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.
Highlights
In the context of animal health control and disease prevention it is fundamental to have access to efficient diagnostic tests capable of detecting early disease outbreaks and/or guaranteeing the absence of disease within large territorial extensions
The results showed that it is feasible to improve a test developed for single serum testing to pooled milk, by switching from an antibody-HRP conjugate to the ANANAS integrated detection method
It is worth noticing that, in other contexts and using model analytes, ANANAS integration was capable of improving sensitivity more than the value obtained here
Summary
In the context of animal health control and disease prevention it is fundamental to have access to efficient diagnostic tests capable of detecting early disease outbreaks and/or guaranteeing the absence of disease within large territorial extensions. Due to the large number of analyses that need to be routinely done within surveillance programs, such tests should be efficient, easy to use and, possibly, economical. High versatility and relatively low cost still make traditional microplate-based Enzyme Linked Immuno Sorbent Assay (ELISA) remaining the platform most widely used. Low antibody titers are common in some relevant veterinary diseases (e.g. during the prolonged phase of seroconversion to bovine paratuberculosis), or in pooled sample (sera or milk). Bovine bulk milk is a typical example of a pooled sample widely used in veterinary diagnostics. Its collection is very cheap and easy, helping to reduce surveillance program costs by permitting to certify, with one single test only, entire herds as “disease-free”
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