro and in vivo diagnostics tools 11 – 13 and have been suggested as potential drug delivery systems 14 to implement more classic monomeric avidin-based targeted delivery strategies 15 . The high affinity for biotin ligands (Kd 10 −15 M) permits stoichiometric control of ANANAS composition, in principle allowing investigation on composition/functionality relationships.…”

Field application of an indirect gE ELISA on pooled milk samples for the control of IBR in free and marker vaccinated dairy herds

“…Indeed, previous studies demonstrated the importance of the within-pool prevalence and of the stage of lactation (variable levels of IgG) in the sensitivity of gE conventional assays [ 13 ]. Nevertheless, new strategies can be adopted to increase sensitivity in BM or PM serological investigations [ 14 ] as well as in the surveillance plan strategies [ 15 ]. In a recent study [ 10 ] we described a new approach for IBR surveillance, based on the use of a novel gE indirect ELISA applied to concentrated/purified BM samples.…”

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): infectious bovine rhinotracheitis (IBR)

“…First, the total number of samples tested was relative low, secondly the report included results for both commercial and in-house ELISA kits (with the former performing better) and finally the indirect kits used in the study have now largely been superseded by a new generation of highly sensitive kits (EFSA, 2006). In regard to gE ELISAs, much higher Se values (99-100%) have been reported (EFSA, 2006) while newer test methodologies, formats and antibody concentration techniques offer the possibility of increased Se values (Bertolotti et al, 2015;Casarin et al, 2016).…”