A New Rapid Indirect ELISA Test for Serological Diagnosis of Feline Immunodeficiency

The aim of this work was the development of a new rapid indirect Enzyme-Linked Immunosorbent Assay (ELISA) assay, called FIVCHECK Ab ELISA, for the detection of antibodies against Feline Immunodeficiency Virus (FIV) in feline serum/plasma samples. FIV is a virus belonging to the Retroviridae family that affects feline immune cells, causing a progressive immunosuppression similar to the acquired immune deficiency syndrome (AIDS) in humans caused by human immunodeficiency virus (HIV). Diagnosis is usually performed by clinicians using rapid ELISA or lateral flow tests that detect FIV antibodies. The new test is the result of an extensive set-up phase carried out by the Research and Development (R&D) team of Agrolabo S.p.A., considering different concentrations of samples and reagents. In addition, the test cut-off was assessed and confirmed through several methods, including the Youden’s index and ROC curve, to achieve the best possible test performance in terms of sensitivity, specificity, accuracy, reliability, and repeatability of results. This study demonstrates that the FIVCHECK Ab ELISA has high sensitivity and specificity, making it also suitable for the analysis of highly lipemic or hemolytic samples, as no interferences were observed on the tested samples. In fact, FIVCHECK Ab ELISA was validated against the ELISA rapid test SNAP FIV/FeLV Combo (IDEXX) considered as reference: it agreed at 100%, with 100% sensitivity (95% confidence interval (CI): 88.5–100%) and 100% specificity (95% CI: 94.0–100%). Moreover, the performance of the new ELISA was compared with two other rapid indirect ELISAs widely used in veterinary practice, INgezim FIV (Gold Standard Diagnostics) and VetLine FIV (NovaTec). The FIVCHECK Ab ELISA agreed at 100% with INgezim FIV and 92.2% with VetLine FIV. Moreover, the new FIVCHECK Ab ELISA is an accurate and precise test with coefficients of variation lower than 10% both in intra- and inter-assays. The new test is fast as it provides correct and reliable results very quickly (25 min), without the need of particular laboratory equipment such as thermostats, because all incubations are performed at room temperature. Only the interpretation of results is performed instrumentally, by reading the OD values at 450 nm using an ELISA microplate reader (Multiskan SkyHigh, Thermo Fisher Scientific, Waltham, MA, USA). The new assay contains ready-to-use and safe reagents for the end-user and has a long-term storage up to 18 months at +2–8 °C. All these features make this test optimal for use in veterinary clinics and practices.